資 料

29

30

31

32

33

34

35

53 巻 11 号（ 2013： 11）

36

37

38

39

40

41

53 巻 11 号（ 2013： 11）

42

43

44

45

Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration, 2016; 17: 38–46

Predictors of impaired communication in amyotrophic lateralsclerosis patients with tracheostomy-invasive ventilation

Downloaded by [Tokyoto Igaku Kenkyu Kiko], [Yuki Nakayama] at 16:22 29 February 2016

Yuki Nakayama1, Toshio Shimizu2, Yoko Mochizuki3,4,
Kentaro Hayashi2, Chiharu Matsuda1, Masahiro Nagao2,
Kazuhiko Watabe5, Akihiro Kawata2, Kiyomitsu Oyanagi6,
Eiji Isozaki2 & Imaharu Nakano2
1Laboratory of Nursing Research for Intractable Disease, Tokyo Metropolitan Institute of Medical Science, Tokyo,

Japan, 2Department of Neurology, Tokyo Metropolitan Neurological Hospital, Tokyo, Japan, 3Department of
Pathology, Tokyo Metropolitan Neurological Hospital, Tokyo, Japan, 4Department of Neurology, Tokyo Metropolitan
Kita Medical and Rehabilitation Center for the Disabled, 5ALS and Neuropathy Project, Tokyo Metropolitan
Institute of Medical Science, Tokyo, Japan, and 6Department of Brain Disease Research, Shinshu University Schoolof Medicine, Nagano, Japan

Abstract
Predictors of communication impairment in patients with amyotrophic lateral sclerosis (ALS) using tracheostomyinvasive ventilation (TIV) were investigated. Seventy-six ALS patients using TIV were enrolled and classified into
three subgroups of communication ability: patients who could communicate with communication devices (Stage I),
patients who had difficulty with communication (Stage II, III, or IV), and patients who could not communicate byany means (Stage V). Predictors of communication impairment were analysed by the Cox proportional hazard
model.
Results demonstrated that there were no significant differences in disease duration between subgroups. Within
24 months after disease onset, patients who needed TIV and tube feeding, developed oculomotor impairment or
became totally quadriplegic and progressed from Stage I to II and V significantly earlier. Multivariate analyses revealed
that within 24 months from onset, the need for TIV and progression to total quadriplegia were significant events inpatients who progressed to Stage II, whereas the development of oculomotor limitation was significant in patients who
progressed to Stage V. In conclusion, TIV, impaired oculomotor movement and total quadriplegia are predictors ofsevere communication impairment. Rapid disease progression might indicate future communication impairment
after the use of TIV. We highly recommend early detection of impaired communication and identification of the best
methods of communication.
Key words: Amyotrophic lateral sclerosis, ventilation, communication ability stage, predictors, communication impairment, totally
locked-in state

Introduction
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of upper and lower motor neurons.
Impaired speech is caused by bulbar or pseudobulbar palsy. Non-verbal communication may also beaffected due to quadriplegia and respiratory compromise. ALS ultimately results in respiratory failureand death, unless treated with mechanical ventilation. Use of non-invasive ventilation (NIV) may
improve survival temporarily (1). However, tracheostomy-invasive ventilation (TIV) supports breathing
and may extend survival for years, well beyond respiratory failure in patients with ALS (2). Therefore,

TIV is referred to as long-term mechanical ventilation (LTMV). Nevertheless, ALS patients using TIV
can achieve a reasonable quality of life (QoL) andare often grateful to be alive (3). Maintaining aneffective method of communication is a primary
requirement for achieving a reasonable QoL in ALS
patients using LTMV (4). In contrast, poor communication decreases QoL under the same circumstances. Difficulty in understanding patients with
impaired communication may also result in theinability to accurately assess their complaints andidentify appropriate care interventions, leading todelay in the effective management of care (5).

Correspondence: Y. Nakayama, Laboratory of Nursing Research for Intractable Disease, Tokyo Metropolitan Institute of Medical Science, 2-1-6
Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan. E-mail: nakayama-yk@igakuken.or.jp(Received 31 December 2014; accepted 19 April 2015)
ISSN 2167-8421 print/ISSN 2167-9223 online © 2015 Informa Healthcare
DOI: 10.3109/21678421.2015.1055276

46

Downloaded by [Tokyoto Igaku Kenkyu Kiko], [Yuki Nakayama] at 16:22 29 February 2016

Predictors of impaired communication in ALS
A study of the clinical profiles of Japanese patientswith sporadic ALS has shown a higher percentage(29.3%) using TIV compared with ALS patients inNorth America or Europe (2.1–5.4%) (6). Use of an
appropriate augmentative and alternative communication (AAC) system is essential for achieving
and maintaining effective communication (7). AAC
includes all forms of communication (other than oral
speech) that are used to express thoughts, needs,
wants, and ideas. Patients with ALS who have severe
speech impairment rely on AAC to supplement
existing speech or replace speech that is not functional. Approximately 13% of ALS patients with TIV
develop progressive immobility (8) and a totally
locked-in state (TLS) (9,10), which refers to a statein which all voluntary movements are lost and communication is impossible by any means. Recently,
various AAC devices and brain-machine interfaces(BMIs) have been developed and used by ALS
patients with severe communication impairment in
an attempt to communicate (7,11,12). Previously,
we determined that the use of an electroencephalography based BMI system was difficult for patientswith severe communication impairment (13).
In a previous study, we proposed a classificationof communication ability ranging from Stage I to
Stage V, and we analysed the clinical characteristics
of patients with ALS (14). Furthermore, the clinicalanalysis of 29 patients with autopsy-confirmed ALS
revealed that those individuals who had reachedStage V had begun to use TIV significantly earlier
than those patients classified with Stage IV or less intheir final assessment, and they frequently had afamily history of ALS. The aim of this study was toestablish the predictors of communication impairment in ALS patients with TIV by analysing the relationship between specific clinical findings and theprognosis for communication impairment.
Subjects and methods
Seventy-six patients with ALS who were using TIV
were retrospectively studied. Patients were selectedfrom two populations. The first group included
29 patients among the 132 in our hospital with
autopsy-confirmed ALS during the period 1980–
2011; they were examined previously and had
survived on TIV without overt dementia, anoxic
encephalopathy, or any other neurological complications (14). The second group included 53 patientswith ALS on TIV who had been continuously followed at our hospital from 2005 to 2012. Of the 53
patients, we excluded two with overt dementia thatappeared at the initial stages of ALS, and four
patients with concomitant Parkinsonism of unknown
cause. Finally, 47 patients were enrolled from thesecond group. All the enrolled patients showedrelentless progressive courses both before and after
ventilator use, and no contributory diseases otherthan ALS were found during follow-up. Among the39

second group of 47 patients, seven died and were notautopsied, and three dropped out of the study atStages I, II and V, respectively, over a period of five
years. Thus, 37 survivors among the 47 patientsremained at the end of March 2012.
The patients’ neurological status and their ability
to communicate were investigated based on a review
of medical and nursing records. Among 76 patientsenrolled, there were none with overt dementia on
routine neurological examination, although full investigations for cognitive and psychiatric function were
not performed in all patients. All patients showednormal orientation, reasonable conversation content,
and satisfactory decision-making. After administration of TIV, many of the patients used a communication board and showed reasonable communication
ability until occurrence of severe ophthalmoplegia,
although a precise evaluation of cognitive function
was not possible in late-stage patients. The ability tocommunicate with any AAC devices was assessed aspreviously reported in patients from each of thestages (I–V) as follows (Table I): Stage I, communicated using sentences; Stage II, communicated using
one word answers only; Stage III, only communicated using non-verbal yes/no responses; Stage IV,
only communicated occasionally with uncertain nonverbal yes/no responses; Stage V: unable to communicate by any means (14). At present, Stage V isindicative of TLS (9,10). When a patient’s ability tocommunicate fluctuated, the stage of communication ability at the time when their communication
remained stable was determined and used.
For clinical assessments, we evaluated the following factors: gender, age at ALS onset, affected body
region at onset, family history of ALS, genetic abnormalities upon examination, and disease duration.
The time from ALS onset to the following four clinical events was determined: need for TIV, feedingtube placement, development of overt oculomotor
limitation, and progression of immobility to total
quadriplegia. The overt oculomotor limitation was
defined as constant abnormal ocular movements
during a bedside neurological examination by atleast two neurologists. The evaluation for ocular
movements at the bedside included examinations ofslow pursuit movement and ocular saccade speed in
the vertical and horizontal gaze. The abnormalities
usually corresponded with severe vertical ophthalmoparesis and slow saccade (slow eye movement).
Table I. A classification of ALS clinical stages focusing on thecommunication ability.
Stage I
Stage II
Stage III
Stage IV
Stage V

communicates using sentences
communicates using one word answers only
only communicates using non-verbal yes/no
responses
only communicates occasionally withuncertain non-verbal yes/no responses
unable to communicate by any means­Revised from Table I in reference 14.

47

40

Y. Nakayama et al.

Total quadriplegia indicated that even slight muscle
movements were no longer possible.

Downloaded by [Tokyoto Igaku Kenkyu Kiko], [Yuki Nakayama] at 16:22 29 February 2016

Statistical analysis
Patients were classified into three subgroups according to their stage of communication ability justbefore death or at the end of the study. Group 1
consisted of patients who could communicate withAAC (Stage I). Group 2 consisted of patients who
had difficulty with communication (Stage II, III,or IV). Group 3 consisted of patients who could notcommunicate by any means (Stage V). Data comparisons among the three groups were performed
using the Kruskal-Wallis test or c2 test.
The analyses for the progression of communication impairment were performed by reclassifying thepatients into two subgroups according to the presence or absence of the aforementioned clinical events
24 months after ALS onset: A) need for TIV; B) needfor feeding tube; C) overt oculomotor limitation; andD) total quadriplegia. The rates of attenuated progression for communication ability from Stage I to
II and to Stage V were assessed for the four clinicalevents (A–D) using the Kaplan-Meier method ofanalysis and the log-rank test.
Finally, univariate and multivariate analyses were
performed for progression from Stage I to II and toStage V using the Cox proportional hazard model toelucidate the predictors for progression of the stageof communication ability, adjusting for age at onset,
affected body region at onset, familial or genetic
abnormalities, and the four clinical events discussed
previously.
All analyses were performed using Predictive
Analytics Software (PASW) Statistics version 21(IBM SPSS Statics) and p-values .05 were considered significant for all the statistical analyses.
This study was approved by the ethics
committees of Tokyo Metropolitan Neurological
Hospital and Tokyo Metropolitan Institute ofMedical Science.
Results
For all 76 patients, the median age at onset was 54.0
years (interquartile range (IQR) 44.3–63.8 years)
and median disease duration was 104.0 months(IQR 61.5–169.3 months). The clinical characteristics of each subgroup are described in Table II and
Figure 1. Among subgroups, there were no significant differences in gender, age at onset, affectedbody region at onset, total disease duration fromonset and TIV duration. Figure 1 shows that there
were many patients whose communication ability
was well preserved even after very long-term ventilator use. There were, however, significant differencesin the times from onset to the need for TIV, the needfor a feeding tube, and progression to total quadriplegia; these times were shortest for group 3(Table II). There were no statistically significant differences for other clinical events among subgroups.
The Kaplan-Meier analysis and log-rank test
(Figures 2, 3) showed that for the clinical eventsassessed 24 months after onset, the subgroup ofpatients who experienced them progressed to both
Stage II and V significantly earlier than the subgroupof patients for which the clinical events were absent
within 24 months after onset.
Using the Cox proportional hazard model, univariate analyses for the progression from Stage I
to II within 24 months after onset showed that age atTable II. Clinical characteristics based on stage of communication ability.

Patients, n (%)
Males, n (%)
Median age at onset, years (IQR)
Bulbar-onset patients, n (%)
Clinical event, n (%)
Familial ALS or sporadic ALS with genetic
abnormalities
Feeding tube
Overt oculomotor limitation
Total quadriplegia
Median disease duration, months (IQR)
Median TIV duration, months (IQR)
Median time from onset to clinical event, months (IQR)
Need for TIV
Need for feeding tube
Development of overt oculomotor limitation
Progression to total quadriplegia

Group 1
(Stage I)

Group 2
(Stages II-IV)

Group 3
(Stage V)

p-value *36 (47.3)
23 (63.9)
54.5 (47.3–61.0)
7 (19.4)

27 (35.5)
19 (70.4)
56.0 (49.0–68.0)
7 (25.9)

13 (17.2)
6 (46.2)
52.0 (38.5–60.5)
1 (7.7)

.328†
.187‡
.397†

4 (30.7).067†

3 (8.3)2 (7.4)32 (88.9)27 (100)
13 (100)
9 (25.0)25 (92.6)
13 (100)
15 (41.7)
21 (77.8)
13 (100)
104.0 (59.3–168.8) 86.0 (57.0–199.0) 126.0 (83.5–171.0)
54.0 (22.0–76.75) 66.0 (38.0–154.0)
102 (67.5–138.0).096†
.0001†
.0001†
.585‡
.061‡

56.5 (29.8–82.0)
56.0 (29.8–93.5)
84.0 (46.0–147.0)
78.0 (50.0–183.0)

.0001‡
.001‡
.107‡
.002‡

21.0 (17.0–36.0)
25.0 (15.0–61.0)
50.0 (29.5–115.0)
65.0 (43.5–126.0)

15.0 (12.5–25.5)
16.0 (12.0–29.0)
33.0 (16.0–69.0)
31.0 (22.0–55.5)­A LS: amyotrophic lateral sclerosis; IQR: interquartile range; TIV: tracheostomy- invasive ventilation; Group 1  patients who could
communicate with augmentative and alternative communication (AAC Stage I); Group 2  patients who had difficulty with communication(Stage II, III, or IV); Group 3  patients who could not communicate by any means (Stage V). * p-value .05 is significant.
†c2 test.
‡ Kruskal-Wallis test.

48

Downloaded by [Tokyoto Igaku Kenkyu Kiko], [Yuki Nakayama] at 16:22 29 February 2016

Predictors of impaired communication in ALS

41Group 1

1 ●
2
3
4 ●
5
6 ●
7
8
9
10
11 ●
12
13
14 ●
15 ●
16 ●
17 ●
18 ●
19 ●
20 ●
21 ●
22 ●
23 ●
24
25 ●
26 ●
27
28 ●
29 ●
30 ●
31 ●
32
33
34
35 ●
36 ●
37 ●
38
39 ●
40 ●
41 ●
42
43
44 ●
45 ●
46
47 ●
48
49
50 ●
51 ●
52 ●
53
54
55
56
57
58
59 ●
60
61
62
63 ●
64
65
66
67
68
69
70
71
72 ●
73
74 ●
75 ●
76

*

Group 2*

Stage I
Stage II
Stage III
Stage IV
Stage VGroup3

*

0

50

100

150

200

250

300

350

400

MonthsFigure 1. Disease duration and communication stage of 76 patients with ALS. Each stage of communication impairment is shownby a different grey colour. White lines in each column represent the initiation time of TIV. Closed circles represent patients who werealive at the end of March 2012, and asterisks represent patients who dropped out of follow-up.

onset, need for TIV, need for a feeding tube, development of overt oculomotor limitation and progression
to total quadriplegia were significant. Multivariate
analyses for the progression from Stage I to II within
24 months after onset showed that the need for TIV
and for development of total quadriplegia were significant (Table III). For progression to Stage V within

24 months after onset (Table IV), the univariate
analyses showed that progression in the stage of communication ability was significantly associatedwith the need for TIV, the need for a feeding tube,
development of overt oculomotor limitation, and theprogression to total quadriplegia. The multivariate
analyses showed that the development of overt
49

Downloaded by [Tokyoto Igaku Kenkyu Kiko], [Yuki Nakayama] at 16:22 29 February 201642

Y. Nakayama et al.

Figure 2. Kaplan-Meier curves showing the rate of attenuated progression to Stage II communication ability in patients with ALS.
Kaplan-Meier curves of the rate of non-progression to Stage II for 76 patients based on the presence (bold line) and absence (dottedline) of clinical events (A–D) 24 months after disease onset.  shows censored patients.

oculomotor limitation within 24 months after onset
had a significant effect on the progression to Stage
V.
Discussion
This study showed that a faster rate of progression
in the early phase of ALS predicted the rate of
impaired communication as the disease advanced.
The patients who reached Stage V not only began
TIV earlier (14) but also had a feeding tube andprogressed to total quadriplegia more rapidly thanpatients who did not reach Stage V. Since our goal
was to determine the predictors of impaired communication in the early phase of ALS, we focused on
specific clinical events within 24 months after ALS
onset. The use of TIV, need for a feeding tube, signsof overt oculomotor limitation and total quadriplegia, indicated by early progression of respiratory dysfunction, bulbar palsy, oculomotor limitation andlimb disability, are strongly related to progression of
impaired communication.
Previous epidemiological studies have shown several factors related to shorter survival in ALS: olderage (6,15,16), bulbar onset (15,16,17), a reduction
in forced vital capacity (16), a rapid reduction inbody mass index (18,19), a rapid decrease in therevised ALS Functional Rating Scale (ALSFRS-R)
score (16,20), and psychosocial factors (21). A high
ALSFRS-R score at TIV initiation also predicts along-term survival after TIV (22). These reported
prognostic factors were for survival of ALS; thus, theendpoints of studies are usually death or the needfor TIV (6,15–22). However, while TIV prolongs50

Downloaded by [Tokyoto Igaku Kenkyu Kiko], [Yuki Nakayama] at 16:22 29 February 2016

Predictors of impaired communication in ALS

43

Figure 3. Kaplan-Meier curves showing the rate of attenuated progression to Stage V communication ability in patients with ALS.
Kaplan-Meier curves of the rate of non-progression to Stage V for 76 patients based on the presence (bold line) and absence (dottedline) of clinical events (A–D) 24 months after disease onset.  shows censored patients.

survival, the disease progression never stops andoften shows a wide and multisystem distribution ofneurodegenerative lesions spreading to areas otherthan the motor system (9). Our patients with
TIV showed much longer survival lengths than previously reported (Table II) (23–28), and we needed
predictors not only for survival but also to aid in themaintenance of care interventions. Seriously disabled patients with TIV cannot be evaluated evenwith the extended version of ALSFRS-R (29). Thus,
our new classification of clinical stages focusing on
the degree of communication impairment is usefulfor such advanced patients with ALS.
In patients with ALS using TIV, TLS is referred
to as the most severe state of ALS, especially due tothe inability to communicate by any means (9,10).
However, only 17.2% of our patients in this study

developed TLS, which is a little higher than the 13%of patients previously reported in Japan (8). In otherwords, about 80% of ALS patients using TIV cancommunicate with appropriate devices for AAC. It isvery important to carefully assess ALS patients using
TIV and develop effective methods for maintaining
communication. Eye movement is the most important communication tool in such patients. Oculomotor limitation was a predictor for developing Stage V
in this study, and about 30% of patients using TIV
for more than nine years were reported to show ophthalmoplegia (6), indicating the importance of theoculomotor evaluation. The oculomotor limitation in
ALS patients with TIV is initially with a supranuclear
mechanism (30–32), but in the advanced stage ofTLS the brainstem oculomotor nucleus is also
involved neuropathologically (9,33), and is the focus

51

44

Y. Nakayama et al.



Table III. Univariate and multivariate analyses for progression to Stage II by Cox proportional hazard model.

Downloaded by [Tokyoto Igaku Kenkyu Kiko], [Yuki Nakayama] at 16:22 29 February 2016

Univariate analysis
GenderAge at onset
Affected body region at onset
Familial or genetic abnormalities

Time from onset to clinical event
Need for tracheostomy-invasive ventilation
Need for feeding tube
Development of overt oculomotor limitation
Progression to total quadriplegia
Multivariate analysis
Age at onset
Time from onset to clinical event
Need for tracheostomy-invasive ventilation
Need for feeding tube
Development of overt oculomotor limitation
Progression to total quadriplegia

Hazardratio

95% CI

p-value *males vs. females
younger vs. older than 65 years
non-bulbar vs. bulbar
non-familial or no genetic
abnormalities vs. familial or
genetic abnormalities

1.236
1.039
1.139
1.410

0.650, 2.350
1.007, 1.073
0.521, 2.491
0.590, 3.372

.517
.015
.745
.440

shorter vs. longer than 24 months
shorter vs. longer than 24 months
shorter vs. longer than 24 months
shorter vs. longer than 24 months

17.00615.2577.959
50.6696.445, 44.876
5.630, 41.344
3.273, 19.357
9.711, 264.369

.0001
.0001
.0001
.0001younger vs. older than 65 years

1.028

0.993, 1.065.116

shorter vs. longer than 24 months
shorter vs. longer than 24 months
shorter vs. longer than 24 months
shorter vs. longer than 24 months

7.762
1.871
1.904
67.0871.660, 36.297
0.434, 8.062
0.721, 5.030
6.634, 707.201

.009
.401
.194
.0001­CI: confidence interval*p  .05 is significant.

of the phosphorylated 43-kDa TAR DNA-binding
protein (pTDP-43) pathology (34,35).
The comparison between subgroups with
Stages I, II–IV and V, however, showed no significant differences in the total disease durations(Table II, Figure 1). This suggests that there might
be no continuity in the disease course between
Stages I and V, i.e. not all patients develop into
TLS, and some populations can remain in Stage I
even with long-term use of a ventilator. Our findings should be useful for those making decisions
about TIV initiation.

One of the limitations of this study is that a genesearch was not conducted in all cases. The univariate
analysis showed a non-significant effect of genemutations or a family history of ALS on progression
to Stage V (Table IV; p  0.051) as suggested by aprevious preliminary study (14). Further study isrequired to elucidate which type of gene abnormalities would cause severe communication impairment.
The other limitation is the lack of full evaluationsof cognitive function. Although we excluded thepatients with overt dementia at the early stages,
severe communication impairment at the advanced

Table IV. Univariate and multivariate analyses for progression to Stage V by Cox proportional hazard model.

Univariate analysis
GenderAge at onset age
Onset body region
Familial or genetic abnormalities
Time from onset to clinical event
Need for tracheostomy-invasive ventilation
Need for feeding tube
Development of overt oculomotor limitation
Progression to total quadriplegia
Multivariate analysis
Familial or genetic abnormalities
Time from onset to clinical event
Need for tracheostomy-invasive ventilation
Need for feeding tube
Development of overt oculomotor limitation
Progression to total quadriplegia

Hazardratio

95% CI

p-valuemales vs. females
younger vs. older than 65 years
non-bulbar vs. bulbar
non-familial or no genetic abnormalities
vs. familial or genetic abnormalities

2.180
0.993
0.378
3.266

0.730, 6.507
0.949, 1.040
0.049, 2.939
0.996, 10.706

.163
.777
.353
.051

shorter vs. longer than 24 months
shorter vs. longer than 24 months
shorter vs. longer than 24 months
shorter vs. longer than 24 months

15.63153.26722.47313.8213.934, 62.108
6.121, 463.532
4.866, 103.785
3.629, 52.633

.0001
.0001
.0001
.0001

non-familial or no genetic abnormalities
vs. familial or genetic abnormalities

1.344

0.239, 7.565.737

shorter vs. longer than 24 months
shorter vs. longer than 24 months
shorter vs. longer than 24 months
shorter vs. longer than 24 months

0.706
38.4037.034
1.964

0.013, 37.528
0.450, 3274.486
1.323, 37.393
0.331, 11.648

.863
.108
.022
.457

­CI: confidence interval.
*p .05 is significant.

52

Predictors of impaired communication in ALS

Downloaded by [Tokyoto Igaku Kenkyu Kiko], [Yuki Nakayama] at 16:22 29 February 2016stages might have masked the underlying dementia.
A considerable number of patients in Stage V show
severe brain atrophy on neuroimaging examinations(unpublished data), suggesting an underlying cognitive decline in Stage V that could not be detected
because of the severe motor disability.
In conclusion, based on our findings of potential
prognostic factors for progression to higher stages ofcommunication impairment, we highly recommend
early detection of any impairment of communicationand identification of optimal methods of communication for achieving the best possible QoL.­­­­
Acknowledgements
The authors are grateful to Pamela Cazzolli, RN at
ALS Care Project for her excellent suggestions inrelation to this study. This work was supported by
JSPS KAKENHI (Grant-in-Aid for Scientific
Research [B] No25293449), Grants-in-Aid from the
Research Committee of CNS Degenerative Diseases(2012 and 2013), the Ministry of Health, Labourand Welfare of Japan, and the Joint Program for ALS
Research, Tokyo Metropolitan Institute of Medical
Science.
Declaration of interest: The authors report noconflicts of interest. The authors alone are responsible for the content and writing of the paper.
References
1. Radunovic A, Annane D, Rafiq MK, Mustfa N. Mechanicalventilation for amyotrophic lateral sclerosis/motor neuron
disease. Cochrane Database Syst Rev. 2013;3: CD004427.
2. Cazzolli PA, Oppenheimer EA. Home mechanical ventilation for amyotrophic lateral sclerosis: nasal compared
to tracheostomy-intermittent positive pressure ventilation.
J Neurol Sci. 1996;139:123–8.
3. Moss AH, Oppenheimer EA, Casey P, Cazzolli PA, Roos RP,
Stocking CB, et al. Patients with amyotrophic lateral sclerosis receiving long-term mechanical ventilation. Advance careplanning and outcomes. Chest. 1996;110:249–55.
4. Cazzolli PA. The perspective of family and caregivers.
In: Ambrosino N, Goldstein R, editors. Ventilatory Support
for Chronic Respiratory Failure. New York: Informa Healthcare USA; 2008. pp 480–500.
5. Nakayama Y, Ogura A, Matsuda C. Physical outcomes ofpatients with amyotrophic lateral sclerosis using long-term
home mechanical ventilation. Jpn J Intract Illness Nurs.
2010;14:179–93 (Article in Japanese).
6. Atsuta N, Watanabe H, Ito M, Tanaka F, Tamakoshi A,
Nakano I, et al. Age at onset influences on wide-ranged
clinical features of sporadic amyotrophic lateral sclerosis.
J Neurol Sci. 2009;276:163–9.
7. Beukelman D, Fager S, Nordness A. Communication support for people with ALS. Neurol Res Int. 2011;2011:
714693.
8. Kawata A, Mizoguchi K, Hayashi H. A nationwide survey of
ALS patients on trachoestomy-positive pressure ventilation(TPPV) who developed a totally lockedin state (TLS) in
Japan. Rinsho Shinkeigaku. 2008;48:476–80 (Article inJapanese).

45

9. Hayashi H, Kato S. Total manifestations of amyotrophic
lateral sclerosis. ALS in the totally lockedin state. J Neurol
Sci. 1989;93:19–35.
10. Hayashi H, Oppenheimer EA. ALS patients on TPPV. Totally
locked-in state, neurologic findings and ethical implications.
Neurology. 2003;61:135–7.
11. Cipresso P, Carelli L, Solca F, Meazzi D, Meriggi P, Poletti B,
et al. The use of P300-based BCIs in amyotrophic lateralsclerosis: from augmentative and alternative communication
to cognitive assessment. Brain Behav. 2012;2:479–98.
12. McCane LM, Sellers EW, McFarland DJ, Mak JN,
Carmack CS, Zeitlin D, et al. Brain-computer interface(BCI) evaluation in people with amyotrophic lateral sclerosis. Amyotroph Lateral Scler Frontotemporal Degener.
2014;15:207–15.
13. Nakayama Y, Matsuda C, Ogura A, Haraguchi M,
Mochizuki Y, Nakamura Y, et al. The usability of anelectroencephalography-based communication device ‘neuro-communicator’ and nursing intervention for severely neurological or neuromuscular disorders. Jpn J Intract Illness
Nurs. 2013;17:187–203 (Article in Japanese).
14. Hayashi K, Mochizuki Y, Nakayama Y, Shimizu T, Kawata A,
Nagao M, et al. Communication disorder in amyotrophic
lateral sclerosis after ventilation: a proposal of staging and astudy of predictive factor. Rinsho Shinkeigaku. 2013;53:
98–103 (Article in Japanese).
15. Chiò A, Logroscino G, Hardiman O, Swingler R,
Mitchell D, Beghi E, et al. Prognostic factors in ALS: acritical review. Amyotroph Lateral Scler. 2009; 10:310–23.
16. Kollewe K, Mauss U, Krampfl K, Petri S, Dengler R,
Mohammadi B. ALSFRS-R score and its ratio: a useful predictor for ALS progression. J Neurol Sci. 2008; 275:69–73.
17. Pinto S, Pinto A, Carvalho DM. Do bulbar-onset amyotrophic lateral sclerosis patients have an earlier respiratory
involvement than spinal-onset amyotrophic lateral sclerosis
patients? Eura Medicophys. 2007;43:505–9.
18. Paganoni S, Deng J, Jaffa M, Cudkowicz ME, Wills AM.
Body mass index, not dyslipidemia, is an independent predictor of survival in amyotrophic lateral sclerosis. Muscle
Nerve. 2011;44:20–4.
19. Shimizu T, Nagaoka U, Nakayama Y, Kawata A,
Kugimoto C, Kuroiwa Y, et al. Reduction rate of body massindex predicts prognosis for survival in amyotrophic lateralsclerosis: a multicenter study in Japan. Amyotroph Lateral
Scler. 2012;13: 363–6.
20. Watanabe H, Atsuta N, Nakamura R, Hirakawa A,
Watanabe H, Ito M, et al. Factors affecting longitudinalfunctional decline and survival in amyotrophic lateral sclerosis patients. Amyotroph Lateral Scler Frontotemporal
Degener. 2014; Early Online.
21. Rabkin JG, Albert SM, Tider T, Del Bene ML, O’Sullivan I,
Rowland LP, et al. Predictors and course of elective longterm mechanical ventilation: a prospective study of ALS
patients. Amyotroph Lateral Scler. 2006;7:86–95.
22. Lo Coco D, Marchese S, La Bella V, Piccoli T, Lo Coco A.
The amyotrophic lateral sclerosis functional rating scale predicts survival time in amyotrophic lateral sclerosis patientson invasive mechanical ventilation. Chest. 2007;132:64–9.
23. Vianello A, Arcaro G, Palmieri A, Ermani M, Braccioni F,
Gallan F, et al. Survival and quality of life after tracheostomy
for acute respiratory failure in patients with amyotrophic
lateral sclerosis. J Crit Care. 2011;26:329.e7–14.
24. Spataro R, Bono V, Marchese S, La Bella V. Tracheostomy
mechanical ventilation in patients with amyotrophic lateralsclerosis: clinical features and survival analysis. J Neurol Sci.
2012;323:66–70.
25. Sanjuán LP, Valiño Ló P, Ricoy GJ, Verea H. Amyotrophic
Lateral Sclerosis: Impact of Pulmonary Follow-Up andMechanical Ventilation on Survival. A Study of 114 Cases.
Arch Bronconeumol. 2014;50:509–13.
26. Dreyer P, Lorenzen CK, Schou L, Felding M. Survival in
ALS with home mechanical ventilation non-invasively and

53

46

27.

28.

29.

Downloaded by [Tokyoto Igaku Kenkyu Kiko], [Yuki Nakayama] at 16:22 29 February 201630.

Y. Nakayama et al.
invasively: a 15-year cohort study in west Denmark. Amyotroph Lateral Scler Frontotemporal Degener. 2014;15:62–7.
Tagami M, Kimura F, Nakajima H, Ishida S, Fujiwara S,
Doi Y, et al. Tracheostomy and invasive ventilation in Japanese ALS patients: decision-making and survival analysis:
1990–2010. J Neurol Sci. 2014;344:158–64.
Veronese S, Valle A, Chiò A, Calvo A, Oliver D. The lastmonths of life of people with amyotrophic lateral sclerosis inmechanical invasive ventilation: a qualitative study. Amyotroph Lateral Scler Frontotemporal Degener. 2014;15:
499–504.
Wicks P, Massagli MP, Wolf C, Heywood J. Measuringfunction in advanced ALS: validation of ALSFRS-EX
extension items. Eur J Neurol. 2009;16:353–9.
Hayashi H, Kato S, Kawada T, Tsubaki T. Amyotrophic
lateral sclerosis: oculomotor function in patients in respirators.
Neurology. 1987;37:1431–2.


31. Shaunak S, Orrell RW, O’Sullivan E, Hawken MB, Lane RJ,
Henderson L, et al. Oculomotor function in amyotrophic
lateral sclerosis: evidence for frontotemporal impairment.
Ann Neurol. 1995;38:38–44.
32. Sharma R, Hicks S, Berna CM, Kennard C, Talbot K, Turner
MR. Oculomotor dysfunction in amyotrophic lateral sclerosis: a comprehensive review. Arch Neurol. 2011;68:857–61.
33. Shimizu T, Kawata A, Kato S, Hayashi M, Takamoto K,
Hayashi K, et al. Autonomic failure in ALS with a novel
SOD1 gene mutation. Neurology. 2000;54:1534–7.
34. Brettschneider J, Arai K, Del Tredici K, Toledo JB,
Robinson JL, Lee EB, et al. TDP-43 pathology and neuronalloss in amyotrophic lateral sclerosis spinal cord. Acta Neuropathol. 2014;128:423–37.
35. Ludolph AC, Brettschneider J. TDP-43 in amyotrophic
lateral sclerosis: is it a prion disease? Eur J Neurol. 2015;
22:753–61.

54

Amyotrophic Lateral Sclerosis

Sympathetic Hyperactivity and Sympathovagal Imbalance inAmyotrophic Lateral Sclerosis
Toshio Shimizu
Director, Department of Neurology, Tokyo Metropolitan Neurological Hospital, Tokyo, Japan

Abstract
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder with progressive loss of upper and lower motor neurons. Autonomic nervous
abnormalities, including sympathetic hyperactivity and sympathovagal imbalance, have been found in both early and advanced stages of ALS. In earlystage, the dysfunction may be subclinical. Occasionally, elevated blood pressure or heart rate and increased sweating may be observed. In advanced
stage when ventilators are required, the sympathetic hyperactivity may lead to hypertensive crisis without counter-regulation of heart rate, followedby the consecutive circulatory collapse, known as the ‘autonomic storm’. The symptoms of ‘autonomic storm’ are similar to that of ‘baroreflex failure’,and ‘autonomic storm’ indicates poor prognosis and may result in sudden death. Careful evaluation and individual treatment are strongly suggested,although appropriate therapeutic approaches have not been established. Causative central nervous lesions remain to be elucidated, although thelimbic system may be involved. The autonomic dysfunction further supports the concept that ALS is a multisystem-degenerative disease.

Keywords
Amyotrophic lateral sclerosis, autonomic nervous system, sympathetic hyperactivity, sympathovagal imbalance, baroreflex
Disclosure: The author has no conflicts of interest to declare.
Received: 2 April 2013 Accepted: 24 April 2013 Citation: European Neurological Review, 2013;8(1):46–50
Correspondence: Toshio Shimizu, Department of Neurology, Tokyo Metropolitan Neurological Hospital 2-6-1 Musashidai, Fuchu, Tokyo 183-0042, Japan.
E: toshio_shimizu@tmhp.jp

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorderwith selective and progressive loss of upper and lower motor neurons.
Although this disease was identified more than 140 years ago by Charcot,
the pathogenesis has yet remained unknown. Recent advancements inneuroimaging, neurophysiology and neuropathology have elucidated
subclinical or clinical involvements of the non-motor systems, in addition
to the upper and lower motor neurons. Accumulating evidence showsthat autonomic nervous abnormalities are found in both early andadvanced stages of ALS.1 The autonomic dysfunction may be clinicallyevident and devastating, especially in advanced stage when ventilators
are required. Here, the autonomic dysfunctions in ALS are reviewed.

In early stage of ALS, the sympathetic hyperactivity may be subclinical.
Patients with ALS do not usually show overt hypertension needing
treatment. However, some patients, especially, the ones with bulbar typeof ALS experience palpitation, facial flushness and hot sensation aroundthe face. These symptoms might be caused by increased sympathetic
tone. Haemodynamic studies have shown that compared to healthy
subjects, patients in early stage of ALS have higher resting heart rate andblood pressure.4–7 Plasma norepinephrine level was also reported to beelevated and not correlated with the extent of motor disability.8–12 Since
plasma norepinephrine level reflects the amount of norepinephrine
released from the peripheral sympathetic nerve terminals, its elevation
is an indirect indicator of increased sympathetic tone. Althoughintravenous norepinephrine infusion test has shown preserved
adrenoceptor function in the peripheral blood vessels, some patientson ventilators exhibit blunted response to infused norepinephrine,
indicating down-regulated hyposensitivity of peripheral adrenoceptors
induced by constantly high level of plasma norepinephrine.13

Clinical and Haemodynamic Studies
The autonomic nervous system is divided into sympathetic andparasympathetic divisions, each with a central and peripheral
component. The central component is known as the central autonomic
network and regulates the balance between the sympathetic andparasympathetic regulations of the visceral organs. The term ‘autonomic
failure’ is mainly used to describe impairment of the sympathetic
vasomotor efferent systems, including the intermediolateral nucleus(IML) of the spinal cord and sympathetic ganglion. Autonomic failure
represents orthostatic or postprandial hypotension or syncope attacks
caused by reduced sympathetic tone, which are often observed in
multiple system atrophy, but patients with ALS rarely develop such
symptoms. Contrary, cardinal autonomic dysfunctions in ALS are
sympathetic hyperactivity and sympathovagal imbalance, the clinicalmanifestations of which often resemble the symptoms of autonomic
overactivity or baroreflex failure.2,3 Differential diagnosis between
autonomic failure and baroreflex failure should be carefully performed,
since the therapeutic approaches for these two are quite different.3

46

Muscle Sympathetic Nerve Activity
Direct measurement of sympathetic activity is possible as muscle
sympathetic nerve activity (MSNA) by recording postganglionic nervous
impulses in peripheral nerves, using the microneurographic recording
technique. MSNA is considered to reflect sympathetic activity related
to cardiovascular control of vascular resistance. Many investigators
have reported that MSNA was increased in patients in early stage of
ALS.5,14–17 MSNA was elevated in ALS when quantified as the numberof sympathetic burst (bursts/min or bursts/100 beats).14,16 Responseof MSNA following head-up tilt, however, is less in patients with ALS
than in control subjects.17 Similarly, various stimulation techniques such
as Valsalva manoeuvre, apnoeic stimulation, painful stimulation, cold55

© To uch M Edical ME dia 2013

Sympathetic Hyperactivity and Sympathovagal Imbalance in Amyotrophic Lateral Sclerosis

Figure 1: Diurnal Variations of Blood Pressureand Heart Rate in a Patient in an Advanced
Stage on Ventilator

water immersion, glucose ingestion and lower body negative pressure,showed blunted responses of MSNA in patients having ALS.5,14 These
reduced responses were attributed to the ‘ceiling effect’ caused by
the constantly elevated sympathetic tone at resting state.14 The levelof MSNA in ALS did not correlate with motor disability, respiratory
function, or disease duration, suggesting a primary phenomenon as
well as motor neuron degeneration. Although Shindo et al. reported that
MSNA level gradually decreased with disease progression in individual
patients,15 chronological variation of MSNA remains in argument.14
In addition, the chronological improvement of MSNA is inconsistent
with the markedly increased sympathetic tone and autonomic storm inpatients at the advanced stage of ALS when ventilators are required.10

Blood pressure (mmHg) & heart rate (bpm)250

Variability of Heart Rate and Blood Pressure
Another effective technique to measure the sympathetic andparasympathetic function is the time and frequency domain analysis ofheart rate and blood pressure, especially the power spectrum analysis.
Heart rate variability (HRV) in the low-frequency (LF) band (0.04–0.15
Hz) is mediated by both sympathetic and parasympathetic influences,whereas oscillation in the high-frequency (HF) band (0.15–0.40 Hz)
is derived from cardiovagal modulation.18 A combination of HRV andblood pressure variability analysis can indicate not only baroreflex
sensitivity (for the LF band), but also functioning of cardiorespiratorytransfer (for the HF band).4

200
blood pressure
heart rate
150

100

50

0
14

15 16 1718 19

20 21 22 23

0

1

2

3

4

5

Time
Daytime hypertension with tachycardia was followed by nocturnal pressure falls without
counter-regulation of hear rate. Source: reproduced with permission from Shimizu et al., 1994.10

the presence of cardiac postganglionic sympathetic denervation.
However, there are no similar reports to date and their conclusionneeds further validation.24

Previous reports on HRV or power spectrum analysis have reported
that the abnormalities in ALS include increased sympathetic
tone or impaired cardiovagal function, both of which lead to thesympathovagal imbalance. Pisano et al. first reported the imbalanceof sympathovagal function due to an increase in LF/HF ratio.6 This
alteration was not related to the clinical features or disease duration.
Similar observations were made by other researchers.4,19–21 For
example, Linden et al. observed reduced baroreflex sensitivity anddiminished cardiorespiratory transfer during normal breathing in ALS
due to decreased LF and HF bands, respectively.4 These findings weresimilar to those reported for essential hypertension sharing a common
central autonomic derangement. By using sinusoidal neck suction
method to stimulate carotid baroreceptors, Hilz et al. showed impaired
cardiovagal response with preserved sympathetic vasomotor control.19
In addition, in early stage of ALS, patients with bulbar involvements
show more predominant cardiovagal dysfunction than patients with
non-bulbar involvement.20

Observations in Advanced Stage of Amyotrophic
Lateral Sclerosis
Although the abovementioned autonomic dysfunctions are usuallysubclinical, patients in most advanced stage of ALS, when ventilators
are used, often show critical autonomic manifestations. Marked
fluctuation of blood pressure and heart rate, namely ‘autonomic storm’,
may occur during clinically stable stage on ventilator. The autonomic
storm presents paroxysmal hypertensive crisis: more than 250 mmHgof systolic blood pressure and tachycardia without counter-regulationof heart rate (see Figure 1).10,13,25 During the hypertensive crisis, plasma
norepinephrine level is usually markedly high, indicating sympathetic
hyperactivity. The presence of tachycardia strongly suggests central
resetting of baroreflex sensitivity. Patients often exhibit facial
flushing, jaw clonus-like involuntary movements, pseudobulbar affect,
and disinhibition of emotional expression during the crisis. Theseobservations may suggest a limbic origin for the autonomic storm,
although the precise pathophysiology remains to be elucidated.

Other Observations
A limited number of reports have shown decreased sympathetic
outflow in patients with ALS. The corrected QT interval (QTc) on
electrocardiography (ECG), a measure of sympathetic influenceon the heart, was reported to be prolonged in length and increased
in dispersion in patients with ALS, indicating reduced sympathetic
activity.22 It should be noted that this report did not evaluate theextent of sympathetic tone and the co-existence of constantly
increased sympathetic tone and QT interval prolongation at theterminal stage of ALS does not seem contradictory. Another evidence
for impaired cardiac sympathetic function was provided by a123
I-metaiodobenzylguanidine (MIBG) uptake study by Druschky etal., which evaluated the function of the postganglionic sympathetic
terminals in the heart. These authors studied MIBG-single photon
emission computed tomography in early stage of ALS and founddecreased heart/mediastinum ratio (mean 1.82 ± SD 0.27) in patientswith ALS compared to normal controls (2.16 ± 0.26).23 They concluded

E u ropean N Eu rolo g ical REVIE W

The hypertensive stage is often followed by successive bloodpressure fall (circulatory collapse) without counter-regulationof heart rate, which may lead to sudden death. 10 The circulatory
collapse is likely to occur during sleep at night and hyposensitivity of
peripheral adrenoceptors might enhance the pressure decrease by asleep-associated decrement of sympathetic tone. 13 The adrenoceptor
hyposensitivity is probably attributed to its down-regulation due to theconstant high-level secretion of norepinephrine from sympathetic
nerve terminals.
All these features resemble symptoms of baroreflex failure, whosecardinal manifestations include hypertensive crisis, volatile or labile
hypertension, orthostatic tachycardia and malignant vagotonia.3
Similar symptoms are also found in the acute stage of Guillain-Barré
syndrome, which involves vagal afferent and sympathetic preganglionicefferent nerves.26,27 Differential diagnosis of baroreflex failure includes

56

47

Amyotrophic Lateral Sclerosis
Figure 2: Schematic Illustrations of theCentral Autonomic Network Inducible ofSympathetic Hyperactivity

patients in early stage of ALS had higher sweating rate in the palms,
potentially supporting the idea of increased central sympathetic tone in
ALS.33 However, they also reported a reduced sweating rate in advanced
stage of ALS, suggesting progressive peripheral sudomotor dysfunction
and resultant atrophy of sweat glands.
In addition, studies have electrophysiologically evaluated the sweat
function by using sympathetic skin response (SSR) and skin sympathetic
nerve activity (SSNA).34–38 SSR was reported to be abolished in 40 %of the patients in early stage of ALS and its latency was prolonged,
suggesting subclinical involvement of sudomotor fibres in ALS.35
Interestingly, in contrast to these reports of SSR, SSNA assessment
by microneurography showed high resting frequency and reduced
response to mental stress, accompanied by slight prolongation of
SSNA reflex latencies.34,38 These findings support basic sympathetic
hyperactivity of central origin. The reduced response may reflect the‘ceiling effect’ as in the case of MSNA.14 Both the vasomotor andsudomotor systems might be hyperactive, even though some patientsshow peripheral sudomotor hypofunction.

Neuropathology
Neuropathological studies on the autonomic dysfunction in ALS
have been limited. IML of the thoracic spinal cord, one of the mostefferent neurons of the baroreflex arc, was reported to be moderately
decreased in number in patients with ALS.39,40 This reduction has been
often considered as a potential cause of the sympathetic dysfunction,although the reduction of IML neurons itself does not explain theincrease in sympathetic tone. It may explain the reduced size ordelayed latency of SSR in ALS and it is not contradictory to the idea ofsympathetic hyperactivity shown in ALS. The reported reduction of IML
neurons in ALS is not severe and the remaining neurons are considered
hyperactive. Consistent with this, IML, dorsal vagus nucleus and solitary
tract nucleus are well preserved in most advanced stage of ALS,41,43
when a considerable number of patients who are on ventilator develop
ophthalmoplegia or a totally locked-in state.41,42

Source: reproduced with permission from Benarroch, 1993.46

central nervous disorders, psychological or metabolic diseases suchas pheochromocytoma and disorders of peripheral baroreflex
deafferentation. A recent study on patients having ALS with circulatory
collapse showed a quantitative preservation of vagal visceral branches,
excluding the possibility of peripheral deafferentation of baroreflex
arc.28 Although there have been reports of pathological involvementof peripheral sensory and small fibers in ALS,29,30 the autonomic storm
cannot be attributed to such peripheral nervous lesions.

There are a few reports on the neuropathology for sympathetic
hyperactivity, especially in the brainstem and the limbic system.
Shimizu et al. and Kato et al. reported that some patients with bloodpressure fluctuation in the advanced stage of ALS showed lesions in
the lateral hypothalamus, the central and basolateral nuclei of theamygdala and the brainstem reticular formation.10,43 It should be
noted that these pathological findings were variable and infrequentacross patients. The primary autonomic centres in the medulla
oblongata, however, were intact. Although the brainstem reticularformation was previously suggested to be primarily involved in ALS,44
Kato et al. observed preservation of catecholaminergic neurons in
the medullary reticular formation in patients with ALS and bloodpressure fluctuation.45

Disease Specificity of Autonomic Storm
The question to be resolved is whether the autonomic storm is primaryor secondary to ALS. The factors that should be considered are thelong-term bedridden state, severe muscle atrophy, long-term ventilator
support and psychological stress.10 At present, there is no evidence
that certainly supports or rules out these potential effects. However,autonomic storm in ALS is a very peculiar phenomenon that seldom
occurs in other neuromuscular disorders. Given that not all patients
who have ALS with a long-term use of ventilator and severe muscle
wasting develop autonomic storm and that ventilator-dependentpatients with Duchenne muscular dystrophy having similar muscle
atrophy do not usually show any circulatory fluctuation, the autonomic
storm may be primary to ALS.10

It will be difficult to neuropathologically elucidate the precise central
lesions corresponding to the clinical autonomic manifestations.
Generally, the central autonomic neural control is very complex andsubtle imbalance between excitatory and inhibitory pathways could
activate various autonomic nuclei and induce autonomic symptoms.46,47
If destruction of nervous tissue is severe, the lesions could produce
constant autonomic symptoms like orthostatic hypotension in multiple
system atrophy, but the fluctuations of symptoms may be attributed tomild alterations that cannot be detected by classic neuropathologicalmethodology. Recent advancement in TDP-43 proteinopathy for

Sudomotor Function
Patients with ALS often complain of increased or reduced sweating intheir hands or feet, altered skin temperature, or skin discolouration.
Studies of sudomotor function have reported that denervation of
sympathetic postganglionic cholinergic nerves could occur in ALS.
The sudomotor axon reflex test showed reduced sweating rate in
ALS, indicating mild postganglionic sudomotor dysfunction.31,32 On theother hand, Beck et al. documented that compared to healthy subjects,

48

57

Eu ropean N Eu rolo g ical RE VIE W

Sympathetic Hyperactivity and Sympathovagal Imbalance in Amyotrophic Lateral Sclerosis

sporadic ALS revealed a wide distribution of the protein aggregatesin the central nervous system,48 including the limbic system, also
called the ‘central autonomic network’ (see Figure 2).46 In addition, thelimbic motor and bulbo-respiratory motor systems are anatomicallyand physiologically linked to each other.49 Furthermore, ALS is often
accompanied by involvement of the frontotemporal lobes, which
overlaps the limbic system. However, there have been no reports on acorrelation between cognitive dysfunction and sympathetic tone, andfurther studies are needed.

with G93S mutation showed paroxysmal hypertension, prominent
sensory disturbances and pressure fall during sleep at night.52 The
norepinephrine level was very high and the bladder function showed
an overactive detrusor type. In addition, Kandinov et al. reported
a higher heart rate at rest and following stress in transgenic micecarrying a SOD1-G93A mutation than in wild-type mice.53 Bloodpressure elevated even before the appearance of motor dysfunction
and gradually decreased with the progress of the disease.54 Thus,
SOD1-related ALS may show variable phenotypes of autonomic
dysfunction across different genotypes.

Prognostic SignificanceTreatments

For patients in early stage of ALS, sympathetic hyperactivity rarely
influences survival. A major cause of death is usually respiratory
muscle paralysis, not circulatory collapse. Pinto et al., however,reported that decreased variability of heart rate might predict sudden
death (cardiac arrest) in ALS.50 Four patients in their report showedvery low values of the heart rate coefficient variation (<0.20), andthree of them died suddenly within the following two months, despite
normal nocturnal oxygenation. The prolongation of QT interval on ECG
may also be an indicator of poor prognosis.22 The autonomic storm
or circulatory collapse in advanced stage of ALS with ventilator use
predicts poor survival prognosis. Patients with hypertensive crisis
may die within several months, if not treated appropriately.10
Ventilator-dependent patients showing blood pressure fluctuation
should be continuously monitored for vital signs, especially at night,
because sudden pressure fall is more likely to occur during sleep.

Therapeutic approaches for the sympathetic hyperactivity in ALS
have not yet been established. Since most patients with ALS die from
respiratory failure, it has not been clarified whether treatment of thesympathetic hyperactivity would bring a better effect on diseaseprogression or survival. If the sympathetic tone is one of the prognostic
indicators, it should be treated by medicine such as beta-adrenergic
antagonists. No evidence, however, has been obtained so far. Avoiding
hypoxic episodes may reduce reflex sympathetic activation.55
Hypertensive crisis or circulatory collapse in advanced stage of ALS
should be immediately treated, given the severe and poor prognosis
it indicates. Direct vasodilators, such as calcium antagonists, however,should be avoided since these may induce critical over-reduction of
the blood pressure. Ohno et al. reported that tamsulosin hydrochloride,by acting as central alpha-adrenergic blocker, might modulate bloodpressure and reduce plasma norepinephrine level.9 Benzodiazepines
might also prove effective for centrally increased sympathetic tones
by potentially mediating central GABAergic functions.3,25 To establish an
appropriate therapy, further studies are needed.

Familial Amytrophic Lateral Sclerosis
Recent development of gene analysis in familial ALS has disclosed
many types of gene abnormalities. Although precise distributions ofdisease-specific central nervous lesions have not been elucidated ineach type of familial ALS, superoxide dismutase 1 (SOD1)-related ALS
may show variable symptoms and lesions across families or locationsof mutation. Some patients with SOD1-related ALS show severe
degenerative lesions in the autonomic nuclei, including IML, dorsal
vagus nucleus and solitary tract nucleus. Autonomic failure, suchas orthostatic or postprandial hypotension and atonic bladder, wasreported to develop in a sporadic case of SOD1-related ALS (V118L).51
Since cases of classical sporadic ALS never showed autonomic
failure, sporadic ALS and SOD1-related ALS must have pathogenic andphenotypic differences. Like classic sporadic ALS, SOD1-related ALS
may also show sympathetic hyperactivity. A ventilator-using patient

1.

2.

3.

4.

5.

6.

7.

8.

9.

Baltadzhieva R, Gurevich T, Korczyn AD, Autonomic
impairment in amyotrophic lateral sclerosis, Curr Opin Neurol,
2005;18:487–93.
Fagius J, Syndromes of autonomic overactivity: clinicalpresentation, assessment, and management. In: Low PA (ed.),Clinical Autonomic Disorders: evaluation and management,
Boston: Little, Brown & Co., 1992;197–208.
Ketch T, Biaggioni I, Robertson R, et al., Four faces ofbaroreflex failure: hypertensive crisis, volatile hypertension,orthostatic tachycardia, and malignant vagotonia, Circulation,
2002;105:2518–23.
Linden D, Diehl RR, Berlit P, Reduced baroreflex sensitivity andcardiorespiratory transfer in amyotrophic lateral sclerosis,
Electroencephalogr Clin Neurophysiol, 1998;109:387–90.
Oey PL, Vos PE, Wieneke GH, et al., Subtle involvement of thesympathetic nervous system in amyotrophic lateral sclerosis,
Muscle Nerve, 2002;25:402–8.
Pisano F, Miscio G, Mazzuero G, et al., Decreased heart ratevariability in amyotrophic lateral sclerosis, Muscle Nerve,
1995;18:1225–31.
Sachs C, Conradi S, Kaijser L, Autonomic function in
amyotrophic lateral sclerosis: a study of cardiovascularresponses, Acta Neurol Scand, 1985;71:373–8.
Chida K, Sakamaki S, Takasu T, Alteration in autonomic
function and cardiovascular regulation in amyotrophic lateralsclerosis, J Neurol, 1989;236:127–30.
Ohno T, Shimizu T, Kato S, et al., Effect of tamsulosin

E u ropean N Eu rolo g ical REVIE W

10.

11.

12.

13.

14.

15.

16.

17.

Conclusion
The cardinal features of the autonomic nervous dysfunction in ALS
are the sympathetic hyperactivity and sympathovagal imbalance.
Their clinical significance is obscured in early stage, but criticalin advanced stage of the disease, when ventilators are required.
Details of the involved central nervous lesions and appropriate
therapeutic approaches remain to be determined. No evidence has
been found to date of the pathophysiological correlation between theneurodegenerative process of ALS and the autonomic abnormalities.
Further studies may be needed to establish the pathognomonic
significance of autonomic dysfunction in ALS. nhydrochloride on sympathetic hyperactivity in amyotrophic
lateral sclerosis, Auton Neurosci, 2001;88:94–8.
Shimizu T, Hayashi H, Kato S, et al., Circulatory collapse andsudden death in respirator-dependent amyotrophic lateralsclerosis, J Neurol Sci, 1994;124:45–55.
Yamashita A, Koike Y, Takahashi A, et al., Relationship between
respiratory failure and plasma noradrenaline levels in
amyotrophic lateral sclerosis, Clin Auton Res, 1997;7:173–7.
Ziegler MG, Brooks BR, Lake CR, et al., Norepinephrine andgamma-aminobutyric acid in amyotrophic lateral sclerosis,
Neurology, 1980;30:98–101.
Shimizu T, Hayashi H, Hayashi M, et al., Hyposensitivity of
peripheral alpha-adrenoceptors in respirator-dependent
amyotrophic lateral sclerosis assessed by intravenous
norepinephrine infusion, Clin Auton Res, 1995;5:165–9.
Nygren I, Fagius J, High resting level and weak response ofbaroreflex-governed sympathetic outflow in amyotrophic
lateral sclerosis, Muscle Nerve, 2011;43:432–40.
Shindo K, Shimokawa C, Watanabe H, et al., Chronologicalchanges of sympathetic outflow to muscles in amyotrophic
lateral sclerosis, J Neurol Sci, 2004;227:79–84.
Shindo K, Tsunoda S, Shiozawa Z, Microneurographic analysis
of muscle sympathetic nerve activity in amyotrophic lateralsclerosis, Clin Auton Res, 1993;3:131–5.
Shindo K, Tsunoda S, Shiozawa Z, Increased sympathetic
outflow to muscles in patients with amyotrophic lateralsclerosis: a comparison with other neuromuscular patients,

58

18.

19.

20.

21.

22.

23.

24.

J Neurol Sci, 1995;134:57–60.
Anonymous, Heart rate variability. Standards of measurement,
physiological interpretation, and clinical use. Task Force of
the European Society of Cardiology and the North American
Society of Pacing and Electrophysiology, Eur Heart J,
1996;17:354–81.
Hilz MJ, Hecht MJ, Mittelhamm F, et al., Baroreflex stimulationshows impaired cardiovagal and preserved vasomotor
function in early-stage amyotrophic lateral sclerosis,
Amyotroph Lateral Scler Other Motor Neuron Disord,
2002;3:137–44.
Merico A, Cavinato M, Autonomic dysfunction in the earlystage of ALS with bulbar involvement, Amyotroph Lateral
Scler, 2011;12:363–7.
Pavlovic S, Stevic Z, Milovanovic B, et al., Impairment ofcardiac autonomic control in patients with amyotrophic
lateral sclerosis, Amyotroph Lateral Scler, 2010;11:272–6.
Asai H, Hirano M, Udaka F, et al., Sympathetic disturbances
increase risk of sudden cardiac arrest in sporadic ALS,J Neurol Sci, 2007;254:78–83.
Druschky A, Spitzer A, Platsch G, et al., Cardiac sympathetic
denervation in early stages of amyotrophic lateral sclerosis
demonstrated by 123I-MIBG-SPECT, Acta Neurol Scand,
1999;99:308–14.
Satoh A, Serita T, Seto M, et al., Loss of 123I-MIBG uptake
by the heart in Parkinson’s disease: assessment of cardiac
sympathetic denervation and diagnostic value, J Nucl Med,49

Amyotrophic Lateral Sclerosis

25.

26.

27.

28.

29.

30.
31.

32.

33.

34.

50

1999;40:371–5.
Shimizu T, Kato S, Hayashi M, et al., Amyotrophic lateralsclerosis with hypertensive attacks: blood pressurechanges in response to drug administration, Clin Auton Res,
1996;6:241–4.
Fagius J, Wallin BG, Microneurographic evidence of excessive
sympathetic outflow in the Guillain-Barré syndrome, Brain,
1983;106:589–600.
Pfeiffer G, Schiller B, Kruse J, et al., Indicators of dysautonomia
in severe Guillain-Barré syndrome, J Neurol,
1999;246:1015–22.
Shimizu T, Hayashi M, Kawata A, et al., A morphometric studyof the vagus nerve in amyotrophic lateral sclerosis
with circulatory collapse, Amyotroph Lateral Scler,
2011;12:356–62.
Hammad M, Silva A, Glass J, et al., Clinical, electrophysiologic,
and pathologic evidence for sensory abnormalities in ALS,
Neurology, 2007;69:2236–42.
Weis J, Katona I, Müller-Newen G, et al., Small-fiber neuropathy
in patients with ALS, Neurology, 2011;76:2024–9.
Kihara M, Takahashi A, Sugenoya J, et al., Sudomotor
dysfunction in amyotrophic lateral sclerosis, Funct Neurol,
1994;9:193–7.
Santos-Bento M, de Carvalho M, Evangelista T, et al.,Sympathetic sudomotor function and amyotrophic lateralsclerosis, Amyotroph Lateral Scler Other Motor Neuron
Disord, 2001;2:105–8.
Beck M, Giess R, Magnus T, et al., Progressive sudomotor
dysfunction in amyotrophic lateral sclerosis, J Neurol
Neurosurg Psychiatry, 2002;73:68–70.
Yamamoto K, Sobue G, Iwase S, et al., Skin sympathetic nerve
activity in amyotrophic lateral sclerosis, Rinsho Shinkeigaku,
1994;34:377–80 (in Japanese with English abstract).

35.

36.

37.
38.

39.

40.

41.

42.

43.

44.

45.

Dettmers C, Fatepour D, Faust H, et al., Sympathetic skin
response abnormalities in amyotrophic lateral sclerosis,
Muscle Nerve, 1993;16:930–4.
Masur H, Schulte-Oversohl U, Papke K, et al., Sympathetic skin
response in patients with amyotrophic lateral sclerosis, Funct
Neurol, 1995;10:131–5.
Miscio G, Pisano F, Sympathetic skin response in amyotrophic
lateral sclerosis, Acta Neurol Scand, 1998;98:276–9.
Shindo K, Watanabe H, Ohta E, et al., Sympathetic sudomotor
neural function in amyotrophic lateral sclerosis, Amyotroph
Lateral Scler, 2011;12:39–44.
Kennedy PG, Duchen LW, A quantitative study ofintermediolateral column cells in motor neuron disease andthe Shy-Drager syndrome, J Neurol Neurosurg Psychiatry,
1985;48:1103–6.
Takahashi H, Oyanagi K, Ikuta F, The intermediolateral nucleus
in sporadic amyotrophic lateral sclerosis, Acta Neuropathol,
1993;86:190–2.
Hayashi H, Kato S, Total manifestations of amyotrophic lateralsclerosis. ALS in the totally locked-in state, J Neurol Sci,
1989;93:19–35.
Hayashi H, Oppenheimer EA, ALS patients on TPPV: totally
locked-in state, neurologic findings and ethical implications,
Neurology, 2003;61:135–7.
Kato S, Oda M, Hayashi H, Neuropathology in amyotrophic
lateral sclerosis patients on respirators: uniformity anddiversity in 13 cases, Neuropathology, 1993;13:229–36.
Nakano I, Iwatsubo T, Hashizume Y, et al., Bunina bodies inneurons of the medullary reticular formation in amyotrophic
lateral sclerosis, Acta Neuropathol, 1993;85:471–4.
Kato S, Oda M, Hayashi H, et al., Decrease of medullarycatecholaminergic neurons in multiple system atrophy andParkinson’s disease and their preservation in amyotrophic

59

46.

47.
48.

49.

50.

51.

52.

53.

54.

55.

lateral sclerosis, J Neurol Sci, 1995;132:216–21.
Benarroch EE, The central autonomic network: functionalorganization, dysfunction, and perspective, Mayo Clin Proc,
1993;68:988–1001.
Loewy AD, Forebrain nuclei involved in autonomic control,
Prog Brain Res, 1991;87:253–68.
Geser F, Brandmeir NJ, Kwong LK, et al., Evidence ofmultisystem disorder in whole-brain map of pathological
TDP-43 in amyotrophic lateral sclerosis, Arch Neurol,
2008;65:636–41.
Holstege G, Descending motor pathways and the spinal
motor system: limbic and non-limbic components, Prog Brain
Res, 1991;87:307–421.
Pinto S, Pinto A, De Carvalho M, Decreased heart ratevariability predicts death in amyotrophic lateral sclerosis,
Muscle Nerve, 2012;46:341–5.
Shimizu T, Kawata A, Kato S, et al., Autonomic failure in
ALS with a novel SOD1 gene mutation, Neurology,
2000;54:1534–7.
Kawata A, Kato S, Hayashi H, et al., Prominent sensory andautonomic disturbances in familial amyotrophic lateralsclerosis with a Gly93Ser mutation in the SOD1 gene,J Neurol Sci, 1997;153:82–5.
Kandinov B, Korczyn AD, Rabinowitz R, et al., Autonomic
impairment in a transgenic mouse model of amyotrophic
lateral sclerosis, Auton Neurosci, 2011;159:84–9.
Kandinov B, Drory VE, Tordjman K, et al., Blood pressure
measurements in a transgenic SOD1-G93A mouse modelof amyotrophic lateral sclerosis, Amyotroph Lateral Scler,
2012;13:509–13.
Hecht MJ, Brown CM, Mittelhamm F, et al., Increased hypoxic
blood pressure response in patients with amyotrophic lateralsclerosis, J Neurol Sci, 2003;213:47–53.

Eu ropean N Eu rolo g ical RE VIE W

bs_bs_banner

Neuropathology 2014; 34, 504–509

doi:10.1111/neup.12130

Cas e Rep o r t

A Japanese patient with familial ALS and a
p.K510M mutation in the gene for FUS (FUS)
resulting in the totally locked-in state
Yoko Mochizuki,1,4 Akihiro Kawata,2 Hirofumi Maruyama,7 Taku Homma,1,5 Kazuhiko Watabe,3Hideshi Kawakami,8 Takashi Komori,1 Toshio Mizutani1,6 and Shiro Matsubara2
1

Department of Pathology, 2Department of Neurology, Tokyo Metropolitan Neurological Hospital, 3ALS and
Neuropathy Project, Tokyo Metropolitan Institute of Medical Science, 4Department of Neurology, Tokyo Metropolitan
Kita Medical and Rehabilitation Centre for the Disabled, 5Department of Pathology, Nihon University School of
Medicine, 6Fuchu Medical Centre for the Disabled, Tokyo, 7Department of Clinical Neuroscience and Therapeutics, and8
Department of Epidemiology, Research Institute for Radiation Biology and Medicine, Hiroshima University,
Hiroshima, JapanWe describe a Japanese patient with familial amyotrophic
lateral sclerosis (ALS) and a p.K510M mutation in thefused in sarcoma gene (FUS). The patient’s condition wascharacterized clinically by an early onset and rapid progression. The patient eventually required mechanical ventilation and progressed to the totally locked-in state.
Neuropathologically, multiple system degeneration withmany FUS-immunoreactive structures was observed. The
involvement of the globus pallidus, subthalamic nucleus,
substantia nigra, cerebellar efferent system, and both
upper and lower motor neurons in the present patient wascomparable to that described for ALS patients with different mutations in FUS, all of whom progressed to the totally
locked-in state. However, the patient also exhibited degeneration of the cerebellar afferent system and posterior
column. Furthermore, the appearance of non-compact
FUS-immunoreactive neuronal cytoplasmic inclusions andmany FUS-immunoreactive glial cytoplasmic inclusions
were unique to the present patient. These features suggestthat the morphological characteristics of the FUSimmunoreactive structures and distribution of the lesions
vary with the diversity of mutations in FUS.
Key words: basophilic neuronal cytoplasmic inclusions,familial amyotrophic lateral sclerosis, fused in sarcoma,
multiple systems involvement, totally locked-in state.

Correspondence: Yoko Mochizuki, MD, Department of Neurology,
Tokyo Metropolitan Kita Medical and Rehabilitation Centre for the
Disabled, 1-2-3 Jujodai, Kita-ku, Tokyo 114-0033, Japan. Email:mochi@nihon-u.ne.jp
Received 5 March 2014; revised and accepted 17 April 2014; published online 19 May 2014.

© 2014 Japanese Society of Neuropathology
60

INTRODUCTION
There have been accumulating reports of autopsiedcases of amyotrophic lateral sclerosis (ALS) in which
neuronal cytoplasmic inclusions (NCIs) and/or glial
cytoplasmic inclusions (GCIs) that are immunoreactive
for fused in sarcoma (FUS) appear in extensive areas
beyond the motor neuron system.1–8 It is noteworthy thatsome of these cases showed neuron and/or fiber loss in
multiple systems.1,3,6,7 In our previous report of two Japanese sisters with a p.P525L mutation in the gene for FUS(FUS),6 the elder sister progressed to a totally locked-instate9 (communication ability stage V10), required
mechanical ventilation, and showed severe degenerationin multiple systems, while the younger sister died in
the early stages of the disease. The findings fromneuropathological examinations revealed that theneuronal loss was preceded by the appearance of FUSimmunoreactive NCIs and that the characteristic featurein the elder sister was severe degeneration of the frontal
lobe and striatum.
Here, we describe another patient with familial
ALS who progressed to the totally locked-in state. The
patient had a p.K510M mutation in FUS and exhibited
degeneration of multiple systems. This is the firstreport of a detailed clinicopathological examinationof an ALS patient with a p.K510M mutation in FUS,
although ALS in a patient with a p.K510E mutationin FUS was reported clinically as early onset with involvement of the proximal muscles, a predominance oflower motor neuron signs, and rapid progression.11
We discuss the differences between previously describedpatients with mutations in FUS who progressed toFamilial ALS with FUS p.K510M mutation
the totally locked-in state and showed multiple systemdegeneration.

CASE REPORT

505
43 kDa (TDP-43), and optineurin (OPTN) were examinedby direct sequencing of PCR products.6
Genetic analysis identified a heterozygous missense
mutation of c.1529A>T at codon 510 (p.K510M) in FUS
but did not identify mutations in the exons or at the exon–
intron boundaries of the other genes.

Clinical summary
A 39-year-old Japanese man (Subject II-5 in Fig. 1) developed weakness in his left leg and then noticed fasciculationof his right leg. He developed quadriparesis, neck weakness, dysphagia and dysarthria. One year and 8 months
after the onset of the disease, he underwent a tracheostomy
with mechanical ventilation and a percutaneous endoscopic gastrostomy. Neurologically, the patient was alert
and the deep tendon reflexes were absent from all extremities. At 6.5 years after appearance of his first symptoms, hebecame quadriplegic with bilateral abduction paresis of theextraocular muscles, and he had difficulty in urinating.
Finally, voluntary movement disappeared totally and he
was unable to communicate by any means (communication
ability stage V10). He died of pneumonia at the age of 47,
8.5 years after onset.
The elder brother of the present patient (Subject II-1 in
Fig. 1) died of ALS, which had a similarly rapid progressive
clinical course, in his third decade.

Genetic analysis
DNA was extracted from the patient’s leukocytes by using
a conventional method after the patient provided informed
consent. All of the exons and exon–intron boundaries of
the gene for FUS, copper/zinc superoxide dismutase(SOD1), transactivation response DNA-binding protein of

METHODS
The specimens from the brain and spinal cord were fixedwith formalin and embedded in paraffin. Neuronal loss
and/or fiber loss and gliosis were assessed in various
regions of the nervous system by using 10-μm-thick sections and HE and KB stains. When necessary, Bodian,
Gallyas–Braak or Holzer staining was performed.
For immunohistochemistry, 6-μm-thick sections wereprepared. Sections from the frontal lobe, medial temporal
lobe of the hippocampus, and pons were immunostainedfor phosphorylated TDP-43 (p-TDP-43), using a rabbit
polyclonal antibody against phosphoTDP-43 (Cat. No.
TIP-PTD-P01 pS409/410-1; CosmoBio, Tokyo, Japan) at adilution of 1:3000. Sections from the cerebral lobe, basal
ganglia, cerebellum, brainstem, and spinal cord were
immunostained for FUS, using a rabbit polyclonal antibody
against FUS (Cat. No. HPA008784; Sigma-Aldrich Japan,
Tokyo, Japan) at a dilution of 1: 100. Prior to antibody
incubation, sections were treated with microwaving incitrate-buffered saline (pH 9.0, 95°C, 500 W, 15 min) for
antigen unmasking.Antibody binding was visualized by thelabeled streptavidin-biotin immunoperoxidase method.
The chromogen and counterstain were diaminobenzidine
and hematoxylin, respectively. Immunoreaction productdeposits were undetectable on sections from which theprimary antibodies were omitted.

Fig. 1 Pedigree of the family. The arrow
indicates the proband. The affected individuals are represented by the solid blacksquare.

© 2014 Japanese Society of Neuropathology
61

506

Y Mochizuki et al.

Pathological findings

lemniscus; (ii) neuronal loss with gliosis in the globus
pallidus, subthalamic nucleus (Fig. 2E), and substantia
nigra; (iii) neuronal loss in the Clarke’s nucleus, accessory
cuneate nucleus, and dorsal root ganglia, and fiber loss inthe middle root zone of the posterior column andspinocerebellar tract (Fig. 2A); (iv) neuronal loss withgliosis in the cerebellar dentate nucleus, red nucleus(Fig. 2C), and inferior olivary nucleus (Fig. 2D), and fiber
loss with many microglia in the superior cerebellar peduncles; and (v) neuronal loss in the pontine nuclei and fiber
loss with many microglia in middle cerebellar peduncles(Fig. 2C) and cerebellar white matter. One to threebasophilic NCIs, identified by HE staining (Fig. 2F), wereobserved in the pontine nuclei, caudate nuclei, cerebellar
dentate nucleus, globus pallidus, and frontal cortex on eachsection. The basophilic NCIs were stained with Bodian
staining. Some of the basophilic NCIs and GCIs weredetected with Gallyas-Braak staining. The basophilic NCIs

The brain weighed 1233 g. While the frontal lobe and cerebellum displayed mild atrophy, the brain stem including
the pontine base and the spinal cord showed marked
atrophy. The brain stem motor nuclei and the anterior hornof the spinal cord showed marked neuronal loss withfibrillary gliosis. Bunina bodies, basophilic NCIs andp-TDP-43-immunoreactive inclusions were undetectable in
the lower motor neurons. Betz cells were markedly
depleted, accompanying a few small groups of microglia in
the precentral cortex. The most remarkable finding of theexamination of the present patient was the involvement ofmultiple systems as follows (Table 1): (i) marked degeneration and atrophy of the anterior and lateral funiculi of the
spinal cord (Fig. 2A) and the brain stem tegmentum(Fig. 2B–D) including the reticular formation, superior
colliculi, superior cerebellar peduncles and medial
Table 1 Neuropathological findings

Degeneration
Motor neurons
Primary motor cortex
Corticospinal tract
Anterior horn of the spinal cord
Hypoglossal nucleus
Oculomotor nucleus
Spinal cord
Clarke’s nucleus
Spinocerebellar tract
Middle root zone of the posterior column
Intermediolateral horn
Anterior and lateral funiculi†
Basal ganglia and substantia nigra
Caudate nucleus
PutamenGlobus pallidus
Subthalamic nucleus
Substantia nigra
Cerebellum and related areas
Purkinje cells
Cerebellar dentate nucleus
Superior cerebellar peduncle
Red nucleus
Central tegmental tract
Inferior olivary nucleus
Pontine nucleus
Middle cerebellar peduncle
Cerebellar white matter
Cerebral cortex
Frontal cortex
Frontal white matter
Insular cortex
Temporal cortex
Primary sensory cortex

FUS immunoreactivities
NCIs

GCIs

+
++
+++
+++
+++

++
n
−
−
−

++
++
+
−
−

+++
+++
++
−
+++

+
n
n
−
n

−
−
−
−
−

−
−
++
+++
+++

+
+
+
n
+

+
++
+++
+++
+++

+
++
++
++
+
+
++
++
++

−
++
n
++
n
++
++
n
n

−
+
++
++
++
++
++
++
++

−
−
−
−
+

+
n
+
+
+

++
++
+
+
++

Degeneration: neuron and/or fiber loss assessed on HE-, KB-stained sections. The degeneration is indicated as absent (−), slight (+), mild (++),
or severe (+++). The FUS immunoreactivities are indicated as none (–), rare (+), occasional (++), or frequent (+++). † The region except thecorticospinal tract. FUS, fused in sarcoma; GCIs, glial cyoplasmic inclusions; n, not evaluated; NCIs, neuronal cytoplasmic inclusions.

© 2014 Japanese Society of Neuropathology
62

Familial ALS with FUS p.K510M mutation

507

Fig. 2 Neuropathological findings. (A) The fifth cervical spinal cord showed marked loss of nerve fibers in the corticospinal tract,spinocerebellar tract, anterolateral funiculus, and middle root zone of the posterior column. The anterior horn showed marked atrophy(arrowheads). (B) Marked degeneration was observed in the tegmentum of the midbrain. (C) Marked nerve fiber loss in the superior andmiddle cerebellar peduncles and transverse fibers, marked degeneration of the tegmentum, and mild atrophy of the corticospinal andcorticopontine tracts in the pons were observed. (D) The medulla oblongata showed marked degeneration. (E) The internal capsule, globus
pallidus (arrow), and subthalamic nucleus (arrowhead) showed fibrillary gliosis. (F) Basophilic neuronal cytoplasmic inclusion (NCI) in the
pontine nuclei. (G) The arrow indicates a fused in sarcoma (FUS)-immunoreactive NCI with a homogeneous morphology. Glial cytoplasmic inclusions (arrowheads) were observed in the pontine base. (H) Filamentous, FUS-immunoreactive NCI in the motor cortex. (A–D)
KB staining; (E) Holzer staining; (F) HE staining; (G, H) Immunostained for FUS. Scale bar = 0.5 cm (A–E) and 50 μm (F–H).

were immunoreactive for FUS. FUS-immunoreactive NCIs
and GCIs were numerous and widespread throughout thebrain (Table 1). Most of the FUS-immunoreactive NCIs
were the non-compact type and consisted of homogeneous(Fig. 2G), granular, or clustered thick filamentous materials(Fig. 2H). No FUS-immunoreactive intranuclear inclusions
were observed.

DISCUSSION
The disease progression in the present patient, who had ap.K510M mutation in FUS, was relatively early-onset withpredominantly lower motor neuron signs. This type of progression is similar to that of ALS patients with different
mutations in FUS.1,6,8,11–15 It is notable that the rapid progression, which led to the patient requiring mechanical ventilation and was followed by totally locked-in state, of thepatient’s clinical course was similar to the progression of
disease in two previously described ALS patients with different mutations in FUS (Table 2).1,6 This feature is consistent with the finding of our previous report,that patients with
ALS who reach communication stage V require mechanicalventilation significantly earlier than patients who remainable to communicate.10© 2014 Japanese Society of Neuropathology
63

508

Y Mochizuki et al.

Table 2 Clinicopathological summary of patients with ALS who progressed to a totally locked-in state and had a mutation in the FUS
Patient [reference number]Present patient[1][6] Patient 1

Mutation in FUS
Family history
GenderOnset age (years)
Start of respiratory assistance (months)
Duration from onset to the totally locked-in state (months)
Disease duration (months)
Initial symptom
Brain weight (g)Neuropathological features (neuron and/or fiber loss)
Motor neurons
Primary motor cortex
Corticospinal tract
Anterior horn of the spinal cord
Frontal cortex
Basal ganglia and substantia nigra
Striatum
Globus pallidus
Subthalamic nucleus
Substantia nigra
Cerebellum and related areas
Cerebellar dentate nucleus (cerebellar efferent system)
Pontine nuclel (cerebellar afferent system)
Middle cerebellar peduncle (cerebellar afferent system)
Limbic area
Subiculum
Entorhinal cortex
Posterior column
Clarke’s nucleus
Middle root zone of the posterior column
Basophilic neuronal cytoplasmic inclusions (NCIs)
Morphology of the FUS-immunoreactive NCIs
FUS-immunoreactive glial cytoplasmic inclusions

p.K510M
Yes
M
39
20
102
102
Lower extremities
1233

p.R521C
Yes
nd
31
9
48
168
Upper extremities
1070

p.P525L
Yes
F
13
18
120
312
Lower extremities
715

+
+++
+++
−

+
+++
+++
+++

++
+++
+++
++

−
+++
+++
+++

+
+++
+++
+++

+++
++
+++
+++

++
++
++

+++
+++
+

+++
++
+

−
−

nd
−

−
−

++
++
Rare
Non-compact
Many

+++
++
Many
Round
Many

−
−
Many
Small compact
Rare

ALS, amyotrophic lateral sclerosis; FUS, fused in sarcoma; nd, not described.

As shown in Table 2, the primary neuropathological features of the three patients was multiple system degeneration with many FUS-immunoreactive structures.1,6 All of
the patients showed involvement of the globus pallidus,
subthalamic nucleus, substantia nigra, and cerebellar efferent system, in addition to severe degeneration of both
upper and lower motor neurons (Table 2).1,6 The degeneration of the posterior column that was observed in thepresent patient was reported as the characteristic featureof an ALS patient with a p.R521C mutation in FUS(Table 2).1,3,7 Degeneration of the cerebellar afferent
system that involved not only the pontine nuclei but also
the middle cerebellar peduncles and cerebellar whitematter was seen only in the present patient. The emergenceof microglia in the cerebellar afferent system suggests thatthe degeneration started later here than in the otherlesions. On the other hand, the degeneration of the frontal
lobe and the striatum was only observed in the patient with
a p.P525L mutation in FUS.6
In the present patient, HE staining identified only a few
basophilic NCIs, known to be a hallmark of ALS with FUS

pathology, whereas many FUS-immunoreactive NCIs wereobserved. In ALS patients with mutations in FUS, who had
many basophilic NCIs,the FUS-immunoreactive NCIs were
small and compact in patients with a p.P525L mutation4,6
and round in patients with a p.R521C mutation (Table 2).1,3,7
In contrast to the basophilic NCIs, the non-compact, FUSimmunoreactive NCIs observed in the patient were difficult
to identify by HE staining. Recently, we examined an ALS
patient with a p.R521G mutation in FUS. The patient had
only a few subtle basophilic NCIs but many non-compact
FUS-immunoreactive NCIs.8 These findings suggest that
not all of the ALS patients with FUS pathology have abundant basophilic NCIs.Furthermore,both the present patient
and the reported patient with a p.R521C mutation in FUS1
had many FUS-immunoreactive GCIs, while the patientwith a p.P525L mutation6 had only a few of these.
It is possible that while multiple system degeneration ischaracteristic of patients who progress to the totally
locked-in state,9,10 the morphological characteristics of the
FUS-immunoreactive NCIs and the occurrence of FUSimmunoreactive GCIs may vary depending on the muta© 2014 Japanese Society of Neuropathology

64

Familial ALS with FUS p.K510M mutation
tion site in FUS. It is well known that the FUS gene islocated on chromosome 16p11.2, and that the gene product
FUS is a 53 kDa (526 amino acids) protein. The mutationsof the patients discussed in this report were all locatedwithin the proline/tyrosine-nuclear localization signal (PYNLS) domain of FUS. Patients with mutation near the
C-terminal of the PY-NLS (p.P525L) seemed to be
younger at the time of onset, progress to respiratory failure
more rapidly, and have more compact NCIs and fewer
GCIs than patients with mutation in the opposite side
(p.R521C, p.K510M).

ACKNOWLEDGMENTS
The authors are grateful to Ms. Yoko Suzuki, Mr. Yasuo
Sato and others in the laboratory at the Tokyo Metropolitan Neurological Hospital for their excellent technicalassistance. This work was supported by JSPS KAKENHI(Grant-in-Aid for Scientific Research (B)) Grant Number
22390429 and by the Joint Program for ALS Research at
the Tokyo Metropolitan Institute of Medical Science.

REFERENCES
1. Tateishi T, Hokonohara T, Yamasaki R et al. Multiple
system degeneration with basophilic inclusions inJapanese ALS patients with FUS mutation. Acta
Neuropathol 2010; 119: 355–364.
2. Rademakers R, Stewart H, Dejesus-Hernandez M
et al. FUS gene mutations in familial and sporadic
amyotrophic lateral sclerosis. Muscle Nerve 2010; 42:
170–176.
3. Kobayashi Z, Tsuchiya K, Arai T et al. Occurrenceof basophilic inclusions and FUS-immunoreactive
neuronal and glial inclusions in a case of familial amyotrophic lateral sclerosis. J Neurol Sci 2010; 293: 6–11.
4. Bäumer D, Hilton D, Paine SML et al. Juvenile ALS
with basophilic inclusions is a FUS proteinopathy with
FUS mutations. Neurology 2010; 75: 611–618.
5. Robertson J, Bilbao J, Zinman L et al. A novel double
mutation in FUS gene causing sporadic ALS.
Neurobiol Aging 2011; 32: 553.e27–553.e30.

509
6. Mochizuki Y, Isozaki E, Takao M et al. Familial ALS
with FUS P525L mutation: two Japanese sisters withmultiple systems involvement. J Neurol Sci 2012; 323:
85–92.
7. Suzuki N, Kato S, Kato M et al. FUS/TLSimmunoreactive neuronal and glial cell inclusions
increase with disease duration in familial amyotrophic
lateral sclerosis with an R521C FUS/TLS mutation. J
Neuropathol Exp Neurol 2012; 71: 779–788.
8. Mochizuki Y, Kawata A, Hashimoto T et al. An
autopsy case of familial amyotrophic lateral sclerosis
with FUS R521G mutation. Amyotroph Lateral
Scler Frontotemporal Degener 2014. doi:10.3109/
21678421.2014.881500
9. Hayashi H, Kato S. Total manifestations of amyotrophic lateral sclerosis. ALS in the totally locked-instate. J Neurol Sci 1989; 93: 19–35.
10. Hayashi K, Mochizuki Y, Nakayama Y et al. Communication disorder in amyotrophic lateral sclerosis after
ventilation. A proposal of staging and a study of predictive factor (in Japanese with English abstract).
Rinsho Shinkeigaku 2013; 53: 98–103.
11. Syriani E, Morales M, Gamez J. FUS/TLS gene mutations are the second most frequent cause of familial
ALS in the Spanish population. Amyotroph Lateral
Scler 2011; 12: 118–123.
12. Chiò A, Restagno G, Brunetti M et al. Two Italiankindreds with familial amyotrophic lateral sclerosis
due to FUS mutation. Neurobiol Aging 2009; 30: 1272–
1275.
13. Blair IP, Williams KL, Warraich ST et al. FUS mutations in amyotrophic lateral sclerosis: clinical, pathological, neurophysiological and genetic analysis. J
Neurol Neurosurg Psychiatry 2010; 81: 639–645.
14. Corrado L, Del Bo R, Castellotti B et al. Mutations of
FUS gene in sporadic amyotrophic lateral sclerosis. J
Med Genet 2010; 47: 190–194.
15. Yamamoto-Watanabe Y, Watanabe M, Okamoto K
et al. A Japanese ALS6 family with mutation R521C in
the FUS/TLS gene: a clinical, pathological and genetic
report. J Neurol Sci 2010; 296: 59–63.

© 2014 Japanese Society of Neuropathology
65

Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration, 2014; 15: 305–308

REPORT

An autopsy case of familial amyotrophic lateral sclerosis
with FUS R521G mutation

YOKO MOCHIZUKI1,5, AKIHIRO KAWATA2, TOMOYO HASHIMOTO1,6,7,
HARUHIKO AKIYAMA3, HIDESHI KAWAKAMI4, TAKASHI KOMORI1,
KIYOMITSU OYANAGI1,7, TOSHIO MIZUTANI1,8 & SHIRO MATSUBARA2
Departments of 1Pathology and 2Neurology,Tokyo Metropolitan Neurological Hospital, 3Project for Dementia,
Tokyo Metropolitan Institute of Medical Science, 4Department of Epidemiology, Research Institute for Radiation
Biology and Medicine, Hiroshima University, 5Department of Neurology, Tokyo Metropolitan Kita Medical
and Rehabilitation Center for the Disabled, 6Department of Neurology, University of Occupational andEnvironmental Health, 7Department of Brain Disease Research, Shinshu University, and8Fuchu Medical Center for the Disabled, Japan

Introduction
To date, several amyotrophic lateral sclerosis (ALS)
patients with the fused in sarcoma (FUS) gene mutation have been reported. Detailed clinicopathological investigations of patients with the FUS R521C
mutation have revealed the following clinical characteristics: weakness of the neck and proximal muscles (1–4) and neuropathological findings withmultiple system involvement, in addition to motor
neuron degeneration (1,4–6). Although the FUS
R521G mutation has been detected in familial ALS
patients (7,8), it is unknown whether their clinicopathological characteristics are the same as thosepatients with the FUS R521C mutation. Here, wereport the first detailed clinicopathological findingsof a familial ALS patient with the FUS R521G
mutation.
Case report
A 67-year-old Japanese female was aware of breathlessness. Eight months later, she developed weaknessof the upper extremities and body weight loss, followed by weakness of the neck. She was admitted toour hospital because of dysphagia and progressionof her symptoms 14 months after onset. Neurological examination revealed bulbar palsy, weakness ofneck muscles, proximal muscle weakness of upper
extremities, patellar hyperreflexia, and respiratory
insufficiency. No movement disorders, including

rigidity, tremor and akinesia were seen. She died of
respiratory failure after 15 months of illness. DNA
was extracted from the patient’s leukocytes withinformed consent. Sequence analysis of the FUS
gene (9) identified an arginine 521 to glycine(R521G) mutation. Her father had died of pneumonia with dysarthria at the age of 36 years, and herbrother died of ALS at the age of 33 years after one
year’s illness.
Post mortem examination showed a mildly swollen brain (weight 1395 g). Spinal cord and ventral
roots were slightly atrophic. Microscopically, theanterior horn (Figure 1A, B) and the hypoglossal
nucleus showed moderate neuronal loss (Table I).
In the precentral cortex, Betz cells were slightly
decreased and small groups of lipofuscin-laden
macrophages (Figure 1C) were scattered. Largesized myelinated fibers were slightly decreased in
the corticospinal tract. Mild neuronal loss was recognized in the substantia nigra (Figure 1D). Clarke’s
nucleus and posterior horn of the spinal cord showed
mild gliosis (Table I). Neither Bunina bodies nor
TAR DNA-binding protein 43-immunoreactive
structures were identified. Few subtle basophilic
neuronal cytoplasmic inclusions (NCIs) wereobserved in the anterior horn of the spinal cord
(Figure 1B) and the subthalamic nucleus. There
were ubiquitin-immunoreactive granular cytoplasmic inclusions (Figure 1E) in the anterior horn of
the spinal cord, while the round NCIs had no ubiq-

Correspondence: Y. Mochizuki, Department of Pathology, Tokyo Metropolitan Neurological Hospital, 2-6-1 Musashidai Fuchu-city, Tokyo 183-0042,
Japan. E-mail: mochi@nihon-u.ne.jp(Received 8 September 2013 ; accepted 6 January 2014 )ISSN 2167-8421 print/ISSN 2167-9223 online © 2014 Informa Healthcare
66
DOI: 10.3109/21678421.2014.881500

306 Y. Mochizuki et al.

Figure 1. Histological evaluation of clinicopathological features. A: Mild atrophy was observed at the eighth cervical cord (C8). The
anterior horn was mildly atrophic (Klüver-Barrera (KB) staining). B: Moderate neuronal loss and gliosis and subtle basophilic neuronalcytoplasmic inclusion (NCI) (arrow) were observed in the anterior horn at C8 (haematoxylin and eosin (HE) staining). C: Betz cells
were mildly decreased and small groups of lipofuscin-laden macrophages (arrow) were scattered (HE staining). D: Mild neuronal loss
was observed in the substantia nigra SN (KB staining). E: Ubiquitin-immunoreactive granular neuronal cytoplasmic inclusion (NCI)
was observed (ubiquitin: DAKO, 1:600). F: Fused in sarcoma (FUS)-immunoreactive Betz cell was observed (FUS: Sigma, 1:100).
G: FUS-immunoreactive NCI and FUS-immunoreactive glial cytoplasmic inclusion were observed. H: Several FUS-immunoreactive
NCIs were seen in the subthalamic nucleus. (E, G: anterior horn at C8; Bar in A and B  1 mm, Bar in B, C, E – H  50 μ m).

uitin immunoreactivity. FUS-immunoreactive NCIs
were found in Betz cells (Figure 1F) in addition tolower motor neurons (Figure 1G) and non-motor
neurons (Figure 1H). Their morphological characteristics included cytoplasmic staining (4) and granular or clustered thick filaments in the cytoplasm(Figure 1F–H). No FUS-immunoreactive intranuclear inclusions were observed.

neuronal loss (Table I). Autopsy reports on affectedfamily members of varying disease duration with
the FUS mutation showed that FUS-immunoreactive structures have an important role in degeneration (4,9). In the present patient, although thedisease duration was only 15 months without ventilatory support, degeneration of multiple systems
had already occurred beyond motor neurons. These
results suggested that degeneration started fromthe substantia nigra, which showed neuronal loss,
and extended to the subthalamic nucleus, which
had many FUS-immunoreactive structures. Furthermore, areas with occasional FUS-immunoreactive structures such as Clarke’s nucleus, themiddle root zone of the posterior column ofthe spinal cord, and the globus pallidus might beinvolved. These lesions were found in patients with
the FUS R521C mutation (Table II). However, the
FUS-immunoreactive NCIs in the present patientwere morphologically different from cytoplasmicround inclusions found in other patients with the

Discussion
The feature of rapid progression of proximal-dominant muscle weakness in our patient was similar
to that of patients with the FUS R521C mutation(Table II) (1–6). Neuropathologically, the present
patient showed mild neuronal loss in the substantia
nigra in addition to severe lower motor neuron
degeneration and mild upper motor neuron degeneration. Furthermore, FUS-immunoreactive NCIs
and glial cytoplasmic inclusions were observed not
only in these areas, but also in other areas with no
67

68


Put, Cau
Thal, LC,
Cerebel

LC
ND
ND

ND
ND
Cytoplasmic
staining

ND
ND
STN, GP, Put,
MRZ
GCIs
FUS immunoreactivities areas
without neuron or fibre loss

Cytoplasmic
staining

ND

307
LMN: lower motor neuron, UMN: upper motor neuron; *Degeneration: neuron and/or fibre loss and/or gliosis; NCIs: neuronal cytoplasmic inclusions; GCIs: glial cytoplasmic inclusions; SN: substantia nigra;
Clark: Clarke’s nucleus; STN: subthalamic nucleus; GP: globus pallidus; Put: putamen; MRZ: middle root zone of the spinal posterior column; ND: not described; Cau: caudate; Thal: thalamus; LC: locus
ceruleus; Cerebel: cerebellum and related areas; CerebC: cerebral cortex; * *Spinal cord: dorsal grey matter and white matter of the spinal cord.


Hippocampus

Cytoplasmic
inclusion
Earlier: Cytoplasmic staining,
Later: Cytoplasmic inclusion

ND
ND







Cytoplasmic
inclusion, or
granular,collections offi laments
Spinal cord* *

SN, STN, GP,
Clark, MRZ.
Cerebel, CerbC
ND
SN, STN, GP, Clark,
MRZ, Cerebel
Cuneate tract

SN, GP,
Put, Cau

SN, MRZ,
Purkinje cell

SN, GP Clark,
MRZ

LMN  UMN
LMN and UMN

Equivocal

The authors are grateful to Yoko Suzuki, Yasuo
Sato and others in the laboratory at Tokyo Metropolitan Neurological Hospital for their excellent
technical assistance. This work was supported by
JSPS KAKENHI (Grant-in-Aid for Scientific
Research (B)) and the Joint Program for ALS
Research, Tokyo Metropolitan Institute of Medical
Science.

Basophilic NCIs
FUS immunoreactivities
NCIs

Acknowledgement

ND

FUS R521C mutation (3,4–6), but were similar to
the early stage NCIs (4).
In this study, we describe several clinical andpathological similarities between patients with
the FUS R521G or FUS R521C mutation. Thesefindings suggest that replacement of amino acid521 arginine (R) to cysteine (C) or glycine (G)does not play a definite role in phenotypic difference in the pathologies seen with the above FUS
mutations.

SN, Clark,
posterior horn

The cerebral cortex except precentral cortex, hippocampus,
thalamus, cerebellum and related areas were preserved with no
FUS-immunoreactive structures.
Neuronal or fibre loss and gliosis were indicated as absent (),mild (), or moderate ().
FUS pathology was indicated as none (), occasional (), or
frequent (), n: unable to evaluate. NCIs: neuronal cytoplasmic
inclusions, GCIs: glial cytoplasmic inclusions.
*Regions except the corticospinal tract.

LMN only







LMN  UMN







LMN and UMN

 /
 /
 /
 /
/

LMN  UMN

Degeneration* besides motor
neuron systemMotor neuron degeneration*

 /

62
41




(5)

n

48
12
ND

/

42
36





30
108


n


28
24
ND

 /
 /

36
12





32
Alive
ND


n

ND
ND
ND

 /
 /

67
15





Onset (years)
Disease duration (months)
Proximally dominant weakness




R521C

/
 /

(4)




(1)

n


(8)

/
/

(7)



R521G



Present patient/FUS mutation
Patient (references)

Motor neurons
Primary motor
cortexCorticospinal tract
Anterior horn of the
spinal cord
Hypoglossal nucleus
Oculomotor nucleus
Spinal cord
Clarke’s nucleus
Middle root zone ofthe posterior
columnSpinocerebellar tract
Intermediolateral
horn
Anterior and lateralfuniculi*
Posterior horn
Basal ganglia andsubstantia nigra
Caudate nucleus
PutamenGlobus pallidus
Subthalamic nucleus
Substantia nigra

GCIs

Table II. Clinicopathological features of patients with fused in sarcoma (FUS) R521G mutation and autopsy confi rmed patients with FUS R521C mutation.

Neuron or fibre
loss/gliosis
NCIs

(6)

FUS
immunoreactivities

31
168


Table I. Distribution of lesions and fused in sarcoma (FUS)
immunoreactivities.

LMN and UMN

FALS with R521G mutation of FUS gene

308 Y. Mochizuki et al.
Declaration of interest: The authors report noconfl icts of interest. The authors alone are responsible for the content and writing of the paper.

5.

References

6.

1. Blair IP, Williams KL, Warraich ST, Durnall JC, Thoeng
AD, Manavis J, et al. FUS mutations in amyotrophic lateralsclerosis: clinical, pathological, neurophysiological andgenetic analysis. J Neurol Neurosurg Psychiatry. 2010;81:
639–45.
2. Corrado L, Del Bo R, Castellotti B, Ratti A, Cereda C, Penco
S, et al. Mutations of FUS gene in sporadic amyotrophic lateral sclerosis. J Med Genet. 2010;47:190–4.
3. Yamamoto-Watanabe Y, Watanabe M, Okamoto K, Fujita Y,
Jackson M, Ikeda M, et al. A Japanese ALS6 family with mutation R521C in the FUS/TLS gene: a clinical, pathological andgenetic report. J Neurol Sci. 2010;296:59–63.
4. Suzuki N, Kato S, Kato M, Warita H, Mizuno H, Kato M,
et al. FUS/TLS-immunoreactive neuronal and glial cell inclusions increase with disease duration in familial amyotrophic

7.

8.

9.

69

lateral sclerosis with an R521C FUS/TLS mutation. J Neuropathol Exp Neurol. 2012;71:779–88.
Rademakers R, Stewart H, Dejesus-Hernandez M, Krieger C,
Graff-Radford N, Fabros M, et al. FUS gene mutations infamilial and sporadic amyotrophic lateral sclerosis. Muscle
Nerve. 2010;42:170–6.
Tateishi T, Hokonohara T, Yamasaki R, Miura S, Kikuchi H,
Iwaki A, et al. Multiple system degeneration with basophilic
inclusions in Japanese ALS patients with FUS mutation. Acta
Neuropathol. 2010;119:355–64.
Kwiatkowski Jr TJ, Bosco DA, Leclerc AL, Tamrazian E,
Vanderburg CR, Russ C, et al. Mutations in the FUS/TLS
gene on chromosome 16 cause familial amyotrophic lateralsclerosis. Science. 2009;323:1205–8.
Sproviero W, La Bella V, Mazzei R, Valentino P, Rodolico C,
Simone IL, et al. FUS mutations in sporadic amyotrophic
lateral sclerosis: clinical and genetic analysis. Neurobiol Aging.
2012;33:837.e1–837.e5.
Mochizuki Y, Isozaki E, Takao M, Hashimoto T, Shibuya M,
Arai M, et al. Familial ALS with FUS P525L mutation: two
Japanese sisters with multiple systems involvement. J Neurol
Sci. 2012;323:85–92.

Clinical Neuropathology, Vol. 34 – No. 5/2015 (267-274)

Marked preservation of the visual andolfactory pathways in ALS patients in a
totally locked-in state©2015 Dustri-Verlag Dr. K. Feistle
ISSN 0722-5091
DOI 10.5414/NP300859
e-pub: June 3, 2015

Kiyomitsu Oyanagi1, Yoko Mochizuki2,3, Yuki Nakayama4, Kentaro Hayashi5,
Toshio Shimizu5, Masahiro Nagao5, Tomoyo Hashimoto6, Mineo Yamazaki7,
Shiro Matsubara5, and Takashi Komori2
1Division of Neuropathology, Department of Brain Disease Research, Shinshu

University School of Medicine, Matsumoto, Nagano, Departments of

2Pathology and 5Neurology, Tokyo Metropolitan Neurological Hospital, Fuchu, Tokyo,
3Department of Neurology, Tokyo Metropolitan Kita Medical and Rehabilitation

Center for the Disabled, Kita-ku, Tokyo, 4Laboratory of Nursing Research for
Intractable Disease, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku,
Tokyo, 6Department of Neurology, University of Occupational and Environmental
Health, Yahatanishi-ku, Kitakyushu, Fukuoka, and 7Department of Neurology,
Nippon Medical School Chiba Hokusoh Hospital, Inzai, Chiba, Japan

Key words
amyotrophic lateralsclerosis – totally
locked-in state – brainmachine interface – optic
pathway – olfactory
pathway

Abstract. Materials and methods: The
present paper examines the brains and spinal cords in 7 patients with amyotrophic
lateral sclerosis (ALS) receiving artificial
respirator support in a totally locked-in state(TLS) neuropathologically in order to clarify
whether any anatomical structures in the central nervous system are preserved. Results
and conclusion: We found that the visual andolfactory pathways, hypothalamus, nucleus
basalis of Meynert, and commissura anterior
were remarkably well preserved, whereas thesomatosensory, auditory, and gustatory pathways in the brain stem and/or spinal cord
showed severe deterioration.

Introduction
Received
March 10, 2015;
accepted in revised form
March 31, 2015
Correspondence toKiyomitsu Oyanagi, MD,
PhD
Division of Neuropathology, Department of Brain
Disease Research,
Shinshu University
School of Medicine,
3-1-1 Asahi, Matsumoto,
Nagano 390-8621,
Japan
k123ysm@shinshu-u.ac.jp

Amyotrophic lateral sclerosis (ALS) is
a disease mainly involving the upper andlower motor neurons in elderly humans.
Many affected patients develop difficulty incommunication due to weakness of the bulbar and skeletal muscles. For such patients,
many types of communication devices, suchas computers controlled by wrinkling thebrow muscles or blinking, and communication boards, have been developed to assist
augmentative and alternative communication (AAC). However, some ALS patients
who require respirator support for many
years fall into a totally locked-in state (TLS)70

as muscle weakness progresses. TLS is astate in which the patients cannot communicate by any means.
Recently, various brain-machine interfaces (BMIs) have been developed in an attempt
to communicate with totally locked-in ALS
patients by detecting alterations of intracerebral blood circulation or changes in electroencephalogram traces [1, 2, 3]. With regard
to input of information to the brains of TLS
patients, little is known about whether any
sensation is preserved in the patients, and noprevious study has investigated which anatomical pathways are preserved in the brain.
The present study was conducted to clarify
whether or not some neural pathways arepreserved in the brains of patients with ALS
who have fallen into a TLS.

Methods
The brains and spinal cords of the patients were fixed in 10% formalin, and multiple tissue blocks were embedded in paraffin.
Histological examinations were performedon 6-µm-thick sections. These sections were
stained with hematoxylin and eosin (H & E),
Klüver-Barrera (K-B), or periodic acid-Schiff(PAS), and immunostained for synaptophysin(mouse monoclonal, clone 171B5, dilution
1 : 200, MBL, Nagoya, Japan), phosphory-

268

V
V
L
U
–
–
FUS*4
n.e.
+
–
Familial ALS
ALS
M
M
47
69
8
5
6
3
41
65
39
64

13
52
57
60
38
1
2
3
4
5

6
7

71

yo = years old; yrs = years; F = female; M = male; ALS = amyotrophic lateral sclerosis; L = lower limb weakness; U = upper limb weakness; B = bulbar paresis; *1Fused in sarcoma P525L mutation,*2Cu/Zn superoxide dismutase (SOD1) Cys146Arg mutation; *3SOD1 Val118Leu mutation; *4Fused in sarcoma K510M mutation [8]; *5Ref. [6].

1,233
1,190

715
783
1,170
1,050
1,019

Pneumonia
Pneumonia
Colon cancer
Colon cancer
Unknown(Sudden death)
Pneumonia
Pneumonia
V
V
V
V
V
L
U
B
U
L
–
–
–
–
–
FUS*1
n.e.
SOD1*2n.e.
SOD1*3
+
–
+
–
–
F
F
M
M
F
40
66
68
69
46

Familial ALS
ALS
Familial ALS
ALS
ALS

GenderDuration of
TPPV
（yrs）
26
13
9
8
8
Start
TPPV
（yo）
14
53
59
61
38
Onset
（yo）
Patient
No.

Table 1.

Examined cases.

Duration ofillness
(yrs)
27
14
11
9
8

Death
（yo）

Clinicaldiagnosis

Familyhistory

Abnormalgene

Dementia

Initialsymptom

Communicative
ability stage*5

Cause of death

Brain weight（g)

Oyanagi, Mochizuki, Nakayama, et al.

lated neurofilament (SMI-31: mouse monoclonal, dilution 1 : 3,000, Covance, Berkley,USA), cystatin C (rabbit polyclonal, dilution
1 : 1000, DAKO, Glostrup, Denmark), SOD1
(mouse monoclonal, dilution 1 : 5,000, MBL,
Nagoya, Japan) or phosphorylated(p)TDP-43(mouse monoclonal, clone TIP-PTD-M01,
dilution 1 : 10,000, COSMO BIO, Tokyo,
Japan).
Immunohistochemical staining was
performed by the avidin-biotin-peroxidase
complex (ABC) method (Vectastain ABC
Elite kit, Vector, Burlingame, CA, USA).
Non-specific binding of the biotin-avidin
system reagents was blocked by pretreating
the sections with 0.3% hydrogen peroxide
in methanol and a normal blocking serum,
and then incubating them with the required
primary antibody overnight (for 17 hours) at
4 °C. The sections were then incubated for
1 hour with the secondary reagent containing
biotinylated anti-rabbit or anti-mouse IgG(diluted 1 : 200) at 37 °C, and finally with
the ABC solution for 1 hour at room temperature (RT). The sections were subjected toperoxidase reaction with 30 µL ImmPACTTMDABChromogen concentrate (Vector, Burlingame, CA, USA) (diluted 1 : 2, by 50 mM
Tris HCl (pH 7.6)) in 1 mL ImmPACTTM Diluent (Vector) for 5 minutes at RT.
Antigenicity was increased for SMI31
immunostaining by boiling the sections in
0.01 M citrate-buffered solution (pH 7.6) ina microwave oven (750 W, 25 min); and for
SOD1 and pTDP-43 immunohistochemistryby autoclaving (121 °C, 20 min) in 0.01 M
citrate buffer solution (pH 6.0).
As antibody controls, the primary antisera were either omitted or were replacedwith normal rabbit or mouse serum. The
preparations were examined by light microscopy.
The nomenclature used for the anatomical areas was basically that of “The HumanCentral Nervous System” [4], and for the detailed areas in the brain stem, that of “Cytoarchitecture of the Human Brain Stem” [5].

Patients
Seven Japanese sporadic and familial
ALS patients were examined neuropathologically. All patients were receiving positive269

Preservation of visual pathway in totally locked-in state ALS

Table 2.

Pathological features of various anatomical structures.
Patient 1

Patient 2

Patient 3

Patient 4

Patient 5

Patient 6

Patient 7

Visual pathway
Retina

N.E.

N.E.

N.E.

N.E.

N.E.

N.E.

N.E.

Nervus opticus

N.E.

N.E.

N.E.

N.E.

N.E.

N.E.

N.E.

Tractus opticus

O

O

O

O

O

O

O

Corpus geniculatum laterale

O

N.E.

O

O

O

O

N.E.

Colliculus superior

O

o

o

O

o

o

O

Radiatio optica

O

O

o

O

O

O

O

Area striata

O

O

O

O

O

N.E.

O

Epithelium olfactorium

N.E.

N.E.

N.E.

N.E.

N.E.

N.E.

N.E.
N.E.

Olfactory (limbic) pathway
Bulbus olfactorius

N.E.

O

O

N.E.

O

N.E.

Amygdala

O

N.E.

O

O

O

O

O

Hippocampus

O

O

O

O

O

O

O

Gyrus parahippocampalis

O

o

O

O

O

O

O

Ggyrus cinguli

o

o

O

o

O

O

O

Corpus mamillare

O

N.E.

O

O

O

O

N.E.

Fornix

O

X

O

O

O

O

O

Nucl. anterior thalami

O

o

O

O

o

O

O

Lobus frontalis

o

o

O

o

O

O

O

General sensory pathway (trigeminal and spinal)
Meissner‘s/pacinian corpuscles

N.E./N.E.

N.E./N.E.

N.E./N.E.

N.E./N.E.

N.E./N.E.

N.E./N.E.

N.E./N.E.

Ganglion trigeminale/ganglion spinale

N.E./N.E.

N.E./N.E.

N.E./N.E.

N.E./N.E.

N.E./N.E.

N.E./N.E.

N.E./N.E.

Radix sensorius nervi trigemini/radix dorsalis nervi spinalis/Nucl. proprius

N.E./O

N.E./O

N.E./N.E.

N.E./N.E.

N.E./N.E.

N.E./N.E.

O/N.E.

/O

/O

/X

/X

/X

/o

/O

/Tractus spinothalamicus/tractus
spinoreticularis/funiculus posterior
Nucl. sensorius principalis nervi trigemini/
nucl. spinalis nervi trigemini/nuclei gracilis
and cuneatus
Lemniscus trigeminalis/lemniscus medialis/X/X/o/X/X/o/X/X/X/X/X/X/X/X/X/o/o/o/o/o/O

X/X/N.E.

X/X/N.E.

X/X/N.E.

X/X/N.E.

X/X/N.E.

o/N.E./N.E.

o/o/N.E.

o/O

X/X

X/X

X/X

X/o

X/X

o/o

Nucl. VPM thalamus/nucl. VL thalamus

X/X

O/O

O/O

o/o

X/X

o/o

O/O

Gyrus postcentralis

o

o

O

o

O

O

O

Auditory pathway
Tympanum

N.E.

N.E.

N.E.

N.E.

N.E.

N.E.

N.E.

Cohlea

N.E.

N.E.

N.E.

N.E.

N.E.

N.E.

N.E.

Nervus cochlearis

N.E.

N.E.

N.E.

N.E.

N.E.

N.E.

N.E.

Nucl. cochlearis dorsalis

O

X

o

o

o

X

o

Corpus trapezoideum

o

X

X

X

X

X

o

Nucl. olivaris superior

o

X

X

X

X

X

o

Lemniscus lateralis

o

X

X

X

X

X

o

Colliculus inferior

o

X

X

X

X

o

N.E.

Corpus geniculatum medialis

o

O

O

O

N.E.

N.E.

X

Gyrus temporalis transversus

o

o

O

O

O

O

O

Other anatomical areas
Commissura anterior

N.E.

O

O

O

O

O

O

Nucl. basalis of Meynert

o

o

O

O

o

O

O

Nucl. paraventricularis

O

o

O

O

o

O

N.E.

Nucl. supraopticus

O

o

O

O

o

O

O

O (large circle) = well preserved; o (small circle) = slight loss of neurons or myelinated fibers; X = severe loss of neurons or myelinated fibers; N.E. = not examined; Nucl. = Nucleus. The nomenclature used for the anatomical areas was basically that of “The HumanCentral Nervous System” [4], and for the detailed areas in the brain stem, that of “Cytoarchitecture of the Human Brain Stem” [5].

72

270

Oyanagi, Mochizuki, Nakayama, et al.

Figure 1. A: Quite well preserved tractus opticus (arrow head), commissura anterior (arrow), and amygdala (asterisk) in patient 4. B: Preserved corpus geniculatum laterale (arrow head) and hippocampus (asterisk), but severely deteriorated thalamus (arrow) in patient 6. C: Well preserved axons in the tractus opticus in patient 3 (immunohistochemistry for phosphorylated neurofilament). D: Preserved geniculatum
laterale in patient 3. E: Well preserved radiatio optica and area striata (asteriks) in patient 7. F: Preserved
presynaptic density in area stiata in patient 1 (immunohistochemistry for synaptophysin). A, B, D and E:
Klüver-Barrera preparation. Bars: 10 mm in A, B, E and F; 20 µm in D, and 50 µm in C.

pressure ventilation support via a tracheostomy (TPPV). The patients were 4 males and3 females, aged between 40 and 69 years at
the time of death. The ages of the patients at
disease onset had ranged from 13 to 64 years(mean 46.1 years) and the duration of the illness had ranged from 5 to 27 years (mean
11.7 years). The duration of respirator use
had ranged from 3 to 26 years. Four patients
had a genetic background for ALS (patients
1, 3, 5, and 6), and 3 had sporadic ALS. All
patients had been in a TLS, i.e., stage V of
Hayashi et al. [6], before death. None of thepatients had shown dementia before entering
the TLS (Table 1). Some of the clinical andneuropathological findings of these patients
have been reported previously [6, 7, 8, 9, 10].
Informed consent was obtained from eachfamily member. This study was performed inaccordance with the provisions of the Declaration of Helsinki (1995), and was approved73

by the Ethics Committees of Shinshu University (No. 1772), and Tokyo Metropolitan
Neurological Hospital.

Results
All patients showed evident degenerationof the anterior and lateral tractus corticospinalis, and severe loss of Betz cells and neurons in the cornu anterioris spinalis, nuclei
facialis, and hypoglossi. The tegmentum of
the brain stem, thalamus, and globus pallidus showed marked atrophy in every patient. In addition, patient 1 showed marked
degeneration of the somatosensory cortices,
frontal and parietal cortices, frontal whitematter, striatum, subthalamic nucleus, substantia nigra, and the Clarke’s nucleus. Posterior column was relatively preserved, but
the other white matter was lost in the spinal

Preservation of visual pathway in totally locked-in state ALS

Figure 2. Well preserved bulbus olfactorius (A:patient 3) and nucleus basalis of Meynert (B: patient
7). A, B: Klüver-Barrera preparation. Bars: 50 µm.

cord. FUS-immunoreactive (ir) neuronal cytoplasmic inclusions (NCIs) were many inthe frontal and motor cortices. Some FUSir glial inclusions were observed in the cerebral cortex and the white matter. Patient 1
did not show dementia in the clinical course,
but the neuropathological subtype should be
FTLD-FUS [10]. In patient 2, the substantia
nigra, subthalamic nucleus, dentate nucleus,
spinal anterolateral funiculus, Clarke’s nucleus, dorsal spinocerebellar tract, posterior
funiculus, and subiculum were degenerated.
Scattered TDP-43-immunopositive glial inclusions were found. TPD-43 pathology isclassified as type 2 [11]. Gene analysis including C9orf72 repeat was not performed.
The patient did not show dementia in theclinical course, but the neuropathologicalsubtype should be FTLD-TDP.
TPD-43 pathology of patients 4 and 7 isclassified as type 1 [11]. Recent gene analysis of patient 6 revealed that K510M mutation in the FUS [9].
Among these patients, the tractus opticus, corpus geniculatum laterale, colliculus
superior, radiatio opticus, and area striata
were quite well preserved (Table 2) (Figure 1), though the retina and nervus opticus
were not available. With regard to the olfactory pathway, the epithelium olfactorium
was not examined, while mitral cells of thebulbus olfactorius, amygdala, hippocampus,
gyrus parahippocampalis, gyrus cinguli, corpus mammillare, fornix and nucleus anterior
74

271

thalami, and lobus frontalis were well preserved (Table 2) (Figure 1, 2). In addition,
the commissura anterior, nucleus basalisof Meynert, nucleus paraventricularis, andnucleus supraopticus were well preserved(Table 2) (Figure 1, 2).
On the other hand, in the somatosensory
system, the funiculus posterior in the spinal cord, nucleus spinalis nervi trigemini
sensibilis principalis, and lemniscus medialis and lemniscus trigeminalis in the brain
stem showed severe degeneration (Table 2)(Figure 3), though relatively well preservation was examined in the thalamus and gyrus postcentralis. In the auditory pathway as
well, the nucleus trapezoideus, nucleus olivaris superior, lemniscus lateralis, and colliculus inferior were deteriorated in most of thepatients (Table 2) (Figure 3), though nucleus
cochlealis dorsalis, corpus geniculatum medialis and gyrus temporalis transversus were
preserved relatively well. In the gustatory
pathway, the nucleus parabrachialis medialis and tractus trigeminothalamicus dorsalis
were degenerated in 6 of the 7 patients (Table
2) (Figure 3).

Discussion
As of 2004, 3% of ALS patients in France
were reported to be receiving TPPV support[12], compared with 28.4% in Japan, amongwhom 13% were in a TLS [13].
Hayashi et al. [6] recently proposed astaging system for classification of communicative ability in ALS patients, stage I:
communicate in sentences; stage II: communicate with one word answers only; stage III:
communicate with nonverbal yes/no response
only; stage IV: cannot communicate occasionally due to uncertain yes/no responses;
stage V: cannot communicate by any means,i.e., a TLS. Clinical analysis showed thatpatients who reached stage V had begun to
use the ventilator significantly earlier thanpatients in whom the final stage was IV or
less. All patients in stage V (TLS) were under TPPV support. In addition, such patientsfrequently had a family history of ALS [6, 7,
8]. All the patients examined in the present
study had stage V ALS.
Preservation of visual evoked potential(VEP) has been reported in ALS patients

Oyanagi, Mochizuki, Nakayama, et al.

272
Figure 3. Severe deterioration of the formatio reticularis (asterisks) in the brain stem. A: Marked
degeneration of the tectum, tegmentum and medialand central portions of the midbrain in patient 1. B
and C: Severe atrophy of the tegmentum of thepons in patient 3. D: Marked degeneration of theformatio reticularis and pyramis in the medulla oblongata of patient 2. E: Deteriolation of the funiculus gracilis and the middle root zone in the funiculus cuneatus in addition to complete degenerationof the funiculi laterale, anterolaterale, and anterior
in the cervical cord in patient 5. A – E: Klüver-Barrera preparation. Bars: 10 mm.

[14, 15], and Lim et al. [16] have examined
steady-state VEP (SSVEP) in an ALS patientwith impaired oculomotor function. Remarkable preservation of the visual pathway in
75

ALS patients in a TLS elucidated in the present study may indicate that photic stimulation
reaches the area striata. Thus, communicationthat exploits visual acuity is considered to be
the best approach for such patients. The visual function of patients with ALS in a TLS
should therefore be examined, and tractography by magnetic resonance imaging (MRI)[17] may disclose well preservation of thevisual pathway in patients of ALS in TLS.
In addition, the present study revealed preservation of the olfactory pathway, suggestingthat olfactory function was also retained.
It has been reported that median nerve
somatosensory evoked potentials (SEP) decrease in accordance with muscle weakness
in ALS patients [18], but no study has investigated such patients in a TLS. The present
and previous results indicate that the somatosensory and auditory pathways are severely damaged in patients in TLS, and thus
somatosensory and auditory function would
appear to be compromised.
It has also been reported that the reticular formation of the brain stem is severely
degenerated in patients with ALS in a TLS[7, 8]. As the reticular formation plays avery important role as the reticular activating system, its degeneration would indicate
difficulty with information processing in the
cerebrum. However, preservation of the limbic area including the hippocampus, gyrus
parahippocampalis, amygdala, and nucleus
basalis of Meynert indicate the probablepreservation of memory acuity; in addition,
preservation of cognitive function has beensuggested in patients with ALS in a TLS
through the use of optical topography [19].
Preservation of the nuclei paraventricularis and supraopticus in patients with ALS

273

Preservation of visual pathway in totally locked-in state ALS

in a TLS may represent a normal degree ofsecretion of antidiuretic hormone. The nucleus suprachiasmaticus in the present patientswas not available for study. Examination of
the circadian rhythm of such patients mightshed new light on these aspects in the TLS
associated with ALS.
The present paper reports a novel findingof the marked preservation of the optic andolfactory system in patients in the TLS due to
ALS. The findings indicate a preservation of
optic and olfactory input systems to the brainof the patients. As for integrating system inthose patients, the number of neurons in the
cerebral cortex was relatively preserved, anda paper reported a possibility to make a communication with those patients by classifying
electroencephalogram data [1]. As for the
output system, there have been no reports on
the electromyography in the patients in TLSALS. Since the course of developing TLS isusually gradual, future advance of brain machine interface (BMI) may elongate the period of communication with the patients, andeven in the end-stage, some BMI may enable
to communicate with the patients and move
extremities by detecting weak signals of output from the patients.

References
[1]

[2]

[3]

[4]
[5]
[6]

[7]

[8]

[9]

Acknowledgments
The authors are indebted to Dr. Toshio
Mizutani, previous head of the Department of
Pathology of Tokyo Metropolitan Neurological Hospital, Tokyo, Japan, and Mr. Mitsuhiro
Ikeda of the same department for their kindand excellent help in the present research.
This work was supported in part by a Grantin-Aid for Science Research from the Ministry of Education, Science, and Culture, Japan(Numbers 22390429 and 25293449 to YN,
and 22300117 to KO), and by the Collaborative Research Project of the Brain ResearchInstitute, Niigata University, Niigata, Japan(No. 2503) to KO.

[10]

[11]

[12]

Conflict of interest
The authors report no conflict of interest.

76

[13]

De Massari D, Ruf CA, Furdea A, Matuz T, vander Heiden L, Halder S, Silvoni S, Birbaumer N.
Brain communication in the locked-in state.
Brain. 2013; 136: 1989-2000. CrossRef PubMed
Halder S, Hammer EM, Kleih SC, Bogdan M,
Rosenstiel W, Birbaumer N, Kübler A. Predictionof auditory and visual p300 brain-computer interface aptitude. PLoS ONE. 2013; 8: e53513.
CrossRef PubMed
Nakanishi Y, Yanagisawa T, Shin D, Fukuma R,
Chen C, Kambara H, Yoshimura N, Hirata M,
Yoshimine T, Koike Y. Prediction of three-dimensional arm trajectories based on ECoG signals recorded from human sensorimotor cortex. PLoS
ONE. 2013; 8: e72085. CrossRef PubMed
Nieuwenhuys R, Voogd J, van Huijzen C. The Human Central Nervous System. A Synopsis andAtlas. 3rd edn. Berlin: Springer-Verlag; 1988.
Olszewski J, Baxter D. Cytoarchitecture of the
Human Brain Stem. Basel: S. Karger; 1954.
Hayashi K, Mochizuki Y, Nakayama Y, Shimizu T,
Kawata A, Nagao M, Mizutani T, Matsubara S.
[Communication disorder in amyotrophic lateralsclerosis after ventilation – a proposal of staging
and a study of predictive factor]. Rinsho
Shinkeigaku. 2013; 53: 98-103. CrossRef
PubMedHayashi H, Kato S. Total manifestations of amyotrophic lateral sclerosis. ALS in the totally lockedin state. J Neurol Sci. 1989; 93: 19-35. CrossRef
PubMedKato S, Oda M, Hayashi H. Neuropathology inamyotrophic lateral sclerosis patients on respirators: uniformity and diversity in 13 cases. Neuropathology. 1993; 13: 229-236. CrossRef
Mochizuki Y, Kawata A, Maruyama H, Homma T,
Watabe K, Kawakami H, Komori T, Mizutani T,
Matsubara S. A Japanese patient with familial
ALS and a p.K510M mutation in the gene for
FUS (FUS) resulting in the totally locked-in state.
Neuropathology. 2014; 34: 504-509. CrossRef
PubMedMochizuki Y, Isozaki E, Takao M, Hashimoto T,
Shibuya M, Arai M, Hosokawa M, Kawata A,
Oyanagi K, Mihara B, Mizutani T. Familial ALS
with FUS P525L mutation: two Japanese sisters
with multiple systems involvement. J Neurol Sci.
2012; 323: 85-92. CrossRef PubMed
Nishihira Y, Tan C-F, Onodera O, Toyoshima Y,
Yamada M, Morita T, Nishizawa M, Kakita A,
Takahashi H. Sporadic amyotrophic lateral sclerosis: two pathological patterns shown by analysis
of distribution of TDP-43-immunoreactive neuronal and glial cytoplasmic inclusions. Acta Neuropathol. 2008; 116: 169-182. CrossRef PubMed
Gil J, Funalot B, Verschueren A, Danel-Brunaud
V, Camu W, Vandenberghe N, Desnuelle C, Guy N,
Camdessanche JP, Cintas P, Carluer L, Pittion S,
Nicolas G, Corcia P, Fleury M-C, Maugras C,
Besson G, Le Masson G, Couratier P. Causes ofdeath amongst French patients with amyotrophic
lateral sclerosis: a prospective study. Eur J Neurol. 2008; 15: 1245-1251. CrossRef PubMed
Kawata A, Mizoguchi K, Hayashi H. [A nationwide survey of ALS patients on trachoestomy
positive pressure ventilation (TPPV) who devel-

Oyanagi, Mochizuki, Nakayama, et al.

[14]

[15]

[16]

[17]

[18]

[19]

oped a totally locked-in state (TLS) in Japan].
Rinsho Shinkeigaku. 2008; 48: 476-480. CrossRef PubMed
Palma V, Guadagnino M, Brescia Morra V, Nolfe
G. Multimodality evoked potentials in sporadic
amyotrophic lateral sclerosis: a statistical approach. Electromyogr Clin Neurophysiol. 1993;
33: 167-171. PubMed
Ghezzi A, Mazzalovo E, Locatelli C, Zibetti A,
Zaffaroni M, Montanini R. Multimodality evoked
potentials in amyotrophic lateral sclerosis. Acta
Neurol Scand. 1989; 79: 353-356. CrossRef
PubMedLim JH, Hwang HJ, Han CH, Jung KY, Im CH.
Classification of binary intentions for individuals
with impaired oculomotor function: “eyes-closed”
SSVEP-based brain-computer interface (BCI). J
Neural Eng. 2013; 10: 026021. CrossRef PubMed
Sarica A, Cerasa A, Vasta R, Perrotta P, Valentino
P, Mangone G, Guzzi PH, Rocca F, Nonnis M,
Cannataro M, Quattrone A. Tractography in amyotrophic lateral sclerosis using a novel probabilistic tool: a study with tract-based reconstruction
compared to voxel-based approach. J Neurosci
Methods. 2014; 224: 79-87. CrossRef PubMed
Hamada M, Hanajima R, Terao Y, Sato F, Okano
T, Yuasa K, Furubayashi T, Okabe S, Arai N, Ugawa
Y. Median nerve somatosensory evoked potentials
and their high-frequency oscillations in amyotrophic lateral sclerosis. Clin Neurophysiol. 2007;
118: 877-886. CrossRef PubMed
Fuchino Y, Nagao M, Katura T, Bando M, Naito
M, Maki A, Nakamura K, Hayashi H, Koizumi H,
Yoro T. High cognitive function of an ALS patientin the totally locked-in state. Neurosci Lett. 2008;
435: 85-89. CrossRef PubMed

77

274

Journal of the Neurological Sciences 363 (2016) 245–248

Contents lists available at ScienceDirect

Journal of the Neurological Sciences
journal homepage: www.elsevier.com/locate/jns

Clinical short communication

ALS patients with ability to communicate after long-term mechanicalventilation have conﬁned degeneration to the motor neuron system
Yoko Mochizuki a,g,⁎, Kentaro Hayashi b, Yuki Nakayama c, Toshio Shimizu b, Masayuki Kamide d, Mieko Ogino e,
Takashi Komori a, Masato Hasegawa f, Eiji Isozaki b, Imaharu Nakano b,1a

Department of Pathology, Tokyo Metropolitan Neurological Hospital, Tokyo, Japan
Department of Neurology, Tokyo Metropolitan Neurological Hospital, Tokyo, Japan
Laboratory of Nursing Research for Intractable Disease, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japand
Emergency Department of Atsugi City Hospital, Kanagawa, Japane
Division of Integrated Care and Whole Person Care, Department of Comprehensive Medicine, Research and Development Center for New Medical Frontiers, Kitasato University School of Medicine,
Kanagawa, Japanf
Department of Dementia and Higher Brain Function, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japang
Department of Neurology, Tokyo Metropolitan Kita Medical and Rehabilitation Center for the Disabled, Tokyo, Japanbca r t i c l e

i n f oArticle history:
Received 28 October 2015
Received in revised form 27 January 2016
Accepted 29 February 2016
Available online 2 March 2016
Keywords:
Amyotrophic lateral sclerosisLong-term survival
Tracheostomy invasive ventilation
Communication ability
Neuropathological ﬁndingsa b s t r a c t
Objective: To clarify the position in the amyotrophic lateral sclerosis (ALS) spectrum, of a subgroup of patients
who maintained the ability to communicate after long-term mechanical ventilation (LTMV) by tracheostomy.
Methods: We undertook a clinicopathological investigation of sporadic ALS in three patients who maintained theability to communicate after approximately 30-year survival on LTMV by tracheostomy.
Results: The age of onset and duration of disease was 48 years and 31 years in patient 1, 55 years and 29 years inpatient 2, and 31 years and 33 years in patient 3, respectively. Each patient displayed slow disease progression. In
all patients, both upper and lower motor neurons were markedly degenerated, while other neuronal systems andthe brainstem tegmentum were spared. A few normal-looking motor neurons remained in the anterior horn of
the spinal cord. There were no TAR DNA-binding protein 43-immunoreactive inclusions in the lower
motor neurons in any patient and only occasional inclusions in the cerebral cortex of one patient.
Conclusion: The clinicopathological ﬁndings of these three patients suggest that there is a distinct subgroupof ALS patients characterized by the above-mentioned features.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction

2. Patients and methods

Most patients with amyotrophic lateral sclerosis (ALS) will die from
respiratory failure 2–4 years after the onset of symptoms unless treatedby mechanical ventilation [13]. In patients with ALS, tracheostomy invasive ventilation (TIV) may extend survival for years [3,4,14]. The use ofTIV may depend on social, cultural, religious, or economic factors [1,5].
Often ALS patients and their close relatives decide not to use TIV due
to the fear of progressing into a totally locked-in state [6], with loss ofall voluntary movement, including eye movement [1,4,9]. Between 8%
and 17.2% of ALS patients using TIV develop a totally locked-in state[9,10]. However, some ALS patients maintain their ability to communicate after long-term mechanical ventilation (LTMV) by tracheostomy[8,15]. The position that the ALS in this patient subgroup occupies inthe disease spectrum has not previously been described.

To clarify the position of ALS in this patient subgroup in the disease
spectrum, we conducted a clinicopathological investigation of sporadic
ALS cases in three Japanese patients who maintained the ability to communicate after approximately 30-year survival on mechanical ventilation by tracheostomy.

⁎ Corresponding author at: Department of Neurology, Tokyo Metropolitan Kita Medical
and Rehabilitation Center for the Disabled, 1-2-3 Jujodai, Kita-ku, Tokyo 114-0033, Japan.
E-mail address: mochi@nihon-u.ne.jp (Y. Mochizuki).
1
Professor Imaharu Nakano died in July 2014.

http://dx.doi.org/10.1016/j.jns.2016.02.068
0022-510X/© 2016 Elsevier B.V. All rights reserved.

78

3. Results
3.1. Clinical features
Table 1 shows the clinical ﬁndings of the three patients [16].
Nerve conduction studies at the time of diagnosis in patient 1 revealed reduced motor response of the median nerve and normal sensory nerve potentials. Needle electromyogram (EMG) showed active
and chronic denervation changes in the upper and lower extremity
muscles, supporting the diagnosis of ALS. EMG records for patients
2 and 3 could not be found and were not available. Patients 1 and 2
showed weakness and muscle atrophy of distal upper extremities

246

Y. Mochizuki et al. / Journal of the Neurological Sciences 363 (2016) 245–248

Table 1
Summary of clinical ﬁndings in three cases with surviving on long-term mechanical ventilation by tracheostomy amyotrophic lateral sclerosis.
Case number

1

2

3

Sex
Age of onset (years)
Disease duration (years)
Initial symptom
Tendon reﬂexes
Progression rate of ALSFRS-R at time of diagnosisa
Duration from the onset (years)
To tracheostomy
To mechanical ventilation
To total quadriplegia
Eye movement
Dementia
Cause of death

Man
48
28
Right hand weakness
Increased
0

Man
55
29
Left hand weakness
Increased
0

Man
30
33
Right hand weakness
Increased
08
13: sometimes, 25: always
26
Preserved–
Malignant lymphoma

14
14
Eventually, knee joints move a little
Mild vertical ophthalmoparesis–b
Sudden death6611
Preserved–
Acute circulatory failureabProgression rate of ALSFRS-R was calculated as: (48-ALSFRS-R at “time of diagnosis”) / duration from onset to diagnosis (month) [16].
Delirium occurred temporarily at the time of 28 years from the onset.

and then showed spasticity of lower extremities 7 months and9 years from disease onset, respectively. Their spasticity continued
after tracheostomy and LTMV. Therefore, the predominant symptoms were considered to be upper motor neuron disorder and aslowly progressing disease course in patients 1 and 2. Patient 3
showed hyperreﬂexia and the ALS progressed earlier than in theother patients and he became quadriplegic. After LTMV by tracheostomy for patient 3, progression of symptoms appeared to stop because his eye and oral movements were functioning well until his
death. All three patients were discharged to their home with ahome support system after tracheostomy and LTMV. They could
communicate via eye movements until their end of life, and could
read newspapers and books.

3.2. Neuropathological features
Before ﬁxation, the brain of patient 1 weighed 1190 g, patient 21030 g, and patient 3 1290 g. Mild frontal lobe atrophy was seen in patient 2; however, neuronal loss in the cortex and gliosis of the whitematter was not observed. All patients showed motor neuron systemconﬁned degeneration, mild brainstem atrophy with preserved tegmental ﬁbers and marked spinal cord atrophy (Fig. 1A–E). There wasmoderate loss of motor neurons in the anterior horn of the spinal cord
(Fig. 1F) and brainstem motor nuclei, with the exception of ocular nuclei, in patients 1 and 2, and the loss was marked in patient 3. However,in all patients, a few normal-looking motor neurons remained in the anterior horn of the spinal cord (Fig. 1G, H). Although some spheroids

Fig. 1. A–E: Cervical cord at the same magniﬁcation. A: patient 1, B: patient 2, C: patient 3, D: A 66-year-old patient with a 13-year-5-month history of amyotrophic lateral sclerosis (ALS)
who progressed to a totally locked-in state, E: A 72-year-old patient with Parkinson disease. The cervical cord of patient 1–3 (A–C) shows marked atrophy, which is similar to that in thepatient with ALS who progressed to a totally locked-in state (D). However, degeneration of the anterior horn, lateral and anterior column is milder in A-C than D, despite the longer disease
duration in patient 1–3 (bar = 5 mm, Klüver–Barrera staining). F–I: Anterior horn of the ﬁfth lumbar cord. F: A small number of motor neurons remained (patient 1, Klüver-Barrera
staining, bar = 200 μm). G and H: Normal-looking motor neurons and a spheroid (arrow) are observed with mild ﬁbrillary astroglial proliferation. However, no hypertrophic
astrocytes, microglia, or macrophages are seen (G: patient 1, H: patient 3, Hematoxylin and eosin staining, bar = 50 μm). I: TAR DNA-binding protein 43-immunoreactive neuronalcytoplasmic inclusions are seen in the granular cells of the hippocampal dentate gyrus (patient 3, bar = 20 μm).

79

Y. Mochizuki et al. / Journal of the Neurological Sciences 363 (2016) 245–248

were seen (Fig. 1G), no hypertrophic astrocytes were observed (Fig. 1 G,
H). Furthermore, no microglia or macrophages were seen in the lower
motor neuron system or in the upper motor neuron system by immunostaining with CD-68 (KP-1) (1:400, Novocastra). There were no TAR
DNA-binding protein 43- (TDP-43) (pS409/410-1 1:3000, CosmoBio)
immunoreactive inclusions or Bunina bodies in the lower motor neurons in any patient. However, in patient 3, TDP-43-immunoreactive
neuronal cytoplasmic inclusions (NCI) were found in the cerebral cortex, hippocampal dentate granule neurons (Fig. 1I), reticular formationof the medulla oblongata, and inferior olivary nuclei. There were no
fused in sarcoma (FUS) or superoxide dismutase 1 (SOD1) immunereactive inclusions seen in any patient.
3.3. Western blotting of TDP-43
Sarkosyl-insoluble TDP-43 prepared from frozen brain tissues of thepatient 2 was investigated by comparing with that in a 78-year-old
patient with a two-year history of ALS. No phosphorylated TDP-43
was detected in the patient 2 (Fig. 2). Due to the lack of available frozen
tissue from patients 1 and 3, this analysis could only be performed on
tissue from patient 2.
4. Discussion
Three patients with ALS showed both upper and lower motor neuron disorder, without ophthalmoplegia, autonomic disorder, cerebellar
signs, extrapyramidal signs, or dementia, and all survived on LTMV bytracheostomy for about 30 years. Prominent upper motor neuron disorder, which is characterized by spasticity in the lower extremities andweakness in the upper extremities, was evident in the early state of disease in patients 1 and 2. These features are different from the features ofprimary lateral sclerosis and may be features of long survival with ALS[17]. The disease duration was extremely long among patients reportedwith ALS on LTMV by tracheostomy [3,4,14]. Survival studies indicatethat advantageous criteria for long-term survival after tracheostomy
were age ≤ 65 years at the time of TIV initiation [14] and patientswith home care [4,14]. These three patients met these criteria: all

247

three patients lived at home and two of them received their tracheostomy at b65 years of age. However, it is important to emphasize thatthe time from onset until the initiation of LTMV by tracheostomy andto become totally quadriplegic was relatively long in all patients. Previously we reported that patients with rapidly progressing ALS before initiation of TIV might progress further to a totally locked-in state(communication stage V) [7]. Furthermore, Nakayama et al. [10] clariﬁed that TIV, impaired oculomotor movement, and total quadriplegia
are predictors of severe communication impairment. Comparing thedisease progression in the present patients with that in the reported patients who progress to a totally locked-in state, suggests that the rate of
disease progression before TIV might predict the long-term prognosis
for communication impairment after LTMV by tracheostomy. Slow disease progression before TIV in ALS patients might predict a better longterm prognosis for communication impairment. Among the present patients with a better prognosis, the ALS progressed very slowly; the ALS
in the third patient apparently stopped progressing at all. The clinicalcourse of the present patients suggests that they fall into a subgroupof patients with slow ALS progression who maintain the ability to communicate after long-term survival on mechanical ventilation.
Neuropathologically, conﬁnement of degeneration to the motor
neuron system in the present patients appears to correspond to theirclinical features, which were similar to those in usual cases of ALS interms of site of onset and symptoms. These features were the same asin reported cases [8,15] and indicated that there are ALS cases that do
not progress beyond the motor neuron system despite extremely
prolonged survival with TIV by tracheostomy. The histopathologicalcharacteristics, in which motor neurons in the anterior horn of the spinal cord and spheroids remained normal looking, were usually observedin ALS cases at an earlier stage [2]. The absence of macrophages and hypertrophic astrocytes indicates that their lesions were not active [15].
Therefore, neuropathologically the ALS in these patients showed slow
progression or likely stopped progressing. Thus, the ALS was different
from usual cases of ALS. Further, Bunina bodies and TDP-43immunoreactive inclusions, which are characteristic of sporadic ALS,
were not observed in the motor neurons. Western blot analysis of
TDP-43 in precenetral tissue from patient 2 revealed that accumulationof abnormal forms of TDP-43 proteins were low or below the detection
limit. However, the distribution and appearance of TDP-43immunoreactive NCI in the third patient was the same as that seen in
the non-motor areas of ALS patients [11]. Therefore, at least the third
patient might have had TDP-43 pathology. The degree of TDP-43immunoreactive NCI inﬁltration is milder in patients with longer disease duration [12]. There is a possibility that we could not ﬁnd TDP43/ubiquitin immunoreactive NCIs because they were so rare.
It is important to note some limitations of this study. First, the study
is a small sample. Second, not all clinical measures were available because of the long duration of disease, and tissue samples from all patients could not be available. However, it is rare for long survival
patients, who stay mainly at home, to provide autopsy studies. Nonetheless, the available clinicopathological ﬁndings in these three patientssuggest that there is a distinct subgroup of ALS patients. This ALS patient
subgroup maintained the ability to communicate after survival on LTMV
by tracheostomy.
Conﬂict of interest statement

Fig. 2. Western blot analysis of the sarkosyl-insoluble fractions extracted fromcryopreserved precenetral tissue. Anti-TAR DNA-binding protein 43- (TDP-43) antibody(Proteintech, 10782-2-AP rabbit polyclonal) detected a strong band for full length
unphosphorylated normal TDP-43 in patient 2 (lane 1), but some abnormal TDP-43
bands at 45kD (arrow head), ~23 kDa fragments and smears in a 78-year-old patientwith a two-year history of ALS (lane 2). Analysis with anti-phospho TDP-43 antibody(pS409/410, Cosmobio, pS409/410-1 rabbit polyclonal) detected the phosphorylated
forms of full length TDP-43 at 45 kDa, C-terminal fragment (CTFs) at 18–25 kDa andsmears, type B CTF banding pattern [18] in the two-year history ALS patient (lane 2). In
contrast, no such band was detected in patient 2 (lane 1). Analysis with anti-tau
antibody (T46, Thermo, mouse monoclonal) showed no tau accumulation in patients 2(data not shown).

80

The authors declare that there are no conﬂicts of interest.
Acknowledgements
The authors are grateful to members in the laboratory at Tokyo Metropolitan Neurological Hospital for their technical assistance. This work
was supported by JSPS KAKENHI (Grant-in-Aid for Scientiﬁc Research(B)) Number 25293449 and the Joint Program for ALS Research at theTokyo Metropolitan Institute of Medical Science, and Grants-in-Aid248

Y. Mochizuki et al. / Journal of the Neurological Sciences 363 (2016) 245–248

from the Research Committee of CNS Degenerative Disease (2011 and2012), the Ministry of Health, Labour and Welfare of Japan.
References[1] N. Atsuta, H. Watanabe, M. Ito, F. Tanaka, A. Tamakoshi, I. Nakano, et al., Age at onset
inﬂuences on wide-ranged clinical features of sporadic amyotrophic lateral sclerosis,
J. Neurol. Sci. 276 (2009) 163–169.
[2] S. Carpenter, Proximal axonal enlargement in motor neuron disease, Neurology 18(1968) 841–851.
[3] P. Dreyer, C.K. Lorenzen, L. Schou, M. Felding, Survival in ALS with home mechanicalventilation non-invasively and invasively: a 15-year cohort study in west Denmark,Amyotroph Lateral Scler. Frontotemporal Degener. 15 (2014) 62–67.
[4] N. Fini, E. Georgoulopoulou, M. Vinceti, M. Monelli, G. Pinelli, P. Vacondio, et al., Noninvasive and invasive ventilation and enternal nutrition for ALS in Italy, Muscle
Nerve 50 (2014) 508–516.
[5] P.H. Gordon, Amyotrophic lateral sclerosis: an update for 2013 clinical features,
pathophysiology, management and therapeutic trials, Aging Dis. 4 (2013) 295–310.
[6] H. Hayashi, S. Kato, Total manifestations of amyotrophic lateral sclerosis. ALS in the
totally locked in state, J. Neurol. Sci. 93 (1989) 19–35.
[7] K. Hayashi, Y. Mochizuki, Y. Nakayama, T. Shimizu, A. Kawata, M. Nagao, et al., Communication disorder in amyotrophic lateral sclerosis after ventilation—a proposal ofstaging and a study of predictive factor, Rinsho Shinkeigaku 53 (2013) 98–103 (Article in Japanese with English abstract).
[8] S. Kato, M. Oda, H. Hayashi, Neuropathology in amyotrophic lateral sclerosis patientson respirators: uniformity and diversity in 13 cases, Neuropathology 13 (1993)
229–236.
[9] A.H. Moss, E.A. Oppenheimer, P. Casey, P.A. Cazzolli, R.P. Roos, C.B. Stocking, et al.,Patients with amyotrophic lateral sclerosis receiving long-term mechanical ventilation. Advance care planning and outcomes, Chest 110 (1996) 249–255.

[10] Y. Nakayama, T. Shimizu, Y. Mochizuki, K. Hayashi, C. Matsuda, M. Nagao, et al., Predictors of impaired communication in patients with amyotrophic lateral sclerosis,
Amyotroph Lateral Scler Frontotemporal Degener 17 (2016) 38–46.
[11] Y. Nishihira, C.-F. Tan, O. Onodera, Y. Toyoshima, M. Yamada, T. Morita, et al., Sporadic amyotrophic lateral sclerosis: two pathological patterns shown by analysis
of distribution of TDP-43-immunoreactive neuronal and glial cytoplasmic inclusions, Acta Neuropathol. 116 (2008) 169–182.
[12] Y. Nishihira, C.-F. Tan, Y. Hoshi, K. Iwanaga, M. Yamada, I. Kawachi, et al., Sporadic
amyotrophic lateral sclerosis of long duration is associated with relatively mild
TDP-43 pathology, Acta Neuropathol. 117 (2009) 45–53.
[13] E. Pupillo, P. Messina, G. Logroscino, E. Beghi, S.L.A.L.O.M. Group, Long-term survival
in amyotrophic lateral sclerosis: a population-based study, Ann. Neurol. 75 (2014)
287–297.
[14] M. Tagami, F. Kimura, H. Nakajima, S. Ishida, S. Fujiwara, Y. Doi, et al., Tracheostomy
and invasive ventilation in Japanese ALS patients: decision-making and survival
analysis: 1990–2010, J. Neurol. Sci. 344 (2014) 158–164.
[15] M. Yoshida, N. Murakami, Y. Hashizume, E. Itoh, A. Takahashi, A clinicopathologicalstudy of two respirator-aided long-survival cases of amyotrophic lateral sclerosis,
Rinsho Shinkeigaku 32 (1992) 259–265 (Article in Japanese with English abstract).
[16] F. Kimura, C. Fujimura, S. Ishida, H. Nakajima, D. Furutama, H. Uehara, et al., Progression rate of ALSFRS-R at time of diagnosis predicts survival time in ALS, Neurology
66 (2006) 265–267.
[17] M. Sabatelli, F. Madia, A. Conte, M. Luigetti, I. Zollino Mancuso, et al., Natural historyof young-adult amyotrophic lateral sclerosis, Neurology 71 (2008) 876–881.
[18] H. Tsuji, T. Arai, F. Kametani, T. Nonaka, M. Yamashita, M. Suzukake, et al., Molecularanalysis and biochemical classiﬁcation of TDP-43 proteinopathy, Brain 135 (2012)
3380–3391.

81bs_bs_banner

Neuropathology 2016; ••, ••–••

doi:10.1111/neup.12303

Case Report

A Japanese familial ALS patient with autonomic
failure and a p.Cys146Arg mutation in the gene for
SOD1 (SOD1)
Kentaro Hayashi,1 Yoko Mochizuki,2,3 Reiji Koide,1,4 Akihiro Kawata,1 Taku Homma,2,5
Toshio Shimizu,1 Takashi Komori2 and Eiji Isozaki1
Departments of Neurology, and 2Pathology, Tokyo Metropolitan Neurological Hospital, 3Department of Neurology, Tokyo
Metropolitan Kita Medical and Rehabilitation center for the disabled, 4Division of Neurology, Department of Medicine, Jichi
Medical University School of Medicine, and 5Department of Pathology, Nihon University School of Medicine
1

We describe a Japanese man with familial amyotrophic
lateral sclerosis (ALS) associated with a p.Cys146Arg mutation in the copper/zinc superoxide dismutase gene (SOD1).
The patient developed bulbar signs followed by rapidly
progressive limb muscle weakness. The prominent clinicalfeature was orthostatic hypotension due to autonomic failure, which occurred after he underwent tracheostomy 1 year
and 3 months after the onset. Thereafter, he required
mechanical ventilation and progressed to communication
stage V (totally locked-in state) 7 years after the onset.
Neuropathology showed ALS with posterior column degeneration and multiple system degeneration. Severe neuronalloss in the intermediolateral nucleus was also observed.
Two previously reported cases of ALS patients with autonomic failure showed severe neuronal loss in theintermediolateral nucleus in addition to degeneration of
the motor neurons. Thus, autonomic failure due to neuronalloss in the intermediolateral nucleus could present in patients with ALS associated with certain mutations in SOD1.
Key words: autonomic failure, familial ALS, orthostatic
hypotension, SOD1, p.Cys146Arg.

INTRODUCTION
The main feature of autonomic dysfunction in patients withamyotrophic lateral sclerosis (ALS) has been reported to be
sympathetic hyperactivity and sympathovagal imbalance.1–3
Autonomic
failure,includingsevereorthostatic

Correspondence: Yoko Mochizuki, MD, Department of Neurology,
Tokyo Metropolitan Kita Medical and Rehabilitation Center for the
Disabled, 1-2-3 Jujodai, Kita-ku, Tokyo 114-0033, Japan. Email:mochi@nihon-u.ne.jp
Received 22 December 2015; revised 4 March 2016 and accepted
5 March 2016.

hypotension, does not usually emerge in ALS, even during
advanced stages with ventilator use. Here we describe an autopsy case of a familial ALS patient who presented withautonomic failure from a relatively early stage of the disease.
The patient had a p.Cys146Arg mutation in the copper/zincsuperoxide dismutase gene (SOD1). The clinical course of
ALS in patients with this mutation has been reported in
one family; however, neither autonomic nervous dysfunction
nor pathological features had been described.4 The present
study is the ﬁrst clinicopathological report related to thismutation. We also compared previously reported cases of
ALS with autonomic failure5,6 with the present case anddiscuss the pathomechanism of autonomic failure in ALS.

CLINICAL SUMMARY
A 58-year-old Japanese man (Subject III-8 in Fig. 1) developed hoarseness, followed by dysarthria. His younger sister,niece and three cousins had also been diagnosed as having
ALS (Fig. 1). His younger sister (Subject III-11 in Fig. 1) developed bulbar palsy at 38 years of age and died without respiratory support at 41 years of age. His niece (Subject IV-9
in Fig. 1) developed right leg weakness at 32 years of age andreceived respiratory support beginning 1 year and 2 months
after the onset; her symptoms progressed without upper motor neuron issues and she currently lives free from autonomic failure. Two cousins (Subjects III-3, 4 in Fig. 1)
developed leg weakness in the third decade and died in the
fourth decade of life. Another cousin (Subject III-14 inFig. 1) developed bulbar palsy in the sixth decade of lifeand died at 65 years of age. Six months after the onset ofthe disease in our patient, neurological examination showed
mild bulbar palsy, mild muscle weakness, and muscle
atrophy affecting the face and all extremities, without
hyperreﬂexia. He had no sensory disturbance, cerebellar
ataxia, cognitive dysfunction or abnormal blood pressure© 2016 Japanese Society of Neuropathology82

K Hayashi et al.

2

Fig. 1 Pedigree of the family. The arrow indicates the proband. Affected individuals are represented by solid black squares and circles.

ﬂuctuation suggesting sympathetic hyperactivity. Nerve conduction studies showed normal conduction velocities in both
sensory and motor nerves. Needle electromyography
showed active denervation potentials in the muscles of theupper and lower extremities and the thoracic paraspinal
muscles. MRI studies of the brain and the cervical spine
were normal. He was diagnosed as having a progressive
muscular atrophy variant of familial ALS. He developed
exertional dyspnea beginning 9 months after the onset, thenunderwent a tracheostomy and percutaneous endoscopic
gastrostomy 1 year and 3 months after the onset. After thetracheostomy, syncope due to orthostatic hypotension frequently occurred. Mechanical ventilation was started 2 years
and 3 months after the onset, and a head-up tilt test showedorthostatic hypotension with a systolic blood pressure reduction of 40 mmHg (from 128/93 mmHg to 85/65 mmHg) without compensatory tachycardia and faintness. The plasma
norepinephrine response was poor, suggesting vasomotor
sympathetic efferent dysfunction. These ﬁndings indicatedthat the patient was experiencing autonomic failure.
Urodynamic analysis showed an uninhibited bladder. Coefﬁcient of variation of R-R intervals on electrocardiogram
showed low value at 1.0%. Clinically, no sweating abnormality was observed. He became a total quadriplegic without
upper motor neuron signs 3 years and 9 months after the onset of the disease. Thereafter he exhibited progressive external ophthalmoplegia, resulting in a complete loss of allvoluntary movement, representing a totally locked-in state7
or stage V of the communication ability stage,8 7 years after
onset. He died of pneumonia at 68 years of age. An autopsy
was conducted 6 h after death.

Genetic analysis
DNA was extracted from the patient’s leukocytes using a
conventional method with informed consent. All exons andexon–intron boundaries of SOD1 were examined by directly

sequencing PCR products. Genetic analysis revealed a heterozygous T-to-C transition at cDNA position 436, resulting
in a Cys146Arg missense mutation within exon 5.

PATHOLOGICAL FINDINGS
Brain weight was 1200 g prior to formalin ﬁxation.
Macroscopically, the spinal cord and brainstem showedmarked atrophy while the frontal and temporal lobes
showed mild atrophy. The globus pallidus, subthalamic nucleus and substantia nigra were brownish in color (Fig. 2A).
The other structures in the cerebrum and the cerebellum appeared normal. Microscopically, the anterior horn of the spinal cord (Fig. 2 M) and all motor nuclei including theoculomotor nucleus of the brainstem showed severe neuronal loss and gliosis. In the very few remaining lower motor
neurons, no Bunina bodies, Lewy body-like hyaline inclusions, phosphorylated transactive response DNA-binding
protein 43 kDa (TDP-43) (pS409/410; CosmoBio, Tokyo,
Japan; 1:5000), neuroﬁlament (DAKO, Tokyo, Japan;
1:500), SOD1 (Proteintech, Tokyo, Japan; 1:100) immunoreactive neuronal cytoplasmic inclusions were found. The primary motor cortex showed a marked loss of Betz cells andpyramidal neurons with gliosis, and the corticospinal tract
showed marked ﬁber loss (Fig. 2H). In the spinal cord, in addition to the corticospinal tract, the dorsal column, posteriorspinocerebellar tract and antero-lateral funiculus also
showed marked ﬁber loss (Fig. 2 K,L). Neurons within
Clarke’s nucleus (Fig. 2 L,O) as well as in theintermediolateral nucleus (Fig. 2 L,N) were markedly decreased with gliosis. Dorsal root ganglion showed slight neuronal loss with a few Nageotte’s nodules. In the brainstemtegmentum, almost all nuclei and ﬁbers including the following structures showed marked loss with gliosis: neurons of
the accessory cuneate nucleus, dorsal motor nucleus of thevagus, solitary tract nucleus, superior colliculus and locus
ceruleus, and reticular formation, as well as ﬁbers of the© 2016 Japanese Society of Neuropathology

83

SOD1 mutant FALS with autonomic failure

3

Fig. 2 Neuropathological ﬁndings. (A) In a coronal section, the subthalamic nucleus and globus pallidus are brownish in color, and the frontal
lobe shows mild atrophy. Bar = 2 cm. (B) Internal segment of the globus pallidus shows moderate neuronal loss and gliosis. (C) Putamen showsslight gliosis. (D) Subthalamic nucleus shows moderate neuronal loss and gliosis with GFAP (Novocastra Laboratories, 1:500) immunostaining.
(E) The midbrain and medulla oblongata shows marked atrophy and the pons shows moderate atrophy. Neurons and ﬁbers of the tegmentum
and corticospinal tract are severely reduced. Bar = 5 mm. (F, G) Neuroﬁlamentous conglomerate inclusions (F; HE) reveal staining for neuroﬁlament (G) in pontine nuclei. (H) Both large and small ﬁbers are lost in the medullary pyramid with Bodian staining. (I, J) Vacuolar
degenerated neuron and gemistocytic astrocytes are observed in the inferior oivary hypertrophy (I; HE, J; Bodian). Marked atrophy and ﬁber
loss are observed in the spinal cord at the seventh cervical cord (K) and 12th thoracic cord (L). Bar = 2 mm. (M) Anterior horn of the seventh
cervical cord shows marked neuronal loss with gliosis. (N, O) Marked neuronal loss is seen in the intermediolateral nucleus (arrow, N) and
Clarke’s nucleus (surrounded by arrowheads, O) at the 12th thoracic cord. (B, C, F, I, M-O) HE staining. (E, K, L) KB staining. (F, G) Bar= 20 μm, (B-D, H-J, M-O) Bar = 50 μm.

central tegmental tract and superior cerebellar peduncles(Fig. 2E). Neurons of the pontine nuclei were atrophied, although the total number was preserved. Several neurons in
the inferior olivary nucleus showed vacuolar degeneration
with gemistocytic astrocytes known as inferior olivary hypertrophy9 (Fig. 2I, J). The middle and inferior cerebellar peduncle and olivocerebellar ﬁbers were relatively wellpreserved. The substantia nigra showed severe neuronal loss
with gliosis, globus pallidus (Fig. 2B), and subthalamic nucleus (Fig. 2D) showed moderate neuronal loss with gliosis,
and the caudate nucleus and the putamen (Fig. 2C) showedslight gliosis. There were no SOD1-immunoreactive

structures in these areas. However, the neuroﬁlament immunoreactive neurons were scattered in the degenerated areas(Fig. 2F, G). Neurons of the hippocampus and amygdala
were preserved. Neuroﬁbrillary tangles (NFTs) were observed in the entorhinal cortex and the hippocampus (Braak
NFT stage II) using anti-phosphorylation-dependent tau(AT8: Innogenetics, Gent, Belgium; 1:5000) immunostaining. There were only a few senile plaques with
Methenamin Bodian staining. In the cerebellum, neuronsof the dentate nucleus and ﬁbers of the hilum of the dentate
nucleus showed a marked loss in addition to a mild decreasein the number of Purkinje cells, especially in the vermis.

© 2016 Japanese Society of Neuropathology
84

K Hayashi et al.

4
There were no Lewy bodies and glial cytoplasmic inclusions
using phosphorylated α-synuclein (WAKO, Tokyo, Japan,
1:2000) immunostaining.

DISCUSSION
We herein reported an autopsy case of Japanese familial
ALS with a p.Cys146Arg mutation in SOD1. Characteristic
clinical features of this patient included bulbar onset, predominant lower motor neuron signs and rapid progression
to communication stage V;8 however, the most distinctive
characteristic was orthostatic hypotension due to autonomic
failure shortly before tracheostomy. Pathologically, this patient had lesions typical of ALS with posterior column involvement; this included both upper and lower motor
neurons, Clarke’s column, the spinocerebellar tract, andthe posterior column of the spinal cord. In addition, the
ALS ﬁndings also showed multiple system degeneration,such as degeneration of the brainstem tegmentum, globus
pallidus, subthalamic nucleus and substantia nigra. Severe
neuronal loss in the intermediolateral nucleus was also
observed.
The clinical courses of affected members of this family
and reported two siblings with a p.Cys146Arg mutation in
SOD14 were variable as follows. (i) Although the majorityof familial ALS patients with a mutation in SOD1 have onset
in the limbs,10 four of eight patients with p.Cys146Arg mutation developed from bulbar palsy and the other four patientsdeveloped from leg weakness. (ii) The age at onset varied
from 32 years old to sixth decades of life. (iii) Three patients
progressed rapidly to die or underwent tracheostomy invasive ventilation (TIV) in 2 years, in contrast to one patient
who had a slowly progressed phenotype. (iv) Only our patient showed autonomic failure. These ﬁndings suggest that
ALS patients with a p.Cys146Arg mutation in SOD1 show
variable clinical features and courses.
Among three reported ALS patients with a mutation in
SOD1 and autonomic dysfunction,2,6,11 only one patient
had autonomic failure.6 This patient showed rapid diseaseprogression and developed to communication stage V8 3
years after the onset, and then showed autonomic failure.
Her autonomic failure developed later than in our patient,although the neuropathological ﬁndings of both the reported patient6 and our patient were the same. Thesepatients showed neuronal loss in the intermediolateral nucleus, which is a sympathetic preganglionic nucleus,12 anddegeneration of the brainstem tegmentum which included
nuclei of the central autonomic network such as the solitary
tract nucleus and the ventrolateral medulla.12 However,these patients did not show symptoms of sympathetic hyperactivity prior to autonomic failure. Their clinical course andpathological features suggest that even in ALS patients,

autonomic failure occurs when the intermediolateral nucleus has severe degeneration.
Another patient with autonomic failure and motor neuron disease5 developed syncope caused by autonomic failure. Neuropathologically, degeneration of both the upperand lower motor neurons was reported. In addition, degeneration of the posterior column of the spinal cord was observed along with intracytoplasmic Lewy body-like hyaline
inclusions and conglomerates and hyaline inclusions in theanterior horns, which are pathological hallmarks of ALS
with a mutation in SOD1 (SOD1-related ALS).13 Marked
neuronal loss in the intermediolateral nucleus of this patient
indicates that neuron loss in the intermediolateral nucleus
can occur when degeneration of the motor neurons is mild
to moderate.
Previous reports have demonstrated that neurons of theintermediolateral nucleus can be preserved in SOD1-related
ALS patients without autonomic failure.13–15 Takahashi
et al.16 reported that in sporadic ALS patients on TIV, neurons of the intermediolateral nucleus were reduced by
60% at the second thoracic segment and 57% at the ninth
thoracic segment. Even in very long duration ALS patientswith severe degeneration of multiple systems, some neurons
remained in the intermediolateral nucleus.17 Therefore, theloss of neurons in the intermediolateral nucleus of the present patient and the two reported patients with autonomic
failure was an unexpected ﬁnding.
Our patient had inferior olivary hypertrophy. Althoughinferior olivary hypertrophy had been reported in progressive supranuclear palsy18 and Creutzfeldt Jakob disease19
in addition to cerebrovascular disease,9 it has not been reported in ALS. However, in our hospital, one other ALS patient who progressed to stage V also showed inferior olivary
hypertrophy (unreported case). These ﬁndings revealed thatinferior olivary hypertrophy may occur also in ALS.
In conclusion, autonomic failure due to neuronal loss in
the intermediolateral nucleus could present in patients with
ALS associated with some SOD1 mutations.

ACKNOWLEDGMENTS
The authors express gratitude to the laboratory members of
the Tokyo Metropolitan Neurological Hospital for theirtechnical assistance. This work was supported by JSPS
KAKENHI, Grant-in-Aid for Scientiﬁc Research (B),
Grant Number 22390429 and by the Joint Program for
ALS Research at the Tokyo Metropolitan Institute ofMedical Science.

REFERENCES
1. Shimizu T, Hayashi H, Kato S, Hayashi M, Tanabe
H, Oda M. Circulatory collapse and sudden death© 2016 Japanese Society of Neuropathology

85

SOD1 mutant FALS with autonomic failure
in respirator-dependent amyotrophic lateral sclerosis. J Neurol Sci 1994; 124: 45–55.
2. Kawata A, Kato S, Hayashi H, Hirai S. Prominent sensory and autonomic disturbances in familial amyotrophic lateral sclerosis with a Gly93Ser mutation in the
SOD1 gene. J Neurol Sci 1997; 153: 82–5.
3. Baltadzhieva R, Gurevich T, Korczyn AD. Autonomic
impairment in amyotrophic lateral sclerosis. Curr Opin
Neurol 2005; 18: 487–93.
4. Ito K, Uchiyama T, Fukutake T, Arai K, Kanesaka T,
Hattori T. Different clinical phenotypes of siblings withfamilial amyotrophic lateral sclerosis showing
Cys146Arg point mutation of superoxide dismutase 1gene (in Japanese with English abstract). Rinsho
Shinkeigaku 2002; 42: 175–7.
5. Noda K, Katayama S, Watanabe C et al. Pure autonomic failure with motor neuron disease: report of aclinical study and postmortem examination of a patient.
J Neurol Neurosurg Psychiatry 1996; 60: 351–2.
6. Shimizu T, Kawata A, Kato S et al. Autonomic failure in
ALS with a novel SOD1 gene mutation. Neurology
2000; 54: 1534–7.
7. Hayashi H, Kato S. Total manifestations of amyotrophic lateral sclerosis. ALS in the totally locked-in state.
J Neurol Sci 1989; 93: 19–35.
8. Hayashi K, Mochizuki Y, Nakayama Y et al. Communication disorder in amyotrophic lateral sclerosis after
ventilation—a proposal of staging and a study of predictive factor (in Japanese with English abstract). Rinsho
Shinkeigaku 2013; 53: 98–103.
9. Goto N, Kaneko M. Olivary enlargement: chronological and morphometric analysis. Acta Neuropathol
1981; 54: 275–82.
10. Orrell RW, Habgood JJ, Gardiner I et al. Clinical andfunctional investigation of 10 missense mutations anda novel frameshift insertion mutation of the gene for5
copper-zinc superoxide dismutase in UK families withamyotrophic lateral sclerosis. Neurology 1997; 48:
746–51.
11. Sakamoto H, Akamatsu M, Hirano M et al. Multiple
system involvement in a Japanese patient with a V31A
mutation in the SOD1 gene. Amyotroph Lateral Scler
Frontotemporal Degener 2014; 15: 312–4.
12. Benarroch EE. The central autonomic network: functional organization, dysfunction, and perspective. Mayo
Clin Proc 1993; 68: 988–1001.
13. Takahashi H, Makifuchi T, Nakano R et al. Familial
amyotrophic lateral sclerosis with a mutation in the
Cu/Zn superoxide dismutase gene. Acta Neuropathol
1994; 88: 185–8.
14. Ince PG, Shaw PJ, Slade JY, Jones C, Hudgson P. Familial amyotrophic lateral sclerosis with a mutation in exon
4 of the Cu/Zn superoxide dismutase gene: pathologicaland immunocytochemical changes. Acta Neuropathol
1996; 92: 395–403.
15. Kobayashi Z, Tsuchiya K, Kubodera T et al. FALS with
Gly72Ser mutation in SOD1 gene: Report of a family
including the ﬁrst autopsy case. J Neurol Sci 2011; 300:
9–13.
16. Takahashi H, Oyanagi K, Ikuta F. The intermediolateral
nucleus in sporadic amyotrophic lateral sclerosis. Acta
Neuropathol 1993; 86: 190–2.
17. Mochizuki Y, Isozaki E, Takao M et al. Familial ALS withFUS P525L mutation: two Japanese sisters with multiple
systems involvement. J Neurol Sci 2012; 323: 85–92.
18. Katsuse O, Dickson DW. Inferior olivary hypertrophy isuncommon in progressive supranuclear palsy. Acta
Neuropathol 2004; 108: 143–6.
19. Mizutani T. Neuropathology of Creutzfeldt-Jakob
disease in Japan. With special reference to thepanencephalopathic type. Acta Pathol Jpn 1981; 31:
903–22.

© 2016 Japanese Society of Neuropathology
86

Hayashi et al. Acta Neuropathologica Communications (2016) 4:107
DOI 10.1186/s40478-016-0379-3

RESEARCH

Open Access

Clinicopathological characteristics ofpatients with amyotrophic lateral sclerosis
resulting in a totally locked-in state(communication Stage V)
Kentaro Hayashi1, Yoko Mochizuki2,3*, Ryoko Takeuchi4,5, Toshio Shimizu1, Masahiro Nagao1, Kazuhiko Watabe6,
Nobutaka Arai7, Kiyomitsu Oyanagi8, Osamu Onodera4, Masaharu Hayashi9, Hitoshi Takahashi5, Akiyoshi Kakita5
and Eiji Isozaki1

Abstract
In the present study, we performed a comprehensive analysis to clarify the clinicopathological characteristics ofpatients with amyotrophic lateral sclerosis (ALS) that had progressed to result in a totally locked-in state(communication Stage V), in which all voluntary movements are lost and communication is impossible. In 11
patients, six had phosphorylated TAR DNA-binding protein 43 (pTDP-43)-immunoreactive (ir) neuronal cytoplasmic
inclusions (NCI), two had fused in sarcoma (FUS)-ir NCI, and three had copper/zinc superoxide dismutase (SOD1)-ir
NCI. The time from ALS onset to the need for tracheostomy invasive ventilation was less than 24 months in ten
patients. Regardless of accumulated protein, all the patients showed common lesions in the pallido–nigro–luysiansystem, brainstem reticular formation, and cerebellar efferent system, in addition to motor neurons. In patients with
pTDP-43-ir NCI, patients with NCI in the hippocampal dentate granule neurons (DG) showed a neuronal loss in the
cerebral cortex, and patients without NCI in DG showed a preserved cerebral cortex. By contrast, in patients with
FUS-ir NCI, patients with NCI in DG showed a preserved cerebral cortex and patients without NCI in DG showedmarked cerebral degeneration. The cerebral cortex of patients with SOD1-ir NCI was preserved. Together, thesefindings suggest that lesions of the cerebrum are probably not necessary for progression to Stage V. In conclusion,
patients with ALS that had progressed to result in communication Stage V showed rapidly-progressed symptoms,
and their common lesions could cause the manifestations of communication Stage V.
Keywords: Amyotrophic lateral sclerosis, Communication Stage V, Totally locked-in state, Pallido-nigro-luysiansystem, Brainstem reticular formation, Cerebral cortical degeneration

Introduction
Patients with amyotrophic lateral sclerosis (ALS)
dependent on tracheostomy with invasive ventilation (TIV)
use mostly nonverbal communication and find it difficult
to communicate as their muscle weakness progresses. In aprevious study [9], we proposed a classification system forthe communication abilities of patients with advanced ALS
that consists of five stages: Stage I, communicates insentences; Stage II, communicates with one-word answers
only; Stage III, communicates with nonverbal yes/no responses only; Stage IV, occasionally cannot communicate
due to uncertain yes/no responses; and Stage V, cannot
communicate by any means. We also analyzed the relationship between clinical findings and the prognosis forcommunication disturbance [9, 19]. At present, communication Stage V is indicative of a “totally locked-in state”[7, 8]. Our previous analysis of 29 autopsies of patientswith ALS who were dependent on TIV showed thatseven patients who progressed to Stage V had begun to* Correspondence: mochi@nihon-u.ne.jp
2
Department of Pathology, Tokyo Metropolitan Neurological Hospital, 2-6-1
Musashidai, Fuchu-shi, Tokyo 183-0042, Japan3
Department of Neurology, Tokyo Metropolitan Kita Medical andRehabilitation Center for the Disabled, 1-2-3 Jujodai, Kita-ku, Tokyo 114-0033,
Japan
Full list of author information is available at the end of the article© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

87

Hayashi et al. Acta Neuropathologica Communications (2016) 4:107

Page 2 of 14

these patients as having phosphorylated TAR DNAbinding protein 43 (pTDP-43)-immunoreactive (ir) neuronalcytoplasmic inclusions (NCI) and copper/zinc superoxide
dismutase (SOD1)-ir NCI respectively. Therefore, we
included them in the present study, and reevaluated theclinicopathological and immunohistochemical featuresof all 11 patients using the same criteria. Of the 11 patients,six (patients 1–6) had pTDP-43-ir NCI, two (patients 7, 8)
had fused in sarcoma (FUS)-ir NCI, and three (patients
9–11) had SOD1-ir NCI (Table 1). This study was approved by the Ethical Review Boards of Tokyo Metropolitan Neurological Hospital and Niigata University.

require TIV significantly earlier than patients who diedin Stage IV or earlier, and the patients who progressedto Stage V frequently had a family history of ALS andgene mutation [9]. Further study showed that need for
TIV, impaired oculomotor movement, and becoming
totally quadriplegic within 24 months of ALS onset
were predictors of severe communication impairment.
Therefore, we recommended early detection of impaired
communication and identification of the best methods ofcommunication [19]. The first neuropathological reportsof two patients with ALS who progressed to Stage V [7]showed severe multisystem degeneration. By contrast,
Oyanagi et al. [22] reported marked preservation of thevisual and olfactory pathways in patients with ALS who
progressed to Stage V. There are reports of patientswith lesions in their primary motor cortex, but a preserved
cerebral cortex [10, 14, 16, 24]. In contrast, patients withmarked cerebral atrophy due to degeneration of the cerebral cortex and white matter have also been reported[11, 17, 20]. Neuroradiologically, a progressive cerebral
atrophy has been shown in siblings with ALS who carried a mutation in the gene for optineurin (OPTN)[28]. However, the distribution and characteristics ofthe cerebral lesions are unclear. To date, clinicopathology of patients with ALS who progressed to communication Stage V has been reported only in patient reports[7, 10, 11, 14, 16, 17, 20, 24, 26, 27]. Therefore, in thepresent study we performed a comprehensive analysis of
the clinicopathological features and immunohistochemical
characterization of patients with ALS who had progressedto communication Stage V.

Histopathology

Specimens from the brain and spinal cord were fixed with
20 % buffered formalin and embedded in paraffin wax.
Loss of neurons or fibers, or both, and gliosis were assessedin various regions of the nervous system using 10 or 4 μm
sections in hematoxylin and eosin and Klüver–Barrera
stains. When necessary, Bodian and Holzer stains wereadditionally used.
For immunohistochemistry, 6 or 4 μm sections wereprepared. Sections from the frontal lobe, temporal lobe,
hippocampus, parietal lobe, occipital lobe, basal ganglia,
thalamus, cerebellum, midbrain, pons, medulla oblongata,and spinal cord were immunostained for pTDP-43, usinga rabbit polyclonal antibody against pTDP-43 (pS409/410;
CosmoBio, Tokyo, Japan) at a dilution of 1:5000 or 1:8000,
FUS, using a rabbit polyclonal antibody against FUS(Sigma-Aldrich St. Louis, MO, USA) at a dilution of 1:
2000, SOD1, using a rabbit polyclonal antibody against
SOD1 (Proteintech, Tokyo, Japan) at a dilution of 1:100.
Required sections were immunostained for phosphorylated tau protein using the mouse monoclonal antibody
AT8 (Innogenetics, Ghent, Belgium) at a dilution of 1:200,α-synuclein using a mouse monoclonal antibody against
α-synuclein (Wako, Osaka, Japan) at a dilution of 1:8000
or 1:10,000, and ubiquitin using a rabbit polyclonal
antibody against ubiquitin (Dako, Glostrup, Denmark)at a dilution of 1:800. Before antibody incubation, sections were treated by microwaving in citrate-bufferedsaline (pH 6.0, 15 min) to unmask antigens. Antibody
binding was visualized using a labeled streptavidin–biotin
immunoperoxidase method. The chromogen and counterstain were diaminobenzidine and hematoxylin, respectively. NCI in each patient was assessed by immunostaining
with pTDP-43, FUS, or SOD1.
We evaluated the degree and extent of the neuronalloss, gliosis, and NCI semiquantitatively. Degeneration wasassessed as the degree of neuronal loss and gliosis and wasindicated as absent (–), slight (+) (as shown in Fig. 3i), mild(++) (as shown in Fig. 3e-g), or severe (+++) (as shown in
Fig. 3a-d, h). The frequency of NCI, evaluated at 200X
magnification, was indicated as follows: none (0), no NCI

Materials and methods
Patients

By examining medical records, we enrolled 11 (3.4 % ofstudied patients with ALS neuropathology) Japanese patients with ALS who had progressed to Stage V (Table 1),
from among 320 patients with ALS neuropathologicallyconfirmed at autopsy at the Tokyo Metropolitan Neurological Hospital between 1980 and 2012 (150 patients),
and in the Department of Pathology Brain Research Institute, Niigata University between 1963 and 2012 (170patients). No patients had clinical manifestations of either cognitive or behavioral impairment before progressing to Stage V. In addition, no patients showed clinicalevidence of anoxia, such as suffocation, artificial ventilator accident, or blood pressure decrease to <80 mmHg
with shock, during their clinical course. Several patientswere reported elsewhere as having ALS that had progressed to Stage V [7, 10, 16, 17, 20, 24]; however, some ofthe older reports did not provide immunohistochemical
characterization of patient specimens. The clinicopathological features of patients 4 and 11 were previously reported as motor neuron disease [27] and we reevaluated

88

89

M

M

9

10

11

52.0

61

57

38

39

13

64

60

73

53

52

119.5

29

128

106

102

312

57

117

78

120

Time from onset to clinical event (months)

UE

B

LE

LE

LE

UE

UE

B

B

UE

LE

17.0

5

21

8

24

18

12

15

5

56

12

12

38.7

6

45

9

66

18

33

31

na

120

25

34

39.4

19

52

11

86

36

33

18

11

120

14

33

69.9

28

84

36

102

120

56

81

29

120

76

37

47.8

1

44

70

0

192

1

36

49

0

85

67

SOD1

SOD1

SOD1

FUS

FUS

TDP-43

TDP-43

TDP-43

TDP-43

TDP-43

TDP-43[27]
patient 1

[10]

[24]

[16]

[17]
patient 1[7] case 2[7] case 1[27]
patient 2[20]

Disease duration Need for tracheostomy Progression to
Development of Resulting inTime from Stage V Accumulated Reference(months)
invasive ventilation
total quadriplegia overt oculomotor communication to death(months) protein
limitation
Stage Va

F female, M male, na not available, TDP-43 TAR DNA-binding protein 43, FUS fused in sarcoma, SOD1 copper/zinc superoxide dismutase 1, LE lower extremity, UE upper extremity, B bulbaraCommunication Stage V is indicative of a “totally locked-in state” [7, 8], in which all voluntary movements are lost and communication is impossible by any means

mean

M

F

8

F

M

5

M

M

4

7

F

3

6

F

2

161

104

F

1

62

Diseaseduration(months)

Patients Sex Age
at onset(years)

Table 1 Clinical characteristics

Hayashi et al. Acta Neuropathologica Communications (2016) 4:107
Page 3 of 14

Hayashi et al. Acta Neuropathologica Communications (2016) 4:107

Page 4 of 14

tract in the spinal cord. The anterior horn of the spinal
cord and the tegmentum of the brainstem including the
hypoglossal nucleus showed severe atrophy (Fig. 2) andsevere neuronal loss with a few NCI in the atrophiedneurons. The facial and the trochlear nuclei, which wereconfirmed in patients 3 and 11 showed severe neuronalloss with gliosis. The oculomotor nucleus showed a difference in the degree of degeneration. We were not able toevaluate the exact occurrence of NCI in the lesions, because of severe neuronal loss.

across the entire section; rare (1), an average of <2 NCI per
5 fields; occasional (2), an average of 2–10 NCI per 5 fields;
and frequent (3), an average of >10 NCI per 5 fields. For
the semiquantitative analysis, two neuropathologists (K.H.
and Y.M.) observed the specimens, and their scores were
almost coincident.

Results
Clinical characteristics

Except for patient 7 with FUS-ir NCI, all patients had
adult onset ALS (Table 1). The time from ALS onset toneed for TIV was less than 24 months in ten patients,and 56 months in patient 3 with pTDP-43-ir NCI. All
the patients progressed to have total quadriplegia anddeveloped overt oculomotor limitation after using TIV.
Development of oculomotor limitation started before orafter they developed total quadriplegia, and the patientsdeveloped vertical gaze palsy followed by horizontal gazepalsy. Subsequently the patients showed slow eye movement and ultimately became ophthalmoplegic. All the patients used only eye movement for communication justbefore progressing to Stage V.

Lesions of the extrapyramidal motor system and nonmotor
systemNeuronal and fiber loss with gliosis were observed in thesubstantia nigra (Fig. 3a–c), globus pallidus (Fig. 3d–f ),subthalamic nucleus, brainstem reticular formation, cerebellar dentate nucleus, superior cerebellar peduncle, red
nucleus, Clarke’s nucleus, and posterior spinocerebellar
tract in all the patients. In particular, the degeneration of
the substantia nigra (Fig. 3a–c), globus pallidus (Fig. 3d–f),subthalamic nucleus, and brainstem reticular formation(Fig. 2) was moderate or severe in all patients regardless ofthe time for progression from Stage V to death, or the typeof accumulated proteins. By contrast, the cerebellar efferent system, which consists of the cerebellar dentate
nucleus, superior cerebellar peduncle, and red nucleus,
showed mild to moderate degeneration in patients 3, 5,
and 6 with pTDP-43-ir NCI, in patient 8 with FUS-ir
NCI, and in patient 11 with SOD1-ir NCI. The time forprogression from Stage V to death was shorter in these
five patients (0–36 months) than in the other six patients(44–192 months). The cerebellar afferent system, which
consists of the pontine nuclei and middle cerebellar peduncle, was relatively preserved. However, in patient 8, who
had a p.K510M mutation in the gene for FUS (FUS) [16],showed severe degeneration in the cerebellar afferent
pathway. Patient 7, who had a p.P525L mutation in FUS
and severe frontal lobe atrophy [17], showed severe degeneration in the caudate nucleus and putamen (Fig. 3h) andmoderate degeneration in the globus pallidus (Fig. 3e).
The caudate nucleus and putamen (Fig. 3g, i) were relatively better preserved than the globus pallidus (Fig. 3d, f),
except for in patient 7. The inferior olivary nucleus in patients 2 and 10 showed vacuolar degeneration in neurons
and an increase of gemistocytic astrocytes, indicating inferior olivary hypertrophy [5, 10]. For pTDP-43, FUS, andSOD1, no patients exhibited accumulation of multiple different proteins. AT8 immunostaining of neurofibrillarytangles revealed a Braak stage of ≤ II [1]. No patients had
α-synuclein-ir structures. In addition, among patients with
pTDP-43-ir NCI, no patients showed displayed ubiquitinir NCI in cerebellar granule cells or hippocampal CA4
subfield neurons.

Macroscopic findings

Brain weight ranged from 610 to 1395 g (mean 1032.3 g)(Table 2) before formalin fixation. Four patients (patients
1, 2 (Fig. 1b), and 3 with pTDP-43-ir NCI, and 7 with
FUS-ir NCI (Fig. 1a)) showed severe cerebral atrophy,
which was a brain weight of <1000 g. They showedmarked atrophy of both the pons and midbrain. The
brain weight of seven patients (patients 4 (Fig. 1c), 5 and6 with pTDP-43-ir NCI, 8 with FUS-ir NCI, and 9–11
with SOD1-ir NCI) was >1000 g, and their pons andmidbrain showed mild to moderate atrophy. All patients
showed severe atrophy of the spinal cord and the medulla oblongata. The optic nerve (Fig. 1) and the lateralgeniculate body of all the patients appeared preserved.
Microscopic findings

Table 2 shows the neuropathological findings of thepatients.
Lesions of the motor system

All the patients showed a severe loss of Betz cells. The
other neurons in the motor cortex were mildly decreased
with a few NCI in the patients with mild cerebral atrophy(patients 4–6, and 8–11). By contrast, the patients with severe cerebral atrophy (patients 1–3, and 7) showed severe
neuronal loss with gliosis and many NCI in layer 2 andthe deeper layer of the motor cortex. Basophilic inclusions
were frequently observed in patient 7 and rarely observedin patient 8. The pyramidal tract showed severe fiber loss,
although a few small fibers remained in the medullary
pyramid and both the lateral and anterior corticospinal

90

Hayashi et al. Acta Neuropathologica Communications (2016) 4:107

Page 5 of 14

Table 2 Neuropathological findings

DEG degeneration assessed degree of neuronal loss and gliosis on the hematoxylin and eosin, and Klüver–Barrera-stained sections
The degeneration was indicated as absent (–); slight (+); mild (++); or severe (+++)
NCI neuronal cytoplasmic inclusions, The NCI was indicated as none (0); rare (1); occasional (2); or frequent (3), yellow means that we were not able to evaluate
the exact occurrence of NCI in the lesions, because of moderate to severe neuronal loss
n not evaluated (or not examined), VPM ventral posterior medial nucleus

91

Hayashi et al. Acta Neuropathologica Communications (2016) 4:107

Page 6 of 14

Fig. 1 Coronal sections of the cerebrum at the subthalamic nucleus. a Patient 7 with fused in sarcoma (FUS)-immunoreactive (ir) neuronal cytoplasmic
inclusions (NCI) showed enlargement of the anterior horn of the lateral ventricle, and atrophy of the caudate nucleus, putamen, and frontal lobe,
particularly in the frontal white matter along with a thin corpus callosum. However, the hippocampus and temporal lobe were preserved. b Patient 2
with phosphorylated TAR DNA-binding protein 43 (pTDP-43)-ir NCI showed temporal predominately frontotemporal atrophy. c Patient 4 with
pTDP-43-ir NCI showed no white matter atrophy nor enlargement of the lateral ventricle. The globus pallidus and subthalamic nucleus werebrownish with mild to severe atrophy, while the caudate nucleus and the putamen were relatively preserved in all patients except for patient
7 (a), and the optic nerve was preserved in all patients. (Bar = 2 cm)

Fig. 2 Brainstem and seventh cervical cord at the same magnification respectively. All patients showed marked nerve fiber loss of the brainstemtegmentum including the brainstem reticular formation and both lateral and anterior funiculus in addition to pyramidal tract, and showed marked
atrophy of the anterior horn of the spinal cord. The medulla oblongata and spinal cords showed severe atrophy and the fourth ventricle was markedly
dilatated with severe degeneration of the hypoglossal nucleus in all patients. The midbrain and pons showed severe atrophy in patients 2
with phosphorylated TAR DNA-binding protein 43 (pTDP-43)-immunoreactive (ir) neuronal cytoplasmic inclusions (NCI) (a) and patient 7 withfused in sarcoma (FUS)-ir NCI (c) in whom brain weight was less than 1000 g, mild to moderate atrophy in patient 5 with pTDP-43-ir NCI (b),patient 8 with FUS-ir NCI (d), and patient 10 with copper/zinc superoxide dismutase (SOD1)-ir NCI (e) in whom brain weight was more than
1000 g. The superior cerebellar peduncle in patients 5 (b) and 8 (d) were degenerated mildly. Loss of transverse fibers of the pons and middle
cerebellar peduncle were observed only in patient 8 (d). The posterior column of the spinal cord showed a marked loss of fibers in patients 5(g) and 10 (j), but they were relatively preserved in patient 7 (h), and fiber loss of the middle root zone in patient 2 (f) and 8 (i). a, f Patient 2
with pTDP-43-ir NCI. b, g Patient 5 with pTDP-43-ir NCI. c, h Patient 7 with FUS-ir NCI. d, i Patient 8 with FUS-ir NCI. e, j Patient 10 with SOD1-ir NCI,
(Bar = 5 mm, Klüver–Barrera staining)

92

Hayashi et al. Acta Neuropathologica Communications (2016) 4:107

Page 7 of 14

Fig. 3 Lesions of the nonmotor system. a–c Substantia nigra showing severe neuronal loss with gliosis. d–f Globus pallidus, g–i putamen. Only
patient 7 showed more severe degeneration in the putamen (h) than in the globus pallidus (e). Other patients showed more severe degeneration in
the globus pallidus (d, f) than in the putamen (g, i). a-d, h neuronal loss and gliosis were severe (+++). e-g neuronal loss and gliosis were mild (++).
i neuronal loss and gliosis were slight (+). a, d, g Patient 2 with phosphorylated TAR DNA-binding protein 43-immunoreactive (ir) neuronal cytoplasmic
inclusions (NCI). b, e, h Patient 7 with fused in sarcoma-ir NCI. c, f, i Patient 10 with copper/zinc superoxide dismutase-ir NCI, (Bar = 50 μm, hematoxylin
and eosin staining)

NCI in the dentate granule neurons in the hippocampus,
demonstrated the occurrence of NCI, limited to thefrontal cortex, in the absence of neuronal loss (Fig. 5d–f).
The pTDP-43-ir pathological pattern of all six patientswas consistent with type B [12].
One (patient 7) with FUS-ir NCI showed frontal andtemporal cortical degeneration with compacted occurrenceof smaller NCI in the absence of NCI in the hippocampal
dentate granule neurons (Fig. 5g–i). The other (patient 8)
had noncompact NCI in the hippocampal dentate granuleneurons, and their cerebral cortex was preserved with rare
NCI (Fig. 5j–l).
The cerebral cortex, with the exception of the motor
cortex, of all three patients with SOD1-ir NCI was preserved. A few NCI were scattered in the temporal cortex
only in patient 9.

Preserved areas

The optic tract and optic radiation were well preservedin all the patients (Fig. 4). The lateral geniculate bodywas examined in nine patients, of which eight showedcomparative preservation. In the striate cortex, although
there were a few NCI in six patients (patients 1–4 with
pTDP-43-ir NCI, and patients 7 and 8 with FUS-ir
NCI), mild neuronal loss and gliosis were observed onlyin patient 4. The nucleus basalis of Meynert was examined in eight patients, showing no neuronal loss althougha few NCI were found in patients 4, 6 with pTDP-43-ir
NCI and in patient 11 with SOD1-ir NCI.

Cerebral cortical findings by accumulated protein

Among six patients with pTDP-43-ir NCI (patients 1–6),
four (patients 1–4) had NCI in the hippocampal dentate
granule neurons and showed neuronal loss and gliosis
with NCI in the hippocampal subiculum, and the frontal
and temporal cortex (Fig. 5a–c). Patients 5 and 6, lacking

Discussion
As noted in our previous reports [9, 19], patients with
ALS that progressed to Stage V showed rapid clinical93Hayashi et al. Acta Neuropathologica Communications (2016) 4:107

Page 8 of 14

Fig. 4 Preserved areas. a Preserved optic tract (arrow) and severe degeneration of the other regions. b Preserved lateral geniculate body (arrow)and severe degeneration of other regions. (Patient 7, Bar = 5 mm, Klüver–Barrera staining)

concluded that the pallido–nigro–luysian degeneration
may be involved in the ALS disease process [13]. Another
patient with ALS who required TIV and did not progress
to Stage V also showed pallido–nigro–luysian degeneration [23]. These two patients who did not progress toStage V showed no apparent degeneration in the brainstem reticular formation or cerebellar efferent system,
although they did show pallido-nigro-luysian degeneration
similar to the patients who progressed to Stage V [13, 23].
Therefore, it is likely that the degeneration of thebrainstem reticular formation and the cerebellar efferent
system in addition to degeneration of the pallido–nigro–
luysian system is necessary for progression to Stage V.
Relatively mild degeneration of the cerebellar efferent
system was seen in patients who died shortly after progressing to Stage V (patients 3, 5, 6, 8, 11) and has beenreported in one patient [26]. The time for progression
from Stage V to death may alter the degree of degeneration in the cerebellar efferent system.
Neuroanatomically, the common lesions of patients with
ALS who progressed to Stage V appeared to be related toeach other through connections with the motor-relatedarea (Fig. 6) [4]. The degeneration of motor neurons,
which are the basic lesions in ALS, is so severe in patients
who progress to Stage V that the degeneration of associated areas appeared to occur secondarily due to degeneration of motor neurons. We could not evaluate
the precise relationship between neuronal loss and occurrence of NCI because of the severe neuronal loss.
Brettschneider et al. described a pTDP-43 staging

deterioration, and tended to need TIV within 24 monthsof ALS onset. The onset of lesions varied, and the onset
lesions and the time from onset to resulting in Stage V did
not appear to be related. Subsequently, their condition
progressed to Stage V due to complete ophthalmoplegia
in addition to total quadriplegia. This clinical course issimilar to that of the previously reported patients who
progressed to Stage V (Table 3) [11, 14, 26, 28]. However,a few patients required TIV over 24 months after the onset, such as our patient 3 with pTDP-43-ir NCI, and threepatients reported in the existing literature, showing
pTDP-43-ir NCI [11], basophilic inclusions [14], and amutation in OPTN [28], respectively (Tables 1 and 3).
These patients showed that patients who required TIV
after more than 24 months from disease onset could notnecessarily avoid progression to Stage V.
Neuropathologically, regardless of the type of accumulated proteins, all the patients shared the feature of severe
degeneration in the substantia nigra, globus pallidus, subthalamic nucleus, brainstem reticular formation, and mild
to severe degeneration of the cerebellar efferent system, in
addition to the severe degeneration in both upper andlower motor neurons. It is likely that the constellation ofsuch lesions is common in patients with ALS who progress to Stage V, because the patients reported in the literature who had progressed to Stage V also demonstrated
a similar constellation (Table 3) [11, 14, 26]. In addition,
Miki et al. [13] reported a patient with ALS who required
TIV and did not progress to Stage V, and discussed thiswith regard to pallido–nigro–luysian degeneration. They

94

Hayashi et al. Acta Neuropathologica Communications (2016) 4:107

Page 9 of 14

Fig. 5 Immunohistochemical features of cerebral lesions. a–c Patient 2 with phosphorylated TAR DNA-binding protein 43 (pTDP-43)-immunoreactive(ir) neuronal cytoplasmic inclusions (NCI) in the hippocampal dentate granule neurons (c) had occasional NCI in the frontal (a) and temporal (b) cortex.
d–f Patient 6 without pTDP-43-ir NCI in the hippocampal dentate granule neurons (f) had rare NCIs in the frontal cortex (d) and no NCIs in thetemporal cortex (e). g–i Patient 7 with occasional fused in sarcoma (FUS)-ir NCI in the frontal cortex (g) and rare NCI in the temporal cortex(h) had no NCI in the hippocampal dentate granule neurons. j–l Patient 8 without FUS-ir NCI in the frontal (j) and temporal cortex (k) had rare
NCI in the hippocampal dentate granule neurons (l). Bar = 50 μm

communication Stage V. At least in the patients with
pTDP-43-ir NCI, we speculate that the ease of occurrence of NCI and the speed of degeneration in the patients with ALS may differ depending on each lesion, as
follows: (1) Lesions that showed a matched degree of
appearance of NCI and neuronal loss, such as in the
motor neurons, globus pallidus, and substantia nigra, (2)lesions that had NCI from an early disease phase without
rapid neuronal loss, such as in the inferior olivary

algorithm, and presented evidence for a possible sequential dissemination of pTDP-43 pathology [2, 3].
However, applying the stages of pTDP-43 to the present
patients with pTDP-43-ir NCI, the inferior olivary nucleus, which was mentioned as an early lesion [2, 3],
showed relatively mild neuronal loss even in communication Stage V, and the subthalamic nucleus, which wasmentioned as a lesion having no NCI until late stages[2, 3], showed moderate to severe degeneration in95

Age at onset(years)

38

38

63

33

35

44

Sex

M

M

M

F

M

M

27

240

288

144

156

60

Disease duration(months)

15

na

6

na

36 ≥

na

132

20

Progression to total
quadriplegia

24

36

60

11

Need for tracheostomy
invasive ventilation

Time from onset to clinical event, months

15

na

na

36

96

20

Development of overt
oculomotor limitation

Table 3 Clinicopathological characteristics of previously reported patients in communication Stage V

26

36

156

na

144

32

resulting in communication
Stage V

1

204

84

na

12

28

Communication Stage V
to death(months)

naa mutation in OPTN

a mutation in OPTN

TDP-43ubiquitina

ubiquitin

Accumulated protein

Hayashi et al. Acta Neuropathologica Communications (2016) 4:107
Page 10 of 14

96

33

35

44

F

M

M

1320

830

1230

1300

Neuropathology
Brain weight

+++

N/A

N/A

+++

+++

+++

Upper and lower
motor neurons

+

N/A

N/A

+++

+++

+++

Substantia nigra

M male, F female, na not available, N/A not applicable, OPTN the optineurin geneaubiquitin-immunoreactive basophilic inclusionsb
Clinical report

38

63

M

38

M

M

Age at onset(years)

Sex

+

N/A

N/A

+

na

+++

Globus pallidus

+

N/A

N/A

na

na

na

Subthalamic nucleus

Table 3 Clinicopathological characteristics of previously reported patients in communication Stage V (Continued)

+++

N/A

N/A

+++

++

+++

Brainstem reticularformation

+

N/A

N/A

+

+++

++

Cerebellar efferent
system[26][28] Case 2b[28] Case 1b[11][14] Patient 2[14] Patient 1

Reference

Hayashi et al. Acta Neuropathologica Communications (2016) 4:107
Page 11 of 14

97

Hayashi et al. Acta Neuropathologica Communications (2016) 4:107

Page 12 of 14

Motor related area

Frontal cortex

ThalamusPutamenGlobus pallidus
Subthalamic
nucleus

Brainstem
reticular
formation

Substantia nigra

PontinenucleiRed nucleus
Cerebellar dentate nucleus
Cerebellar cortex
Inferior olivary nucleus

Spinal cord
Fig. 6 The connections of motor neuron-related area. The common lesion of the ALS patients who progressed to Stage V is emphasized. All thecommon lesions related to the motor neurons and pyramidal tract. Shaded areas: lesions that showed moderate or severe degeneration in all thepatients regardless of the time for progression to Stage V to death or type of accumulated protein. Stippled areas: lesions that showed a different
degree of degeneration depending on the time from progression to Stage V to death

degeneration showed a contrasting trend in patientswith pTDP-43-ir NCI and FUS-ir NCI in this study as
follows: patient 7 [17], who lacked NCI in the hippocampal dentate granule neurons showed severe atrophy of thecerebrum, whereas patient 8 [16], who had NCI, showedno atrophy of the cerebrum (Table 2, Fig. 5g–l). Second,the cerebral cortex of all 3 patients with SOD1-ir NCI
was preserved with rare NCI. Patients with a mutationin SOD1 were reported as having frontotemporal dementia (FTD) and decline of cognitive function wasrare [25, 29]. Only one patient with a mutation in
SOD1 showing frontotemporal lobar degeneration(FTLD) underwent a detailed clinicopathological analysis [18]. Taking these findings together, it is likely thatthe cerebrum of patients with SOD1-ir NCI may be preserved even in the patients who progressed to Stage V. That
there were three patients who showed a preserved cerebrum (patients 9–11), and that cerebral lesions shared by
all patients and severe neuronal loss were missing in thepresent patients, suggest that cerebral cortical involvement
was not necessary to progress to Stage V.
It is important to differentiate the present patients frompatients showing FTLD. There is a limitation in that the
decline of cognitive function cannot be assessed in thepatients who progressed to Stage V. However, none ofthe patients had any clinical manifestation of cognitiveor behavioral impairment while they could communicate,so they were not diagnosed as having FTD [25]. Furthermore, it was revealed that the patients who progressed toStage V did not always show pathological characteristics
of FTLD.

nucleus, (3) lesions although having rare NCI in early
disease phases, subsequently showed severe neuronalloss, such as in the subthalamic nucleus, and (4) lesions
that had rare NCI without obvious neuronal loss, suchas in the lateral geniculate body and striate cortex in thevisual pathway, which were preserved from lesions inpatients who progressed to Stage V [22].
In this study, patient 2 with pTDP-43-ir NCI and patient
10 with SOD1-ir NCI [10] had inferior olivary hypertrophy.
It is noteworthy that inferior olivary hypertrophy canoccurs in ALS, regardless of accumulated protein, likely
to progressive supranuclear palsy [6], Creutzfeldt–Jakob
disease [15], or cerebrovascular disease [5].
A distinct pattern of protein accumulation was foundin the cerebral cortex. First, in patients with pTDP-43-ir
NCI (Table 2, Fig. 5a–f ), patients with no NCI in the
hippocampal dentate granule neurons (patients 5, 6)
showed limited degeneration in the frontal cortex, andhad relatively preserved brain weight. By contrast, patients with NCI in the hippocampal dentate granule neurons (patients 1–4) showed marked degeneration in thetemporal cortex and hippocampal subiculum; and NCI
were observed in the frontal, temporal, and occipital cortex. The characteristics of the cerebral cortical featuresin the present patients could be classified according to
the presence of NCI in the hippocampal dentate granuleneurons as described by Nishihira et al. [21]. Moreover,
we identified that the patient with a Nishihira’s Type 1
distribution pattern also progressed to Stage V. Interestingly, the relationship between NCI in the hippocampal
dentate granule neurons and cerebral cortical98Hayashi et al. Acta Neuropathologica Communications (2016) 4:107

Page 13 of 14

Conclusion
In the present study, we evaluated only a small number ofpatients because the patients who progressed to Stage V
were limited to 3.4 % of all patients with ALS who underwent autopsy. Nevertheless, we identified that the patients
who progressed to Stage V, including the patients reported
previously, had common lesions in the pallido–nigro–luysian system, brainstem reticular formation, cerebellar dentate nucleus, superior cerebellar peduncle, and red
nucleus, besides motor neurons. Moreover, we clarified specific characteristics in the relationship between accumulated protein and the cerebral cortical lesions. Thus, there
may be common lesions that can identify patients with
ALS who progress to Stage V. We consider that attention
to the occurrence of symptoms of the extrapyramidal
motor system and nonmotor system, and a radiologicalassessment of common lesions are important for predicting communication disability, especially in the patients
who progressed rapidly from disease onset to TIV.

References
1. Braak H, Alafuzoff I, Arzberger T, Kretzschmar H, Tredici KD. Staging of
Alzheimer disease-associated neurofibrillary pathology using paraffin
sections and immunocytochemistry. Acta Neuropathol. 2006;112:389–03.
doi:10.1007/s00401-006-0127-z.
2. Brettschneider J, Arai K, Del Tredici K, Toledo JB, Robinson JL, Lee EB,
Kuwabara S, Shibuya K, Irwin DJ, Fang L, Van Deerlin VM, Elman L,
McCluskey L, Ludolph AC, Lee VM, Braak H, Trojanowski JQ. TDP-43
pathology and neuronal loss in amyotrophic lateral sclerosis spinal cord.
Acta Neuropathol. 2014;128:423–37. doi:10.1007/s00401-014-1299-6.
3. Brettschneider J, Del Tredici K, Toledo JB, Robinson JL, Irwin DJ, Grossman
M, Suh E, Van Deerlin VM, Wood EM, Beak Y, Kwong L, Lee EB, Elman L,
McCluskey L, Fang L, Feldengut S, Ludolph AC, Lee VM, Braak H,
Trojanowski JG. Stages of pTDP-43 pathology in Amyotrophic lateralsclerosis. Ann Neurol. 2013;74:20–38. doi:10.1002/ana.23937.
4. Brodal P. Motor systems. In: Brodal P, editor. The central nervous
system—structure and function. 3rd ed. New York: Oxford University;
2004. p. 241–329.
5. Goto N, Kaneko M. Olivary enlargement: chronological and morphometricanalysis. Acta Neuropathol. 1981;54:275–82. doi:10.1007/BF00697000.
6. Hanihara T, Amano N, Takahashi T, Itoh Y, Yagishita S. Hypertrophy of theinferior olivary nucleus in patients with progressive supranuclear palsy. Eur
Neurol. 1998;39:97–102. doi:10.1159/000007915.
7. Hayashi H, Kato S. Total manifestations of amyotrophic lateral sclerosis.
ALS in the totally locked-in state. J Neurol Sci. 1989;93:19–35. doi:10.1016/
0022-510X(89)90158-5.
8. Hayashi H, Oppenheimer EA. ALS patients on TPPV: totally locked-in state,neurologic findings and ethical implications. Neurology. 2003;61:135–7.
doi:10.1212/01.WNL.0000069925.02052.1F.
9. Hayashi K, Mochizuki Y, Nakayama Y, Shimizu T, Kawata A, Nagao M,
Mizutani T, Matsubara S. Communication disorder in amyotrophic lateralsclerosis after ventilation. A proposal of staging and a study of predictive
factor. Rinsho Shinkeigaku. 2013;53:98–103. doi:10.5692/clinicalneurol.53.98[article in Japanese with English abstract].
10. Hayashi K, Mochizuki Y, Koide R, Kawata A, Homma T, Shimizu T, Komori T,
Isozaki E. A Japanese familial ALS patient with autonomic failure and a p.
Cys146Arg mutation in the gene for SOD1 (SOD1). Neuropathology. 2016.
doi:10.1111/neup.12303 [Epub ahead of print].
11. Kimura T, Jiang H, Konno T, Seto M, Iwanaga K, Tsujihata M, Satoh A,
Onodera O, Kakita A, Takahashi H. Bunina bodies in motor and non-motor
neurons revisited: A pathological study of an ALS patient after long-term
survival on a respirator. Neuropathology. 2014;34:392–7. doi:10.1111/neup.12105.
12. Mackenzie IR, Neumann M, Baborie A, Sampathu DM, Plessis DD, Jaros E,
Perry RH, Trojanowski JQ, Mann DM, Lee VM. A harmonized classification
system for FTLD-TDP pathology. Acta Neuropathol. 2011;122:111–3. doi:10.
1007/s00401-011-0845-8.
13. Miki Y, Mori F, Nunomura J, Ookawa K, Yajima N, Yagihashi S, Wakabayashi K.
Sporadic amyotrophic lateral sclerosis with pallido-nigro-luysian degeneration:
A TDP-43 immunohistochemical study. Neuropathology. 2010;30:149–53.
doi:10.1111/j.1440-1789.2009.01046.x.
14. Mizutani T, Sakamaki S, Tsuchiya N, Kamei S, Kohzu H, Horiuchi R, Ida M,
Shiozawa R, Takasu T. Amyotrophic lateral sclerosis with ophthalmoplegia
and multisystem degeneration in patients on long-term use of respirators.
Acta Neuropathol. 1992;84:372–7. doi:10.1007/BF00227663.
15. Mizutani T. Neuropathology of Creutzfeldt-Jakob disease in Japan. With
special reference to the panencephalopathic type. Acta Pathol Jpn.
1981;31:903–22. doi:10.1111/j.1440-1827.1981.tb02006.x.
16. Mochizuki Y, Kawata A, Maruyama H, Homma T, Watabe K, Kawakami H,
Komori T, Mizutani T, Matsubara S. A Japanese patient with familial ALS anda p.K510M mutation in the gene for FUS (FUS) resulting in the totally
locked-in state. Neuropathology. 2014;34:504–9. doi:10.1111/neup.12130.
17. Mochizuki Y, Isozaki E, Takao M, Hashimoto T, Shibuya M, Arai M, Hosokawa M,
Kawata A, Oyanagi K, Mihara B, Mizutani T. Familial ALS with FUS P525L
mutation: two Japanese sisters with multiple systems involvement. J
Neurol Sci. 2012;323:85–92. doi:10.1016/j.jns.2012.08.016.
18. Nakamura M, Bieniek KF, Lin WL, Graff-Radford NR, Murray ME,
Castanedes-Casey M, Desaro P, Baker MC, Rutherford NJ, Robertson J,
Rademakers R, Dickson DW, Boylan KB. (2015) A truncating SOD1 mutation, p.
Gly141X, is associated with clinical and pathologic heterogeneity, including
frontotemporal lobar degeneration. Acta Neuropathol. 2015;130:145–57.
doi:10.1007/s00401-015-1431-2.

Acknowledgments
The authors are grateful to the laboratory members at the Tokyo Metropolitan
Neurological Hospital, Tokyo Metropolitan Institute of Medical Science, and Brain
Research Institute, Niigata University for their excellent technical assistance. This
work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (B))
Grant Number 22390429, by the Joint Program for ALS Research at the Tokyo
Metropolitan Institute of Medical Science, and by the Collaborative Research
Project (2626) of the Brain Research Institute, Niigata University.

Authors’ contributions
KH and YM designed the study and performed semiquantitative analysis. KH,
YM, RT, KW, NA, KO, MH, HT, and AK performed the pathologicalobservations and evaluations. KH, ST, MN, OO, and EI performed clinicalevaluations. KW, NA, OK, MH, HT, AK, and EI supervised the whole process ofthe study. KH and YM wrote the manuscript. All authors read and approved
the final manuscript.

Competing interests
The authors declare that they have no competing interests. The authors
alone are responsible for the content and writing of the paper.
Author details
1
Department of Neurology, Tokyo Metropolitan Neurological Hospital, 2-6-1
Musashidai, Fuchu-shi, Tokyo 183-0042, Japan. 2Department of Pathology,
Tokyo Metropolitan Neurological Hospital, 2-6-1 Musashidai, Fuchu-shi, Tokyo
183-0042, Japan. 3Department of Neurology, Tokyo Metropolitan Kita Medical
and Rehabilitation Center for the Disabled, 1-2-3 Jujodai, Kita-ku, Tokyo
114-0033, Japan. 4Department of Neurology, Brain Research Institute, Niigata
University, 1-757 Asahimachidori, Chuo-ku, Niigata 951-8585, Japan.
5
Department of Pathology, Brain Research Institute, Niigata University, 1-757
Asahimachidori, Chuo-ku, Niigata-shi, Niigata 951-8585, Japan. 6Department
of Sensory and Motor System, Tokyo Metropolitan Institute of Medical
Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.
7
Department of Laboratory Neuropathology, Tokyo Metropolitan Institute ofMedical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.
8
Division of Neuropathology, Department of Brain Disease Research, Shinshu
University School of Medicine, 3-1-1 Asahi, Matsumoto-shi, Nagano 390-8621,
Japan. 9Department of Brain Development and Neural Regeneration, Tokyo
Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku,
Tokyo 156-8506, Japan.
Received: 11 August 2016 Accepted: 22 September 201699

Hayashi et al. Acta Neuropathologica Communications (2016) 4:107

Page 14 of 14

19. Nakayama Y, Shimizu T, Mochizuki Y, Hayashi K, Matsuda C, Nagao M,
Watabe K, Kawata A, Oyanagi K, Isozaki E, Nakano I. Predictors of impaired
communication in amyotrophic lateral sclerosis patients with tracheostomyinvasive ventilation. Amyotroph Lateral Scler Frontotemporal Degener.
2016;17:38–46. doi:10.3109/21678421.2015.1055276.
20. Nishihira Y, Tan C-F, Toyoshima Y, Yonemochi Y, Kondo H, Nakajima T,
Takahashi H. Sporadic amyotrophic lateral sclerosis: Widespread multisystemdegeneration with TDP-43 pathology in a patient after long-term survival
on a respirator. Neuropathology. 2009;29:689–96. doi:10.1111/j.1440-1789.
2008.00999.x.
21. Nishihira Y, Tan CF, Onodera O, Toyoshima Y, Yamada M, Morita M,
Nishizawa M, Kakita A, Takahashi H. Sporadic amyotrophic lateral sclerosis: twopathological patterns shown by analysis of distribution of TDP-43-immunoreactive
neuronal and glial cytoplasmic inclusions. Acta Neuropathol. 2008;116:169–82.
doi:10.1007/s00401-008-0385-z.
22. Oyanagi K, Mochizuki Y, Nakayama Y, Hayashi K, Shimizu T, Nagao M,
Hashimoto T, Yamazaki M, Matsubara S, Komori T. Marked preservation of
the visual and olfactory pathways in ALS patients in a totally locked-in state.
Clin Neuropathol. 2015;34:267–74. doi:10.5414/NP300859.
23. Sasaki S, Tsutsumi Y, Yamane K, Sakuma H, Maruyama S. Sporadic amyotrophic
lateral sclerosis with extensive neurological involvement. Acta Neuropathol.
1992;84:211–5. doi:10.1007/BF00311398.
24. Shimizu T, Kawata A, Kato S, Hayashi M, Takamoto K, Hayashi H, Hirai S,
Yamaguchi S, Komori T, Oda M. Autonomic failure in ALS with a novel SOD1
gene mutation. Neurology. 2000;54:1534–7. doi:10.1212/WNL.54.7.1534.
25. Strong MJ, Grace GM, Freedman M, Lomen-Hoerth C, Woolley S, Goldstein LH,
Murphy J, Shoesmith C, Rosenfeld J, Leigh PN, Bruijn L, Ince P, Figlewicz D.
Consensus criteria for the diagnosis of frontotemporal cognitive andbehavioural syndromes in amyotrophic lateral sclerosis. Amyotroph
Lateral Scler. 2009;10:131–46. doi:10.1080/17482960802654364.
26. Tabuchi Y, Takahashi K, Tanaka J. Familial amyotrophic lateral sclerosis with
ophthalmoplegia. Rinsho Shinkeigaku. 1983;23:279–87 [article in Japanesewith English abstract].
27. Takeda S, Yamada M, Kawasaki K, Oyanagi K, Ikuta F, Arai M, Inuzuka T, Yuki N,
Yuasa T, Sato S, Tsuji S, Miyatake T. Motor neuron disease with multisystem involvement presenting as tetraparesis, ophthalmoplegia andsensori-autonomic dysfunction. Acta Neuropathol. 1994;88:193–200.
doi:10.1007/BF00293393.
28. Ueno H, Kobatake K, Matsumoto M, Morino H, Maruyama H, Kawakami H.
Severe brain atrophy after long-term survival seen in siblings with familial
amyotrophic lateral sclerosis and a mutation in the optineurin gene: a caseseries. J Med Case Rep. 2011;5:573. doi:10.1186/1752-1947-5-573.
29. Wicks P, Abrahams S, Papps B, Al-Chalabi A, Shaw CE, Leigh PN, Goldstein LH.
SOD1 and cognitive dysfunction in familial amyotrophic lateral sclerosis.
J Neurol. 2009;256:234–41. doi:10.1007/s00415-009-0078-0.

Submit your next manuscript to BioMed Central
and we will help you at every step:• We accept pre-submission inquiries• Our selector tool helps you to find the most relevant journal• We provide round the clock customer support• Convenient online submission• Thorough peer review• Inclusion in PubMed and all major indexing services• Maximum visibility for your research
Submit your manuscript at
www.biomedcentral.com/submit100