Open Access Case
Report

DOI: 10.7759/cureus.6920

Contribution of Transferrin and
Ceruloplasmin Neurotransmission andOxidant/Antioxidant Status to the Effects ofEverolimus: A Case Series
Kunio Yui 1 , George Imataka 2 , Hitomi Sasaki 1 , Yohei Kawasaki 3 , Shigemi Yoshihara 2
1. Department of Urology, Fujita Health University Hospital, Toyoake, JPN 2. Department of Pediatrics,
Dokkyo Medical University Hospital, Mibu, JPN 3. Clinical Research Center, Chiba University Hospital,
Chiba, JPN
Corresponding author: Kunio Yui, yui16@bell.ocn.ne.jp

Abstract
Tuberous sclerosis complex (TSC) is a genetic disorder with a high prevalence of associatedautism spectrum disorder (ASD). The pathophysiology of TSC mainly involves thehyperactivation of mammalian target of rapamycin (mTOR) induced by TSC1 (hamartin) and
TSC2 (tuberin) heterozygosity. The mTOR inhibitor, everolimus, is a therapeutic target for TSCrelated ASD. The efficacy of everolimus may be affected by iron and copper neurotransmission
and oxidant-antioxidant systems. Creatine has an antioxidant activity related to thecytoprotective paradigm. Additionally, TSC-related epileptic activity may influence thedevelopment of autistic symptoms. This case series examined the efficacy of everolimus in
relation to the serum levels of the iron mediator (transferrin (Tf)), the copper mediator(ceruloplasmin (Cp)), the oxidant marker (oxidized low-density lipoprotein (oxLDL)), theantioxidant marker (total antioxidant power (TAP)), and creatine in four cases of TSC
accompanied with autism. Everolimus improved autistic symptoms with increased serum Cp
and Tf levels in all four cases. Serum TAP and creatine levels showed positive correlations
with decreased total Aberrant Behavior Checklist (ABC) and Social Responsiveness Scale (SRS)
scores, respectively. As everolimus regulates iron homeostasis and increased copper levels
suppress mTOR signaling, everolimus improved autism symptoms with increased serum levelsof Cp and Tf via homeostatic control of mTOR activity, accompanied with the considerable
overlap of oxidant-antioxidant systems, such as TAP and creatine. Everolimus had no effect on
TSC-related epileptiform discharges; thus, the autistic symptoms and epileptic activity may be
two independent end results of a common central nervous system including mTOR
hyperactivity.

Received 02/11/2019
Review began 04/02/2019
Review ended 12/24/2019
Published 02/08/2020© Copyright 2020
Yui et al. This is an open accessarticle distributed under the terms of
the Creative Commons Attribution
License CC-BY 4.0., which permits
unrestricted use, distribution, andreproduction in any medium, provided
the original author and source arecredited.

Categories: Neurology, Pediatrics
Keywords: tuberous sclerosis, autism, everolimus, oxidant/antioxidant systems, creatine,
ceruloplasmin transferrin

Introduction
Tuberous sclerosis complex (TSC) is a multisystem disorder with a high prevalence of
associated autism spectrum disorders (ASDs). As mutations in TSC1 (hamartin) and TSC2
(tuberin) proteins lead to mammalian target of rapamycin (mTOR) hyperactivity in thepathophysiology of TSC, mTOR inhibitors are the therapeutic targets for TSC-related autistic
symptoms [1]. Nevertheless, treatment with the mTOR inhibitor, everolimus, showed only a
30% improvement in 35 patients with TSC in one study [2]. Moreover, a prospective, double-

How to cite this article
Yui K, Imataka G, Sasaki H, et al. (February 08, 2020) Contribution of Transferrin and Ceruloplasmin
Neurotransmission and Oxidant/Antioxidant Status to the Effects of Everolimus: A Case Series. Cureus
12(2): e6920. DOI 10.7759/cureus.6920

blind randomized, placebo-controlled study revealed that treatment with everolimus (4.5mg/m2 of body surface area) did not significantly improve neurocognitive function or abnormal
behavior [3]. Thus, there is a critical need for alternative medical approaches for the treatment
of TSC-related ASD.
Importantly, mTOR regulates cellular iron homeostasis by modulating the transferrin (Tf)receptor [4]. Furthermore, increased intracellular copper levels suppress mTOR signaling [5].
Thus, mTOR activity may regulate iron and copper homeostasis with Tf acting as an ironmediator [6] and ceruloplasmin (Cp) as a copper mediator [7]. Oxidized low-density lipoprotein(oxLDL) suppresses the P13K/AKT/mTOR signaling pathway as the oxidative stress marker [8].
Additionally, creatine has antioxidant activity and emerges as an additional mechanism [9].
Moreover, mTOR stimulates the creatine transporter through mechanisms partially shared withthe glucocorticoid-inducible kinase in protein synthesis [10]. Thus, there is a close relationshipbetween mTOR activity and creatine.
As mTOR regulates neuronal excitability in established neural circuits, mTOR hyperactivation
enhances neural excitability related to seizures [1], inducing epileptiform discharges (referred
to as spikes) on electroencephalograms (EEGs) and may contribute to progressive brain
dysfunction, including autistic symptoms [11].
In this study, to examine the therapeutic contribution of serum levels of creatine, oxidant(oxLDL)/antioxidant status (total antioxidant power (TAP)), and neurotransmission of Tf and
Cp to the efficacy of everolimus, we assayed the serum levels of these variables in four cases of
TSC-associated ASD. Subsequently, the main findings on the association between autistic
assessment scores and plasma variables are presented. Furthermore, this study also examined
the association between epileptiform discharges and TSC-related autistic symptoms.

Case Presentation
Technical information
Our study included four cases of TSC-associated ASD. ASD was diagnosed by one psychiatrist
and one pediatric ASD specialist using the Autism Diagnostic Interview-Revised (ADI-R) andthe Autism Diagnostic Observation Schedule (ADOS). ADI-R was usually used as a diagnostic
instrument [12], while ADOS was useful for studying the longitudinal changes of core autism
symptom severity.
Four cases were treated using the mTOR inhibitor, everolimus (1.47 mg/m 2 of body surfacearea) for 24 weeks. Social impairment and quantitative autistic social impairment were
evaluated using the Social Responsiveness Scale (SRS) and Social Communication
Questionnaire (SCQ), respectively. Abnormal behaviors were evaluated using the Aberrant
Behavior Checklist (ABC). These evaluations were conducted at baseline and 4, 8, 12, 16, 20,
and 24 weeks after initiation of the everolimus treatment. Table 1 details the clinicalcharacteristics of the four patients. TAP and serum levels of Tf, Cp, creatine, and oxLDL wereassayed at baseline, 8, 16, and 24 weeks after initiation of the everolimus treatment. Serumeverolimus levels were measured at 12, 16, and 24 weeks after the treatment initiation.

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Patient

Case 1Case 2

Case 3

Case 4Age

Female, 8 years

Male, 10 years

Male, 11 years

Female, 6 years

6 years

6 years

6 years

7 months

Age atonset ofASD
SEGA in the right

AML in bilateral
MRI orEchokidneys, SEN left onanterior horn of lateral
ventricle

AML in the right side,
SEN in the left frontal
area

SEGA located beside in theforamen of Monroforamen of Monro,
SEN in the bothinterventricularforamina
Deviated social

Wrapping up doll play

Fantastic confabulation,with repeated finger-stereotyped imitation ofsucking, lost temper

TV announcers)

67

57

67

61

2.5 mg/day for 24

3.6 mg/day for 24

4.4 mg/day for 24 weeks,

1.9 mg/day for 24

weeks, ABC score: 70%

weeks, ABC score: 67%

ABC score: 54% decrease,

weeks, ABC score: +2

decrease, SRS score:
Everolimus
27% decrease, social
doses
response and repetitive
Results
behaviors weredecrease, SRS score:
9% decreased, repeated

SRS score: 40% decrease,
impaired social% change, SRS score:
45% decrease,behaviors and social

communication and repetitive

impaired social

impairment were

behaviors were apparently

communication was

improved

improved

improved

Serum Tf and Cp levels

Serum Tf and Cp
levels increased inincreased in accordance withaccordance withsymptom improvement.

decreased ABC andASD
features

WISC IQ

improved

Neologisms, restricted,
fixated interests

drawing faces

Serum Tf and Cp levels
Serumlevels of Tf
and Cpincreased from baseline

Serum Tf and Cp levels

to 24 weeks oftreatment in accordance

increased in accordancewith symptom

with symptom

improvement.

SRS scores

improvement.
Serum

Serum TAP levels

levels ofshowed opposite

TP andreaction to decreased

oxLDL

ABC scoresinteraction, functional
tales, stereotyped

Serum oxLDL levels

Serum TAP levels increased

showed an opposite
reaction to serum TAP

in accordance with decreased
ABC scores

Serum TAP levels inaccordance withdecreased SRS
scoresTABLE 1: Patients’ Clinical and MRI Features
ABC: Aberrant Behavior Checklist; ADI-R: Autism Diagnostic Interview, Revised; AML: angiomyolipomas; ASD: autism spectrum
disorder; Cp: ceruloplasmin; Echo: echocardiogram; IQ: intelligence quotient; MRI: magnetic resonance imaging; oxLDL: oxidized lowdensity lipoprotein; SEGA: subependymal giant cell astrocytoma; SEN: subependymal nodule; SRS: Social Responsiveness Scale;
TAP: total antioxidant power; Tf: transferrin; TV: television; WISC-5: Wechsler Intelligence Scale for Children, 5th Ed.

Brain magnetic resonance imaging (MRI) and EEG measurements were conducted immediately
before and after everolimus treatment.

2020 Yui et al. Cureus 12(2): e6920. DOI 10.7759/cureus.6920

3 of 16Case 1The patient was an eight-year-old girl born of a non-consanguineous marriage and a full-term,
normal delivery with TSC accompanied by abnormal speaking without expression or intonation,
delayed language development, poor socialization, poor eye contact, no imitation and pretend
play, stereotypical movements, repetitive play, and temper tantrums (Table 1). At the age offour months, she experienced focal seizures with impaired awareness for the first time,
followed by another one at two years of age. Although she received anticonvulsant therapy,
these seizures sometimes reoccurred. Her EEG recording indicated high voltage, irregular slow
waves intermixed with spikes and polyspikes (hypsarrhythmia) (Figure 1). The presence of such
epileptogenic lesions represents epileptiform discharges [11], which are brief abnormalities thatstand out from the EEG background, usually due to a peak or sharp appearance, including
hypsarrhythmia.

FIGURE 1: Electroencephalography of Case 1 before treatment

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4 of 16(a) and after treatment (b)At the age of three years, abdominal ultrasonography revealed small angiomyolipomas (AMLs)
on both kidneys.
At the age of six years, she developed the autistic symptoms described above, with the primaryones being repetitive play, wrapping up in doll play alone with repeated finger-sucking, anddelayed motor milestones. She sometimes lost her temper whenever things did not go the way
she wanted. A pediatric opinion was sought in view of intractable epilepsy and autistic
symptoms, and ASD was clinically diagnosed using the ADI-R and the ADOS.
As shown in Table 1, her scores clearly exceeded the cut-off limits for autism in all three ADI-R
domains (social interaction domain: 21, cutoff = 10; communication domain: 19, cutoff = 15;
restricted/repetitive domain: 3, cutoff = 3), indicating mild ASD. Her total ADOS score (total
score was 16.0 in communication, imagination/creativity, and reciprocal social interaction) was
greater than the mean total scores (females - 8.94, males - 9.71) reported in a recent study of 33
adolescents with ASD [13]. The SRS and SCQ scores were used to evaluate social cognition [14].
Her SRS total score (107) was comparable to and the total ABC score (50) was greater than themean score of 29 individuals (age range: 13 - 27 years) with ASD (total SRS and ABC scores
were 120.21 and 60.14, respectively) [15]. A total SRS score greater than 76 indicated severe
levels of social reciprocity difﬁculties [16]. As the SCQ cutoff score is ≥ 14 [17], her total SCQ
score of 6 was within the normal range. Intelligence assessment using the Wechsler Intelligence
Scale for Children (WISC-IV) (total intelligence quotient (IQ) was 76) suggested normal levels
of intellectual functioning (Table 1). No abnormalities were observed on ophthalmologic anddental examinations.
At the age of seven years, MRI revealed a subependymal nodule (SEN) on the left lateral
ventricle. These findings confirmed the diagnosis of clinically definite TSC (Table 1).
At the age of seven years, she received everolimus treatment (2.5 mg/day for 24 weeks).
Everolimus treatment improved the impaired social interaction and repetitive finger-sucking.
Moreover, her ability to comprehend the intentions and feelings of others improved; thus, she
exhibited an effective response to her mother’s scolding.
As shown in Table 2, her SRS, ABC, SCQ, and ADOS scores gradually decreased in parallel withsymptom improvement. Particularly, the total ABC, SRS, and SCQ scores decreased by 70%,
27%, and 50%, respectively, at the end of the 24-week treatment as compared with baseline
scores, indicating an improvement of her behavioral symptoms and social impairment (Table
2). According to the previous studies, a 20% reduction in initial states is defined as the response
criteria in psychiatric clinical trials [18]. Thus, her social and behavioral symptoms showed a
response to everolimus treatment. Her small AMLs disappeared; however, no changes wereobserved in the SEN after the treatment.

Baseline

ADI-R Social
interaction Domain

ADI-R
Communication
DomainADI-R Restricted/Total Scores Repetitive

Total

Behavior Domain

Score

Case 1

21

19

3

43

Case 2

25

14

12

51

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5 of 16Case 3

23

14

12

49

Case 4

18

14

4

36

ADI-R Scores at the Baseline
BaselineCommunication

Reciprocal Social
Interaction

Imagination/Creativity

Stereotyped Behavior andRestricted Interests

Total
ScoresCase 1

5

10

1

0

16

Case 2

7

10

1

6

24

Case 3

6

10

2

5

23

Case 4

5

5

1

0

11

Case 1 %decrease

4 (20.0)

2 (80.0)

1 (0.0)0 (0.0)7(56.3)

Case 2 %decrease

3 (57.1)

1 (90.0)

1 (0.0)4 (33.3)

9(62.5)

4 (20.0)

100.0 (44.0)0 (100.0)

2 (60.0)5 0.0

1 (80.0)

1 (0.0)0 (0.0)24 weeks

Case 3 %decrease
Case4 %
decrease

6(30.4)
7(36.4)ADOS Scores at the Baseline and 24 Weeks of Treatment
Case

Case 1

Case2

Case 3

Case 4

ABC

50

74

113

36

SRS

107

119

126

80

SCQ

6

16

17

10

ABC % decrease

15 (70.0)67 (9.5)

52 (54)37 +2.8%

SRS % decrease78 (27.1)

97 (9.3)

76 (39.6)

44 (45.0)

SCQ % decrease

3 (50)

13 (18.8)

11 (35.3)

8 (20.0)

Baseline

24 weeks

TABLE 2: Scores of ADI-R, ADOS, ABC, SRS, and SCQ at Baseline and Changes of
These Scores at 24 Weeks
ABC: Aberrant Behavior Checklist; ADI-R: Autism Diagnostic Interview-Revised; ADOS: Autism Diagnostic Observation Schedule; SCQ:
Social Communication Questionnaire; SRS: Social Responsiveness Scale2020 Yui et al. Cureus 12(2): e6920. DOI 10.7759/cureus.6920

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Serum Tf and Cp levels at baseline gradually increased till 24 weeks, in correlation withimprovements in behavioral and social symptoms (the decreased total scores of ABC, as well as
SRS). The serum TAP levels gradually decreased from baseline to the lowest value at 16
weeks and then increased at 24 weeks, showing a trend similar to that for the decreased total
ABC scores (Figure 2). The serum creatine and oxLDL levels showed no definite alteration incorrelation with the total ABC and SRS scores. As her epilepsy and epileptiform discharges did
not improve, she received a complete corpus callosotomy at the age of nine years, which
resulted in the disappearance of her epilepsy and autism.

FIGURE 2: Total scores of SRS, ABC, SCQ and ADOS andserum levels of Tf, Cp, oxLDL, TAP, and creatine in Case 1
ABC: Aberrant Behavior Checklist; ADOS: Autism Diagnostic Observation Schedule; Cp:
ceruloplasmin; oxLDL: oxidized low-density lipoprotein; SCQ: Social Communication Questionnaire;
SRS: Social Responsiveness Scale; TAP: total antioxidant power; Tf: transferrin

Case 2
A 10-year-old male presented with TSC associated with autistic verbal stereotypy (Table 1). At
the age of five months, the patient experienced a severe epilepsy syndrome of the triad of focal
epileptic spasms (West syndrome), which was characterized by clusters of epileptic spasms,
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psychomotor delay, and a specific interictal electroencephalogram (EEG) pattern known as
hypsarrhythmia.
At the age of six years, his ASD symptoms gradually began to develop. Important features of
ASD symptoms included repeatedly asking the same questions on his own matter of interest,
frequently imitating television announcers, and sometimes exhibiting fantastic confabulations.
Moreover, he sometimes exhibited flashbacks of memories of frightening experiences, resulting
in psychomotor excitation. His mean ADI-R domain scores exceeded the cut-off limits for
autism (social interaction domain - 25, communication domain - 14, and restricted/repetitive
domain - 12). His ADOS total score (24.0) was greater than the mean total score (females - 8.94,
males - 9.71) reported in a recent study of 33 adolescents with ASD [13]. His scores for the
ADOS and ADI-R confirmed the diagnosis of ASD consisting mainly of reciprocal interaction
and communication. The patient’s total SRS score (119) was comparable to the mean score of
29 individuals with ASD (age range: 13 - 27 years) where the total SRS was 120.2 [15]. His total
SCQ score of 16 indicated impaired social interaction and social-communicationdifficulties [17]. His total IQ on the WISC-IV was 61, suggesting a borderline IQ (Table 1).
A SEN in the left frontal area and AMLs on the right kidney were detected at the age of nine
years. As antiepileptics were ineffective, a focal resection of the SEN was performed with theaim of eliminating his seizures. However, epileptiform discharges persisted.
At the age of nine years, he received everolimus treatment (3.6 mg/day for 24 weeks). After thetreatment, he started to understand and follow some verbal commands issued by his mother,and the repetitive patterns of asking questions disappeared. However, the epileptiformdischarges still persisted after the treatment (Figure 3). There was a slight size reduction in thepatient’s SEN.

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FIGURE 3: Electroencephalography of Case 2 before treatment(a) and after treatment (b)At the age of 10 years, pediatric opinion was sought in view of intractable epilepsy, and he wasdiagnosed as TSC with autistic social and behavioral symptoms. His EEG was suggestive of
hypsarrhythmia (Figure 3), which was considered as epileptiform discharge (Table 1).
His SRS, ABC, SCQ, and ADOS scores gradually decreased. The SRS and SCQ decreased by 18.5%
and 18.8%, respectively, at the end of treatment as compared with baseline total scores. As
there was about a 20% reduction of the SRS total scores, it may be considered as a response totreatment [18]. Thus, the patient’s social symptoms showed a response to everolimus (Table 2).
Serum Cp levels gradually increased during the treatment, and serum Tf levels increased at 162020 Yui et al. Cureus 12(2): e6920. DOI 10.7759/cureus.6920

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weeks in parallel with an improvement of social and behavioral symptoms as indicated by adecrease in the total SRS and ABC scores. The serum TAP levels gradually increased until 16
weeks in correlation with decreased total SRS scores, indicating an improvement in social
impairment. Serum oxLDL levels gradually increased from baseline to the peak level between 16
to 24 weeks, showing a trend toward negative correlation with decreased total ABC scores(Figure 4).

FIGURE 4: Total scores of SRS, ABC, SCQ, and ADOS andserum levels of Tf, Cp, oxLDL, TAP, and creatine in Case 2
ABC: Aberrant Behavior Checklist; ADOS: Autism Diagnostic Observation Schedule; Cp:
ceruloplasmin; oxLDL: oxidized low-density lipoprotein; SCQ: Social Communication Questionnaire;
SRS: Social Responsiveness Scale; TAP: total antioxidant power; Tf: transferrin

Case 3
An 11-year-old male patient, born of a full-term, normal delivery, had TSC associated with
developed impairment of social cognition, which primarily included impaired social interaction
and communication.
He experienced infantile spasms at the age of 13 months which were treated using2020 Yui et al. Cureus 12(2): e6920. DOI 10.7759/cureus.6920

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antiepileptics; thereafter, his epileptic seizures did not recur.
From the age of six years, he developed autistic symptoms. His important clinical features werethat he often showed delayed speech and sometimes created neologisms and idiosyncratic
language. His speech was confined to narrow topics of expertise. He had repetitive patterns of
behavior, such as washing his face in the morning and maintained this habit. When his routine
was disturbed by others, he usually entered a state of panic. Thus, he gradually withdrew into
his repetitive play and behavior (Table 1).
At the age of eight years, brain MRI showed a subependymal giant cell astrocytoma (SEGA) at
the right foramen of Monro, for which he received a keyhole craniotomy. Eight months after
this operation, an MRI of the brain and kidney showed a small SEGA on the left foramen of
Monro and two AMLs on both kidneys. An EEG at the age of 11 years showed slow-wave andfast generalized small spindle-like ictal epileptiform discharges in the anterior-temporal area.
As shown in Table 1, his ADI-R scores were above the diagnostic cutoff scores in three domains(social interaction, 23; communication, 14; restricted/repetitive domain, 12). His total ADOS
score was 23.0, which was greater than the mean total ADOS score (8.94 ± 9.71) of 33
adolescents with high-functioning autism [17]. His SRS and ABC total scores were 126 and 113,
respectively, which were greater than those of individuals with ASD (age, 13 - 27 years) [15]. As
shown in Table 2, his total SCQ score (17.0) was greater than the established cutoff for ASD
screening (scores ≥ 14) [13]. At the age of 10 years, his total IQ on the WISC-IV was 67,
indicating a borderline IQ (Table 1). Other evaluations revealed no abnormalities.
At the age of 10 years, he received 4.4 mg/day of everolimus for 24 weeks. Everolimus
remarkably improved social communication and behavioral symptoms without severe side
effects. Three months after the treatment, he was able to understand and follow some verbal
commands issued by classmates and communicated with them. After four months, repetitive
motions disappeared.
His SRS, ABC, SCQ, and ADOS total scores gradually decreased in parallel with symptom
improvement. Particularly, the SRS and ABC total scores decreased by 39.6% and 54.0%,
respectively, as compared with the baseline scores, indicating improvement of social
impairment and behavioral symptoms (Table 2). Also, the SCQ and ADOS total scores
decreased by 35.3% and 30.4%, respectively, as compared with the baseline scores, indicating
improvement of social impairment and behavioral symptoms. However, epileptiform discharges
persisted. A slight reduction in the sizes of the SEGA (-0.3 cm) and AML (-0.4 cm) weredetected.
Serum levels of Cp, as well as Tf, increased from baseline until eight and 16 weeks incorrelation with symptom improvement as indicated by decreased total scores of SRS, as well
as ABC. Serum oxLDL levels gradually increased to the peak level at 16 weeks, showing a trend
opposite to that of the decreased total SRS scores. By contrast, serum TAP levels gradually
decreased to the lowest level at 16 weeks and then increased at 24 weeks, showing a trend
similar to that of the decreased total ABC and SRS scores. Serum creatine levels increased fromweeks eight to 16, showing a trend similar to that of decreased total SRS and ABC scores (Figure
5).

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FIGURE 5: Total scores of SRS, ABC, SCQ, and ADOS andserum levels of Tf, Cp, oxLDL, TAP, and creatine in Case 3
ABC: Aberrant Behavior Checklist; ADOS: Autism Diagnostic Observation Schedule; Cp:
ceruloplasmin; oxLDL: oxidized low-density lipoprotein; SCQ: Social Communication Questionnaire;
SRS: Social Responsiveness Scale; TAP: total antioxidant power; Tf: transferrin

Case 4
A six-year-old previously healthy girl presented with poor social interaction, delayed motor
milestones, and repetitive behaviors with a persistent sensorimotor or ritualistic quality (Table
1). At the age of six months, an MRI showed a SEGA located at the right foramen of Monro and a
SEN located beside the interventricular septum. There were no AMLs in her kidneys.
From the age of seven months, she gradually developed autistic symptoms. Her important
autistic clinical features included impaired social interaction, and she often engaged in“parallel play” at the edge of a group and did not engage in pretend play. It was difficult for her
to form and sustain interactions with other children, resulting in a withdrawal into repetitiveplay and behaviors. She also created fictional tales and talked about them to herself in a loudvoice at home and in kindergarten. She was very sensitive to the sound of fireworks and astarting pistol (Table 1).

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At the age of 13 months, she experienced focal seizures with impaired awareness. Various
anticonvulsants were ineffective. Her EEG frequently showed spikes and spike-and-wave
complexes in the central areas (Cz and C3), which are sometimes recognized in patients withcomplex partial seizures and considered as epileptiform discharge. Ictal activity was suspected
near the SEGA in the right frontal lobe; therefore, everolimus treatment was initiated to control
her partial seizures.
Her ADI-R scores were above the autism diagnostic cutoff scores (social interaction domain, 18;communication domain, 14; restricted repetitive/stereotyped domain, 4), indicating repetitive
behavior and mild impairment in social communication. Her total score on the ADOS module 2
algorithm showed a communication score of 11.0, which was greater than the mean total scoreof the ADOS (females - 8.94, males - 9.71) reported in 33 adolescents with high-functioning
autism [13]. Her SRS and ABC total scores were 80 and 37, respectively (Table 2). These scores
were lower than those recently reported for a group of individuals with ASD (age: 13 - 27 years)[15]. Collectively, her behavioral ASD symptoms appeared not to be very severe. At the age ofsix years old, her total IQ on the WISC-IV was 61, suggesting a borderline IQ (Table 1).
Collectively, her social and behavioral symptoms were within the moderate range of ASD
symptoms. Other evaluations revealed no additional abnormalities.
At the age of five years, she received 1.9 mg of everolimus every day for 24 weeks. The
everolimus treatment induced apparent improvements in social interaction and verbal andnon-verbal communication. She gradually showed the development of language. She started tocommunicate appropriately with classmates and showed pretend play skills.
As shown in Table 2, her total SRS and SCQ scores gradually decreased. Compared with thebaseline scores, her SRS and SCQ total scores decreased by 45% and 20%, respectively,
indicating an improvement of social impairment (Table 2). However, the total ABC scores at theend of the treatment were only slightly increased (2.8%), indicating no improvement of
behavioral symptoms. Thus, the everolimus treatment induced remarkable improvements inher social impairment. However, her epileptiform discharges persisted.
The size of the SEGA was also slightly reduced (0.2 cm) after the everolimus treatment, but theSEN was unchanged.
Serum Cp and Tf levels gradually increased during the treatment in parallel with social andbehavioral symptom improvement (decreased total scores of SRS, as well as ABC). Serum TAP
levels gradually increased from that at baseline to that at 24 weeks in correlation with total SRS
scores, indicating an improvement in social impairment. The serum levels of oxLDL andcreatine levels showed no definite alteration in correlation with the total ABC and SRS scores(Figure 6).

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FIGURE 6: Total scores of SRS, ABC, SCQ, and ADOS andserum levels of Tf, Cp, oxLDL, TAP, and creatine in Case 4
ABC: Aberrant Behavior Checklist; ADOS: Autism Diagnostic Observation Schedule; Cp:
ceruloplasmin; oxLDL: oxidized low-density lipoprotein; SCQ: Social Communication Questionnaire;
SRS: Social Responsiveness Scale; TAP: total antioxidant power; Tf: transferrin

Discussion
Everolimus treatment improved autism symptoms in all four cases with increased serum Cp
and Tf levels. As described above, mTOR activity regulates iron and copper homeostasis
through modulating Tf as an iron mediator [6] and Cp as a copper mediator [7]. Thus, thepresent findings suggest that everolimus-induced attenuation of mTOR hyperactivity results inincreased serum Tf and Cp levels to induce the homeostatic balance between mTOR activity. In
brief, mTOR hyperactivity may be attenuated with everolimus treatment, resulting in increased
serum Tf and Cp levels to induce the homeostatic balance between mTOR activity and serum Tf
and Cp levels. The previous findings that copper treatment downregulated mTOR signaling
supports these findings [5]. The present finding that serum TAP levels gradually increased fromits baseline value at 24 weeks in accordance with decreased total SRS scores, suggested thatantioxidant properties may partially overlap with the aforementioned homeostaticmechanisms. Oxidative stress is one pathological mechanisms leading to cognitive impairment
via the pro-oxidant pathway [19]. The plasma TAP levels have an inverse correlation with2020 Yui et al. Cureus 12(2): e6920. DOI 10.7759/cureus.6920

14 of 16cognitive function [20]. Thus, a gradual increase in the serum TAP may reflect the decreased
SRS total scores.
Everolimus treatment had no effect on epileptiform discharges in all four cases. A recent review
article proposed the possibility that these two conditions may be different end-results ofcommon brain dysfunction [11] or that they may co-occur depending on shared mTOR
hyperactivation [1]. The present findings suggest that TSC-related autistic symptoms and TSC
epileptiform discharges may be different end-results of mTOR hyperactivity.

Conclusions
Everolimus treatment improved autistic symptoms through increased activity of Cp and Tf to
induce homeostatic balance. This occurred through the attenuation of mTOR hyperactivationand the associated increase in serum Tf and Cp levels in four cases. Importantly, thesealterations were accompanied by the overlapping of the antioxidant properties of creatine andTAP systems. Furthermore, TSC-related autistic symptoms were not influenced by TSC-related
epileptiform discharges.

Additional Information
Disclosures
Human subjects: Consent was obtained by all participants in this study. Ethical Standards
Committee of Dokkyo Medical University issued approval NO27014. Human subjects: Consent
was obtained by all participants in this study. The Ethical Standards Committee of Dokkyo
Medical University confirms that it has reviewed the research in accordance with the Japanese
Medical Association, Clinical Trial Registration (JMAS-IIA00258). . Conflicts of interest: In
compliance with the ICMJE uniform disclosure form, all authors declare the following:
Payment/services info: Financial support for this work was provided by the Japan Agency for
Medical Research and Development (AMED) (15Aek0109015h0002). . Financial relationships:
All authors have declared that they have no financial relationships at present or within theprevious three years with any organizations that might have an interest in the submitted work.
Other relationships: All authors have declared that there are no other relationships oractivities that could appear to have influenced the submitted work.

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