Yonago Acta medica 1997;40:53–61

A < 10 Kilodalton Fraction of Cerebrospinal Fluid from Patients with
Parkinson’s Disease Decreases the Survival of Rat Mesencephalic
Dopaminergic Neurons in Culture
Katsuko Mishima, Takao Takeshima and Kenji Nakashima
Division of Neurology, Institute of Neurological Sciences, Tottori University, Faculty of
Medicine, Yonago 683, Japan
Parkinson’s disease (PD) is characterized by loss of dopaminergic neurons of the pars
compacta in the substantia nigra. It has been suggested that environmental or intrinsic
toxic factors for mesencephalic dopaminergic neurons are associated with this progressive neuronal loss. Using a primary rat mesencephalic dopaminergic culture method,
we tested the impact of the cerebrospinal fluid (CSF) obtained from patients with PD
on the survival of mesencephalic dopaminergic neurons. Specimens of CSF wereobtained by lumbar puncture from 6 patients with PD (including 2 patients never treatedwith L-dopa) and control patients with other neurological diseases. On day 4 of in vitro
culture of the CSF specimens, 20% of the growth medium was exchanged with the < 10
kDa fraction of CSF. The survival of dopaminergic neurons was evaluated by tyrosinehydroxylase (TH) staining on day 6 of in vitro culture. The percentage of TH-positive
neurons significantly decreased in cultures treated with the < 10 kDa fraction of CSF
from PD patients (3.0 ± 1.4%; mean ± SD) than in those from control subjects (6.2 ±
2.5%, P < 0.05), without any significant difference to the total cell number. The toxicity
of CSF from PD patients not treated with L-dopa was significantly higher than thatfrom PD patients treated with L-dopa. A dose-response curve of 1-methyl-4-phenylpyridinium ion (MPP+) toxicity on this culture was tested in a range from 0.1 to 20 µM(median effective dose, 1.0 µM). The mean effect of the 20% medium replacement withthe < 10 kDa fraction of CSF from PD patients was equivalent to that of 5.0 µM MPP+.
It is suggested that a somewhat toxic factor exists in CSF of PD patients, which is one of
the causes of the development of PD.
Key words: cerebrospinal fluid; mesencephalic dopaminergic neurons; microisland cultures; neurotoxicity; Parkinson’s disease

Parkinson’s disease (PD) is characterized byprogressive degeneration of the mesencephalicdopaminergic neurons (MDNs) from the pars
compacta in the substantia nigra, which causes
tremors, muscle rigidity and akinesia. The
cause and mechanism of the selective
degeneration of MDNs in PD has not yet been
fully understood. At present, the involvementof genetic factors (Smith et al., 1992; Shoffner
et al., 1993), endogenous or exogenous toxic
factors (Nagatsu and Yoshida, 1988; Drucker etal., 1990; Matsubara et al., 1992, 1993) and oxidative stress (Edwards, 1993) are hypothesized as
variables in the development of PD. Since a
neurotoxin, 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), was found to cause

Abbreviations: 10 kDa CTL-CSF, 10 kDa fraction of CSF from control subjects; 10 kDa PD-CSF, 10 kDa fractionof CSF from PD patients; CSF, cerebrospinal fluid; CYP2D6, cytochrome p-450 deprisoquine spulteine gene; FCS,
fetal calf serum; MDN, mesencephalic dopaminergic neuron; MPP+, 1-methyl-4-phenylpyridinium ion; MPTP, 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PBS, phosphate buffered saline; PD, Parkinson’s disease; TH,tyrosine hydroxylase; TIQ, 1,2,3,4-tetrahydroisoquinoline53

K. Mishima et al.

Table 1. Patient characteristics
Case
No.

Age*

Sex Duration* Yahr Period* afterofstage
L-dopaillness
therapy

PD 1
PD 2

70
63

M
M

6
2

III
III

6 (200)2 (300)

PD 3
PD 4
PD 5
PD 6

77
69
66
66

F
F
F
F

10
3
1
3

III
IV
III
III

2 (400)1 (300)Free
Free

Other anti-Parkinson drugs

Amantadine (100)Amantadine (100), pergolide [450],droxydopa (300), trihexyphenidyl (2)Amantadine (300)
Bromocriptine (5), droxydopa (300)
Free
Free

Mean ± SD 68.2 ± 5.2
4.2
F, female; M, male; PD, Parkinson’s disease.
* year(s).
( ), mg/day; [ ], µg/day.

Parkinsonism in human (Langston et al., 1983)
Materials and Methods
and nonhuman primates (Davis et al., 1979;
Langston et al., 1984), endogenous andexogenous toxic factors similar to MPTP have
CSF of PD patients and control subjects
been sought in the cause of PD.
Recently, a microisland culture method for The CSF was collected from 6 patients with PD
the ventral mesencephalon of 14-embryonic- and from 7 patients with other neurological disday-old rats, which centers on the A8, A9 and eases as controls. A lumbar puncture was perA10 dopaminergic nuclei, has been established formed under routine clinical conditions: the(Takeshima et al., 1994a, 1996). In this culture, initial 4 mL CSF was evaluated with the routine
95% of the cells are stained positive for neu- CSF test including cell counts and a protein andronal markers, and 20% for thyrosine hydro- glucose assay, and the remaining 6 mL CSF was
xylase (TH), which is a marker for dopa- used in this study. Table 1 summarizes theminergic neurons, at the early stage of culture. backgrounds of patients with PD, and Table 2,Less than 5% of the cells are stained positive to the backgrounds of control subjects. Informed
glial markers. This culture technique involving consent was fully obtained after the nature anda high percentage of TH-positive dopaminergic scope of the study were explained.
neurons is suitable for the bioassay of toxic
or trophic factors for dopaminergic neurons(Takeshima et al., 1994a, 1994b, 1996).
Table 2. Control characteristics
Using this technique, we verified the imDiagnosis
pact of the < and > 10 kDa fractions of CSF Case No. Age* Sexfrom patients with PD and other neurological
CTL 1
75
M Vascular dementia
diseases on the survival of mesencephalic CTL 2
57
M Abducens nerve palsy
dopaminergic neurons in vitro. In addition, CTL 3
78
FMultiple cerebral infarction
we tested the dose-response toxicity of MPP+ CTL 4
75
F
Meige syndrome
71
F
Alzheimer disease
in a serum-priming serum-free microisland CTL 5
CTL
6
51
F
Motor neuron diseaseculture, and quantified the toxicity of the <
CTL 7
68
F
Abducens nerve palsy
10 kDa fraction of CSF from patients withPD.
Mean ± SD 67.9 ± 10.1
CTL, control subject; F, female; M, male.
*years.

54

CSF from PD decreases dopaminergic neurons

Following the collection, the CSF samples
were immediatly centrifuged (1000 × g, 3 min)and the supernatants were frozen at –80˚C.
Later, the CSF samples were thawed at room
temperature and diluted twice with Dulbecco’s
phosphate buffered saline (PBS). The CSF
samples were fractionated at 10 kDa with
Centricon 10 microconcentrators (Amicon,
Grace Co., Bevely, MA), then sterilized withlow protein binding filters (0.22 µm) (SLGV
025 LS; Millipore, Bedford, MA) and frozen at–80˚C until tested in culture.

Cell viability was evaluated with a 2-color
fluorescence cell viability assay kit (Live/Dead
Assay Kits, Molecular Probes, Inc., Eugene,
OR). Cell viability just before plating wasalways > 90%. The cells were resuspended in a
final density of 5.0 × 10 5/mL. A 25 µL dropletof suspension (1.25 × 10 4 cells) was plated on 8well chamber slides (Nunc, Inc, Naperville, IL).
Before use, the 8-well chamber slides were
coated overnight with poly-D-lysine (Sigma
Chemical Co., St. Louis, MO). The droplet
occupied an area up to 6.2 mm2, for a final density of 2.0 × 105 cells/cm2. The cultures in the
covered chamber slides were incubated at 37˚C,
in 5% CO2 at 100% humidity, for 4 h to allow
cells to attach to the coated surface. Then, 375µL of the serum-supplement medium wasadded to each well. The cultures were primed
with serum for 24 h, and grown in serum-free
medium: Dulbecco’s modified Eagle medium/
Ham’s F-12 (GIBCO), 1.0 mg/mL bovine albumin fraction V (Sigma), 0.1 mg/mL apo-transferrin (Sigma), 15 µg/mL insulin (Sigma), 30
nM L-thyroxine (Sigma), 20 nM progesterone(Sigma), 30 nM sodium selenite (Sigma), 100
U/mL penicillin and 100 µg/mL streptomycin(GIBCO). Every 2nd day, 50% of the medium
was exchanged with the serum-free medium.

Serum-priming serum-free microisland
culture of MDNs
All procedures were carried out in accordancewith the standardized methods of bioassayneurotrophic factors for dopaminergic neurons(Takeshima et al., 1996). In brief, pregnant
Sprague-Dawley rats were obtained from Shimizu
Jikken Doubutsu Co. (Shizuoka, Japan), andexposed to CO2 on the 14th gestational day. A
laparotomy was done as described previously,
the uterus was removed and transferred to cold,
clean PBS at pH 7.4 without Ca 2+ and Mg 2+.
Subsequently, the intact brain was removed, thebrain stem was isolated, and then the ventral
mesencephalic region (ventral medial region;.
1.0 × 1.5 × 0.75 =. 1.0 mm3) was isolated. Thisregion centered on A8, A9 and A10 dopaminergic nuclei. Dissected tissue blocks werepooled in cold fresh medium kept at 4˚C. The
serum-supplemented medium was composed ofDulbecco’s modified Eagle medium/Ham’s F12 (1:1) (GIBCO, Grand Island, NY), 10% fetal
calf serum (FCS; GIBCO), 100 U/mL penicillinand 100 µg/mL streptomycin (GIBCO). The
tissue was triturated with a 1000 µL pipet with ablue tip, and then with a 21 G needle fitted to a
1.0 mL plastic syringe. Special care was taken
to prevent the cells from touching the rubber tipof the plunger, so as not to create bubbles in thecell suspension. The dispersed cells weretransfered to 1.5 mL Eppendolf tubes (1.0 mL/tube), and centrifuged at 300 × g for 10 min.
The medium was carefully removed, and thecells were resuspended in fresh medium andcounted using a hemocytometer.

Treatment of test CSF
On day 4 of in vitro culture, 20% (80 µL) of themedium was replaced with either the < or > 10
kDa fraction of the CSF from PD patients andcontrols, and with PBS as a vehicle. To verify
the possible effect of the vehicle, the control
culture without any supplementation other thanthe medium was also served.

Immunostaining of TH and evaluation ofsurvival of MDNs in vitro
The cultures were evaluated on day 6 of in vitro
culture for the survival of MDNs using TH
immunostaining as an assay. The cultures were
washed with cold PBS, fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized
in 1% CH3COOH/95% C2 H5OH at –20˚C for 5min, and then washed with PBS twice. Non55

K. Mishima et al.

Fig. 1. Total number of neurons cultured in the < 10
kDa fraction of the cerebrospinal fluid (CSF)
obtained from patients with Parkinson’s disease(PD-CSF) and controls (CTL-CSF), and inphosphate-buffered saline (PBS). The serumpriming serum-free microisland culture ofmesencephalic dopaminerigic neurons obtained
from 14-embryonic-day-old rats was treated withtest CSF (20% of medium was replaced with the < 10
kDa fraction of CSF) on day 4 of in vitro culture, andevaluations were done on day 6 of in vitro culture.
Columns and bars show mean ± SD. The data wereanalyzed with the analysis of variance. Differencesamong the 3 groups are not significant.

NS
NS

NS

100

Cell number

90
80
70
60
50
40
30
20
10
0

PBS

CTL-CSF

PD-CSF

Culturespecific binding was blocked with 1% bovine
serum albumin (Sigma) in PBS for 15 min.
After aspiration of blocking buffer, 50 µL of
anti-TH antibody (× 100) (Boehringer Mannheim,
Mannheim, Germany) was applied, and thechamber slides were incubated for 30 min in adark and humid chamber. After washing twicewith blocking buffer, 50 µL of fluorescein
isothiocyanate-conjugated anti-mouse immunoglobulin G (× 100) (Vector Laboratories,
Burlingame, CA) was applied, and slides wereincubated for 30 min. After washing twice with
PBS, the excess fluid was aspirated, thechamber walls were taken off and 2 drops of
Vectashield mounting medium (Vector) wereapplied, followed by a cover glass sealed with

nail polish. An Olympus BX60 microscope
equipped with the appropriate filters forfluorescence microscopy was used to visualize
the cells. The TH-positive neurons were blindly counted in a 0.4 mm2 area (2.6% of the platearea) using an eye glid. Three fields werecounted, and the percentage of TH-positive
neurons was calculated as (number of THpositive neurons/number of total cells in cultures) × 100 (%). An objective of 20 magnifications was used to count cells.

Dose-response toxicity of MPP+
Serum-priming serum-free MDN cultures were
set up as described above. On day 4 of in vitro
culture, 0.5 to 20 µM MPP+ was added in culture, and TH staining and counting were carriedout on day 6 of in vitro culture.

P < 0.001%TH-positive neurons

NS
14

NS

P < 0.001
P < 0.05

NS

12

Statistical analysis

10

Statistical analyses were carried out with theone-way analysis of variance, followed by appropriate post hoc tests.

8
6
4
2
0

Standard

PBS

CTL-CSF

PD-CSF

CultureFig. 2. Impact of the < 10 kDa fraction of the CSF from PD patients (< 10 kDa PD-CSF) on the survival oftyrosine hydroxylase (TH)-positive dopaminergic neurons in culture. The < 10 kDa PD-CSF decreased thesurvival of TH-positive dopaminergic neurons in culture. The percentage of TH-positive neurons wascalculated as (number of surviving TH-positive neurons/number of total neurons) × 100 (%). Columns andbars show mean ± SD. The data were analyzed with the analysis of variance (P < 0.05, 0.001). NS, notsignificant. CTL-CSF, < 10 kDa fraction of CSF from control subjects; PBS, phosphate-buffered saline;
standard, standardized control culture of serum-priming serum-free microisland mesencephalic dopaminergic
neurons.

56

CSF from PD decreases dopaminergic neurons

Fig. 3. TH immunostaining of the cultures exposed to the < 10 kDa fraction of the CSF from control subjects(< 10 kDa CTL-CSF) (A) and PD patients (< 10 kDa PD-CSF) (B), showing the growth and survival of THpositive neurons on day 6 of in vitro culture. The number of TH-positive neurons decreases in cultures treatedwith < 10 kDa PD-CSF. Scale bar = 20 µm.

PD treated with L-dopa, and 1.6 ± 0.2% when
cultured in that obtained from 2 patients with
PD never treated with L-dopa (de novo PD),
respectively. Although the statistical comparison was not applicable because of the limitationof given CSF samples, the mean percentage ofTH-positive neurons of CSF obtained frompatients with de novo PD was apparently lower
than that from patients with PD treated with Ldopa.

Results

Impact of the < 10 kDa fraction of CSF
from PD patients and controls and in
PBS
The total cells in culture were exposed to the <10 kDa fraction of CSF from PD patients (< 10kDa PD-CSF) or control subjects (< 10 kDa
CTL-CSF), and to PBS (Fig. 1). There were no
significant differences among the 3 test groups.
Figure 2 shows the percentage of TH-positive
neurons cultured in the < 10 kDa PD-CSF, < 10
kDa CTL-CSF, PBS and the standardized
serum-priming serum-free medium. The ratio
in the mean percentage of TH-positive neurons
cultured in the < 10 kDa PD-CSF, < 10 kDa
CTL-CSF and PBS to that in the standard medium, were 39.2%, 79.8% and 109.3%, respectively. The mean percentage of TH-positive
neurons cultured in the < 10 kDa PD-CSF was
significantly lower than that of those cultured in
the < 10 kDa CTL-CSF (P < 0.05), PBS (P <0.001) and standard medium (P < 0.001). Figure
3 shows the surviving TH-positive neurons
cultured in the < 10 kDa PD-CSF or < 10 kDa
CTL-CSF.
The mean percentage of TH-positive
neurons was 3.8 ± 1.1% when cultured in the <10 kDa PD-CSF obtained from 4 patients with

Dose-response toxicity of MPP+
The dose-response toxicity of MPP + in theserum-priming serum-free microisland MDN
culture is shown in Fig. 4. After exposed to
MPP+ , TH-positive neurons selectively decreased. The median effective dose of MPP+ in
this culture model was 1.0 µM. The mean toxicity of the 20% medium replacement with the <10 kDa PD-CSF was determined to be equivalent to that of 5.0 µM MPP +.

Impact of the > 10 kDa fraction of CSF
from PD patients and controls
The total cell number was 74.8 ± 21.7 cells/field(mean ± SD) when cultured in the > 10 kDa PDCSF, and 83.9 ± 9.2 cells/field when cultured
in the > 10 kDa CTL-CSF, respectively (not57

Ratio of %TH-positive neurons

K. Mishima et al.

(%)
100

80

60

40

20

0

5.0

10.0

15.0

MPP+

20.0

(µM)

significant). The percentage of TH-positive
neurons was 4.9 ± 2.1% when cultured in the >10 kDa PD-CSF, and 6.4 ± 0.6% when cultured
in the > 10 kDa CTL-CSF, respectively (notsignificant).

Fig. 4. The dose-response toxicity curve of 1methyl-4-phenylpyridinium ion (MPP+) from
0.1 to 20.0 µM on thyrosine hydroxylase (TH)positive neurons cultured in serum-priming
serum-free mesencephalic dopaminergic
neurons (MDNs). The vertical axis represents
the ratio to the survival of TH-positive neurons
without MPP + treatment on day 6 of in vitro
culture. The median effective dose of MPP+
was 1.0 µM in this culture model.

contains both trophic and toxic factors. This
could be possible, because toxic factors and adamaged brain can upregulate the production ofneurotrophic factors. We fractionated the CSF
of 10 kDa because most trophic factors have amolecular mass higher than 10 kDa; for example, the brain-derived neurotrophic factor has a
135 kDa molecular mass (Hyman et al., 1991),
and the striate-derived neurotrophic factor has a
14 kDa molecular mass (Dal Toso et al., 1988).
Hao and colleagues (1995a, 1995b) reported
that the < 10 kDa PD-CSF from Caucasian patients was toxic to MDNs in culture. Yu andcolleagues (1994), using CSF specimens obtained at autopsy, have shown that the < 10 kDa
fractions of CSF from PD patients, Alzheimer’s
patients and age-matched healthy controls
inhibited dopaminergic neuronal growth inculture. Dying and postmortal changes in thebrain might bring different results. We foundthat control CSF specimens did not show toxic
effects on dopaminergic neuronal growth, andconfirmed the toxicity of < 10 kDa PD-CSF in
Japanese people. The existence of racial difference was reported in the etiology of PD, and theprevalence ratio of PD is higher in Caucasians
than in Japanese (Schoenberg et al., 1985; Kusumi
et al., 1996). However, we detected that the
toxicity of the < 10 kDa PD-CSF in Japanesepeople was the same as the reported toxicity in
Caucasians (Hao et al., 1995b). Increase of toxic factor(s) is considered to play an importantrole of development of PD in both races.
In this study, the impact of < 10 kDa PDCSF on the survival of the total cell number inculture was not different from the impact of <Discussion
PD is one of the most common neurodegenerative diseases, which is characterized by systematic neuronal death. The cause and mechanismof such systematic neuronal death have notbeen fully understood. Since MPTP was foundto induce Parkinsonism (Langston et al., 1983),
neurotoxins like MPTP have been suspected as
the cause of PD. Rat mesencephalic cultures
have been used for the bioassay of neurotoxins
to dopaminergic neurons. Defazio and colleagues (1994) tested specimens of serum from
PD patients using a culture of rat MDNs, andfound the complementary-dependent toxicity ofserum from PD to rat MDNs. Serum might be agood test material, because serum is obtained
relatively easily with minimum pain to patients.
However, serum contains numerous factors
from many organs and may not always reflect
alterations in the brain. CSF is more relevant
than serum for the study of changes in the brain
because the brain exists within the CSF.
Although some attempts were made to test the
CSF in MDN cultures, some found toxicity andothers found trophic effects of CSF from PD
patients with MDNs in culture (Carvey et al.,
1993; Yu et al., 1994; Hao et al., 1995a, 1995b).
One possibility is that the CSF from PD patients58

CSF from PD decreases dopaminergic neurons

10 kDa CTL-CSF, PBS and standardized
serum-priming serum-free culture. Treatment
with the < 10 kDa PD-CSF more significantly
decreased the number of surviving TH-positive
neurons in culture than the treatment with the <10 kDa CTL-CSF and PBS did. These results
clearly mean that the toxicity of < 10 kDa PDCSF is selective for dopaminergic neurons.
Judging from the MPP+ dose-response toxicity
curve (Fig. 4), we determined that the 20%
medium replacement with the < 10 kDa PDCSF was equivalent in toxicity to 5.0 µM MPP+in this culture model. MPP + is produced by
MPTP oxygenated with monoamine oxidase-B,
whose inhibitor deprenyl improves parkinsonian symptoms in monkeys, and is regarded
to delay the progression of PD (Choen et al.,
1985; Youngster et al., 1989). In in vitro experiments, monoamine oxidase-B inhibitor
selegiline increased the number of TH-positive
neurons cultured in the < 10 kDa PD-CSF (Haoet al., 1995a). Therefore, the neurotoxin in the< 10 kDa PD-CSF may act like MPP +.
MPP + or MPTP has never been found invivo or from natural sources; however, MPTPlike neurotoxins such as 1,2,3,4-tetrahydroisoquinoline (TIQ) have been found in thehuman brain (Kohno et al., 1986; Niwa et al.,
1989; Ohta et al., 1987). An oxidation productof TIQ by monoamine oxidase-B can damage
the mitochondrial respiratory functions andcause neuronal death. Kotake and colleagues(1995) reported that the administration of 1benzyl-TIQ, which has the same skeleton as
TIQ, induced abnormal behavior in rats such as
MPP + treatment, and a significantly highconcentration of 1-benzyl-TIQ was found in the
CSF from PD patients. The toxic factor(s) in
the < 10 kDa PD-CSF might be TIQ or similar
substances.
The CSF from patients with de novo PD
never treated with L-dopa was more toxic to
MDNs in vitro than that from patients with PD
treated with L-dopa. Administration of L-dopa
relieves parkinsonian symptoms in patients.
Long-term or high-dose administration of Ldopa causes the down-regulation of dopamine
receptors in the striatum to go down, and decreases the clinical effects of L-dopa. Steece

and colleagues (1990) reported that L-dopa has
a dose-dependent toxicity to MDNs in culture.
Hao and colleagues (1995b) measured dopamine levels in the CSF from PD patients, andfound no significant relation between L-dopa
concentration in the CSF and toxicity to MDNs.
Although L-dopa is toxic to MDNs in culture,
the CSF from patients receiving L-dopa wasless toxic to MDNs in culture. This finding isnew. Based on our data, L-dopa treatment may
have favorable effects not only on improving
symptoms but also on preventing MDN degeneration.
Our data and earlier observations imply thatsomewhat neurotoxic factor(s) for MDNs exist
in the CSF of PD patients. Regarding the pathogenesis of PD, such neurotoxic factors might beendogenous. Allelic associations in PD patientswith cytochrome P-450 deprisoquine spulteine
gene (CYP2D6) (Smith et al., 1992; Kurth et al.,
1993; Sandy et al., 1996) and monoamine oxidase(Kurth et al., 1993; Shoffner et al., 1993) havebeen reported. CYP2D6 and monoamine oxidase are related to the degradation of MPTP and
MPTP-like substances in vivo. It is possiblethat the abnormal metabolism of MPTP-like
substances in vivo increases the concentrationof neurotoxins in the brain, and that it increases
the risk of PD.
The standardized microisland MDN culture
technique used in this study is useful andpromises further understanding of the pathogenesis of PD. The toxicity of the < 10 kDa PDCSF, which was confirmed in this study, should
be related to the development of PD. Treatment
with L-dopa may protect MDNs from degeneration. Further studies are necessary to clarify
the advantage of L-dopa therapy in PD progression.

Acknowledgments: The authors thank Prof. M.
Oshimura, Dept. of Molecular and Cell Genetics andProf. K. Ohno, Dept. of Neurobiology, School ofLife Sciences, Faculty of Medicine, Tottori University, for their kind advice and criticism in thismanuscript.

59

K. Mishima et al.

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