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Glutamate dehydrogenase deficiency in three patients with spinocerebellar syndrome.

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ORIGINAL ARTICLES
Glutamate Dehydrogenase
uehciency in .Ihree Yatients
with Spinocerebellar Syndrome
Andreas Plaitakis, MD, William J. Nicklas, PhD, and Robert J. Desnick, PhD, MD
Four nicotinamide-adenine dinucleotide phosphate-requiring enzymes were measured in disrupted cultured skin
fibroblasts from a 19-year-old patient with juvenile onset of a spinocerebellar and extrapyramidal syndrome. There
was marked reduction in the activity of glutamate dehydrogenase (GDH) (22% of mean control activity); GDH
activity was also decreased in homogenates of leukocytes from this patient (38% of mean control activity). GDH
activity was measured in the leukocytes of two siblings afflicted with adult-onset spinocerebellar syndrome and
found to be decreased in both (29% and 31% of mean control activity); an unaffected sibling had normal GDH
activity. Mixing experiments with control fibroblast and leukocyte homogenates did not show the presence of a
GDH inhibitor in cells from these patients. This allosterically regulated enzyme was stimulated by adenosine 5 ‘ M) and inhibited by guanosine 5‘-triphosphate
M) in both fibroblast and leukocyte homogediphosphate
nates; these changes occurred in equal proportions in the patients and controls. The decreased fibroblast and leukocyte GDH activity persisted at different concentrations of the enzyme’s substrates and with successive passages of
cultured fibroblasts. GDH may have an important role in the metabolism of glutamate, a putative neurotransmitter
in cerebellum, brainstem, and spinal cord. A genetic deficiency of GDH may underlie some forms of spinocerebellar
ataxias.
Plaitakis A, Nicklas WJ, Desnick RJ: Glutamate dehydrogenase deficiency in three patients with
spinocerebellar syndrome. Ann Neurol 7:297-303, 1980
The inherited spinocerebellar degenerations form a
heterogeneous group of disorders, some of which
have been classified by clinical and pathological criteria [ 5 , 13, 19, 291. Specific biochemical defects have
been described in patients with clinically defined
hereditary ataxias (Refsum’s disease and perhaps
some patients with Friedreich’s ataxia and its variants
[ 171) and in patients with degenerative neurological disorders in which ataxia is a clinical feature
(Maple syrup urine disease, Hartnup disease, yglutamylcysteinyl transferase deficiency, cytochrome
6 deficiency, abetalipoproteinemia, and lysosomal
storage disorders [ 171). Despite these advances,
the majority of clinicopathologically defined spinocerebellar syndromes have not been characterized biochemically. These include the cerebelloolivary ataxias (according t o the classification by
Refsum and Skre [29]), olivopontocerebellar atrophies, spastic ataxia, spastic paraplegia, and those
cases of Friedreich’s ataxia in which there does not
appear t o be pyruvate dehydrogenase deficiency [ 331.
While searching for animal models for cerebellar
ataxias, we became aware that the nicotinamide antagonist 3-acetylpyridine (3-AP) can produce long-
lasting equilibrium disturbances (gait ataxia) in rats
[8, 141. The animals experience degeneration of
the inferior olives, olivocerebellar fibers, lower cranial nerve nuclei, and areas of the pons and nigra [8].
These morphological changes resemble those of the
olivopontocerebellar atrophies (OPCA), particularly
OPCA type IV, according to the Koningsmark and
Weiner classification [ 191. The mechanism by which
administration of 3-AP results in degeneration of
selective neuronal systems is not understood; however, it is known that the toxic agent can form abnormal nucleotides which inhibit several enzymes
that require nicotinamide-adenine dinucleotide phosphate (NADP) [6, 141. It is therefore possible
that the neuronal systems which degenerate following administration of 3-AP are selectively sensitive to
inhibition of NADP(H)-dependent enzymes. The
possibility can also be raised that patients with
OPCA may be deficient in one of these enzymes.
T o explore this hypothesis, the activities of the
NADP(H)-requiring enzymes glucose 6-phosphate
dehydrogenase (G-6-PDH), isocitrate dehydrogenase (ICDH), glutathione reductase (GSSGRD), and
glutamate dehydrogenase (GDH) were measured in
From the Departments of Neurology and Medical Genetics,
Mount Sinai School of Medicine, New York, NY.
Address reprint requests to Dr Plaitakis, Department of Neurology, Mount Sinai School of Medicine, Fifth Ave and 100th St,
New York, N Y 10029.
Accepted for publication Aug 30, 1979.
0364-5134/80/040297-07$01.25@ 1979 by Andreas Plaitakis 297
cultured skin fibroblasts from a patient with a
juvenile-onset degenerative neurological d i s o r d e r
compatible with OPCA s y n d r o m e . Because t h e s e
studies revealed a marked reduction in the activity of
GDH, t h e same e n z y m e was t h e n m e a s u r e d in this
addition, GDH was deterpatient’s leukocytes.
patients (siblings) afmined in leukocyres from
fected by a n adult-onset spinocerebellar s y n d r o m e
consistent with OPCA. The results of these studies
have b e e n p r e s e n t e d i n p a r t [25al.
Case Reports
Patient 1
This 19-year-old man has been the subject of a previous
report by Kilroy et a1 [ 181. Dysarthria and dysphagia had
appeared insidiously around age 11. One year later he developed abnormal posturing, tremor, and cogwheeling
rigidity of his left arm as well as facial hypomimia with a
positive glabellar sign. He was reported to improve on
levodopa therapy and was throught to suffer from juvenile
parkinsonism. However, by age 17, ataxia of gait and incoordination of hand movements appeared. The patient
had no siblings, and both parents were healthy. There was
no reported consanguinity in the family, and no relatives of
the patient are known to have neurological disorders.
The general physical and mental status examinations
were normal. O n neurological examination he exhibited
orofacial dyskinesias and tremor involving the head. His
speech was nasal, slow, and dysrhythmic. There was virtually no movement of the soft palate on phonation, and attempts to swallow liquids frequently resulted in endotracheal aspiration. Muscle power and tone were normal,
and the muscle stretch reflexes were active throughout.
The plantar responses were flexor. Coordination was severely impaired in all four extremities; there was dysmetria
and intention tremor on finger-to-nose and heel-to-shin
tests. Rapid alternating movements were impaired bilaterally. He walked with a broad-based, staggering, ataxic gait,
and the Romberg sign was positive.
Laboratory investigation revealed normal complete
blood counts (CBC) and routine blood chemistry determinations. The cerebrospinal fluid (CSF) protein level was 57
mg/dl with normal glucose and no cells. The electroencephalogram
showed diffuse slowing. Computerized tomography of the head showed a mild to moderate degree of
diffuse cortical atrophy. Electromyography (EMG) of the
right vastus medialis and right biceps brachii muscles revealed motor units of prolonged duration and an increased
incidence of complex polyphasic potentials. There was
slowing in motor but not sensory conduction velocity (left
peroneal nerve)‘ These EMG findings suggested a
neuropathic process. Visual evoked potentials (2.3 cycles
per degree at 1 Hz rate) were within normal limits.
SPECIAL STUDIES. The fasting blood pyruvate level was
within normal limits (0.73 mg/dl), and there was no rise
after glucose administration (1.75 gm/kg). The serum copper and ceruloplasmin levels were normal. No storage cells
could be detected in a bone marrow aspirate. The activities
298 Annals of Neurology Vol 7 No 4
April 1980
of arylsulfatase A, p-hexosaminidase A and B, and p galactosidase in the leukocytes were within normal limits.
CSF amino acid levels were n ~ m d .
Patient2
A 64-year-old man had noted the insidious development of
gait ataxia at around age 49. Because of his slowly progressive disability he became unable to work at age 56. Slurred
speech was noted at age 55, and a few years later he complained of diplopia and intermittent dizziness, Urine incontinence and impotence developed after age 60.
T h e general physical examination was unremarkable.
Mental status was within normal limits. There was tremor
involving the head. The pupils were irregular and reacted
poorly to light. H e had downbeat nystagmus on both lateral
and downward gaze. Speech was dysrhythmic, slurred, and
slow. There was a mild degree of generalized muscle atrophy, especially of the interosseous muscles of both
hands. Muscle fasciculations were noted in the upper and
lower extremities. Muscle power was normal. The muscle
stretch reflexes were active throughout, and plantar responses were flexor. Coordination was markedly impaired
(especially in the lower extremities), with intention tremor,
dysmetria, and dysdiadochokinesia. The Romberg sign was
positive and his gait was severely ataxic (broad based, small
stepped, and jerky); ambulation was impossible without
support. Position sense was impaired in the toes and
fingers, and vibration sense was diminished below both iliac
crests and in the fingers. Pinprick sensibility was impaired
over the entire body, including the face.
Laboratory investigations showed normal CBC and
routine blood chemistry determinations. CSF studies revealed a protein concentration of 4 1 mgldl with a normal
glucose level and no cells. Computerized tomography of
the head showed motion artifact; no abnormalities could be
detected in this suboptimal study. Visual evoked potentials
(2.3 cycles per degree at 1 Hz rate) gave borderline abnormal latencies, 140 msec for the right eye and 136 msec for
the left (normal, up to 120 msec).
SPECIAL STUDIES. The activities of P-hexosaminidase A
and B, arylsulfatase A, and p-galactosidase were normal in
the patient’s leukocytes. Blood and CSF amino acid levels
were within normal limits.
Patient
A 71-year-old man, brother of Patient 2, experienced diplopia at age 50 and thereafter developed progressive
difficulty with walking because of leg weakness and unsteadiness. He became confined to a wheelchair by age 69.
Urine incontinence developed at age 5 5 , impotence at 62,
and stool incontinence at 69. Dysarthria appeared at 65
years of age and progressed steadily to a severe degree of
speech impairment. Visual acuity declined progressively
after age 68 and could not be corrected with new eyeglasses.
There was a mild degree of organic mental syndrome and
severely dysarthric speech (slurred, slow, and dysrhythmic). His face was somewhat immobile, and the
glabellar sign was positive. Visual acuity was 20/100 in both
I
@&!
I
I
I
ti%
II
51
71
69
67
64
40
F i g 1. Pedigree of Patients 2 (11-6) and 3 (11-3). Leukocytes of
these two family members and a brother (11-Si were assayedfor
glutamate dehydrogenase activity. Patient 2 has no offspring.
Patient 3 had one daugher (111-6),who committed suicide at
age 22:apparently she was free of neurological maniyestations
at death. I r = individuals examined by the authors;@=
deceased family members without neurological illness at death.
Ages, in years, are indicated below the symbols.)
eyes with corrective lenses. Funduscopy revealed welldemarcated and perhaps pale discs. Divergent strabismus in
the primary position and impaired convergence were
noted. The volitional eye saccades were impaired, and opticokinetic responses were defective in both the horizontal
and vertical planes. There was symmetrical atrophy of leg
muscles below the knees as well as atrophy of the intrinsic
muscles of both hands. Occasional muscle fasciculations
were noted in the lower extremities. There was marked
paraparesis and weakness of all finger movements. H e had
difficulty making a fist, especially with the right hand, and
exhibited dystonic posturing of his right fingers together
with random choreoathetoid movements. There was
marked dysmetria and intention tremor in the right arm
with a less marked degree of dystaxia in the left arm. Rapid
alternating movements were slow and irregular. Position
sense was impaired in the toes and left fingers. Vibratory
sense was decreased in the lower and upper extremities,
anterior iliac crests, and lower vertebral processes. Pinprick
sensibility was defective throughout, except perhaps on the
face. He felt the stimulus more strongly in the proximal
than in the distal parts of his extremities.
A pedigree of Patients 2 and 3 is shown in Figure 1. Apparently, both parents were free of neurological affliction at
death. There was no documented consanguinity in the
family although both parents came from the same small
Russian village. Patient 2 (11-6) has no offspring. Patient 3
(11-3) had only one daughter; she committed suicide at age
22. Apparently, she was free of neurological manifestations
at death.
Materials and Methods
The controls used in these studies were healthy individuals
as well as patients with neurological disorders due to
known defects in lipid metabolism (metachromatic
leukodystrophy and GM2-gangliosidosis).
Skin was obtained by biopsy, and primary cultures of
skin fibroblasts were established, cultured, and passed as
previously described [2]. Enzyme studies were carried out
on fibroblasts from Patient 1 and the controls after a comparable number of passages. At specific intervals after subculture (7 and 14 days) the cultured flasks were washed
with physiological saline solution containing 0.02%
ethylene diamine tetraacetic acid (EDTA), and the cells
were harvested by treatment with 10 to 20 ml of a 0.25%
trypsin solution for 2 minutes at 22°C. The cell suspension
was centrifuged at 4°C (270g for 10 minutes), and the pellet was washed twice by resuspension in 2 0 to 30 ml of
physiological saline solution and recentrifugation. If intact
cells were stored at -2O"C, GDH activity did not decrease
even after several months.
The cells were disrupted by four freeze-thaw cycles since
their disruption by sonication resulted in decreased GDH
activity. The cells were then homogenized with a glass
homogenizer in 0.05 M triethanolamine (TRA) buffer (pH
8.0). Aliquots of this whole homogenate were used for the
enzyme assays.
For white blood cell preparation, heparinized venous
blood (20 to 30 ml) was mixed with one-fifth volume of 6%
dextran in physiological saline solution. The blood was
then left in an inverted syringe for 4 5 to 60 minutes at
room temperature. Supernatant was collected through a
bent needle and centrifuged at 4°C for 10 minutes at 480g.
The pellet was suspended in 0.5 ml of physiological saline
solution, and red cells were lysed by addition of 2 ml of
0.87% ammonium chloride. Five minutes later the suspension was centrifuged, and the pellet was washed twice by
resuspension in 20 to 30 ml of physiological saline solution
and recentrifugation. Disruption of cells and preparation of
homogenates were carried out as for the fibroblast preparation.
Glutamate dehydrogenase (E.C. 1.4.1.3) was assayed (in
the direction of glutamate formation) by a modification
[25] of previously described methods [ll]. The activity of
the enzyme was measured fluorometrically by determining
the rate of N A D P o r N A D production from N A D P H o r
N A D H , respectively. Internal standard curves relating
N A D P H o r N A D H concentration to the actual reading on
the fluorometer were used to calculate the rate of N A D P H
o r N A D H utilization. The sensitivity of the apparatus was
set so that full-scale reading on the recorder would be obtained by 10 nmol of N A D H or N A D P H . The reaction
Plaitakis et al: Glutamate Dehydrogenase in Ataxia
299
mixture contained 1.8 ml of 50 mM TRA buffer ( p H 8.0),
0.03 ml of 2 to 5 mM N A D P H or N A D H , 0.07 ml of 3 M
ammonium acetate, 0.02 ml of 0.26 M EDTA, and 2.5 pM
rotenone (to inhibit N A D H oxidase). Addition of Triton
X-100 was found to inhibit G D H . Twenty to 50 pl of cell
homogenate (5 to 10 mg of protein per milliliter) was
added and baseline N A D P H or N A D H oxidation was
noted. Then 40 pl of 0.4 M sodium a-ketoglutarate (pH
6.5) was added to initiate GDH activity. Initial rates were
recorded at 22°C. The enzyme activity was linear under
these experimental conditions. The effect of adenosine
5'-diphosphate (ADP) and guanosine-5 -triphosphate
(GTP) o n GDH activity was studied by adding these nucleotides (20 pl of 0.1 M ADP or 20 pl of 0.1 M GTP)
before or after initiation of the reaction by a-ketoglutarate.
The effect of diethylstilbestrol (DES) on GDH activity was
studied by adding this hormone (40 pl of 2 x lop4M) before the reaction was initiated by a-ketoglutarate; the drug
was ineffective if it was added after initiation. Kinetic
studies using different substrate concentrations were also
carried out. The assays were performed by varying one
substrate and keeping the other two constant at the concentrations already indicated. a-Ketoglutarate was varied
between 1 and 8 mM, N A D P H between 20 and 110 p M ,
and ammonium acetate between 10 and 100 mM.
Glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) was
measured spectrophotometrically according to the method
described by Liihr and Waller [20]. In a cuvette of the
Gilford spectrophotometer was placed 2.9 ml of 50 m M
TRA buffer (pH 7.5), 0.05 ml of cell homogenate, and 0.05
ml of 30 mM N A D P solution. The reaction was initiated by
adding 0.05 ml of 40 m M glucose-6-phosphate.
NADP-dependent isocitrate dehydrogenase (E.C.
1.1.1.42) was measured spectrophotometrically according
to the procedure of Plaut [26] as modified by Salganicoff
and Koeppe [30].The reaction mixture of 2 ml contained
50 mM tris-hydrochloride buffer (pH 7.35), 2 mM manganese chloride, 0.025 m M N A D P , 1 m M DL-isocitrate
(tris-sodium), and 0.07 mM EDTA. Triton X-100 in buffer
was added to homogenates before assay (0.16% final concentration).
Glutathione reductase (E.C. 1.6.4.2) was measured
fluorometrically by a modification of the method described
by Horn Ll5]. The reaction mixture consisted of 0.067 M
phosphate buffer ( p H 6.6), N A D P H (lO-jM), and 2.5 p M
rotenone. After 0.05 ml of cell homogenate was added to
the cuvette, baseline N A D P H oxidation was noted. T h e
reaction was initiated by adding oxidized glutathione ( lop5
M final concentration).
Lactate dehydrogenase (E.C. 1.1.1.27) and aspartate
aminotransferase (E.C. 2.6.1.1) (cytoplasmic and mitochondrial) were measured as previously described [251.
In all assays the enzyme activities were expressed as
nanomoles o r micromoles of substrate utilized (or product
formed) per hour per milligram of protein. Protein was
measured by the method of Lowry et al [21].
Results
Studies on Cultured Fibroblasts
GDH activity was markedly decreased in cultured
skin fibroblasts from Patient 1, to 225% of the mean
activity in fibroblasts from 10 controls (Table 1).This
activity was less than half that of the lowest value
found in controls. As indicated by Stumpf and Parks
[ 3 3 ] , great care must be taken in comparing the mean
determinations in a single patient to a mean of many
controls. A statistical range of control values was
obtained by using the mean k 1.96 SD. The GDH
activity for the patient was outside this range (Table
1). The activities of the other NADP(H)-requiring
enzymes, G-6-PDH, ICDH, and GSSGRD, did not
differ in cells from this patient compared with controls. Two additional metabolic enzymes, lactate dehydrogenase and aspartate aminotransferase (cytoplasmic and mitochondrial), were also found to be
normal.
The deficient activity of GDH in homogenates of
cultured fibroblasts from Patient 1 persisted through
six passages of the cells. GDH activity did not change
in fibroblasts from either the patient or controls between the second and sixth passages in culture. Also,
there was essentially no difference in GDH activity
after 7 o r 14 days of growth in culture (data not
shown). The GDH activity in fibroblasts was linear
over the range of fibroblast protein added.
The decrease in GDH activity was present at different concentrations of enzyme substrates ( N A D P H
Table 1 . Activities of Various Enzymes i n Fibroblastsfrom Patient 1 and Controlsa
Subiect
GDH
Controls 0.454 2 0.11 1
(R = 0.236-0.672)
(N = 20)
Patient 1 0.090 f 0.025
(N = 10)
G-6-PDH
GSSGRD
ICDH
LDH
AAT-cvt.
AAT-mit.
1.35 2 0.17
(N = 2)
0.112k 0.027
(N = 2)
0.499? 0.057
(N = 2)
14.53? 1.60
(N = 3)
2.05
0.572 f 0.065
(N = 2)
1.10 f 0.03
(N = 1)
0.137 f 0.011
(N = 1)
0.654f 0.022
(N = 1)
17.60f 1.05
( N = 1)
1.51 f 0.10
(N = 1)
f 0.58
(N = 2)
f 0.035
( N = 1)
0.520
"Enzyme activity IS expressed in Fmol/mg proteidhr (mean k SD). Enzymes were measured in disrupted fibroblasts as described in the
methods section. GDH activity was determined without additions using 8 rnM a-ketoglutarate, 100 mM ammonium acetate, and 70 p M
NADPH or NADH (final concentrations). For GDH, ten cultures were studied for Patient 1 and at least two cultures for each of the 10
controls. The activity in each culture was determined at least in duplicate.
GDH = glutamate dehydrogenase; G-6-PDH = glucose 6-phosphate dehydrogenase; GSSGRD = glutathione reductase; ICDH = isocitrate dehydrogenase; LDH = lactate dehydrogenase; AAT-cyt. = aspartate aminotransferase, cytoplasmic; AAT-mit. = aspartate arninotransferase, mitochondrial; R = statistical range of control values obtained by using the mean f'1.96 SD; N = number of cultures.
300 Annals of Neurology Vol 7 No 4
April 1980
0 No Additions
H +ADP
r
r
0 +DES
m +GTP
[L
1000
2
000
W
c
f
3 0
(3
0
40
80 123 0 2 4 6 8 1 0 1 2
pM NADPH
mM d,KG
0
40 80 I20
mM NH4
3
400
I
Fig 2. Glutamate dehydrogenase ( G D H ) activity i n disrupted
skin fibroblasts from Patient I (A) and two controlsubjects (B)
at different substrate concentrations of N A D P H , aketoglutarate (a-KG), and ammonium acetate (NH,).
The activity of glutamate dehydrogenase was measured without
additions, using different concentrations of N A D P H , aketoglutarate, or ammonium acetate as described in the methods
section. Each point represents the mean of duplicate determinations. Bracketed bars represent standard deviation.
or NADH, a-ketoglutarate, and ammonium) over the
ranges shown in Figure 2. There were no differences
in the apparent Michaelis-Menton constant (K,)
from the patient and a typical control (2.4 and 2.1
mM, respectively). Mixing experiments with a control fibroblast homogenate showed no evidence for
the presence of a GDH inhibitor in the patient's
cells.
This allosterically regulated enzyme [ l , 22, 27, 321
was markedly stimulated by ADP (by 70 to 15096)
and inhibited by G T P (by > 90%) and DES (by 83 to
86%). The effects on GDH activity occurred in equal
proportions in the cells from Patient 1 and controls
(Fig 3).
600
200
a
0
PH
SI
SB
DG
SA
F i g 3 . Effect of adenosine diphosphate (ADP), gaanosine
triphosphate ( G T P ) , and diethylstilbestrol (DES)on glubamate dehydrogenase ( G D H ) activity i n fibroblastsfrom controls
( P H , SI, SB, SA) and from Patient I (DG). G D H was assayed with either no additions or i n the presence of A D P
M), GTP
M), or DES (4 x
Mi as described in the
methods section.
Studies on Leukocytes
GDH activity in leukocytes from Patient 1 was 38%
of the mean control activity in leukocytes from 10
controls (Table 2). GDH activity in the leukocytes of
the two affected siblings (Patients 2 and 3) was 29%
and 31%, respectively, of the mean control activity.
The activity of this enzyme in the three patients was
outside the statistical range of control values. The
leukocytes from an unaffected brother of Patients 2
and 3 (11-5 in Fig 1) had normal GDH activity.
The enzyme activity was proportional to the
amount of leukocyte protein added. Mixing experiments with a control leukocyte homogenate did not
show the presence of a GDH inhibitor in the leuko-
Table 2. Glutamate Dehydrogenase Activity in Leukocytes from Patients I , 2 , and 3 and Controlsa
With ADP
With GTP
Subiect
Without Additions
(10-3 M)
(10-3 M)
Controls (N = 10)
264 ? 69 (R = 149-379)
(N = 20)
101 L 22
(N = 2)
78 ? 28
(N = 4)
83 ? 20
(N = 4)
200 ? 25
(N = 2)
378 ? 69 (R = 243-513)
(N = 20)
155 ? 24
(N = 2)
117 ? 31
(N = 4 )
122 ? 24
(N = 4 )
351 ? 36
(N = 2 )
22 -+ 11
(N = 2)
12 r 6
(N = 2)
10 ? 8
(N = 2)
10 ? 9
(N = 2)
18 -+' 12
(N = 2)
Patient 1
Patient 2
Patient 3
Asymptomatic
brother of
Patients 2 and
(11-5 in Fig 1)
3
*
aEnzyme activity is expressed in nmoVmg proteidhr (mean SD). GDH was measured as described in the methods section with either no
additions or with 1 mM ADP or 1 mM GTP. The final leukocyte protein concentration ranged between 100 and 400 pdrnl; GDH activity
was linear under these conditions. All assays were carried out with 8 mM a-ketoglutarate, 100 mM ammonium acetate, and 70 p M NADPH
or N A D H (final concentrations). The activity of GDH in each preparation was measured at least in duplicate.
ADP = adenosine 5'-diphosphate; GTP = guanosine 5-triphosphate; G D H = glutamate dehydrogenase; R
values obtained by using the mean % 1.96 SD; N = number of leukocyte homogenates.
=
statistical range of control
Plaitakis e t al: Glutamate Dehydrogenase in Ataxia
301
cyte suspension from any of the three patients. The
decrease in leukocyte GDH activity persisted over a
tenfold range of a-ketoglutarate concentration (0.8
to 8 mM final concentration). Similar to the studies
with fibroblast G D H , the apparent K, for aketoglutarate did not differ in leukocyte preparations
from patients and controls (2.2,2.2,2.3, and 2.5 mM
for a control and Patients 1, 2, and 3, respectively).
GDH activity in leukocytes from all three patients
and the controls was stimulated by A D P and inhibited by GTP (Table 2).
Discussion
Three patients with slowly progressive spinocerebellar ataxia, lower cranial nerve dysfunction,
and various extrapyramidal manifestations were
found to have decreased activity of the N A D P H requiring enzyme glutamate dehydrogenase. Two of
these patients are siblings, suggesting that the disorder is familial and that the neurological affliction and
enzyme deficiencies might be causally related. Since
the parents of these patients were apparently not affected neurologically, the disorder appears to be
transmitted as an autosomal recessive or X-linked
trait. The constellation of clinical findings (particularly in the two siblings, Patients 2 and 3) is consistent with OPCA type IV (Schut-Haymaker type [3 13)
according to the classification of Koningsmark and
Weiner [19]. However, this type of OPCA appears to
be inherited as an autosomal dominant trait [19]. A
firm diagnosis of OPCA in these patients will require
pathological confirmation. Nevertheless, there is
general agreement that classification of patients with
spinocerebellar ataxias is difficult (except perhaps for
patients with Friedreich’s ataxia [29]) because clinical
and even pathological syndromes frequently overlap,
and that the classification should be regarded as provisional until it can be related to a specific biochemical marker [29].
The defect in G D H , shown in cultured skin
fibroblasts and leukocytes from Patient 1 and in
leukocytes from Patients 2 and 3, did not appear to
result from altered binding capacity of the enzyme
with its substrates, but rather was consistent with a
decreased amount of available enzyme activity. Such
a defect could result from genetic mutation in these
patients. For example, a structural gene mutation rendering the enzyme less stable would decrease the
maximum velocity but not the K,. Further studies are
needed to test this hypothesis.
GDH in vertebrates is a hexameric molecule with
only one type of polypeptide chain [32] and no
known isoenzymes [24]. The enzyme is allosterically
regulated by nucleotides (ADP and GTP) and can be
inhibited by steroid hormones [l, 22, 27, 321. Evi-
302
Annals of Neurology
Vol 7 N o 4
April 1980
dence suggests that the enzyme’s regulatory sites
originated from partial gene duplication during the
evolution process [9]. Because modulation of the enzyme activity by nucleotides (ADP and GTP) and its
inhibition by DES seemed not to differ in the patients compared with controls, the genetic mutation
in these patients did not appear to be associated with
qualitative changes in the regulatory sites of the residual enzyme.
This study shows that GDH deficiency may occur
in some forms of spinocerebellar ataxia, but whether
or how this enzyme defect relates to the pathogenesis
of the clinical syndrome is not clear at present. Little
is known about the exact role of GDH and the consequence of its inhibition in nervous tissue function.
The enzyme has a central role in glutamate metabolism, linking this amino acid with the tricarboxylic
cycle through a-ketoglutarate. This reaction may be
important not only for the synthesis of glutamate, but
also for ammonia metabolism [3, 4, 281. GDH activity shows regional variation in the central nervous
system (CNS) that correlates to some degree with
free glutamate content [ 161. In addition, distribution
of the enzyme may be compartmented in the brain,
and it is perhaps related to neuronal pools of glutamate [28]. This possibility, however, is not universally accepted [4]. From these neuronal pools glutamate is thought to be released as neurotransmitter,
especially in the cerebellar cortex [23, 341, spinal
cord [121, brainstem, and possibly other areas of the
C N S [ 7 , 101. It is therefore conceivable that a defect
in GDH could lead to alterations in glutamate and
perhaps ammonia metabolism in certain areas of the
CNS, which could result in dysfunction and possible
pathological changes in these areas.
Additional studies are needed to investigate these
possibilities and to explore further the importance of
these findings in patients with OPCA or other types
of system atrophies.
Supported in part by Grant NS-1163 1 from the Clinical Center for
Research on Parkinson’s and Allied Diseases.
Presented at the 104th Annual Meeting of the American Neurological Association, St. Louis, MO, Oct 4-6, 1979.
The authors wish to thank Dr S. Berl and Dr R. Duvoisin for constructive suggestions and assistance in the preparation of this
manuscript. We are indebted to Safiana Katz for technical assistance in fibroblast culture and to Dr W. A. Paulsen for supplying
the leukocytes from Patient 1.
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