Brain Ammo Acid Reductions in One F d y with Chromosome 6p-Lnked Dominantly Inherited Olivopontocerebellar Atrophy ~

Stephen J. Kish, PhD," Yves Robitaille, MD,? Munir El-Awar, MD,S Joseph Gilbert, MD,B John Deck, MD,B Li-Jan Chang, MSc," and Lawrence Schut, MD""

We measured the levels of aspartate, glutamate, gamma-aminobutyric acid (GABA), and other amino acids in autopsied brain of 6 patients from one family (Pedigree S) with dominantly inherited olivopontocerebellar atrophy. A previous demonstration of reduced aspartate concentration in plasma of affected members of this family suggested the possibility of a generalized disorder of amino acid metabolism affecting the brain. As compared with the control levels, mean levels of aspartate and glutamate were markedly reduced by about 70 and 40%, respectively, in the degenerated cerebellar cortex from the patients. Since the cerebellar aspartate reduction likely exceeds the amount that could be explained by neuronal loss, other factors such as abnormal aspartate metabolism, neurotransmitter turnover, or both are probably involved. Mean aspartate, glutamate, and GABA levels were also reduced by about 10 to 30% in most of the 16 examined extracerebellar brain areas in which no or, at most, mild neuronal cell loss was observed by semiquantitative estimation. Concentrations of taurine, glutamine, and o-phosphoethanolamine were normal in all brain areas examined. Our biochemical data provide support to the presence of a generalized, but quantitatively mild, disturbance in amino acid metabolism in patients with olivopontocerebellar atrophy from Pedigree S. The regionally widespread -amino acid reductions in the brain, of as yet unknown pathophysiological significance, could be due to a failure of one or more enzymes involved in aspartate and glutamate metabolism. Kish SJ, Robitaille Y , El-Awar M, Gilbert J, Deck J, Chang L-J, Schut L. Brain amino acid reductions in one family with chromosome 6p-linked dominantly inherited olivopontocerebellar atrophy. Ann Neurol 1991;30:780-784

Although the cause of the brain neurodegeneration in patients with inherited and sporadic disorders of progressive cerebellar ataxia is not understood, circumstantial evidence suggests that an abnormal excitatory amino acid mechanism may be involved. This evidence is based on the results of experimental animal studies demonstrating the excitotoxic action of glutamate and related compounds in mammalian brain (see El]) and the observations of reduced levels of a key glutamatemetabolizing enzyme, glutamate dehydrogenase (EC 1.4.1.3.), in platelets and leukocytes of many, but not all, patients with olivopontocerebellar degenerations 12- 131. Taken together, these observations suggest that increased synaptic levels of glutamate, as a result of impaired metabolism, could play an important role in the degenerative process. Evidence implicating the excitatory amino acid aspartate in one inherited cerebellar ataxia disorder was

obtained by Plaitakis and coworkers 114, 157 who demonstrated reduced levels of aspartate and altered aspartate metabolism in the plasma of affected members of one family (Pedigree S Cl6J) with dominantly inherited olivopontocerebellar atrophy (OPCA). These data suggest that a generalized defect in aspartate metabolism affecting susceptible areas of the central nervous system could be responsible for the brain degeneration in at least this one family affected by OPCA. In contrast with the extensive investigations of the behavior of excitatory amino acid levels and metabolism in the blood of patients with degenerative cerebellar disorders, the actual status of the excitatory amino acids and their related enzymes in the brain of such paticnts has received comparatively much less attention, thus leaving the clinical relevance of the biochemical changes demonstrated in blood elements uncertain. Moreover, in the relatively few biochemical investiga-

From the "Clarke Institute of Psychiatry, Toronto, Ontario; +Mom treal Neurological Institute, Montreal, Quebec; $University of Pitcsb u s h , Pittsburgh, PA; §Victoria Hospital, London, Ontario; TITOronto General Hospital, Toronto, Ontario, Canada; and ""University of Minnesota and VA Hospital, Minneapolis, MN.

Received Apr 3, 1991, and in revised form May 14. Accepted for publication May 21, 1931. correspondence Dr Clarke Institute of Psychiatry, 250 Street, OIltario,Canada M5T 1R8,

780 Copyright 0 1991 by the American Neurological Association

tions on the brain that have addressed this question, the brain neurochemical findings have been fragmentary in terms of either the number of patients or the brain regions examined {17-21], with most attention directed only to the degenerated brain areas. In this regard, a regionally comprehensive biochemical analysis of both neuronally affected and unaffected brain areas in OPCA is required to assess the presence of any “generalized” disorder of amino acid metabolism. We examined the behavior of the amino acid neurotransmitter aspartate as well as five other amino compounds (glutamate, GABA, glutamine, o-phosphoethanolamine, and taurine) in 17 brain areas of 6 patients from one family with end-stage OPCA (Pedigree S { 161).This family, in which the affected gene has been localized to the short arm of chromosome 6 {22, 231, was specifically selected in view of the earlier observations of reduced plasma aspartare levels and altered metabolism [14, 151 in affected members and markedly reduced aspartate and glutamate levels in several examined brain areas of 2 patients in the family [lS]. We reasoned that a regionally comprehensive biochemical examination of the brain would provide information regarding the likelihood of an actual generalized amino acid metabolic disorder affecting the central nervous system in this OPCA pedigree.

Patients and Methods Autopsied brains were obtained from 6 severely ataxic patients with end-stage OPCA from Pedigree S [16}, in which eight successive generations have been affected, and 10 control subjects who had died without evidence of neurological or psychiatric illness. The mean ages and postmortem times (interval between death and freezing at - 80” C) of the control subjects (43 +- 4 years; 10 * 2 hours; mean standard error {SE}) and patients with OPCA (37 ? 3 years; 9 * 3 hours) did not differ significantly (p > 0.05, Student’s twotailed t test). Diagnosis of OPCA in this established family was made on the basis of clinical signs (imbalance,dysarthria, dysphagia, and limb ataxia} and characteristic neuronal cell loss in the cerebellum, pons, and inferior olives. An anatomically extensive semiquantitative histological analysis was performed on numerous representative sections stained with hematoxylin-eosin,Kliiver-Barrera,and modified Bielschowsky stains. This revealed, in all of the patients, severe inferior olivary cell loss and gliosis and, in the lateral cerebellar hemispheres, a severe loss of Purkinje cells with extensive gliosis in the Purkinje cell and molecular layers. The superior vermis showed a slightly increased number of remaining Purkinje cells and the flocculonodular lobe, slightly more. The granule cell layer was graded according to the following scale: mild, single focus of neuronal depletion; moderate, continuous bands or multiple foci of neuronal loss; and severe, extensive continuous loss of granule cells in the folium. In most of the samples assessed, the granule cell loss was graded mild, with only signs of some focal neuronal loss. In addition, in 5 of the 6 patients for which spinal cord was available, moderate to severe cell loss was observed in the anterior horn.

*

Semiquantitative histological analysis of cerebral cortex, basal ganglia, and diencephalon on numerous representative sections revealed no, or only mild neuronal cell loss or gliosis. Complete neuropathological details will be published elsewhere. Amino compounds were quantitated in ethanolic brain extracts using minor modifications of the procedure of Fernstrom and Fernstrom 124).

Results The Figure shows the concentrations of aspartate, glutarnate, and GABA in the brain of the patients with OPCA. Quantitatively, the most severe amino acid changes occurred in the cerebellar cortex, with marked and statistically significant reductions in mean aspartate (-67G/o), glutamate (-43%), and GABA ( - 36%) concentrations Cp < 0.01), as compared with the control levels. The mean levels of aspartate, glutamate, and GABA were reduced by about 10 to 30% in all of the 16 extracerebellar brain regions analyzed, with the exception of the Ammon’s horn (aspartate, glutamate, and GABA) and the midline thalamus (GABA). These differences were found to be statistically significant (p < 0.05 or less) for aspartate (frontal, parietal, and occipital cortices; internal globus pallidus), glutamate (frontal, temporal, parietal, occipital, and hippocampal cortices; intermediate caudate nucleus; internal globus pallidus), and GABA (temporal, parietal, and occipital cortices; intermediate caudate; rostral putamen; internal and external globus pallidus; medial dorsal thalamus). A sign test confirmed the generalized trend for aspartate, glutamate, and GABA reductions in the 17 brain areas (p < 0.01). In the OPCA group no statistically significant abnormalities in the concentrations of glutamine, o-phosphoethanolamine, o r taurine were observed in either the cerebellar cortex or extracerebellar brain areas Cp > 0.05, data not shown).

Discussion As mentioned in the Introduction, Plaitakis and coworkers suggested the presence of a systemic defect in neuroexcitatory amino acid metabolism in patients from Pedigree S who have OPCA, on the basis of their observations of reduced plasma aspartate levels in affected members of this family E14, 157. The results of our investigation extend this observation to the central nervous system and demonstrate, in this family, a deficit of aspartate, as well as the metabolically related amino acids glutamate and GABA, throughout the brain of the patients. These data generally confirm and extend, by an additional 6 patients studied, the observations of Perry and coworkers {18], who reported a severe (>70o/c)depletion of asparrate and glutamate in cerebellar cortex and two extracerebellar brain regions in 2 patients with OPCA from Pedigree S. The results of our regionally more comprehensive amino acid anal-

Kish et al: Brain Amino Acids in OPCA

781

30 f rn

75

1T

I

CONTROL OPCA

CBL 10 21 7b 17 GD GH CNr CNi PTr PTi GPi GPe AH NAMMD ML

BRAIN AREA 120

0 2BL 10 21 76 17 GD GH CNr CNi PTr PTi GPiGPe AH NAMMD ML

BRAIN AREA CONTROL

'

-

U-

CBL' 10 ' 21 ' 7b ' 17 'GD 'GH 'CNr'CNi-PTr-PTi'GPiGPe AH NAM MD 'ML

BRAIN AREA

ysis in a larger number of Pedigree S patients disclosed, however, that the reductions in aspartate and glutamate in the extracerebellar brain regions are, on average, only quantitatively mild (10 to 30% reduction). The amino acid reductions observed in both cerebellar cortex and extracerebellar brain areas are unlikely to be explained in toto by loss of neuronal elements containing these substances. Thus, in the cerebellar cortex, although part of the reduction in aspartate concentration is probably explained by a loss of inferior olivary climbing fiber neurons terminating in the cerebral cortex and utilizing aspartate as a neurotransmitter (see 125, 26]), the magnitude of the loss (-67%) greatly exceeds the cerebellar aspartate reduction observed in experimental animal brain following total inferior olive destruction (about 20%; see E27)). Similarly, loss of granule cells utilizing glutamate as a neurotransmitter [25 } appears unlikely to explain the glutamate reduction (approximately 40%) in the cerebellar cortex of our patients with OPCA, since the degree of loss of granule cell neurons in these patients was only mild. The observations that glutamate, aspartate, and GABA levels were reduced, on average, in most of the extracerebellar brain areas examined, for which semiquantitative histopathological analysis revealed no, or only mild cell loss, also suggest that factors other than neuronal loss are likely to be involved. The possibility, however, has to be considered that part of the amino acid reduction could, in some brain areas, be secondary to nerve terminal loss (which was not assessed in our neuropathological analysis) without loss of perikarya. The amino acid reductions could be explained by increased neuronal activity of neurons utilizing these amino acids as neurotransmitters, and consequent lowered steady-state levels, or an actual failure of one or more enzymes involved in glutamate and aspartate metabolism. Interestingly, a regionally widespread reduction in brain glutamate concentration has also been reported in patients with amyotrophic lateral sclerosis (ALS) E28-30). This condition is characterized by a loss of cells in the anterior horn of the spinal cord, as

Lmeh of aspartate, glutamate, and G A B A in autopsied brain regions of ten neurologically normal control subjects and 6 patients from olivopontocerebellar atrophy (OPCA) Pedigree S. Values represent mean SE. CBL = cerebellar cortex; 10 = Brodmann frontal cortex area 10; 21 = Brodmann temporal cortex area 21; 7b = Broa’mann parietal cortex area 76; 17 = Brodmann occipital cortex area 17; GD = dentate grus: G H = hippocampal gyrus; CNr and C N i = ro.rtral and intermediate caudute bead; PTr and P T i = rostral and intermediate putumen; GPi and GPe = internal m d external globus pallidus; A H = Ammon’s horn; NAM = amygdula; M D = medial-dorsal thalamus; M L = midline thalamus; a = p < 0.05; b = p < 0.01 (Student’s two tailed t test).

*

occurs in patients from OPCA Pedigree S (see Patients and Methods). However, the actual functional significance of the brain amino acid deficits in ALS and in our OPCA study is uncertain and may, in both conditions, represent an epiphenomenon (see 13 1)). Certainly, our observation in OPCA brain of excitatory amino acid deficits provides no direct evidence for excessive, possibly neurotoxic excitatory neurotransmitter levels at the synaptic cleft. One interesting possibility is that the amino acid reductions are a biochemical consequence of an etiologically significant metabolic error that renders some neurons susceptible to neurotoxic damage. Such damage might, but not necessarily, be mediated through excessive activity at the excitatory amino acid receptor. The biochemical defect is unlikely to involve the enzyme glutamate dehydrogenase, which is normal in autopsied brain of patients from Pedigree S E32341. An interesting candidate is a-ketoglutarate dehydrogenase, an enzyme involved in both glutamate and aspartate metabolism and energy production. Reduced activity of such an enzyme, as part of the disease process, and consequent depletion of neuronal energy stores could explain both the lowered brain glutamate and aspartate levels C351 and also lead to greatly increased neuronal susceptibility to endogenous excitatory amino acid-induced neurotoxicity [36} (see [37)). Measurement of this and other key glutamateand aspartate-metabolizing enzymes in OPCA brain is currently in progress in our laboratory (Clarke Institute of Psychiatry, Toronto).

This study was supported by US National Institutes of Health grant R 0 1 NS26034. Dr Kish is a Career Scientist of the Ontario Ministry of Health. A portion of this study was presented at the 116th annual meeting of the American Neurological Association, Seattle, 1991.

References 1. Schwarcz R, Foster AC, French ED, Whatsell WO Jr, Kohler C. Excitotoxic models for neurodegenerative disorders. k f e Sci 1984;35:19-32 2. Plaitahs A, Nicklas WJ, Desnick RJ. Glutamate and rnalate dehydrogenase deficiency in three patients with spinocerebellar syndrome. Ann Neurol 1980;7:297-303 3. Yamaguchi T, Hayashi K, Murakami H, Ota K, Kanazawa I. Glutamate dehydrogenase deficiency in spinocerebellar degenerations. Neurochem Res 1982;7:627-636 4. Duvoisin RC, Chokroverty S, Lepre F, Nicklas W. Glutamate dehydrogenase deficiency in patients with oiivopontocerebellar atrophy. Neurology 1983;33:1322-1326 5. Plaitakis A, Berl S, Yahr MD. Neurological disorders associated with deficiency uf glutamate dehydrogenase. Ann Neurol 1984; 15~144-153 6. Konagaya Y , Konagaya M, Takayanagi T. Glutamate dehydrogenase and its isozyme activity in olivopontocerebeflar atrophy. J Neurol Sci 1986;74:231-236 7. Sorbi S, Tonini S , Giannini E, Piacentini S, Marini P, Amaducci L. Abnormal platelet glutamate dehydrogenase activity and activation in dominant and nondominant olivopontocerebellar atrophy. Ann Neurol 1986;19:239-245

Kish et al: Brain Amino Acids in OPCA

783

8. Aubby D , Sasgu HK, Jenner P, Quinn NP, Hardirig AE, Marsden CD. Leukocyte glutamate dehydrogenase activity in patients with degenerative neurological disorders. J Neurol Neurosurg Psychiatry 1988;5 1:893-902 9. Kajiyama K, Ueno S, Tarsumi T, Yorifugi S, Tachashi M, T 6 S. Decreased glutamate dehydrogenase protein in spinocerebellar degeneration. J Neurol Neurosurg Psychiatry 1988;5 1: 1078- 1080 10. Orsi L, Brignolio F, Chi0 A, er al. Glutamate dehydrogenase (GDH) deficiency in different types of progressive hereditary cerebellar ataxia. Acta Neurol Scand 1988;78:394-400 11. Kostic VS, Mojsilovic LJ, Stojanovic M. Degenerative neurological disorders associated with deficiency of glutamare dehydrogenase. J Neurol 1989;236:111-114 12. Tatsumi C, Yorifuji S,Kajiyama K, Ueno S, Takahashi M, Tarui S. Glutamare metabolism of leukocytes and skin fibroblasts in spinocerebellar degeneration with lowered glutamate dehydrogenase activity. Acta Neurol Scand 1989;79:468-475 13. Kaakkola S, Marneb K-M, Oja SS, Icen A, Palo J. Leukocyte glutamare dehydrogenase and CSF amino acids in late onset ataxias. Acra Neurol Scand 1990;82:225-229 14. Plaitakis A, Berl S, Schut LJ, Yahr &ID. Abnormal asparrate: malate metabolism in dominantly inherited olivopontocerebellar degeneration. Ann Neurol 1982,12:79 15. Plaitakis A. Abnormal merabolism of neuroexcitatory amino acids in olivopontocerebellar atrophy. In: Duvoisin RC, Plaitakis A, eds. The olivopontocerebellar atrophies. New York: Raven Press, 1984:225-243 16. Schut JW. Hereditary ataxia: clinical study through six generations. Arch Neurol Psychiatry 1950;168:75-95 17. Perry TL, Currier RD, Hansen S, MacLean J. Aspartare-tarrrine imbalance in dominantly inherited olivopontocerebellar atrophy. Neurology 1977;273:257-261 18. Perry TL, Kish SJ, Hansen S, Currier RD. Neurotransmitter amino acids in dominantly inherited cerebellar disorders. Ncurology 1981;3 1:237-242 19. Kanazawa I, Kwak S, Sasaki H, et al. Studies on neurotransmitter markers and neuronal cell density in the cerebellar system in olivoponrocerebellar arrophy and cortical cerebellar atrophy. J Neurol Sci 1985;71:193-208 20. Whitehouse PJ, Muramoto 0,Tronsco JC, Kanazawa I. Neurotransmitter receptors in olivopontocerebellar atrophy: an auroradiographic study. Neurology 1986;36:1.93-197 21. Bebin EM, Bebin J, Currier RD, Smith EE, Perry TL. Morphometric studies in dominant olivopontocerebellar atrophy: comparison of cell losses with amino acid decreases. Arch Neurol 1990;4?:189-1 92 22. Rich SS, Wilkie PJ, Schut L, et al. Spinocerebellar ataxia: localization of an autosomal dominant locus between rwo markers on human chromosome 6. Am J Hum Genet 1987;41:524-531

784 Annals of Neurology Vol 30 No 6 December 1991

23. Ranum LPW, Duvick LA, Rich SS, Schut LJ, Litr M,Orr HT. Localization of the autosomal dominant HLA-linked spinocerebellar ataxia (SCA1) locus, in two kindreds, within an 8cM subregion of chromosome 6p. Am J Hum Genet 1991;49:31-41 24. Fernstrom MH, Fernsrrom JD. Rapid measurement of free amino acids in serum and CSF using high performance liquid chromatography. Life ScJ 1981;29:2119-2 140 25. Tiemeyer MJ, Singer HS, Tronsco JC, Cork LC, Coyfe JT, Price DL. Synaptic neurochemical alterations associated with neuronal degeneration in an inherited cerebellar ataxia of Gordon setters. J Neuropathol Exp Neurol 1984;43:580-591 26. Vollenweider FX, Cuenod M, Do KQ. Effect of climbing fiber deprivation on release of endogenous asparrate, gluramate, and homocysteate in slices of rat cerebellar hemispheres and vcrmis. J Neurochem 1990;54:1533- 1540 27. McBride WJ, Ghetti B. Changes in the content of glutamate and GABA in the cerebellar vermis and hemispheres of the Purkinje cell degeneration (pcd) mutant. Neurochem Res 1988;13:121- 125 28. Perry TL, Hansen S, Jones K Brain glutamate deficiency in amyotrophic lateral sclerosis. Neurology 1987;37: 1845-1848 29. Plaicakis A, Constantakakis E , Smith J. The neuroexcitotoxic amino acIds glutamate and asparrate are altered in the spinal cord and brain in amyotrophic lateral sclerosis. Ann Neurol 1988;24:446-449 30. Malessa S, Leigh N, Hornykiewicz 0. Branched chain amino acids in amyotrophic lateral sclerosis. Lancet 1988;2:681-682 3 1. Young AB. What’s the excitement about excitatory amino acids in amyotrophic lateral sclerosis? Ann Neurol 1990;28:9- 10 32. Grossman A, Rosenberg RN, Warmoth L. Glutamare and malare dehydrogenase acrivities in Joseph disease and olivopontocerebellar atrophy. Neurology 1987;37:106-111 33. Kish SJ, Robiraille Y , Chang L, Dixon LM. Brain gluramare and metabolizing enzymes in dominantly-inherited olivoponrocerebeliar atrophy (OPCA). Eur J Neurosci 1989;l(suppl 21239 34. Hussain MM, Zannis VI, Plaitakis A. Characterization of glutamate dehydrogenase isoproteins purified from the cerebellum of normal subjects and patients with degenerative neurological disorders, and from human neoplastic cell lines. J Biol Chem 1989;264:20730-20735 35. Butterworth RF, Heroux M. Effect of pyrithiamine treatment and subsequent thiamine rehabrlitxrion o n regional cerebral amino acids ,and thiamine-dependent enzymes. J Neurochem 1989;52:1079-1084 36. Novelli A, Reilly JA, Lysko PG, Henneberv RC. Glutamate becomes neurotoxic via the N-methyl-maspartate receptor when intracellular energy levels are reduced. Brain Res 1988;451: 205-212 37 Greenamyre JT, Young AB. Aurhors’ response to commentaries. Neurobiol Aging 1989;10:618-620