28 Wilson, H. R., Blake, R. and Halpern, D. L. (1991) J. Opt, Soc. Am. 8, 229-236 29 Nishihara, H. K. (1984) Opt. Eng. 23, 536-545 30 Quam, L. H. (1984) in Readingsin Computer Vision (Fischler, M. A. and Firschein, 0., eds), pp. 80-86, Kauffman 31 Anderson, C. H. and van Essen, D. C. (1987) Proc. NatlAcad. Sci. USA 84, 6297-6301 32 Yeshurun, Y. and Schwartz, E. L. (1989) IEEETrans. Pattern Anal. Mach. Intell. 11,759-767

33 Ohzawa, I., DeAngelis, G. C. and Freeman, R. D. (1990) Science 249, 1037-1041 34 Mayhew, J. E. W. and Longuet-Higgins, H. C. (1982) Nature 297, 376-379 35 Pouget, A. and Sejnowski, T. J. (1990) Invest. Ophthalmol. Visual Sci. 31, 96 36 Blake, R. (1989) Psychol. Rev. 96, 145-167 37 Ross, J. (1974) Nature 248, 363-364 38 Rogers, B. and Cagenello, R. (1989) Nature 339, 135-137

Autoimmunityto glutamRaciddecarboxylase(GAD)in StiffMan syndromeand insulin-dependentdiabetesmellitus Michele Solimena and Pietro De Camilli

MicheleSolimenaand PietroDe Camil/iare at the Deptof Cell Biology, Yale UniversitySchoolof Medicine,333 Cedar Street, New Haven, CT06510, USA.

Stiff-Man syndrome (SMS) is a disorder of the CATS, characterized by rigidity of the body musculature, which has been hypothesized to result from an impairment of GABAergic neurotransmission. GABA is the main inhibitory neurotransmitter of the brain. It is also a putative signal molecule in the pancreas, where it is produced by ~ cells (insulin-secreting cells) - the autoimmune target in insulin-dependent diabetes mellitus (IDDM). Autoantibodies to the GABAsynthesizing enzyme glutamic acid decarboxylase (GAD) have been found in SMS and in IDDM. This review summarizes evidence suggesting that SMS may be an autoimmune disease and discusses the possible significance of the autoimmune response to GAD in SMS and IDDM. Until recently, it was thought that anatomical barriers and functional properties normally protect neurons of the CNS from becoming the target of autoimmunity. Now, however, increasing evidence suggests that this is not the case. Paraneoplastic neurological disorders were the first neuronal diseases of the CNS for which an autoimmune origin was proposed 1. Evidence supporting an autoimmune pathogenesis has now been provided for a rare and severe neuronal CNS disease, Stiff-Man syndrome (SMS).

contraction of antagonist muscles. It was proposed that an imbalance between excitatory (catecholaminergic), and inhibitory (GABAergic) pathways controlling a-motoneuron activity causes the manifestations of the disease 8. High doses of various agonists of GABAergic neurotransmission, such as baclofen, sodium valproate and primarily benzodiazepines, are generally effective in ameliorating rigidity by reducing a-motoneuron firing. These drugs, however, do not affect the course of the disease s. More recently, steroid treatment has been shown to be effective in some cases 3'9-u. The few autopsies carried out so far have failed to demonstrate pathological lesions of specific areas of the CNS. However, evidence for an inflammatory process in the spinal cord and brainstem has been reported in some cases (Ref. 6 and Refs listed there).

The autoimmune hypothesis of SMS An autoimmune pathogenesis of SMS was suggested by the observation of sporadic cases in which this condition was associated with autoimmune diseases including IDDM 12'13'~7. IDDM is due to an autoimmune destruction of insulin-secreting [~ cells of pancreatic islets 1< 15. The study of one SMS patient 13, and subsequently of an additional 32 patients 16 (who fitted the criteria for the diagnosis of the disease established by Gordon et al. 5,7) addressed directly the Stiff-Man syndrome SMS was originally described by Moersch and possibility that CNS autoimmunity was involved in Woltman in 1956, who reported 14 cases observed at SMS. In many of these patients, levels of IgG the Mayo Clinic2. Since then, more than a hundred antibodies in the cerebrospinal fluid (CSF) were cases of the disease have been reported in the elevated or had an oligoclonal pattern or both, literature. SMS, also recently referred to as Stiff- suggesting that IgG antibodies were produced locally Person syndrome3, is characterized by rigidity of within the blood-brain barrier. Furthermore, in 60% skeletal muscles, primarily of the trunk and limbs, of these patients, autoantibodies directed against with superimposed painful spasms 3-5. It usually GABAergic neurons, and primarily their nerve terappears in adulthood and generally has a fluctuating, minals, were identified by an immunocytochemical slowly progressive course. In a few cases, sudden assay in the serum and the available CSFs (Fig. 1). By death has been reported 6. western blotting13'16 and immunoprecipitation16"'1 7, SMS resembles a chronic form of tetanus in many the dominant autoantigen recognized by these autoways, although some differences, such as the absence antibodies in brain tissue was found to be the GABAof trismus in SMS, differentiate the two conditions4. synthesizing enzyme, glutamic acid decarboxylase Symptoms result from the simultaneous activation of (GAD) (Figs 2,3). Sera and CSFs found to be negative agonist and antagonist muscles. Several character- by immunocytochemistry were also found to be istics of the disease indicate its CNS origin5'7. negative for GAD antibodies by western blotting and Neurophysiological studies have demonstrated the immunoprecipitation. A few sera were found to be presence of a continuous discharge of motor-unit positive by immunocytochemistry and immunoprecipipotentials resembling a normal voluntary contraction. tation but negative by western blotting 16' 18. This can This activity cannot be inhibited by the voluntary probably be explained by the variable specificity of 452

© 1991.ElsevierSciencePublishersLtd,(UK) 0166-2236/91/$02.00

TINS, Vo/. 14, No. 10, 1991

GAD antibodies. Some GAD epi- A B topes might be preserved by aldehyde fixation (immunocytochemistry) and immunoprecipitation (non-ionic detergent extraction17), but disrupted by the strong denaturation involved in the western blot procedure [ionic detergent (SDS) extraction]. It has previously been reported that several monoclonal antibodies raised against GAD recognize the protein by immunocytochemistry and immunoprecipitation but not by western blotting 19. We have so far tested a total of 76 SMS patients and found GAD antibodies in 43 of these patients (56%) (Solimena, M., Folli, F., Cofiell, R. and De Camilli, P., unpublished observations). GAD is so far the only protein known to be specific for GABAergic neurons in the CNS. Other proteins involved in GABA neurotransmission, such as GABA transaminase and the plasmalemma GABA transporter (Fig. 3), are present at high concentrations in Fig. 1. Comparison of immunostains produced by (A) a sheep antiserum raised against glutamic GABAergic neurons, but are also acid decarboxylase (GAD) (fluorescein) and by (B) the serum of a Stiff-Man syndrome patient (rhodamine) on the same frozen section of rat cerebellar cortex (sagittal section). Immunoreactivity expressed in other cells of the appears white. Note that the two staining patterns are identical. White spots visible in both fields CNS'°'zl. The occurrence of a represent GABAergic nerve terminals. The prominent accumulation of immunoreactivity around dominant (i.e. strong and highly perikarya of the four Purkinje cells shown in the micrographs represent the GABAergic nerve specific) humoral autoimmune re- terminals of basket-cell axons. No specific fluorescence was observed with human control sera. sponse directed against GAD in a Scale bar is 30 ~m. (The sheep serum was kindly supplied by W. Oertel and E. Mugnaini2S'37.) high proportion of SMS patients appears to be very relevant to the manifestations of the disease, because an impairment cases, SMS might have an autoimmune pathogenesis, of GABAergic neurotransmission had been proposed and point to GAD as the major autoantigen. It will be to occur in SMS (see above). Epilepsy is another of interest to establish whether GAD-antibodycondition in which an impairment of GABAergic negative cases of SMS have different pathogenetic neurotransmission might be involved 22. Epilepsy, like mechanisms. Occasional cases of SMS may have a SMS, is treated by drugs that potentiate GABAergic paraneoplastic origin 1°'26. neurotransmission. It is interesting that epilepsy was present in 5 of the 43 GAD-antibody-positive SMS The biochemistry of GAD patients that we studied (Refs 13,16, and unpublished GAD is the enzyme that catalyses the conversion of observations). The presence of epilepsy in about 10% glutamic acid to GABA. The identity of the GAD of SMS cases has previously been reported '~a. molecule(s) has been controversial. While a putative A comparison of the available case histories of GAD protein of about 80 kDa has been reported 27, it GAD-antibody-positive and GAD-antibody-negative is now generally accepted that GAD activity in the SMS patients does not reveal obvious clinical differ- adult mammalian brain is primarily accounted for by ences in the neurological symptoms. However, a a doublet of proteins 28-al - referred to here as 65 kDa striking difference between the two patient sub- and 67 kDa GAD isoforms 31 (their estimated molpopulations is defined by the occurrence of associated ecular mass has been reported as ranging from 55 to diseases. One or more organ-specific autoimmune 67 kDa2~-31). Both proteins have enzymatic diseases, including vitiligo, pernicious anemia, hypo- activity :~1. They have very similar biochemical propor hyperthyroidism, and primarily IDDM, were pres- erties and are known to be antigenically related ent in 25 out of 43 (58%) of the GAD-antibody- because polyclonal antibodies raised against the positive patients, but only in 2 out of 33 (6%) of the 67 kDa protein a2 or against some of its fragments GAD-antibody-negative patients (Refs 12,13,16, and (Ref. 17 and Solimena, M. and De Camilli, P., ununpublished observations). It is well known that published observations) also recognize the isoform of organ-specific autoimmune disorders can occur to- lower molecular mass. However, antibodies specific gether in the same patient. This association has been for each of the two proteins have also been a useful clue for identifying the autoimmune patho- obtained 3°,31. genesis of other diseases, for example of LambertThe 67 kDa isoform has been sequenced in many Eaton myasthenic syndrome ~4'ea. Taken together, all species, including cat, rat and mouse, and has been these findings strongly suggest that, in the majority of found to be remarkably conserved during evolTIN& Vol. 14, No. 10, 1991

453

1

2

3

4

5

6

Q

~'ii i!!i i i !~ii

! ,~ii!!iii!,i

,

Fig. 2. Specificity of glutamic acid decarboxylase (GAD) antibodies of Stiff-Man syndrome (SMS) patients as revealed by western blotting. Identical amounts of Triton-X 114 extracts of rat brain were added to each of the lanes shown in the figure. Individual lanes were then labeled with the following antibodies: lane 1, polyclonal rabbit serum raised against the mouse recombinant 67 kDa GAD isoform that recognizes both the 67 kDa and 65 kDa isoforms (gift of Z. Katarova and R. J. Greenspan32); lane 2, polyclonal rabbit serum raised to a 17-met peptide corresponding to the carboxy terminus of the 67 kDa isoform; lane 3, mouse monoclonal antibody GAD-6 (gift of D. Gottlieb3°); lanes 4-6, sera of different SMS patients. GAD-6 and the human sera recognize only the 65 kDa GAD isoform (Reetz, A., Solimena, M. and De Camilli, P., unpublished observations).

ution 3e-35. The amino acid sequence predicts a highly hydrophilic protein with no transmembrane regions and with a consensus sequence for pyridoxal phosphate binding, a cofactor required for the enzymatic activity aa. The 65 kDa GAD isoform is the product of another gene 36, but so far the complete sequence of this isoform has not been reported. Stretches of amino acid sequences of the 65 kDa GAD isoform, obtained by Edman degradation a° and by nucleotide sequencing of PCR products (Solimena, M., De Camilli, P. and Hayday, A., unpublished observations), have indicated a high degree of similarity between the two isoforms. In western blot assays, autoantibodies of SMS patients recognize primarily the 65 kDa isoform 4.54

(Fig. 2). When SMS antibodies that in western blots recognize only the 65 kDa isoform are used to immunoprecipitate GAD from brain extracts prepared by non-ionic detergents, a pool of the 67 kDa isoform is found in the immunoprecipitate along with the 65 kDa protein. Similar results have previously been obtained with a monoclonal antibody specific for the 65 kDa isoform in a western blot assay. This finding might be explained by the ability of the two isoforms to form heterodimers eg':~°. Both GAD isoforms are selectively expressed by GABAergic neurons and are primarily concentrated in their nerve terminals 37, although some differences in their intracellular distribution have been reported :~1. In nerve terminals, GAD immunoreactivity is concentrated at the cytoplasmic surface of synaptic vesicles 3s. Both isoforms can be dissociated from membranous organelles by. media of low ionic strength 3s, a finding that is in agreement with the lack of transmembrane regions of the molecule. However, a significant pool of GAD was found to partition in the detergent phase after Triton-X 114 extraction ~7:~'~, suggesting the existence of hydrophobic posttranslational modifications of the protein. Outside the brain, GAD has a very restricted distribution. It is expressed at high levels only by pancreatic 13 cellsU~ (Fig. 4), epithelial cells of the fallopian tube 11 and spermatozoa le. Indeed, all GAD-antibody-positive sera and CSFs were found to selectively stain [3 cells in pancreatic islets. GAD in [3 cells is biochemically and irnmunologically indistinguishable from brain GAD ~r':~ and is concentrated at the cytoplasmic surface of synaptic-like microvesicles :~. These vesicles are closely related to neuronal synaptic vesicles and are thought to be involved in the storage and release of GABA from [3 ceils :~s. GAD is a key antigen of IDDM The high concentration of GAD in pancreatic [3 cells is of special interest since IDDM is the organ-specific

glutamicacid

s•uccinic emi-aldehyde1GAD GABA

/ plasmalernma GABAtransporter

"~

H" G A B ~ p r o t o n pump carrier

Fig. 3. Schematic drawing of a GABAergtc synapse. Glutamic acid decarboxylase (GAD) converts glutamic acid to GABA. A GABA carrier coupled to a proton pump mediates the uptake of GABA into synaptic vesicles. GABA is secreted by synaptic vesicle exocytosis and acts on ionotropic GABAA receptors as weft as on metabotropic GABAe receptors. A plasmalemma GABA transporter reuptakes GABA into the presynaptic terminal. GABA transaminase (GABA-T) converts GABA to succinic semialdehyde. TINS, Vol. 14, No. 10, 1991

autoimmune disease most frequently associated with SMS a'5'le'l:~'16''~3. In our study of a total of 76 patients (43 being GAD-antibody positive and 33 being GADantibody negative), 24% of GAD-antibody-positive patients (10/43) had IDDM, and at least four other GAD-antibody-positive SMS patients not affected by IDDM were found to have an abnormal glucose tolerance, as revealed by oral or intravenous glucosetolerance tests (ReL 16 and unpublished observations). An abnormal glucose tolerance is typically seen in pre-diabetic patients, and has been shown to correlate with a reduction in the [3-cell mass due to autoimmune destruction ~8. These considerations have led to the hypothesis that GAD autoimmunity in SMS and IDDM might be interrelated 1:~'l~. Interestingly, in a few cases of IDDM without SMS, autoantibodies directed against GAD were found, which had biochemical properties similar to those observed in SMS ~. A clue supporting this hypothesis has come from comparing the properties of GAD with those of putative autoantigens in IDDM. A protein widely regarded as a key autoantigen in IDDM is the '64 kDa antigen'. Autoantibodies directed against a 64 kDa protein of pancreatic islets (anti-64 kDa antibodies) were originally discovered in the sera of eight out of ten newly diagnosed IDDM patients, using an immunoprecipitation assay involving non-ionic detergent extracts of metabolically labeled islets tl. Later studies demonstrated that these autoantibodies can be detected many years before the onset of IDDM and are present in 80% of the patients at its onset ;1:>.7. Anti-64 kDa antibodies were also detected in animal models of IDDM, such as the NOD mouse and the BB rat '~7. In a survey of several tissues, which did not include the brain, the 64 kDa antigen was found to be a [3-cell-specific protein :~9. Like GAD, the 64 kDa antigen is represented by a doublet of bands in SDS gels *s. Since the known properties of the 64 kDa doublet closely matched those of the GAD doublet, the possible identity of the 64 kDa antigen as GAD was investigated. By a variety of stringent immunological and biochemical criteria, the two proteins were found to be identical 17 (Fig. 5). It was also found that GAD antibodies in SMS have a higher titer and a different epitope specificity compared with GAD antibodies in IDDM. Only a few of the IDDM sera positive for antibodies to the 64 kDa antigen or GAD in the immunoprecipitation assay 44 were found to detect GAD by immunocytochemistry or western blots ~7. This suggests that IDDM autoantibodies recognize primarily epitopes that are highly sensitive to aldehyde fixation or ionic detergent (SDS) denaturation, but are preserved by the non-ionic detergents used in the immunoprecipitation procedure.

Fig. 4. Color double-immunofluorescence micrograph of rat pancreatic islet, showing the fi-cell specific localization of glutamic acid decarboxylase (GAD). The section was stained for GAD with the mouse monoclonal antibody GAD-6 (Ref. 30) (green) and for glucagon with rabbit antibodies (orange). GAD immunoreactivity is restricted to the core of the islet, which consists of fl cells (insulin-secreting cells) and is not present in mantle cells consisting primarily of o: cells (glucagon-secreting cells). Scale bar is 13/Lm.

neurons (i.e. GAD-expressing neurons) has been postulated ~ but not yet demonstrated. Furthermore, a variety of criteria required to conclusively establish the autoimmune origin of SMS remains to be fulfilled. Notwithstanding these limitations, it is interesting to discuss some possible implications of the presence of GAD antibodies in both IDDM and SMS. It is unlikely that GAD antibodies alone have a direct pathogenetic role, because GAD is a cytoplasmic antigen not exposed at the extracellular milieu, and therefore not accessible to antibodies. In IDDM, strong evidence indicates that the disease is mediated by T cells rather than by antibodies 4"~. An antibodymediated pathogenesis of SMS cannot be ruled out, but, if this was the case, autoantibodies directed against surface antigens of GABAergic neurons, rather than against cytoplasmic antigens, should come into play. Functional and cytolytic studies on living neurons might help to verify this hypothesis. A positive effect of plasmapheresis in the treatment of SMS has been observed in two cases 5°''~1, but not in another six52 s.l _ although steroid therapy has been effective in several instances :~'u 11. On the other hand, it is also unlikely that the strong Possible significance of GAD autoimmunity humoral response to GAD is an epiphenomenon not Is the presence of a dominant humoral response to closely related to pathogenetic mechanisms in SMS GAD in both SMS and IDDM related to the patho- and IDDM. It could be hypothesized that lysis of genesis of the two diseases? From the information GAD-expressing cells leads to the production of available so far, a close parallelism between the antibodies to cytoplasmic antigens normally not exmechanisms of the two diseases cannot be drawn. In posed to the immune system, including GAD. This IDDM, an autoimmune destruction of [3 cells is well possibility seems unlikely for SMS, because GAD established 1"~''1"'67. In the case of SMS, it is unknown antibodies are not seen in a variety of other neurologiwhether symptoms are derived from functional im- cal disorders including degenerative neuronal dispairment or from cellular damage of selected neuronal eases us. Furthermore, in GAD-antibody-positive SMS subpopulations. A specific impairment of GABAergic patients, GAD antibodies are by far the dominant TINS, Vol. 14, No. 10, 1991

455

7.1

6.4

A islets

125- I

B brain

125-1

C 35- S

4NIbo

islets

D 3s'S

islets

Fig. 5. Autoradiographs showing an identical mobility of the 64 kDa antigen and of GAD in two-dimensional gel electrophoresis. (A,B) Western blots of rat islet and brain fractions, respectively, probed with antibodies directed against glutamic acid decarboxylase (GAD). (C,D) Fluorographs of immunoprecipitates of [35S]methionine-labeled rat islet extracts obtained with an anti-64 kDa IDDM serum (C) or with a human control serum (D). (Modified from Ref. 17.)

ones, although antibodies directed against other nervous system antigens are present in a few cases 16'5'~. In IDDM, antibodies to a variety of [3-cell antigens have been identified56''~7. However, the antibodies directed against GAD have been reported to be the ones present with highest frequency and the first ones to be identified in the pre-diabetic stage 4~'47. They may also be present in first-degree relatives of IDDM patients and, occasionally, in patients with organ-specific autoimmune disorders other than IDDM 16'45'46. This is consistent with the possibility that humoral immunity directed against GAD might be a predisposing factor, or a marker of a predisposition, to develop IDDM, rather than being the direct consequence of [3-cell destruction. The hypothesis that GAD autoimmunity might have a direct relationship to the two diseases, and particularly to SMS, is therefore very attractive. The intracellular localization of GAD does not argue necessarily against this hypothesis because in several well-established autoimmune disorders, the immune attack appears to be primarily directed against dominant autoantigens that have an intracellular localization5~'s~. Although no known mechanisms allow antibodies to interact with intracellular antigens 456

in living cells, T cells specifically recognize peptide fragments of intracellular antigens presented at the cell surface by major histocompatibility complex (MHC) molecules 59. GAD-specific CD8 + T cells could destroy GAD-expressing cells, while GAD-specific CD4 + T cells could play a role in activating or helping such killer cells, as well as B cells (which make antibodies to GAD). Recent studies carried out in transgenic mice have shown that experimentally induced immunity directed against an antigen expressed in the cytoplasm of [3 cells can lead to [3-cell destruction 6°'61. Irrespective of the significance of GAD autoimmunity in SMS and IDDM, the presence of a dominant autoimmune response to GAD in the two diseases raises many questions. IDDM usually develops in childhood, although in some SMS patients IDDM has a late onset (Ref. 13 and Solimena, M. and De Camilli, P., unpublished observations), while SMS always appears in adulthood. IDDM affects both sexes equally, while SMS affects primarily women. IDDM has a high prevalence (more than 500 000 patients in the USA), while SMS is extremely rare 2'4'5. Some of these differences might be due to the significant extent of immunological 'privilege' of the CNS. The presence of the blood-brain barrier limits free access of elements of the immune system to the CNS, although it is known that activated lymphocytes can cross the blood-brain barrier 6~. Neurons and [3 cells also differ in their capacity to present antigens. Pancreatic [3 cells normally express class I MHC molecules, and might also express class II MHC molecules ~6. In contrast, expression of class I MHC molecules by neurons has never been demonstrated and only one incidence of the expression of one of the class II MHC molecules is known 63. Antigen processing might differ outside and inside the CNS so that different fragments of GAD might be preferentially presented to the immune system in the nervous tissue and in the pancreas respectively. Loss of tolerance against a specific fragment of GAD might doom one GAD-expressing cell, but not another. The modulation of the immune response can vary from one tissue to another according to local factors. Finally, environmental agents, which have been implicated in the pathogenesis of IDDM ~4, might also have a differential impact on the brain and on the pancreas. To answer all these questions, the most immediate future goals will be to elucidate the cellular basis of SMS, to further investigate pathogenetic mechanisms in IDDM, and to characterize the molecular mechanisms of GAD autoimmunity. The identification of the epitope specificity of GAD antibodies in SMS and IDDM might provide important clues. The broader epitope specificity of SMS antibodies compared with IDDM antibodies is intriguing. In other autoimmune diseases characterized by humoral immunity, the immune response is directed at first against a single dominant epitope of the antigen, and spreads subsequently to cover other portions of the antigen65, A similar process could occur in the case of GAD autoimmunity. It could be that SMS occurs only when loss of tolerance to GAD extends to specific epitopes. It still remains to be determined whether, at the beginning, autoantibodies recognize a single dominant epitope, and whether this epitope is the same in both diseases. TINS, Vo/. 14, No. 10, 1991

It will also be important to characterize GADresponsive T cells (CD8 ÷ cells or CD4 ÷ cells or both) and to determine if they have homogeneous T-cell receptors, as has been demonstrated for other autoimmune diseases 66. It would be of interest to determine whether SMS is preferentially associated with certain MHC phenotypes. The identification of linear GAD fragments recognized by T cells in an MHCrestricted fashion would open up the possibility of searching for molecular mimicry with foreign antigens (viruses and micro-organisms), which might be implicated in the disease. An animal model of SMS, similar to the existing animal models for IDDM, will greatly help in the elucidation of pathogenetic mechanisms in SMS. The diagnosis of SMS is often questionable and frequently its symptoms are attributed to hysteria. Whether or not GAD is directly involved in the pathogenesis of the disease, autoantibodies to GAD can be used as a useful marker in the diagnosis of SMS. Likewise, the identification of the 64 kDa antigen as GAD means that it is now possible to develop a widely applicable immunoassay to help in the diagnosis of IDDM and, more importantly, of the pre-diabetic stage. Being able to diagnose the prediabetic stage will be very useful if effective ways to stop {3-cell destruction are identified. If future work demonstrates that GAD autoimmunity is involved in the pathogenesis of either IDDM or SMS or of both diseases, it may be possible to develop a specific immunotherapy for these conditions 58.

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Acknowledgements We thank the many colleagues who referred their patienG to us. We thank Dr Folli for discussions, Drs £. Janeway, B. Sherwin, A. Hayday and M. Butler for critical reading of the manuscript. We also thank Dr F. Folli and R. Cofiell for performing some o f the unpublished GAD-antibody assays. The work carried out in our laboratory was supported by the Klingenstein Foundation, the Multiple Sclerosis Society, the Diabetes Researchand Education Foundation, the Muscular Distrophy Association, Fidiaand NIH.

Note added in pioof: the fuii amino acid sequence of the 65 kDa GAD isoform was recently reported by Erlander, M. G. et al. [Neuron (1991) 7, 91-100]. 457

Autoimmunity to glutamic acid decarboxylase (GAD) in Stiff-Man syndrome and insulin-dependent diabetes mellitus.

Stiff-Man syndrome (SMS) is a disorder of the CNS, characterized by rigidity of the body musculature, which has been hypothesized to result from an im...
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