Role of synaptic vesicle proton gradient and protein phosphorylation on ATP-mediated activation of membrane-associated brain glutamate decarboxylase.

Previously, we have shown that the soluble form of brain glutamic acid decarboxylase (GAD) is inhibited by ATP through protein phosphorylation and is activated by calcineurin-mediated protein dephosphorylation (Bao, J., Cheung, W. Y., and Wu, J. Y. (1995) J. Biol. Chem. 270, 6464-6467). Here we report that the membrane-associated form of GAD (MGAD) is greatly activated by ATP, whereas adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP), a non-hydrolyzable ATP analog, has no effect on MGAD activity. ATP activation of MGAD is abolished by conditions that disrupt the proton gradient of synaptic vesicles, e.g. the presence of vesicular proton pump inhibitor, bafilomycin A1, the protonophore carbonyl cyanide m-chorophenylhydrazone or the ionophore gramicidin, indicating that the synaptic vesicle proton gradient is essential in ATP activation of MGAD. Furthermore, direct incorporation of (32)P from [gamma-(32)P]ATP into MGAD has been demonstrated. In addition, MGAD (presumably GAD65, since it is recognized by specific monoclonal antibody, GAD6, as well as specific anti-GAD65) has been reported to be associated with synaptic vesicles. Based on these results, a model linking gamma-aminobutyric acid (GABA) synthesis by MGAD to GABA packaging into synaptic vesicles by proton gradient-mediated GABA transport is presented. Activation of MGAD by phosphorylation appears to be mediated by a vesicular protein kinase that is controlled by the vesicular proton gradient.     

Oxygen-induced seizures and inhibition of human glutamate decarboxylase and porcine cysteine sulfinic acid decarboxylase by oxygen and nitric oxide

The recombinant forms of the two human isozymes of glutamate decarboxylase, GAD65 and GAD67, are potently and reversibly inhibited by molecular oxygen (Ki = 0.46 and 0.29 mM, respectively). Inhibition of the vesicle-associated glutamate decarboxylase (GAD65) by molecular oxygen is likely to result in incomplete filling of synaptic vesicles with gamma-aminobutyric acid (GABA) and may be a contributing factor in the genesis of oxygen-induced seizures. Under anaerobic conditions, nitric oxide inhibits both GAD65 and GAD67 with comparable potency to molecular oxygen (Ki = 0.5 mM). Two forms of porcine cysteine sulfinic acid decarboxylase (CSADI and CSADII) are also sensitive to inhibition by molecular oxygen (Ki = 0.30 and 0.22 mM, respectively) and nitric oxide (Ki = 0.3 and 0.2 mM, respectively). Similar inhibition of glutamate decarboxylase and cysteine sulfinic acid decarboxylase by two different radical-containing compounds (O2 and NO) is consistent with the notion that these reactions proceed via radical mechanisms.     

Protein phosphorylation of human brain glutamic acid decarboxylase (GAD)65 and GAD67 and its physiological implications

Previously, we reported that protein phosphorylation plays an important role in regulating soluble l-glutamic acid decarboxylase (GAD) [Bao, J. (1995) J. Biol. Chem. 270, 6464-6467] and membrane-associated GAD activity [Hsu, C. C. (1999) J. Biol. Chem. 274, 24366-24371]. Here, we report the effect of phosphorylation on the two well-defined GAD isoforms, namely, GAD65 and GAD67, using highly purified preparations of recombinant human brain GAD65 and GAD67. GAD65 was activated by phosphorylation, while GAD67 was inhibited by phosphorylation. The effect of phosphorylation on GAD65 and GAD67 could be reversed by treatment with protein phosphatases. We further demonstrate that protein kinase A (PKA) and protein kinase C isoform epsilon are the protein kinases responsible for phosphorylation and regulation of GAD67 and GAD65, respectively. Direct phosphorylation of GAD65 and GAD67 was demonstrated by incorporation of [(32)P] from [gamma-(32)P]ATP into purified GAD65 and GAD67 and immunoblotting assay using anti-phosphoserine/threonine antibodies. We have identified one specific phosphorylation site, threonine 91 (T91), in hGAD67 that can be phosphorylated by PKA using MALDI-TOF. Site-directed mutation of T91 to alanine abolished PKA-mediated phosphorylation and inhibition of GAD activity. Furthermore, mutation of T91 to aspartic acid or glutamic acid mimics the effect of phosphorylation. A model depicting the effect of phosphorylation on GAD activity upon neuronal stimulation is also proposed.     

Two Forms of the γ-Aminobutyric Acid Synthetic Enzyme Glutamate Decarboxylase Have Distinct Intraneuronal Distributions and Cofactor Interactions

Glutamate decarboxylase (GAD) catalyzes the production of gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter. The mammalian brain contains two forms of GAD, with Mrs of 67,000 and 65,000 (GAD67 and GAD65). Using a new antiserum specific for GAD67 and a monoclonal antibody specific for GAD65, we show that the two forms of GAD differ in their intraneuronal distributions: GAD67 is widely distributed throughout the neuron, whereas GAD65 lies primarily in axon terminals. In brain extracts, almost all GAD67 is in an active holoenzyme form, saturated with its cofactor, pyridoxal phosphate. In contrast, only about half of GAD65 (which is found in synaptic terminals) exists as active holoenzyme. We suggest that the relative levels of apo-GAD65 and holo-GAD65 in synaptic terminals may couple GABA production to neuronal activity.     

Demonstration of functional coupling between dopamine synthesis and its packaging into synaptic vesicles

We have previously shown that the membrane-associated form of the GABA-synthesizing enzyme, glutamate decarboxylase 65 (GAD₆₅), is activated by synaptic vesicle proton gradient-mediated protein phosphorylation. We now report that the rate-limiting enzyme in dopamine (DA) biosynthesis, tyrosine hydroxylase (TH), is regulated similarly to GAD₆₅. The membrane-associated form of TH (MTH) was activated by conditions favoring protein phosphorylation (e.g. ATP) and was inhibited by phosphatase (e.g. calf intestine phosphatase). Furthermore, the ATP-mediated activation of MTH was abolished by conditions that disrupted the proton gradient of synaptic vesicles, e.g. the presence of carbonyl cyanidem-chorophenylhydrazone, gramicidin, or the V-type ATPase inhibitor (bafilomycin), but not the P-type ATPase inhibitor (vanadate). Moreover, DA newly synthesized from tyrosine by MTH and membrane-associated aromatic amino acid decarboxylase was taken up preferentially rather than pre-existing DA. Therefore, the previously proposed model showing close coupling between GABA synthesis and GABA packaging into synaptic vesicles by vesicular GABA transporters is also applicable to the DA system. Hence, it is concluded that there is a general coupling mechanism between neurotransmitter synthesis and packaging of transmitter into synaptic vesicles.     

Two distinct mechanisms target GAD67 to vesicular pathways and presynaptic clusters

The inhibitory neurotransmitter γ-amino butyric acid (GABA) is synthesized by two isoforms of the enzyme glutamic acid decarboxylase (GAD): GAD65 and GAD67. Whereas GAD67 is constitutively active and produces >90% of GABA in the central nervous system, GAD65 is transiently activated and augments GABA levels for rapid modulation of inhibitory neurotransmission. Hydrophobic lipid modifications of the GAD65 protein target it to Golgi membranes and synaptic vesicles in neuroendocrine cells. In contrast, the GAD67 protein remains hydrophilic but has been shown to acquire membrane association by heterodimerization with GAD65. Here, we identify a second mechanism that mediates robust membrane anchoring, axonal targeting, and presynaptic clustering of GAD67 but that is independent of GAD65. This mechanism is abolished by a leucine-103 to proline mutation that changes the conformation of the N-terminal domain but does not affect the GAD65-dependent membrane anchoring of GAD67. Thus two distinct mechanisms target the constitutively active GAD67 to presynaptic clusters to facilitate accumulation of GABA for rapid delivery into synapses.     

The hydrophilic isoform of glutamate decarboxylase, GAD67, is targeted to membranes and nerve terminals independent of dimerization with the hydrophobic membrane-anchored isoform, GAD65

GAD67, the larger isoform of the gamma-aminobutyric acid-synthesizing enzyme glutamic acid decarboxylase, is a hydrophilic soluble molecule, postulated to localize at nerve terminals and membrane compartments by heterodimerization with the smaller membrane-anchored isoform GAD65. We here show that the dimerization region in GAD65 is distinct from the NH(2)-terminal membrane-anchoring region and that a membrane anchoring GAD65 subunit can indeed target a soluble subunit to membrane compartments by dimerization. However, only a fraction of membrane-bound GAD67 is engaged in a heterodimer with GAD65 in rat brain. Furthermore, in GAD65-/- mouse brain, GAD67, which no longer partitions into the Triton X-114 detergent phase, still anchors to membranes at similar levels as in wild-type mice. Similarly, in primary cultures of neurons derived from GAD65-/- mice, GAD67 is targeted to nerve terminals, where it co-localizes with the synaptic vesicle marker SV2. Thus, axonal targeting and membrane anchoring is an intrinsic property of GAD67 and does not require GAD65. The results suggest that three distinct moieties of glutamate decarboxylase localize to membrane compartments, an amphiphilic GAD65 homodimer, an amphiphilic GAD65/67 heterodimer, tethered to membranes via the GAD65 subunit, and a hydrophilic GAD67 homodimer, which associates with membranes by a distinct mechanism.     

Membrane anchoring of the autoantigen GAD65 to microvesicles in pancreatic beta-cells by palmitoylation in the NH2-terminal domain

Pancreatic beta-cells and gamma-aminobutyric acid (GABA)-secreting neurons both express the enzyme glutamic acid decarboxylase (GAD) which is a major target of autoantibodies associated with beta-cell destruction and impairment of GABA-ergic neurotransmitter pathways. The predominant form of GAD in pancreatic beta-cells, GAD65, is synthesized as a soluble hydrophilic molecule, which is modified to become firmly membrane anchored. Here we show by immunogold electron microscopy that GAD65 is localized to the membrane of small vesicles which are identical in size to small synaptic-like microvesicles in pancreatic beta-cells. The NH2-terminal domain of GAD65 is the site of a two-step modification, the last of which results in a firm membrane anchoring that involves posttranslational hydroxylamine sensitive palmitoylation. GAD65 can be released from the membrane by an apparent enzyme activity in islets, suggesting that the membrane anchoring step is reversible and potentially regulated. The hydrophobic modifications and consequent membrane anchoring of GAD65 to microvesicles that store its product GABA may be of functional importance and, moreover, significant for its selective role as an autoantigen.     

Activation of Brain L-glutamate Decarboxylase 65 Isoform (GAD65) by Phosphorylation at Threonine 95 (T95)

Protein phosphorylation plays an important role in regulating soluble L-glutamic acid decarboxylase (GAD) and membrane-associated GAD activity. Previously, we reported the effect of phosphorylation on the two well-defined GAD isoforms, namely, GAD65 and GAD67, using highly purified preparations of recombinant human brain GAD65 (hGAD65) and GAD67. GAD65 was activated by phosphorylation, while GAD67 was inhibited by phosphorylation. The effect of phosphorylation on GAD65 and GAD67 could be reversed by treatment with protein phosphatases. We further demonstrated that protein kinase A (PKA) and protein kinase C isoform ε were the protein kinases responsible for phosphorylation and regulation of GAD67 and GAD65, respectively. In the current study, using MALDI-TOF, a total of four potential phosphorylation sites were identified in GAD65, two of which (threonine-95 (T-95) and Ser-417) were not reported previously. We have identified one specific phosphorylation site, (T95), in hGAD65 that can be phosphorylated by kinase C ε (PKCε) using MALDITOF. When T95 is mutated to alanine, hGAD65 could no longer be phosphorylated by PKCε, and the effect of PKC-mediated activation on hGAD65 is abolished. However, when T95 is mutated to glutamic acid, which mimics the phosphorylation status of hGAD65, the activity was greatly increased. An increase of GAD65 activity by 55 % compared to the wild type hGAD65 was observed indicating that mutation of T95 to glutamic acid mimics the effect of phosphorylation. A model depicting the role of phosphorylation of GAD65 in regulation of GABA neurotransmission is presented.     

Cysteine string protein (CSP) inhibition of N-type calcium channels is blocked by mutant huntingtin

Cysteine string protein (CSP), a 34-kDa molecular chaperone, is expressed on synaptic vesicles in neurons and on secretory vesicles in endocrine, neuroendocrine, and exocrine cells. CSP can be found in a complex with two other chaperones, the heat shock cognate protein Hsc70, and small glutamine-rich tetratricopeptide repeat domain protein (SGT). CSP function is vital in synaptic transmission; however, the precise nature of its role remains controversial. We have previously reported interactions of CSP with both heterotrimeric GTP-binding proteins (G proteins) and N-type calcium channels. These associations give rise to a tonic G protein inhibition of the channels. Here we have examined the effects of huntingtin fragments (exon 1) with (huntingtin(exon1/exp)) and without (huntingtin(exon1/nonexp)) expanded polyglutamine (polyQ) tracts on the CSP chaperone system. In vitro huntingtin(exon1/exp) sequestered CSP and blocked the association of CSP with G proteins. In contrast, huntingtin(exon1/nonexp) did not interact with CSP and did not alter the CSP/G protein association. Similarly, co-expression of huntingtin(exon1/exp) with CSP and N-type calcium channels eliminated CSP's tonic G protein inhibition of the channels, while coexpression of huntingtin(exon1/nonexp) did not alter the robust inhibition promoted by CSP. These results indicate that CSP's modulation of G protein inhibition of calcium channel activity is blocked in the presence of a huntingtin fragment with expanded polyglutamine tracts.     

Amino acid residues 24-31 but not palmitoylation of cysteines 30 and 45 are required for membrane anchoring of glutamic acid decarboxylase, GAD65

The smaller isoform of the GABA synthesizing enzyme glutamic acid decarboxylase, GAD65, is synthesized as a soluble protein that undergoes post-translational modification(s) in the NH2-terminal region to become anchored to the membrane of small synaptic-like microvesicles in pancreatic beta cells, and synaptic vesicles in GABA-ergic neurons. A soluble hydrophilic form, a soluble hydrophobic form, and a hydrophobic firmly membrane-anchored form have been detected in beta cells. A reversible and hydroxylamine sensitive palmitoylation has been shown to distinguish the firmly membrane-anchored form from the soluble yet hydrophobic form, suggesting that palmitoylation of cysteines in the NH2-terminal region is involved in membrane anchoring. In this study we use site-directed mutagenesis to identify the first two cysteines in the NH2-terminal region, Cys 30 and Cys 45, as the sites of palmitoylation of the GAD65 molecule. Mutation of Cys 30 and Cys 45 to Ala results in a loss of palmitoylation but does not significantly alter membrane association of GAD65 in COS-7 cells. Deletion of the first 23 amino acids at the NH2 terminus of the GAD65 30/45A mutant also does not affect the hydrophobicity and membrane anchoring of the GAD65 protein. However, deletion of an additional eight amino acids at the NH2 terminus results in a protein which is hydrophilic and cytosolic. The results suggest that amino acids 24-31 are required for hydrophobic modification and/or targeting of GAD65 to membrane compartments, whereas palmitoylation of Cys 30 and Cys 45 may rather serve to orient or fold the protein at synaptic vesicle membranes.     

Compartmentalization of GABA Synthesis by GAD67 Differs between Pancreatic Beta Cells and Neurons

The inhibitory neurotransmitter GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD) in neurons and in pancreatic β-cells in islets of Langerhans where it functions as a paracrine and autocrine signaling molecule regulating the function of islet endocrine cells. The localization of the two non-allelic isoforms GAD65 and GAD67 to vesicular membranes is important for rapid delivery and accumulation of GABA for regulated secretion. While the membrane anchoring and trafficking of GAD65 are mediated by intrinsic hydrophobic modifications, GAD67 remains hydrophilic, and yet is targeted to vesicular membrane pathways and synaptic clusters in neurons by both a GAD65-dependent and a distinct GAD65-independent mechanism. Herein we have investigated the membrane association and targeting of GAD67 and GAD65 in monolayer cultures of primary rat, human, and mouse islets and in insulinoma cells. GAD65 is primarily detected in Golgi membranes and in peripheral vesicles distinct from insulin vesicles in β-cells. In the absence of GAD65, GAD67 is in contrast primarily cytosolic in β-cells; its co-expression with GAD65 is necessary for targeting to Golgi membranes and vesicular compartments. Thus, the GAD65-independent mechanism for targeting of GAD67 to synaptic vesicles in neurons is not functional in islet β-cells. Therefore, only GAD65:GAD65 homodimers and GAD67:GAD65 heterodimers, but not the GAD67:GAD67 homodimer gain access to vesicular compartments in β-cells to facilitate rapid accumulation of newly synthesized GABA for regulated secretion and fine tuning of GABA-signaling in islets of Langerhans.     

Enzymatic characterization of a recombinant isoform hybrid of glutamic acid decarboxylase (rGAD67/65) expressed in yeast

BACKGROUND AND AIMS: Glutamic acid decarboxylase (GAD, EC 4.1.1.15) catalyses the conversion of glutamate to gamma-aminobutyric acid (GABA). The 65 kDa isoform, GAD65 is a potent autoantigen in type 1 diabetes, whereas GAD67 is not. A hybrid cDNA was created by fusing a human cDNA for amino acids 1-101 of GAD67 to a human cDNA for amino acids 96-585 of GAD65; the recombinant (r) protein was expressed in yeast and was shown to have equivalent immunoreactivity to mammalian brain GAD with diabetes sera. We here report on enzymatic and molecular properties of rGAD67/65. METHODS: Studies were performed on enzymatic activity of rGAD67/65 by production of 3H-GABA from 3H-glutamate, enzyme kinetics, binding to the enzyme cofactor pyridoxal phosphate (PLP), stability according to differences in pH, temperature and duration of storage, and antigenic reactivity with various GAD-specific antisera. RESULTS: The properties of rGAD67/65 were compared with published data for mammalian brain GAD (brackets). These included a specific enzyme activity of 22.7 (16.7) nKat, optimal pH for enzymatic activity 7.4 (6.8), K(m) of 1.3 (1.3) mM, efficient non-covalent binding to the cofactor PLP, and high autoantigenic potency. The stability of rGAD67/65 was optimal over 3 months at -80 degrees C, or in lyophilized form at -20 degrees C. CONCLUSIONS: Hybrid rGAD67/65 has enzymatic and other properties similar to those of the mixed isoforms of GAD in preparations from mammalian brain as described elsewhere, in addition to its previously described similar immunoreactivity.     

Glutamate decarboxylase of the parasitic arthropods Ctenocephalides felis and Rhipicephalus microplus: Gene identification,cloning, expression,assay development,identification of inhibitors by high throughput screening and comparison with the orthologs from Drosophila melanogaster and mouse

Glutamate decarboxylase (l-glutamate 1-carboxylyase, E.C. 4.1.1.15, GAD) is the rate-limiting enzyme for the production of γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in vertebrates and invertebrates. We report the identification, isolation and characterization of cDNAs encoding GAD from the parasitic arthropods Ctenocephalides felis (cat flea) and Rhipicephalus microplus (cattle tick). Expression of the parasite GAD genes and the corresponding Drosophila melanogaster (fruit fly) GAD1 as well as the mouse GAD₆₅ and GAD₆₇ genes in Escherichia coli as maltose binding protein fusions resulted in functional enzymes in quantities compatible with the needs of high throughput inhibitor screening (HTS). A novel continuous coupled spectrophotometric assay for GAD activity based on the detection cascade GABA transaminase/succinic semialdehyde dehydrogenase was developed, adapted to HTS, and a corresponding screen was performed with cat flea, cattle tick and fruit fly GAD. Counter-screening of the selected 38 hit substances on mouse GAD₆₅ and GAD₆₇ resulted in the identification of non-specific compounds as well as inhibitors with preferences for arthropod GAD, insect GAD, tick GAD and the two mouse GAD forms. Half of the identified hits most likely belong to known classes of GAD inhibitors, but several substances have not been described previously as GAD inhibitors and may represent lead optimization entry points for the design of arthropod-specific parasiticidal compounds.     

Coatomer-bound Cdc42 regulates dynein recruitment to COPI vesicles

Cytoskeletal dynamics at the Golgi apparatus are regulated in part through a binding interaction between the Golgi-vesicle coat protein, coatomer, and the regulatory GTP-binding protein Cdc42 (Wu, W.J., J.W. Erickson, R. Lin, and R.A. Cerione. 2000. Nature. 405:800-804; Fucini, R.V., J.L. Chen, C. Sharma, M.M. Kessels, and M. Stamnes. 2002. Mol. Biol. Cell. 13:621-631). The precise role of this complex has not been determined. We have analyzed the protein composition of Golgi-derived coat protomer I (COPI)-coated vesicles after activating or inhibiting signaling through coatomer-bound Cdc42. We show that Cdc42 has profound effects on the recruitment of dynein to COPI vesicles. Cdc42, when bound to coatomer, inhibits dynein binding to COPI vesicles whereas preventing the coatomer-Cdc42 interaction stimulates dynein binding. Dynein recruitment was found to involve actin dynamics and dynactin. Reclustering of nocodazole-dispersed Golgi stacks and microtubule/dynein-dependent ER-to-Golgi transport are both sensitive to disrupting Cdc42 mediated signaling. By contrast, dynein-independent transport to the Golgi complex is insensitive to mutant Cdc42. We propose a model for how proper temporal regulation of motor-based vesicle translocation could be coupled to the completion of vesicle formation.     

A combination of three distinct trafficking signals mediates axonal targeting and presynaptic clustering of GAD65

The signals involved in axonal trafficking and presynaptic clustering are poorly defined. Here we show that targeting of the gamma-aminobutyric acid-synthesizing enzyme glutamate decarboxylase 65 (GAD65) to presynaptic clusters is mediated by its palmitoylated 60-aa NH(2)-terminal domain and that this region can target other soluble proteins and their associated partners to presynaptic termini. A Golgi localization signal in aa 1-23 followed by a membrane anchoring signal upstream of the palmitoylation motif are required for this process and mediate targeting of GAD65 to the cytosolic leaflet of Golgi membranes, an obligatory first step in axonal sorting. Palmitoylation of a third trafficking signal downstream of the membrane anchoring signal is not required for Golgi targeting. However, palmitoylation of cysteines 30 and 45 is critical for post-Golgi trafficking of GAD65 to presynaptic sites and for its relative dendritic exclusion. Reduction of cellular cholesterol levels resulted in the inhibition of presynaptic clustering of palmitoylated GAD65, suggesting that the selective targeting of the protein to presynaptic termini is dependent on sorting to cholesterol-rich membrane microdomains. The palmitoylated NH(2)-terminal region of GAD65 is the first identified protein region that can target other proteins to presynaptic clusters.     

A novel method for expression and large-scale production of human brain l-glutamate decarboxylase

l-Glutamate decarboxylase (GAD; EC 4.1.1.15) is the rate-limiting enzyme involved in the synthesis of gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain. Imbalance in the conversion of glutamate to GABA has been implicated in a host of human diseases. Studies on the structure, function, and therapeutic use of GAD have been precluded by insufficient quantities of purified active enzyme. Here we report a novel methodology for the expression and large-scale production of enzymatically active, pure, recombinant human GAD65 and GAD67. This method circumvents the sequestering of expressed protein into insoluble inclusion bodies and reduces production of truncated proteins. The availability of sufficient quantities of purified HGAD65 and HGAD67 has allowed for the production of specific polyclonal antibodies that discriminate between the two isoforms. This methodology, in addition to providing key human brain enzymes, may be generally applicable to other systems.     

Antibodies to Inhibitory Synaptic Proteins in Neurological Syndromes Associated with Glutamic Acid Decarboxylase Autoimmunity

Antibodies to glutamic acid decarboxylase (GAD-ab) associate to different neurological syndromes. It is unknown if the diversity in syndrome association represents epitopes in different immunodominant domains or co-existence of antibodies to other proteins of the inhibitory synapsis. We examined the serum and CSF of 106 patients with anti-GAD related syndromes (39 cerebellar ataxia, 32 stiff-person syndrome [SPS], 18 epilepsy, and 17 limbic encephalitis [LE]). GAD65-ab titres were quantified by ELISA. Immunoblot was used to determine if the antibody-targeted epitopes of GAD65 and GAD67 were linear. A cell-based assay (CBA) with HEK293 cells expressing the GAD65 N-terminal, central catalytic domain, or C-terminal was used to investigate the immunodominant domains. Antibodies to GAD67, gamma-aminobutyric acid A receptor (GABAaR), glycine receptor (GlyR), GABAaR-associated protein (GABARAP), and gephyrin were determined with CBA. GAD-ab internalization was investigated using cultured rat hippocampal neurons. CSF GAD65-ab titres were higher in patients with cerebellar ataxia and LE compared to those with SPS (p = 0.02). GAD67-ab were identified in 81% of sera and 100% of CSF. GAD65-ab recognized linear epitopes in 98% of the patients and GAD67-ab in 42% (p<0.001). The GAD65 catalytic domain was recognized by 93% of sera, and the three domains by 22% of sera and 74% of CSF (p<0.001). Six patients had GABAaR-ab and another 6 had GlyR-ab without association to distinctive symptoms. None of the patients had gephyrin- or GABARAP-ab. GAD65-ab were not internalized by live neurons. Overall, these findings show that regardless of the neurological syndrome, the CSF immune response against GAD is more widespread than that of the serum and that there is no specific association between clinical phenotype and the presence of antibodies against other proteins of the inhibitory synapsis.