Monoclonal antibodies to 65kDa glutamate decarboxylase induce epitope specific effects on motor and cognitive functions in rats

Abstract

BackgroundStiff Person Syndrome (SPS) is a rare autoimmune movement disorder characterized by the presence of autoantibodies specific to the smaller isoform of glutamate decarboxylase (GAD65). A pathological role of these antibodies has been suggested by their capacity to inhibit GAD65 enzyme activity and by the observation that rats receiving cerebellar injections of GAD65Ab showed cerebellar motor hyperexcitability. To assess the effect of epitope-specific GAD65Ab on cognitive and motor functions, we conducted behavioral experiments in rats that received cerebellar injections with two distinct monoclonal GAD65Ab (b96.11 and b78).MethodsRats received three injections of GAD65Ab b96.11 (5 or 7 μg), GAD65Ab b78 (5 or 7 μg), or saline at the level of three cerebellar nuclei. Animals were submitted to neurological evaluation and Morris Water Maze (MWM) test. Cellular internalization of GAD65Ab was analyzed by Flow Cytometry, Fluorescence and Bright Field microscopy.ResultsMonoclonal GAD65Ab induced dose-dependent and epitope-specific effects on motor and cognitive functions. Injections of the higher dose altered motor and spatial procedural behaviors, while the lower dose induced only modest cerebellar motor symptoms and did not affect MWM performances. While b96.11 provoked immediate severe effects, which rapidly decreased, b78 induced moderate but prolonged effects. Both GAD65Ab were taken up by live cells in a dose-dependent manner.ConclusionsOur findings support the hypothesis that epitope-specific GAD65Ab induce cerebellar dysfunction impairing motor and procedural abilities. This is the first demonstration of a critical role of cerebellar nuclei GAD65 enzyme in procedural spatial functions.

GABA and Glutamate: Their Transmitter Role in the CNS and Pancreatic Islets

“…Specifically, the pathologic effects of anti-GAD65Abs on GABA synthesis and release were specific to antiGAD65Ab b78-representing SPS-and CA-associated anti-GAD65Ab-and could not be reproduced by monoclonal anti-GAD65Ab b96.11, representing an epitope specificity associated with type 1 diabetes mellitus [80][81][82]. This epitope dependence might explain the differences in neurological phenotypes.…”

“…Specifically, IgGs obtained from the CSF of patients with anti-GAD65Ab-associated CA depressed GABA release in cerebellar brain slices [77,78,82,85], and their intracerebellar injection interfered with cerebellar control of the motor cortex and resulted in ataxic gait in rats [79]. These actions were reproduced by human anti-GAD65 monoclonal Ab b78, which binds to an epitope similar to that recognized in SPS patients positive for anti-GAD65Ab [80][81][82].…”

“…Second, studies in animals have shown internalization of antibodies by cerebellar neurons [81,86], demonstrating that anti-GAD65Ab can access their intracellular target. Together, these results indicate the possibility that anti-GAD65Ab may damage sufficient numbers of GABAergic neurons to result in the appearance of frank neurological symptoms [87].…”

The Role of Pathogenic Autoantibodies in Autoimmunity

“…GAD65 conformational Catalytic region [223] Catalytic region [222] GAD65 N-terminal linear None ascertained [210,224] Amino acids 4-22 [210,224,225] GAD65 C-terminal linear Rare Amino acids 475-585 [222] GAD67 conformational GAD67 or GAD65 [216] GAD67 specific [210,224] Cross-inhibition (see text) b96.11 > b78 [226] b78 > b96.11 [210] Anti-GAD65 transfer to animals (see text) Diabetes: no Not recorded Neurological: yes [227][228][229][230][231] Other autoimmune diseases Thyrogastric cluster [232] Diabetes 30%-60% Thyrogastric cluster [207,208] IvIg Plasmapheresis Rituximab ……..…”

“…SPS has been successfully treated with intravenous Ig, and is the treatment of choice for intractable SPS [234,235], suggesting that antibodies may be important in pathogenesis. Although both GAD65 and GAD67 are intracellular antigens, the mAbs b78 and b96.11 have been shown to penetrate the AF5 rat CNS cell line [228], possibly being internalised by Fc receptors that are widely distributed in neural tissue including Purkinje cells [192,193]. As Purkinje cells are the major source of GABA in the brain, uptake of anti-GAD could disrupt function in these cells.…”