Presynaptic impairment of cerebellar inhibitory synapses by an autoantibody to glutamate decarboxylase

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Abstract

Glutamic acid decarboxylase (GAD), the enzyme responsible for converting glutamate to gamma-aminobutyric acid (GABA), is a target of humoral autoimmunity in stiff-man syndrome and subacute cerebellar ataxia. Recently, we found that an anti-GAD autoantibody in the CSF of an ataxic patient selectively suppressed GABA-mediated transmission on cerebellar Purkinje cells without affecting glutamate-mediated transmission. Here, we examine the mechanism by which the autoantibody impaired the inhibitory transmission, using immunohistochemistry and whole-cell recording in rat cerebellar slices. The present results indicate that CSF immunoglobulins prepared from an ataxic patient acted on the presynaptic terminals of GABAergic interneurons and decreased GABA release onto Purkinje cells.

Introduction

Glutamic acid decarboxylase (GAD) catalyzes the conversion of glutamic acid to gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter. Anti-GAD autoantibodies are present in stiff-man syndrome (SMS), a rare disease characterized by progressive muscle rigidity and superimposed painful muscle spasm [1], [2]. Anti-GAD autoantibodies are also present in a subgroup of patients with subacute cerebellar ataxia [3], [4], [5], [6]. GABA-mediated synaptic transmission is thought to be functionally impaired by autoantibodies to GAD, which results in the appearance of various neurological symptoms [2]. In support of this hypothesis, we found that immunoglobulins (IgGs) present in the CSF of an ataxic patient selectively suppressed GABA-mediated transmission from basket cells, GABAergic interneurons in the cerebellar cortex, to Purkinje cells, the sole output neurons from the cortex, and that this suppression was presumably mediated by an anti-GAD autoantibody in the CSF [7]. In contrast, IgGs did not affect glutamate-mediated transmission on Purkinje cells [7].

It is not clear whether the anti-GAD autoantibody suppresses the inhibitory transmission through a presynaptic or postsynaptic mechanism. This is important for our understanding of the molecular pathophysiology of SMS and subacute cerebellar ataxia. We have recently demonstrated that CSF IgGs reduced the frequency of synaptic activities caused by spontaneous firing of presynaptic basket cells [7]. Furthermore, nerve terminals immunoreactive to patient’s CSF surrounded Purkinje cell somata, and were also found in the axon hillock where the terminals of basket cells are located. These observations suggested that the GAD autoantibody acted on basket cell nerve terminals and reduced GABA release onto Purkinje cells. However, to establish that humoral autoimmune responses to GAD in presynaptic terminals cause a decrease in GABA release, direct methods are necessary to demonstrate the following two conditions: (1) immunoreactivity localized on presynaptic terminals of basket cells rather than postsynaptic somata of Purkinje cells; and (2) GABA release from nerve terminals, which is evoked by a nerve impulse invading the terminals, is depressed by CSF IgGs.

In the present study, we localized CSF-immunoreactive nerve terminals and examined the action of CSF IgGs on GABA release. The first series of experiments was carried out to compare immunoreactivities of CSF with those of an antibody to spot 35 (calbindin-D28K) protein, which is specifically expressed in Purkinje cells [8]. Then, we analyzed the effects of the CSF IgGs on the paired-pulse ratio (PPR) in the nerve stimulation-induced inhibitory transmission from the basket cell terminals on Purkinje cells, using whole-cell recording in rat cerebellar slices. PPR, defined as the ratio of the first response over the second response to paired-pulse nerve stimuli, is a parameter utilized to examine electrophysiologically whether the release of a transmitter from nerve terminals is depressed [9]. It has been well documented that, following cerebral excitatory synaptic transmission, PPR increases when glutamate release is depressed [9].

Studies were performed using CSF IgGs obtained from a 66-year-old woman who developed progressive truncal and limb ataxia within 8 months. The same patient was documented in our previous report [7].

Section snippets

Methods

The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Tokyo Medical and Dental University, School of Medicine.

Immunoreactivities of the patient’s CSF compared with those of a Purkinje cell-specific anti-spot 35 antibody

Nerve terminals with punctate immunoreactivity to the patient’s CSF were observed surrounding the somata and in the axon hillock of Purkinje cells (Fig. 1A,C). Immunoreactivity was also observed along the dendritic shaft. On the other hand, anti-spot 35 antibody intensely labeled the somata of Purkinje cells and their dendrites and axons (Fig. 1B). Double immunostaining revealed that the immunoreactivities of CSF and anti-spot 35 antibody were not co-localized at the light microscopic level as

Discussion

The present immunohistochemical experiment showed that the immunoreactivities of a CSF sample containing a GAD autoantibody, prepared from an ataxic patient, and anti-spot 35 antibody, an antibody specific to Purkinje cells, were not co-localized. Taken together with our previous findings that immunoreactivities of the CSF were co-localized with those of anti-GAD antibody [7], it is likely that the CSF autoantibody recognized GAD in the terminals of basket and stellate cells, GABAergic

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