Human stiff-person syndrome IgG induces anxious behavior in rats

Abstract

Background: Anxiety is a heterogeneous behavioral domain playing a role in a variety of neuropsychiatric diseases. While anxiety is the cardinal symptom in disorders such as panic disorder, co-morbid anxious behavior can occur in a variety of diseases. Stiff person syndrome (SPS) is a CNS disorder characterized by increased muscle tone and prominent agoraphobia and anxiety. Most patients have high-titer antibodies against glutamate decarboxylase (GAD) 65. The pathogenic role of these autoantibodies is unclear.

Methodology/principal findings: We re-investigated a 53 year old woman with SPS and profound anxiety for GABA-A receptor binding in the amygdala with (11)C-flumazenil PET scan and studied the potential pathogenic role of purified IgG from her plasma filtrates containing high-titer antibodies against GAD 65. We passively transferred the IgG fraction intrathecally into rats and analyzed the effects using behavioral and in vivo electrophysiological methods. In cell culture, we measured the effect of patient IgG on GABA release from hippocampal neurons. Repetitive intrathecal application of purified patient IgG in rats resulted in an anxious phenotype resembling the core symptoms of the patient. Patient IgG selectively bound to rat amygdala, hippocampus, and frontal cortical areas. In cultured rat hippocampal neurons, patient IgG inhibited GABA release. In line with these experimental results, the GABA-A receptor binding potential was reduced in the patient's amygdala/hippocampus complex. No motor abnormalities were found in recipient rats.

Conclusion/significance: The observations in rats after passive transfer lead us to propose that anxiety-like behavior can be induced in rats by passive transfer of IgG from a SPS patient positive for anti-GAD 65 antibodies. Anxiety, in this case, thus may be an antibody-mediated phenomenon with consecutive disturbance of GABAergic signaling in the amygdala region.

Figure 1. Bilateral reductions of (11)C-FMZ binding potential in amygdala regions in the SPS patient with GAD 65 autoantibodies.

This figure shows the results of the statistical parametric mapping (SPM) with voxel-based analysis and thus the comparison of GABA-A receptor binding potential in the patient when compared to normal controls. Note that the (11)C-FMZ binding potential is significantly reduced bilaterally in the patient's amygdala region.

Figure 2. SPS patient IgG induces anxiety-like behavior.

(A and B) Animals after intrathecal passive transfer of IgG were tested on an elevated plus maze (EPM) to analyze anxiety-like behavior. Rats treated with SPS patient IgG (SPS IgG; n = 6) moved normally but spent significantly less time in the open arms and explored significantly less arms of the EPM in testing period compared to controls (control IgG: n = 9; saline n = 7), indicating increased anxiety-like behavior (* p<0.05; Mann-Whitney Test). (C and D) Locomotor activity, assessed as time spent in the periphery of the open field (OF) and total distance moved during the observation period, was not different between the experimental groups and did not contribute to the behavioral observations in the EPM. (E) Representative tracks of a rat treated with saline (left panel) and SPS IgG (right panel) in the EPM. Whereas the control animal explored 3 arms of the EPM including one open arm with bright illumination, the rat treated with SPS IgG stayed in the closed, darker arms and avoided entries into the open arms.

Figure 3. Purified SPS-IgG binds selectively to defined areas in rat brain.

(A) Incubation of naïve rat brain sections with purified patient SPS IgG resulted in distinct labeling of different brain areas, such as the rostral and temporobasal part of the cortex (CTXro, CTXtb), hippocampus (HC, CA1), nucleus paraventricularis thalami (PVT), lateral and basolateral amygdala (LA, BLA), whereas in other areas, e.g. most of the thalamus, parts of the cortex and midbrain regions, staining was absent or less pronounced (scale bar: 2.0 mm). (B) At higher magnification, the staining pattern differed within the labeled brain regions. In the HC strongest immunoreactivity was detected in the CA1 region just below the pyramidal cell layer with clear labeling of single neurons resembling GABAergic basket neurons. In the immunoreactive areas of the cortex (temporobasal part: CTXtb), labeling of single neurons with extensive staining of the dendrites was observed. The strongest immunoreactivity was found in the region of the amygdala nuclei, most pronounced in the lateral and basolateral parts (basolateral part: BLA) with dense reticular staining and strongly immunoreactive small cell bodies showing a dense network within the amygdala nuclei. No specific staining was detectable after incubation with control IgG (scale bar: 25 µm). (C) Western blotting of patients purified IgG on rat CNS tissue revealed a single band at 65 kDa (lane a) while the characteristic double band at 65 and 67 kDa was seen when a commercial polyclonal GAD 65/67 antibody was used (lane b). (D) Western blotting of patient purified IgG (lane a) and a rabbit polyclonal GAD 65 antibody (lane b) on a rat GAD 65 fusion protein displayed specific binding with a single band at the expected molecular weight of 90 kDa.

Figure 4. Binding properties of purified SPS - IgG on human CNS tissue.

Tissue of normal human cerebellum, amygdala, frontal cortex, and spinal cord was immunoreacted with purified control IgG or IgG preparations containing high titer of GAD 65 antibodies (scale bar 200 µm). Incubation with control IgG (A) resulted in unspecific perivascular staining (arrowheads), whereas SPS IgG (B) gave intense labeling of the molecular (arrows) and granular layer (arrowheads) in the cerebellum, of the amygdala core region, and the grey matter of the frontal cortex (arrows). SPS IgG immunoreactivity in the ventral horn (VH) of the spinal cord (arrowheads) was less pronounced as compared to the highly immunoreactive brain regions, particularly the amygdala and frontal cortex. (C) Higher magnification revealed (1) strongly positive staining of GABAergic basket cell fibers around Purkinje cells in the cerebellum (thin arrows), (2) a very intense staining of the densely packed reticular network in the amygdala and some small sized interneurons (arrowheads), (3) positive immunoreactivity of single interneurons (thick arrows) in the deeper layers of the frontal cortex, and (4) less strong staining of perineuronal dendrites and proximal dendrites of a motor neuron (open arrowheads) in the ventral horn of the spinal cord (scale bar: 25 µm).

Figure 5. SPS IgG accumulates in interneurons of rat amygdala complex and reduces GABA release from hippocampal neurons.

In rats treated intrathecally with SPS IgG (A) but not in those treated with control IgG (B), immunoreaction against human IgG showed positive staining of neurons in the amygdala complex (scale bar: 10 µm). (C) Dissociated hippocampal neurons from E18 mouse embryos were incubated with SPS or control IgG. The increase of GABA release into the supernatants induced by stimulation with 90 mmol KCl was absent in SPS IgG treated neuronal cell cultures as demonstrated by HPLC analysis (* p<0.05, Student's t-test).