Membranglykoprotein M6a: Expression und Regulation im Gehirn unter Stress

Abstract

Glycoprotein M6a is a neuronally expressed transmembrane protein belonging to the myelin proteolipid protein (PLP) family. In vitro studies have identified M6a as a key modulator of neurite outgrowth and spine formation, however, the precise location of M6a within neurons in the adult brain remains obscure. M6a was previously identified as a stress-responsive gene in the hippocampus of psychosocially stressed animals, but it is not known whether stress also regulates M6a expression in other brain regions. It is generally accepted that stress induces aberrant neuronal plasticity and it has been hypothesised that stress-induced morphological changes in the hippocampal formation and prefrontal cortex may contribute to the development of stress-related neuropathologies such as depression. Therefore, the aim of the present thesis was to characterise the regional neuronal expression of membrane glycoprotein M6a in the brain and to investigate the effects of stress on M6a expression within the prefrontal cortex of chronically restrained rats. A combination of in situ hybridization and immunocytochemistry was performed to characterize the expression of M6a within the hippocampal formation, prefrontal cortex and cerebellum. In situ hybridization confirmed that M6a is abundantly expressed in pyramidal and granule neurons in the hippocampal formation, in cortical pyramidal and in cerebellar granule neurons. Neurons bearing the morphological characteristics of inhibitory interneurons do not express M6a. Confocal laser microscopy demonstrated that M6a immunoreactivity colocalizes with synaptophysin and the vesicular glutamate transporter (VGLUT1) indicating that the glycoprotein is targeted to the terminals of glutamatergic axons. M6a immunoreactivity was not detected within neuronal somata and did not colocalise with MAP-2 in any brain region investigated, demonstrating that M6a is not expressed in dendrites. In the hippocampus M6a immunoreactivity was visualised as focal puncta localised to distinct sites within the terminal regions of granule cell mossy fibre axons that were visualised by immunoreactivity for the cytoplasmic calcium-binding protein calbindin. Analysis of giant mossy fibre terminals, which contact dendrites of CA3 pyramidal neurons in the hippocampal stratum lucidum, revealed that M6a immunoreactivity was associated primarily with the membrane of axon fibres and their terminals, but not with synaptic vesicles. M6a and the vesicular GABA transporter (VGAT) exhibited largely contrasting patterns of immunoreactivity and colocalization was observed only rarely, indicating that M6a is not expressed in inhibitory neurons but that colocalisation might be observed when GABAergic terminals are situated in close proximity to glutamatergic axons. The second part of the thesis investigated the effects of stress on M6a expression in the brains of rats exposed to 21 days chronic restraint stress. Stress experiments were performed to investigate the effects of chronic restraint on M6a expression using: i) quantitative real-time RT-PCR and ii) quantitative in situ hybridization. RTPCR analysis revealed that M6a is regulated in a region-specific manner in the brains of chronically restrained rats. M6a was significantly downregulated in the hippocampus, whereas the prefrontal cortex demonstrated a non-significant increase in expression. No effect of stress was observed in the cerebellum. Since the prefrontal cortex comprises several anatomically and functionally distinct areas, of which only some may be responsive to stress, regional changes in M6a expression may be masked in RT-PCR analyses performed on prefrontal samples containing multiple subregions. Therefore, quantitative in situ hybridization was performed to provide a means of localising potential stress-induced changes in M6a expression to specific neuronal populations. M6a was found to be significantly upregulated in layer II/III pyramidal neurons in the infralimbic and prelimbic, but not in the anterior cingulate cortex. In conclusion, the present data show that M6a is expressed in pyramidal and granule cells and that the glycoprotein is targeted to distinct sites within the axonal plasma membrane of these excitatory neurons. Stress was found to regulate M6a expression in a region-specific manner. Moreover, changes in M6a expression correlate with brain regions exhibiting maladaptive alterations in neuronal morphology in response to chronic stress. Stress-induced changes in M6a expression may influence the structural integrity of presynaptic terminals and impair the induction of neuroplastic mechanisms designed to protect against the deleterious effects of chronic stress.