Dissection of astrocyte-mediated cerebral homeostasis in synucleinopathies

Language
en
Document Type
Doctoral Thesis
Granting Institution
Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Naturwissenschaftliche Fakultät
Issue Date
2024
Authors
Schneider, Yanni
Editor
Abstract

Multiple system atrophy (MSA) represents a rare atypical parkinsonian disorder characterized by a rapid and fatal course currently lacking causal therapies. Clinical features of MSA com- prise classical parkinsonism, cerebellar ataxia and pronounced autonomic failure occurring in the mean of the 6th decade of life. So far, the disease is classified as a sporadic disease with rare cases of familial MSA. Data concerning environmental factors causing the disease is still controversial. Dependent on the clinical manifestation, MSA is subdivided into a parkinsonian or a cerebellar type. Due to the low frequency of the disease and the fact that a definitive diagnosis is based on post mortem, animal models have been developed to mirror the molec- ular and motor phenotype of the disease. The MBP29-haSyn mouse model expresses human α-synuclein (aSyn) under the myelin basic protein (MBP) promotor specifically in oligodendro- cytes thus resembling MSA-related molecular pathology, such as glial cytoplasmic aSyn inclu- sions, severe demyelination, and microgliosis. Moreover, MBP29-haSyn mice display motor impairments, gait instability and a reduced lifespan. So far, comprehensive molecular profiling of astrocytes in MSA is lacking. Astrocytic expres- sion of glial fibrillary acidic protein (GFAP) was assessed as an indicator for astrocytic re- sponse to alterations in the microenvironment of MBP29-haSyn mice. Reactive astrogliosis was observed in the cortex and striatum on protein and RNA level analyzing expression levels of GFAP and vimentin. Moreover, focusing on cortex and striatum, an impaired capacity of upregulation of homeostasis-associated aquaporin-4, growth-associated protein 43, and glu- tamine synthetase was observed in the striatum of MBP29-haSyn-Syn mice compared to cor- tex. Additionally, expression of glutamate reuptake transporters was downregulated in the stri- atum of MBP29-haSyn mice, but not altered in the cortex. Based on these differences, a magnetic-activated cell sorting protocol for astrocyte isolation was adapted to ensure a high yield of pure astrocytes and bulk RNA sequencing was per- formed subsequently. Using these datasets, a transcriptomic landscape was generated high- lighting altered gene expression in astrocytes in the cortex and the striatum of MBP29-haSyn mice. Comprehensive analysis of both astrocyte populations revealed a dichotomous profile of astrocytes in the regions analyzed. Striatal astrocytes exhibit a pronounced immuno-active profile with upregulated pro-inflammatory transcripts involved in cytokine-cytokine interaction, toll-like receptor signaling, and phagocytosis. Conversely, analysis of the cortical astrocyte population demonstrated a decreased expression of transcripts associated with the inflamma- tory response, such as cytokine-cytokine interaction, and chemokine signaling. Since an increased presence of oligodendrocyte-associated transcripts was found upregulat-ed, the cellular components were further analyzed using gene set enrichment. Notably, the results indicated an enrichment of upregulated transcripts predominantly in the myelin sheath and the maintenance of axon implying a supportive role of astrocytes in the cortex of MBP29-haSyn mice. To examine the presence of oligodendroglial transcripts in astrocytes, in situ RNA hybridization was performed targeting Sry-related HMG-box 10 (SOX10) and myelin regulatory factor (MYRF) as prototypical oligodendroglial transcription factor. SOX10 and MYRF were identified in the proximity of astrocytic processes suggesting a potential oligoden-droglia-astroglia interaction in the cortex of MBP29-haSyn mice. Finally, reactive astrogliosis was investigated in human MSA-P patients post mortem. MSA-P patients display a strong reactive astrogliosis in the cortex, the striatum, and the substantia nigra. Moreover, expression of excitatory amino acid transporter 2 (EAAT2) was investigated expand the analysis of astrocytic characteristics. While no general decrease in expression of EAAT2 was detected, astrocytes in MSA-P patients show a re-distribution of EAAT2 from the branches towards the soma. The re-distribution of EAAT2 implies altered astrocyte functions in MSA-P patients, with EAAT2 as a potential target for a more detailed investigation of the glutamate metabolism in MSA. In conclusion, the findings of this thesis strongly demonstrate the heterogeneity of astrocytes within the CNS dependent on the region and the predominant pathology. The MBP29-haSyn mouse model provided a powerful tool to generate a transcriptomic landscape, thus highlight-ing the dynamic characteristic of astrocytes, displaying distinguished molecular profiles de-spite showing a similar upregulation pattern of GFAP. This work highlights the necessity of a comprehensive profiling of astrocytes to unravel the molecular complexity of this cell type to identify dysregulated pathways. Identification of potential interventional targets may be a promising approach to modulate the astrocytic phenotype in MSA-related pathology and thus slowing disease progression.

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