Mechanisms of neuronal pathology in a model of grey matter inflammation

Ullrich Gavilanes, Emily Melisa (2023): Mechanisms of neuronal pathology in a model of grey matter inflammation. Dissertation, LMU München: Graduate School of Systemic Neurosciences (GSN)

Abstract

Multiple sclerosis (MS) is a chronic, inflammatory, and demyelinating disease of the central nervous system (CNS). It is characterized by formation of lesions both in white and grey matter. Upon disease evolution into progressive stages, grey matter pathology plays a larger role and permanent disability ensues. Grey matter pathology of MS has been widely characterized through histopathological studies in terms of demyelination, neuronal pathology, and inflammation. However, the mechanisms that play a role in pathology development and progression are not fully understood. Furthermore, treatment options for progressive stages of MS are limited and we have no way of effectively blocking ongoing cortical neurodegeneration. Thus, my PhD project focused on modeling, visualizing, understanding, and therapeutic targeting of cortical grey matter pathology. To address these questions, I used a combination of confocal microscopy, multiphoton in vivo microscopy, bioinformatic transcriptomic analysis, CRISPR/Cas9 gene editing, and PET imaging. The first part of my thesis aimed to establish a mouse model of grey matter pathology that resembled cortical pathology in MS. This model was induced in BiozziABH mice, which is a strain characterized by high antibody response and susceptibility to chronic CNS inflammation. Mice were immunized with MOG, followed by an intracerebral injection of pro-inflammatory cytokines to induce cortical lesions. Results suggest that our mouse model indeed presents with cortical grey matter demyelination, synapse loss and inflammatory lesions, which in turn, resembles previously described grey matter pathology in MS. Moreover, an age effect was observed in pathology resolution, with older mice displaying a more sustained neuroinflammatory response while younger mice spontaneously resolved inflammation. Further analysis of grey matter lesions revealed a potential role of synaptic calcium accumulation and phagocyte engulfment in neuronal pathology. In the second part of the thesis, the focus was on investigating pathways and mechanisms underlying neuronal pathology in grey matter of MS. For this purpose, we utilized single nuclei transcriptomic analysis of our mouse model, which was further mapped together with data sets from MS patients. We aimed at determining a MS specific gene signature that was present both in our model as well as in patients with MS. To ensure MS specificity, we further analysed the enrichment of our cortical MS-related gene signature in an Alzheimer’s disease patient data set. Our results demonstrated a species conservation of a cortical MS-related gene signature with five genes that are highly upregulated in neurons in the inflamed cortex of mice and humans that are interesting targets for further mechanistic analysis. We subsequently aimed to establish a CRISPR/Cas9 system for neuron-specific gene knockout which could then be used for investigation of mechanisms and pathways by which our candidate genes might play a role in MS pathology. Using two of the target genes, we demonstrated that the CRISPR/Cas9 system was successful in knocking out genes in neurons. However, we were up to now not able to conclusively resolve the role our selected genes played in MS grey matter pathology. In the next part of my thesis, we aimed to test different therapeutic strategies in our mouse model to determine if they would inhibit neuronal pathology in the inflamed grey matter or could rescue existing pathology. We tested immunomodulatory therapies targeting microglia activation as a strategy to limit the induction of neuronal pathology and could show that CSF1R inhibition can prevent synapse loss in the cortical MS model. Finally, we investigated imaging based approaches that could be used to track synaptic pathology in MS. For this purpose we performed a preclinical study with a SV2a specific PET tracer in our cortical MS model. PET imaging of mice demonstrated that the PET tracer was able to sensitively detect synapse loss in our model with the reduction in tracer uptake corresponding to the synaptic density decrease that was observed by histological examinations in situ. Overall, the results obtained of my thesis provide new insights into the pathomechanisms undelying neuronal pathology in the grey matter, the therapeutic strategies that can be used to prevent it and the imaging strategies that can be used to track it in MS patients.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Grey matter inflammation, Multiple sclerosis, Neuronal pathology, CRISPR/Cas9, PET/CT
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Graduate School of Systemic Neurosciences (GSN)
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 23. Oktober 2023
• 1. Bericht­erstatter:in: Kerschensteiner, Martin