Tufano, Michele (2024) Temporal evolution and phenotypic diversity of glial cells in A53T Transgenic mice: insights into Parkinson’s disease progression. [Tesi di dottorato]

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Abstract

Parkinson’s disease (PD) is a complex neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the presence of alpha-synuclein (α-Syn) aggregates, and Lewy bodies. Neuroinflammation is a hallmark of PD, with glial cells, particularly astrocytes and microglia, playing dual roles in neuroprotection and neurotoxicity. However, whether glial activation is a primary event in dopaminergic neurodegeneration or a consequence of it remains unclear. This study investigated: 1. The temporal evolution of glial phenotypes in the midbrain and striatum during disease progression. 2. The genetic mechanisms driving the shift from neuroprotective to detrimental glial states. 3. The relationship between changes in glial phenotype and dopaminergic dysfunction in those brain areas mainly involved in PD motor and non-motor symptoms. To address these issues, Western blot analysis was performed in the midbrain and striatum of 4-6-12-16-month-old mice expressing the human mutation A53T of α-Synuclein to characterize the pro- and the anti-inflammatory glia subtypes. This analysis demonstrated that in A53T-α-Syn transgenic mice, glial cells exhibited an age-related increase in neurotoxic markers, with 12 months identified as the critical time point for the transition to neurotoxic phenotypes. At this stage, gliosis was prominent in the midbrain, while neurotoxic microglia predominate in the striatum, persisting into 16 months. To study the molecular determinants of glial phenotype switching in brain regions related to PD, a pre-existing single-nucleus RNA-Seq database on the same mouse model was analysed. The in-silico analysis found that the GNAO-1 gene is selectively dysfunctional in mesencephalic but not in striatal astrocytes. GNAO-1 encodes a Gαo protein that controls the synthesis of cAMP-modulating inhibitory and stimulatory inputs to the adenylyl-cyclase-5. GNAO-1 mutations are linked to neurological disorders such as developmental disorders, hypotonia, epilepsy, and movement disorder, suggesting a potential relationship with the motor symptoms observed in PD. Molecular and functional analysis of post-natal astrocytes from Wild-type and A53T mouse pups confirmed these findings, prompting further investigations into GNAO1’s role in the astrocytes phenotypic transition in A53T mice.

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