Doctor of Philosophy
The cerebellum is a region of the central nervous system widely known for its role in motor learning and coordination. In the past, mutant mice have been critical in discerning key pathways associated with cerebellar dysfunction and motor deficits. Although a variety of mouse models exist that model the symptoms and pathogenesis of cerebellar ataxia, some have yet to be characterized at the molecular level. Nitric oxide (NO), specifically derived from neuronal nitric oxide synthase (nNOS), is a well-established regulator of synaptic transmission in Purkinje neurons (PNs), governing fundamental processes such as motor learning and coordination. Previous morphological analyses showed similar gross cerebellar structures between neuronal nitric oxide null (nNOS-/-) and wild-type (WT) adult male mice, despite prominent ataxic behaviour within nNOS-/- mice. However, a study has yet to characterize potential differences in cerebellar network formation during development in nNOS-/- mice. This thesis study is the first to determine morphological and functional deficits within the cerebellum of nNOS-/- mice using immunostaining, immunoblotting, ex vivo slice culturing, calcium- and sodium-imaging, colourmetric assays and ELISAs. Results from Chapter 2 showed stark PN dendritic abnormalities in nNOS-/- mice compared to WT across development. Specifically, we noted that PN dendritic abnormalities are associated with elevated levels of intracellular calcium via overactivation of metabotropic glutamate receptor type 1 (mGluR1)-initiated store operated calcium entry. Chapter 3 analyses linked the overactivation of mGluR1 in nNOS-/- mice with decreased glutamate uptake via glutamate aspartate transporters (GLAST) on Bergmann glia (BG). Specifically, a lack of NO production resulted in decreased GLAST expression on BG and decreased glutamate uptake. Importantly, Chapter 4 results further demonstrated that the effects of mGluR1 overactivation on PNs specific to nNOS-/- mice resulted in increases in endocannabinoid levels. Specifically, our group noted increases in enzymes downstream of mGluR1 activation and subsequent increases in the endocannabinoid 2-arachidonoylglycerol, as well as decreases in endocannabinoid hydrolyzing enzymes in nNOS-/- cerebella compared to WT. Together, these results are foundational in establishing the nNOS-/- mouse as a model of cerebellar ataxia. Understanding the role of nNOS/NO signaling in cerebellar development may be beneficial in uncovering novel therapeutics for cerebellar disorders.
Summary for Lay Audience
The cerebellum is a brain region associated with the fine-tuning of various movements such as walking, blinking, and balance. In neurodegenerative diseases such as cerebellar ataxia, principal neurons of the cerebellum called Purkinje neurons (PN) begin to malfunction and degenerate, leading to symptoms such as tremors, uncoordinated walking, and loss of balance. Studies on genetically modified mice have helped to gather information on crucial disturbances in cerebellar development that can result in motor deficits similar to those displayed in individuals with cerebellar ataxia. However, some mouse models that present with symptoms of cerebellar ataxia have yet to be characterized at the molecular level. Within the cerebellum, nitric oxide (NO), produced by neuronal nitric oxide synthase (nNOS) is an important gaseous molecule responsible for facilitating PN synaptic firing and motor learning. Despite being abundantly expressed in the cerebellum, previous reports analyzing the nNOS knockout (nNOS-/-) mouse revealed no differences in cerebellar anatomy, despite exhibiting deficits in motor behaviour. However, a study has yet to characterize protein-level differences within the cerebellum of nNOS-/- mice across development in comparison to wildtype (WT) mice. The results within this thesis are the first to show differences in nNOS-/- cerebellar development in comparison to WT mice. Specifically, we discovered stark delays in PN dendritic growth in nNOS-/- cerebella across development in comparison to WT. PN dendritic deficits were associated with increases in PN calcium entry caused by overactivation of metabotropic glutamate receptor type 1 (mGluR1) within nNOS-/- mice compared to WT. Overactivation of glutamate receptors is often caused by excess glutamate within the synaptic cleft, and our group showed that nNOS-/- cerebellar astrocytes uptake less glutamate compared to WT cerebellar astrocytes. Specifically, we showed that the glutamate/aspartate transporter (GLAST) is significantly decreased in nNOS-/- cerebella compared to WT across development. Overactivation of mGluR1 is not only responsible for causing increases in intracellular calcium levels within the PN but is also needed for endocannabinoid production. Likewise, our group discovered increases in endocannabinoid concentrations within nNOS-/- cerebella compared to WT. Together, these results are an important first step in understanding the role of nNOS/NO signaling in cerebellar disorders.
Tellios, Vasiliki, "The Role of Neuronal Nitric Oxide Synthase in Regulating Cerebellar Network Formation Across Murine Development" (2021). Electronic Thesis and Dissertation Repository. 7858.
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