Development and characterisation of new mouse models for parkinson's disease based on alpha-synuclein and pink1 using viral vectors and RNA interference

Parkinson’s disease (PD) is a common neurodegenerative movement disorder that affects 1% of the population aged over 65. Clinically, the diagnosis of PD is based on the presence of motor symptoms such as bradykinesia (slowness of movement), cogwheel rigidity (increased muscular tone), resting tremor and postural instability. The neuropathology observed post-mortem in these patients comprises both the degeneration of the dopaminergic neurons in a specific region of the brain involved in motor control, the substantia nigra, and the prevalence of α-synuclein-rich intracellular inclusions called Lewy bodies. Still today, PD is an incurable disorder whose molecular aetiology is not yet fully understood. Nonetheless, over the past decade, research on PD has increased tremendously by the identification of genes that are linked to rare familial forms of the disease. These inherited forms account for 5 to 10% of the total number of PD patients and cause in most cases early-onset (To date, much effort has been devoted tothe development of pharmacological and surgical therapies for PD that might provide some relief from the motor symptoms but are not curative. The discovery of these genes linked to hereditary PD has impelled the development of animal models, which aim at elucidating the mechanisms at the origin of the disease and at finding new therapeutic treatments. However, up till now, transgenic rodent models for α-synuclein and PINK1have failed to reproduce the prominent progressive neurodegeneration seen in patients. Therefore, a good mouse model for PD is a major requirement now to elucidate mechanisms of pathophysiology and for preclinical testing of new therapies. In the present thesis, we optimised the overexpression of α-synuclein by means of viral vector-mediated gene delivery to the mouse brain. This resulted in gradual loss of dopaminergic neurons in the substantia nigra and concomitant degeneration of the projecting fibers of the striatum in a time- and dose-dependent manner. In addition, we observed α-synuclein-related pathology such as the presence of α-synuclein-positive aggregates as well as increased insoluble and phosphorylated α-synuclein in the surviving neurons. To summarise, we successfully developed and characterised an α-synuclein mouse model for PD which recapitulates several indispensable features of PD’s neuropathology. The next major goal of our research was the generation of a new mouse model for PD based on the silencing of PINK1 in dopaminergic neurons. In order to knockdown PINK1 in adult mouse brain, we first optimised the use of RNA interference in the context of a viral vector by delivering gene specific microRNA-based short-hairpin viral vectors in the midbrain dopaminergic neurons. We achieved potent and specific gene silencing in mouse substantia nigra by meansof our viral vector and RNA interference technologies. After successfulvalidation of the newly generated knockdown constructs, we managed to efficiently knockdown PINK1 in both striatum and substantia nigra of mouse. However, down-regulation of PINK1 in dopaminergic neurons induced no overt behavioural impairment or neurodegenerative process in adult mice in a long-term study up to ten months. Finally, we aimed at studying α-synuclein-related pathology in the absence of PINK1. Forthis approach, we delivered the α-synuclein overexpression viral vector in the substantia nigra of PINK1 knockout and knockdown mice generated in our laboratory. In PINK1 knockout mice, we observed a significantly higher loss of dopaminergic neurons and an enhanced increase in the number of phosphorylated α-synuclein-positive cells in the substantia nigra when compared to age-matched control littermates. In contrast, no significant differences were observed between PINK1 knockdown and control mice, indicating that total, but not partial, loss of PINK1 leads to an increased vulnerability to α-synuclein-induced neuropathology and cell death in vivo .status: publishe