Parkinson’s disease (PD) is a common neurodegenerative disorder of unknown origin mainly characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the presence of intraneuronal proteinaceous cytoplasmic inclusions, called Lewy bodies (LB), in several affected brain areas. Although LB were identified a century ago, their significance to the pathogenic process in PD remains unknown. Mounting evidence suggest that α-synuclein, a major protein component of LB, may be responsible for initiating and spreading the pathological process in PD. Supporting this concept, intracerebral inoculation of synthetic recombinant α-synuclein fibrils can trigger α-synuclein pathology in mice. However, it remains uncertain whether the observed pathogenic effects of recombinant synthetic α-synuclein can actually apply to PD-linked human α-synuclein and occur in species closer to humans. In this thesis, we addressed this question by assessing the potential pathogenic effects of inoculating α-synuclein-containing nigral LB extracts from PD patients into the brains of wild-type mice and macaque monkeys. Nigral LB containing pathological α-synuclein were purified from post-mortem PD brains by sucrose gradient fractionation and subsequently inoculated into the SNpc or striatum of wild-type mice and macaque monkeys. In both mice and monkeys, intranigral or intrastriatal inoculations of PD-derived LB extracts resulted in progressive nigrostriatal neurodegeneration starting at striatal dopaminergic terminals. In LB-injected animals, exogenous human α-synuclein was quickly internalized within host neurons and triggered the pathological conversion of endogenous α-synuclein. At the onset of LB-induced neurodegeneration, host pathological α-synuclein diffusely accumulated within nigral neurons and anatomically interconnected brain regions. LB-induced pathogenic effects required both human α-synuclein present in LB extracts and host expression of α-synuclein. These results indicate that human α-synuclein species contained in PD-derived LB are pathogenic and have the capacity to initiate a PD-like pathological process. Further supporting a pathogenic role of α-synuclein in PD, increased levels of this protein have been described in PD patients. Therefore, molecular tools able to reverse abnormal α-synuclein expression back to physiological levels might provide therapeutic benefit in PD. Based on this hypothesis, in the second aim of this thesis we assessed the feasibility and safety of downregulating α-synuclein expression in vivo specifically in PD-vulnerable neuronal populations by intransal administration of cell-targeted small interfering RNA (siRNA) directed against α-synuclein. To achieve this goal, we performed first an in vitro screening of various siRNA sequences to select those able to downregulate basal or overexpressed α-synuclein without decreasing β- or γ-synuclein levels. Once identified, the selected molecule (SNCA499-siRNA) was then confirmed to be able to downregulate nigral α-synuclein mRNA in vivo by its local infusion in the SN of mice. This molecule was then chemically modified to enhance its biostability and conjugated to the cell-specific ligand indatraline (IND) to promote its selective delivery into aminergic neurons, the latter being validated in rat ventral midbrain primary cultures. Finally, intranasal administration of IND-SNCA499-siRNA to mice was able to selectively downregulate α-synuclein SNpc expression, both at mRNA and protein levels, without affecting the integrity of the dopaminergic nigrostriatal pathway. These results set the stage for future studies aimed at assessing the disease-modifying potential of intranasally delivered IND-SNCA499-siRNA in experimental PD models associated with increased α-synuclein levels.
|Date of Award||16 Jun 2014|
|Supervisor||Miquel Vila Bover (Director)|
- Lewy bodies