386. rAAV-Mediated Reduction of Alpha-Synuclein in the Substantia Nigra of the African Green Monkey Results in Neurodegeneration
Authors: Fredric P. Manfredsson, D. Eugene Redmond, Jack W. Lipton, Ryan M. Malpass, Timothy J. Collier
Journal: Molecular Therapy (2015)

Parkinson Disease (PD) is characterized by the loss of midbrain nigrostriatal dopaminergic neurons and the presence of proteinacious inclusions known as Lewy bodies. Lewy bodies are highly enriched in the protein alpha-synuclein (α-syn). Mutations or multiplication of the α-syn gene are also implicated in some familial forms of PD. Thus, α-syn has been proposed to directly contribute to dopamine (DA) neuron loss in sporadic and familial forms of PD. The predominant hypothesis posits that α-syn-mediated pathology arises due to a toxic gain-of-function. Hence, some therapeutic strategies are aimed at reducing α-syn protein from DA neurons. However, our own efforts to reduce α-syn load in midbrain DA neurons of rodents caused enhanced DA neurodegeneration. This suggested that the reduction of α-syn protein can also be toxic, and that a certain level of α-syn is crucial to DAergic neuron survival. As a result of this observation we propose that α-syn is not a toxic species in PD per se; but rather that α-syn aggregation decreases the pool of soluble α-syn within a neuron to sufficiently impair cellular function and produce pathology. In the current study we expanded our investigation to the non-human primate. African Green monkeys (n=1) were injected unilaterally in the substantia nigra (SN) with low (5×1012 vector genomes (vg)/ml) or high (2×1013vg/ml) titer AAV2/5 expressing a shRNA designed against a-syn or scrambled shRNA as control (total of 4 subjects). Animals were monitored for behavioral deficits indicative of nigrostriatal denervation for three months until sacrifice. Following sacrifice, brains were analyzed for striatal catecholamine content and neuropathology of the SN. The subject treated with high-dose a-syn shRNA exhibited a progressive deficit in a summary score of healthy behaviors, but no increase in overt parkinsonian signs. Analysis of tissue indicated that all a-syn shRNA treated animals exhibited reduced striatal DA levels as compared to the intact hemisphere. A stereological count of midbrain tyrosine hydroxylase positive (TH+) neurons indicated that neurodegeneration was preferentially present in the ventral tier of a-syn shRNA treated SNs, whereas the dorsal tier displayed modest neurodegeneration. The ventral tegmental area remained intact. Scrambled shRNA treated subjects showed no behavioral or neural pathology. Transduction, as measured by the marker GFP (transduction marker), was observed throughout the SN in control subjects. In contrast, GFP was only seen in dorsomedial neurons of a-syn shRNA treated animals, ostensibly due to the loss of DA neuron loss in the ventral tier. Additionally, a-syn shRNA treatment caused a loss of TH phenotype in some surviving neurons as indicated by the observation of several TH-/neuromelanin+ neurons. Our results indicate that non-human primate nigral dopamine (particularly ventral tier) neurons require a-syn for survival, and that the experimentally-induced neuron loss is not a product of nonspecific shRNA toxicity.