We will test the hypothesis that caspase-1 inhibition will down- regulate α-synuclein (α-SYN) at mRNA and/or protein levels within the SN and restore impaired function of the dopamine system in two pre-clinical models of Parkinson’s disease (PD). In this application, we will use transgenic pre-clinical models that over express the α-SYN, a molecule believed to be central to PD pathology. We will also use a virus to over-express α-SYN in pre-clinical models, as this model also induces the expression of α-SYN aggregates and has a more predictable, more PD- like motor phenotype, as well as frank cell death, relative to α-SYN over-expressing pre-clinical models.
We will test the hypothesis the caspase-1 inhibitor VX-765 will prevent or delay (pre-treatment) or reverse (post-treatment) α-SYN accumulation and aggregation and prevent (pre-treatment) or reverse (post-treatment) motor disability in α-SYN over-expressing pre-clinical models.
Research Design: A total of 105 pre-clinical models (α-SYN over expressors, α-SYN knockouts, and wild-type) will comprise Experiment Groups (1-2) of one month old transgenic over-expressing pre-clinical models who will receive caspase-1 inhibitors (10 (low dose) or 50 (high dose) mg/kg, based on similarities to the Vertex compound) for 8 weeks. Groups 3-4 will be α-SYN know-out pre-clinical models receiving similar doses, Group 5 will be α-SYN transgenics receiving vehicle. One, four, eight and 12 weeks following drug treatments, pre-clinical models will be tested for motor function (see below) after which they will be sacrificed. Groups 6-10 will be treated identically as Groups 1-5, except treatment will be initiated at 4 months of age, when the presence of behavioral deficits and α- SYN aggregates have already been established.
Our outcome measures are: 1) behavioral data from the rotorod test;2) counts of nigral DAT- and TH-ir neurons and α-SYN-ir cells in both aggregated and non- aggregated forms within the SN; 3) Neuochemical measurements of dopamine and its metabolites, 4) measurement of the optical density of TH-ir within striatum; 5) Western Blot Analysis for α- SYN protein expression; and 6) Quantitative RT-PCR analysis for α-SYN mRNA expression within SN.
130 young adult Sprague Dawley pre-clinical models will comprise Experiment 2. For one week, pre-clinical models will receive daily injections of the caspase-1 inhibitor VX-765 or vehicle. Then, pre-clinical models in groups 1-7 above will receive vector injections comprised of 2ul of equally titered AAV6- α-SYN or AAV6-GFP. All pre-clinical models will continue to receive caspase-1 or vehicle treatment for 6 weeks, after which time they will be sacrificed for histological and biochemical analysis. Statistical analysis will be similar to Experiment 1
Our outcome measures are: Same as Experiment 1, except “analysis of behavioral data of motor function will be performed on the cylinder test.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
α-synuclein is believed to be integral in the killing of dopamine cells in PD. We believe that it is the cleaved product of α-synuclein that is the culprit. Co-P.I. Dr. Petsko demonstrated previously that inhibition of caspase 3 protect nigral neurons in culture. The present study will see whether we can get similar neuroprotection of nigral neurons in two pre-clinical models of PD.
In our first experiment, we expect that caspase-1 inhibition will 1) reduce the expression of α-SYN mRNA and aggregated protein within SN, 2) block nigrostriatal dopaminergic degeneration (pretreatment experiment), 3) prevent or delay the onset of motor disability (pretreatment experiment), 4) reverse dopamine degeneration (post-treatment experiment) 5) reverse motor disability (post-treatment experiment) in transgenic α-SYN over expressing pre-clinical models. We believe that all of these effects will occur in a dose dependent manner. We believe none of these effects will be seen in α-SYN knock out pre-clinical models attesting to the specificity of the effects on reduced cleavage of the α-SYN protein.
In experiment 2, we expect that caspase-1 inhibition will 1) reduce α-SYN mRNA and protein levels within SN, 2) protect against nigrostriatal dopaminergic degeneration, and 3) reverse or retard motor disability in this AAV6-SYN induced pre-clinical PD model. We also are interested to see if inhibition of synuclein aggregation leads to down-regulating the expression of α-SYN at both mRNA and protein level.