Caspase- 1 Inhibition to Reduce Cleaved Alpha-synuclein in Pre-clinical Models of Parkinson's Disease
MJFF Research Grant, 2011
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.
Arthur J. Mahon Professor of Neurology and Neuroscience at Weill Cornell Medical College
Tauber Professor of Biochemistry and Chemistry, Emeritus at Brandeis University
Location: New York, New York, United States
The Jean Schweppe-Armour Professor of Neurological Sciences and Professor of Neurosurgery at Rush University Medical Center
Director of the Research Center for Brain Repair and Neuroscience Section Head at Rush University Medical Center
Location: Chicago, Illinois, United States