Construction of Lentiviral Vectors Coding for Mutant and Wild Type α-Synuclein
Parkinson's Disease (PD) is the second most prevalent neurodegenerative disease in the world and is clinically characterized by motor control problems. These problems are a result of dopaminergic neuron loss in the brain's substantia nigra pars compacta region, which participates in motor and sensory responsiveness as part of the nigrostriatal pathway. Neuropathologically, the disease is characterized by the presence of Lewy bodies (LB), intracytoplasmic inclusions found in the degenerating neurons of PD sufferers. The main component of LB' s is the protein α-synuclein, which has been implicated in a variety of cellular functions, but whose definite physiological functions have yet to be determined. In addition to the protein's presence in LB' s, familial PD has been linked to two α-synuclein point mutations, A30P and A53T. Further research in this area is necessary, as conflicting studies have made the connection between α-synuclein and LB formation more uncertain than ever. During the course of this project the beginning of a vector delivery system was constructed, which will eventually be used to observe the effects of α.-synuclein expression (its wild type and mutant forms) in rats. Three shuttle plasmids were produced, all of which contained the CMV promoter: HR'CMV α-synuclein, HR'CMV A30P and HR'CMV A53T. Protein expression was then verified in vitro using immunocytochemistry, which showed α.-synuclein expression only in cells transfected with the α-synuclein plasmids. These plasmids will be used to make lentiviral vectors and will then be injected into live rat brains. Subsequent observations on the effect(s) of α.-synuclein expression (wild type, A30P and A53T) on rat dopaminergic neurons as well as rat behavior will be made.
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