We are pursuing studies on the normal physiological role of the protein alpha-synuclein in which missense mutations cause autosomal dominant, early onset Parkinson's disease. In knock-out mice we generated a lack of alpha-synuclein; electron microscopic examination of synapses revealed a striking deficiency of the vesicle cluster located more than one vesicle diameter away from docked vesicles. Field recording of hippocampal slices showed that CA1 synapses from the mutant mice have a normal response to a train of high frequency stimulation (100 Hz, 40 pulses) sufficient to exhaust docked synaptic vesicles, but their response to a lower frequency stimulation (12.5 Hz, 300 pulses) was impaired. These data suggest that the reserve pool of vesicles may be depleted more quickly in the mutant than in normal mice. We have also studied mice expressing normal alpha-synuclein and mice expressing the mutant form of the protein associated with PD in humans.The substantia nigra of mice expressing mutant a-synuclein displayed pathological somatodendritic distribution of two of the major Lewy body components, a-synuclein and phosphorylated neurofilament. Electron microscopy of the substantia nigra and locus ceruleus revealed spherical inclusion bodies in the neuronal cytoplasm. Cultured hippocampal neurons demonstrated a-synuclein aggregates associated with lysosomes in proximity to primary dendrites. Mice expressing mutant a-synuclein had progressive reduction of spontaneous motor activity, particularly vertical movements, and may be a valuable model to assess pathogenesis and therapeutic strategies in Parkinson's disease. Finally, we have extended our studies on control of alpha-synuclein expression and the effect of a polymorphic microsatellite, NCAP-Rep1, upstream of the alpha-synuclein promoter. In order to study the regulation of the human a-synuclein gene, we cloned a 10.7 kb fragment upstream of the translational start site and used restriction sites to construct 7 additional constructs extending varying distances upstream of the gene; all 8 constructs were inserted in a luciferase expression vector and transfected into 293T cells and the neuroblastoma cell line SH-SY5Y. The shortest fragment, 400 bp upstream of the transcriptional start site, was sufficient for transcription in both cell lines. The other constructs led to variable expression levels, with some showing maximum expression and others showing nearly complete extinction of expression. These results illustrate that different regions of the promoter might contribute to tissue specific regulation of a-synuclein. An 880 bp fragment located ~10 kb upstream of the gene and containing the NACP-Rep1 polymorphism, was shown to be necessary for normal expression in the luciferase reporter assays. Four alleles carrying different repeat lengths at the NACP-Rep 1 site were cloned into the luciferase expression vector. The four constructs were transfected into the 293T and the SH-SY5Y cells; expression levels using the different NACP-Rep1 alleles varied very significantly over a 3 fold range in the SH-SY5Y cells but showed little or no significant variation in the 293T cells. Additional analysis of the NACP-Rep1 and the DNA flanking it supports a functional role for the NACP-Rep1 polymorphism in the regulation of a-synuclein expression in neuronal cells. Given that even small changes in expression may, over many decades, increase the risk for disease, allelic differences at this site might contribute to sporadic PD risk.
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