alpha-synuclein and 3-aminobenzamide

Alleles at NACP-Rep1, the polymorphic microsatellite repeat located approximately 10 kb upstream of the alpha -synuclein gene (SNCA), are associated, in some reports, with differing risks of sporadic Parkinson disease (PD). We showed previously that NACP-Rep1 acts as a negative modulator of SNCA transcription, with an effect that varied threefold among different NACP-Rep1 alleles. Given that duplications and triplications of SNCA have been implicated in familial Parkinson disease (PD), even a 1.5-2-fold increase in alpha -synuclein expression may, over many decades, contribute to PD. Thus, the association of different NACP-Rep1 alleles with PD may be a consequence of polymorphic differences in transcriptional regulation of SNCA. Here we aimed to identify the factor(s) that bind to NACP-Rep1 and potentially contribute to SNCA transcriptional modulation, by pulling down proteins that bind to NACP-Rep1 and identifying them by mass spectrometry. One of these proteins was poly-(ADP-ribose) transferase/polymerase-1 (PARP-1), a DNA-binding protein and transcriptional regulator. Electrophoresis mobility shift and chromatin immunoprecipitation assays showed specific binding of PARP-1 to NACP-Rep1. Inhibition of PARP-1's catalytic domain increased the endogenous SNCA mRNA levels in cultured SH-SY5Y cells. Furthermore, PARP-1 binding to NACP-Rep1 specifically reduced the transcriptional activity of the SNCA promoter/enhancer in luciferase reporter assays. This down-regulation effect of PARP-1 depended on NACP-Rep1 being present in the construct and was abrogated by inhibiting PARP-1's catalytic activity with 3-aminobenzamide. The association of different NACP-Rep1 alleles with PD may be mediated, in part, by the effect of PARP-1, as well as other factors, on SNCA expression.

Topics: Alleles; alpha-Synuclein; Base Sequence; Benzamides; Binding Sites; Cell Line; DNA; Enzyme Inhibitors; Gene Expression Regulation; Humans; Microsatellite Repeats; Nerve Tissue Proteins; Parkinson Disease; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Polymorphism, Genetic; Protein Binding; RNA, Messenger; Synucleins

Amyloid beta peptide (A beta) and non-A beta component of Alzheimer's disease amyloid (NAC) are involved in pathomechanism of Alzheimer's Disease (AD) and are deposited in the AD brain in the form of senile plaques. However, the mechanism of their neurotoxicity is not fully understood. In this study the sequence of events involved in NAC and A beta peptides evoked toxicity was investigated in brain slices, synaptosomes and in subcellular fractions. Radio-, immunochemical, spectrophotometrical methods and DNA electrophoresis were used in this study. Our data indicated that A beta 1-40 (25 microM) and NAC (10 microM) peptides induced liberation of free radicals and massive DNA damage that lead to activation of DNA bound enzyme poly(ADP-ribose) polymerase-1 (PARP-1). In consequence of these processes apoptosis-inducing factor (AIF) was released from mitochondria and was translocated to nucleus. The inhibitor of PARP, 3-aminobenzamide significantly decreased AIF release from mitochondria and its translocation. Both peptides under the investigational conditions had no effect on caspase-3 activity. Our data indicated that A beta and NAC peptides stimulate AIF-dependent apoptotic pathway that seems to be caspase independent process. The inhibition of PARP-1 may protect the brain against A beta and NAC toxicity.

Topics: alpha-Synuclein; Amyloid beta-Peptides; Animals; Apoptosis Inducing Factor; Benzamides; Brain; Caspase 3; Cell Nucleus; DNA Damage; Enzyme Inhibitors; In Vitro Techniques; Male; Mitochondria; Peptide Fragments; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Protein Transport; Rats; Rats, Wistar; Reactive Oxygen Species; Synaptosomes