| Summary: | 碩士 === 國立陽明大學 === 腦科學研究所 === 101 === One of the pathological hallmarks of Alzheimer’s disease (AD) is the accumulation of senile plaques, which are mainly composed of amyloid beta-peptide (Aβ). Aβ-induced cytotoxicity directly initiate formation of free radicals and reactive oxygen species (ROS), cellular dysfunction, and subsequent neuronal death. Nicotinamide adenine dinucleotide (NAD) is a necessary cofactor for cellular energy production and signaling transduction. Recent studies indicated that cellular NAD replenishment confers neuroprotection against neuronal death induced by oxygen-glucose deprivation, an in vitro model for ischemic stroke. However, whether the cellular NAD replenishment could have neuroprotective effect against Aβ-induced DNA damage has never been explored. The goal of the present study is therefore to test whether NAD treatment may protect neurons against Aβ-induced neurotoxicity and DNA damage in rat cortical culture. Cells were treated with Aβ25-35 or Aβ1-42 to mimic AD in vitro. The APPswe/PS1dE9 mice were sacrificed at 4, 6, 9, and 12 months of age for immunohistochemistery. Cell survival was determined by MTT assay, Hoechst staining, and immunocytochemistery. The extents of oxidative DNA damage were quantified by 8-hydroxy-2'-deoxyguanosine (8-OH-dG) staining and apurinic/apyrimidinic (AP) sites. DNA single-stranded breaks (SSBs) were quantified by the DNA polymerase I–mediated biotin-dATP nick-translation (PANT). The extents of DNA double-stranded breaks (DSBs) and neuronal apoptosis were quantified by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. We found that 50 mM NAD can enhance neuronal survival and neuronal morphology against Aβ-induced neurotoxicity in rat cortical culture. We observed an increase of DNA single- and double-stranded breaks expression in the cortical region of APPswe/PS1dE9 mice at 12 month old. Further in vitro studies confirmed that Aβ-induced DNA damage is decreased by NAD treatment in neurons. Rat cortical cultures treated with 50 mM NAD have significantly reduced extents of DNA base modifications (8-OHdG) upon Aβ exposure for 16 hours. Moreover, we found that the same concentrations of NAD also efficiently reduced the numbers of AP sites induced by Aβ exposure for 16 hours, indicating NAD effectively attenuates Aβ-induced DNA abasic site formation. Furthermore, NAD cotreatment significantly attenuated the heightened extent of PANT- and TUNEL- positive cells induced by Aβ exposure for 40 and 48 hours, indicating that NAD effectively alleviate Aβ-induced DNA SSBs and DSBs formation. In conclusion, these results provide evidence that exogenous NAD has neuroprotective effect against Aβ-induced neurotoxicity and
DNA damage in vitro.
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