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    • Neurodegeneration, Autophagy, and Amyloid-beta metabolism in Alzheimer’s disease

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  • Alzheimer’s disease (AD) is a complex neurodegenerative disease presenting with senile plaques, neurofibrillary tangles, extensive cell death, and dysregulation of the cell’s internal processes. Out of AD’s many molecular hallmarks, we here focus on autophagy’s involvement in this pathology. To investigate autophagy’s role in AD we studied specifically mouse models with the Swedish and Beyreuther mutations in the App gene with either normal or conditional deficient autophagy (due to the knock-out of Atg7) in the excitatory neurons (Atg7 cKO). Using laser microdissection (LMD), we analyzed the proteome of the pyramidal cell layer of CA1 from wildtype, AppNL-F, Atg7 cKO, and Atg7 cKO x AppNL-F mice using label-free mass spectrometry (MS). We initially identified 228 proteins using MS from a LMD CA1 20 µm thickness sample from AppWT mice. To increase the number of identified proteins, we established a protocol to cut 30 µm thick CA1 tissue sections. The raw MS results for the whole cohort of mice identified 1130-2046 proteins per mouse. The proteomic analysis showed a significant downregulation of synaptic proteins between Atg7 cKO x APPNL-F versus AppWT, Atg7 cKO versus AppWT, Atg7 cKO x APPNL-F versus AppNL-F mice. Therefore, we next focused on assessing synaptic loss using immunofluorescence (IF), by staining for a specific presynaptic protein, synaptophysin1, to quantify the number of synapses between the mouse groups, until the final MS results arrive. We observed significant differences in the intensity of synaptophysin1 in CA3 between the mouse groups (Welch’s ANOVA test p<0.05) at 10x magnification and a similar trend but not significant differences comparing the autophagy-deficient and autophagy-competent mice at 63x magnification. The high number of proteins identified by MS can provide a steppingstone for understanding the role of intracellular Aβ and autophagy in AD and for identifying new candidate proteins for the study of AD. 
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