MethodologyStudy DesignThis study will utilize both in vitro and in vivo models to investigate the role of autophagy in cellular stress responses and neurodegenerative diseases.In Vitro StudiesCell Culture: Human neuroblastoma SH-SY5Y cells and primary neuronal cultures will be used.Induction of Stress: Cells will be exposed to various stress conditions, including oxidative stress (hydrogen peroxide), nutrient deprivation, and hypoxia.Autophagy Assays: Autophagy will be monitored using techniques such as LC3 immunoblotting, fluorescence microscopy of GFP-LC3, and quantification of autophagic flux using inhibitors like bafilomycin A1.Protein Aggregation: Assessment of protein aggregates (beta-amyloid, tau, alpha-synuclein, and mutant huntingtin) through immunocytochemistry and Western blotting.Cell Viability: MTT and LDH assays will be performed to assess cell viability under stress conditions.In Vivo StudiesAnimal Models: Transgenic mouse models of Alzheimer’s (APP/PS1), Parkinson’s (alpha-synuclein overexpression), and Huntington’s disease (R6/2 mice) will be used.Induction of Autophagy: Pharmacological agents (e.g., rapamycin) and genetic tools (e.g., ATG5/7 knockdown/knockout) will be employed to modulate autophagy.Behavioral Tests: Cognitive and motor functions will be evaluated using tests such as the Morris water maze, rotarod, and open field test.Histological Analysis: Brain sections will be examined for protein aggregates and neurodegeneration markers using immunohistochemistry and electron microscopy.Biochemical Assays: Brain tissues will be analyzed for autophagy markers (LC3, p62), inflammatory cytokines, and oxidative stress markers.Data AnalysisStatistical Analysis: Data will be analyzed using appropriate statistical methods (e.g., ANOVA, t-tests) to determine significance.Software Tools: ImageJ for image analysis, GraphPad Prism for statistical analysis, and other relevant bioinformatics tools for data interpretation.
