Evaluating Triptolide Effects on Hippocampal Gephyrin, Collybistin, and Autophagy in an Aβ1-42 Mouse Model

Alzheimer's disease is associated with synaptic dysfunction, but standardized procedures for evaluating how Aβ-induced pathology affects inhibitory synapse-associated proteins and autophagy-related signaling after candidate intervention remain limited. This protocol describes a workflow for establishing an Aβ1-42-induced Alzheimer's disease-like mouse model and assessing the effects of triptolide on hippocampal Gephyrin, Collybistin, PI3K/Akt/GSK-3β signaling, and autophagy-related markers. Adult C57BL/6J mice receive bilateral intracerebroventricular injection of Aβ1-42 prepared under aggregation-inducing conditions, followed by daily intraperitoneal administration of triptolide with or without the PI3K inhibitor LY294002. Spatial learning and memory are evaluated using Morris water maze testing. Hippocampal neuronal injury and Aβ deposition are assessed by hematoxylin and eosin staining and Aβ immunohistochemistry, and hippocampal lysates are analyzed by Western blotting to quantify Gephyrin, phosphorylated Gephyrin, Collybistin, PI3K/Akt/GSK-3β signaling proteins, LC3-II/I, and p62. Key procedural considerations include standardized Aβ1-42 preparation, accurate stereotaxic injection, consistent behavioral testing conditions, blinded region-of-interest selection, and standardized image and densitometry analysis. Using this workflow, Aβ1-42 administration produced spatial learning and memory deficits, hippocampal neuronal injury, Aβ deposition, reduced Gephyrin and Collybistin expression, altered PI3K/Akt/GSK-3β signaling, and increased LC3-II/I and p62 accumulation. Triptolide partially reversed these behavioral, histological, and molecular changes, whereas LY294002 attenuated its effects. This protocol can be used to evaluate Aβ-induced hippocampal molecular alterations and candidate interventions, while recognizing that this model does not reproduce the chronic, multifactorial progression of human Alzheimer's disease.