siRNA Mediated GSK3β Knockdown Targets Insulin Signaling Pathway and Rescues Alzheimer’s Disease Pathology: Evidence from In Vitro and In Vivo Studies

Sporadic Alzheimer's disease (sAD) is a progressive neurodegenerative disorder with dysfunctional insulin signaling and energy metabolism. Emerging evidence suggests impairments in brain insulin responsiveness, glucose utilization, and energy metabolism may be major causes of amyloid precursor protein mishandling. The support for this notion comes from the studies wherein streptozotocin (STZ) induced brain insulin resistance in rodent model resulted in sAD-like neuropathology with cognitive decline. Our previous study showed a compromised insulin signaling pathway, glucose uptake, glucose metabolism, and energy homeostasis in STZ-induced glial-neuronal coculture and in vivo model of sAD. Various components of insulin signaling pathway were examined to understand the metabolic correlation, and GSK3β was selected for gene knockdown strategy to reverse sAD pathology based on the data. In the present study, we have synthesized carboxylated graphene oxide (GO) nanosheets functionalized with PEG and subsequently with polyethylenimine (PEI) to provide attachment sites for GSK3β siRNA. Our results showed that siRNA mediated knockdown of the GSK3β gene reduced expression of amyloid pathway genes (APP and BACE1), which was further confirmed by reduced amyloid beta (Aβ) levels in the in vitro STZ-induced sAD model. GSK3β knockdown also restored insulin signaling, AMPK and Mapk3 pathway by restoring the expression of corresponding candidate genes in these pathways (IR, Glut1/3, Prkaa1/2, Mapk3, BDNF) that reflected improved cellular energy homeostasis, neuronal proliferation, differentiation, maturation, and repair. Behavioral data from Morris water maze (MWM), open field (OF), novel object recognition (NOR), Y maze, and radial arm maze (RAM) tests showed that 0.5 μg nanoformulation (GOc-PP-siRNAGSK3β) intranasally for 7 days improved spatial memory, rescued anxiety like behavior, improved visual and working memory, and rescued exploratory behavior in STZ-induced sAD rats. GSK3β silencing resulted in decreased BACE1 expression and prevented accumulation of Aβ in the cortex and hippocampus. These molecular findings with improved behavioral performances were further correlated with reduced amyloid beta (Aβ) and neurofibrillary tangle (NFTs) formation in the cortex and hippocampus of GOc-PP-siRNAGSK3β administered sAD rats. Therefore, it is conceivable from the present study that nanoparticle-mediated targeting of GSK3β in the sAD appears to be a promising strategy to reverse sAD pathology.