Behavioural and neuropathological effects of intranasal insulin treatment in a triple-transgenic mouse model of Alzheimer's disease (3xTg-AD)

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a progressive cognitive impairment and by the appearance of non-cognitive or behavioural symptoms. Currently there is no cure for AD and, to this regard, it’s growing the interest in the identification of novel therapeutic targets for the development of new promising drugs. Recently, it was observed that insulin is able to play a neuroprotective and a neurotrophic action within the central nervous system (CNS) (Craft and Watson, 2004; Hoyer, 2004; Stockhorst et al, 2004). In particular, recent evidence have shown that CNS insulin is widely involved in cognition, especially in memory and learning processes, suggesting a strict relation between AD and diabetes (Ott et al, 1996; Arvanitakis et al, 2004; Banks et al, 2012). In fact, type 2 diabetes mellitus (T2DM) patients present several alterations of insulin signaling that lead to cognitive and no cognitive symptoms, such as those manifested by AD patients. To this regard, diabetes mellitus (DM) is fully recognized as an additional risk factor of AD (Janson et al, 2004; Craft and Watson, 2004). Although it’s not yet entirely clear what is the neuropathological link between AD and DM, in support of this hypothesis it was demonstrated that AD patients show a central insulin-resistance condition (Steen et al, 2005; De La Monte, 2008), characterized by a reduced brain insulin receptors (IR) sensitivity, hyperphosphorylation of IR and insulin receptor substrate-1 and 2 (IRS-1/2), as well as decreased expression of insulin and insulin-like growth factor (IGF) receptor (Bedse et al, 2015). On the other hand, it cannot be excluded that these alterations may be involved in the neuropathology of AD by one of the following mechanisms: increasing expression of the amyloid precursor protein (APP), excessive formation and accumulation of the amyloid-β peptide (Aβ), by impairing Aβ clearance and or by increasing phosphorylation of tau protein (Steen et al, 2005; Zhao et al, 2009). Based on these assumptions, the aim of the present study was to analyse the behavioural and neurochemical effects induced by insulin treatment in triple-transgenic mouse model of AD (3×Tg-AD). 3×Tg-AD model reproduces the cognitive impairment and the neuropathological alterations observed in human patients. In particular, 3×Tg-AD mice develop age-dependent Aβ and tau pathology that is also associated with cognitive decline and a depressive-like phenotype (Romano et al, 2014). In this study, 4- and 10-month-old 3×Tg-AD mice and non-transgenic control mice (Non-Tg) were treated intranasally every other day for 2 months with human insulin (Humulin R U-100) or with vehicle (saline). The effects of insulin were evaluated on mice at 6 or 12 months of age, that represents respectively the early- and the late-stage of AD-like pathology. To evaluate the effects of the treatment on the cognitive domain, all mice were tested by the morris water maze test and the novel object recognition test. Instead to evaluate the effects of insulin on the depressive-like phenotype exposed by 3×Tg-AD mice, animals were tested by the forced swimming test and the tail suspension test. Results of Morris water maze and Novel object recognition test have shown that insulin is able to improve the short-term memory mainly in 7-months-old 3xTg-AD mice while forced swimming test and tail suspension test results have shown that insulin can ameliorate the depressive-like phenotype showed by 7- and 13-months-old 3×Tg-AD mice. The expression levels of the main neuropathological hallmarks of AD (APP, Aβ and tau) were evaluated in frontal cortex and hippocampus of treated mice by western blotting and immunohistochemical experiments. Moreover, these results are accompanied by a reduction of Aβ oligomers and hyperphosphorylated tau levels mainly in frontal cortex and hippocampus of 13-month-old 3×Tg-AD mice.