Effects of high-fat diet on nutrient metabolism and cognitive functions in young APPKINL-G-F/NL-G-F mice.

AIM: Type 2 diabetes mellitus (T2DM) is an increased risk factor for Alzheimer's disease (AD); however, the relationship between the 2 conditions is controversial. High-fat diet (HFD) causes cognitive impairment with/without Aß accumulation in middle-aged or aged transgenic (Tg) and knock-in (KI) AD mouse models, except for metabolic disorders, which commonly occur in all mice types. Alternatively, whether HFD in early life has an impact on nutrient metabolism and neurological phenotypes in young AD mouse models is not known. In the present study, we examined the effects of HFD on young APPKINL-G-F/NL-G-F mice, one of the novel KI-AD mouse models. METHODS: The mice were categorized by diet into 2 experimental groups, normal diet (ND) and HFD. Four-week-old wild-type (WT) and APPKINL-G-F/NL-G-F mice were fed ND or HFD for 9 weeks. Both types of mice on ND and HFD were examined during young adulthood. RESULTS: HFD caused T2DM-related metabolic disturbances in both young WT and APPKINL-G-F/NL-G-F mice, whereas impaired thermoregulation and shortage of alternative energy sources specifically occurred in young APPKINL-G-F/NL-G-F mice. However, HFD had no impact on the cognitive function, Aß levels, and phosphorylation of hippocampal insulin receptor substrate 1 (IRS1) at all the 3 Ser sites in both types of mice. CONCLUSION: HFD is effective in causing metabolic disturbances in young WT and APPKINL-G-F/NL-G-F mice but is ineffective in inducing neurological disorders in both types of mice, suggesting that the aging effects, along with long-term HFD, facilitate neurological alterations.

Irs2 deficiency alters hippocampus-associated behaviors during young adulthood.

Type 2 diabetes mellitus (T2DM), characterized by hyperglycemia and insulin resistance, has been recognized as a risk factor for cognitive impairment and dementia, including Alzheimer's disease (AD). Insulin receptor substrate2 (IRS2) is a major component of the insulin/insulin-like growth factor-1 signaling pathway. Irs2 deletion leads to life-threatening T2DM, promoting premature death in male mice regardless of their genetic background. Here, we showed for the first time that young adult male mice lacking Irs2 on a C57BL/6J genetic background (Irs2-/-/6J) survived in different experimental environments and displayed hippocampus-associated behavioral alterations. Young adult male Irs2-/-/6J mice also exhibit aberrant alterations in energy and nutrient sensors, such as AMP-activated protein kinase (AMPK) and glucose transporter3 (GLUT3), and reduced core body temperature accompanied by abnormal change in the temperature sensor in the brain. These results suggest that Irs2 deficiency-induced impairments of brain energy metabolism and thermoregulation contribute to hippocampus-associated behavioral changes in young adult male mice.

Serine Phosphorylation of IRS1 Correlates with Aß-Unrelated Memory Deficits and Elevation in Aß Level Prior to the Onset of Memory Decline in AD.

The biological effects of insulin signaling are regulated by the phosphorylation of insulin receptor substrate 1 (IRS1) at serine (Ser) residues. In the brain, phosphorylation of IRS1 at specific Ser sites increases in patients with Alzheimer's disease (AD) and its animal models. However, whether the activation of Ser sites on neural IRS1 is related to any type of memory decline remains unclear. Here, we show the modifications of IRS1 through its phosphorylation at etiology-specific Ser sites in various animal models of memory decline, such as diabetic, aged, and amyloid precursor protein (APP) knock-in NL-G-F (APPKINL-G-F) mice. Substantial phosphorylation of IRS1 at specific Ser sites occurs in type 2 diabetes- or age-related memory deficits independently of amyloid-ß (Aß). Furthermore, we present the first evidence that, in APPKINL-G-F mice showing Aß42 elevation, the increased phosphorylation of IRS1 at multiple Ser sites occurs without memory impairment. Our findings suggest that the phosphorylation of IRS1 at specific Ser sites is a potential marker of Aß-unrelated memory deficits caused by type 2 diabetes and aging; however, in Aß-related memory decline, the modifications of IRS1 may be a marker of early detection of Aß42 elevation prior to the onset of memory decline in AD.

Involvement of insulin receptor substrates in cognitive impairment and Alzheimer's disease.

Type 2 diabetes-associated with impaired insulin/insulin-like growth factor-1 (IGF1) signaling (IIS)-is a risk factor for cognitive impairment and dementia including Alzheimer's disease (AD). The insulin receptor substrate (IRS) proteins are major components of IIS, which transmit upstream signals via the insulin receptor and/or IGF1 receptor to multiple intracellular signaling pathways, including AKT/protein kinase B and extracellular-signal-regulated kinase cascades. Of the four IRS proteins in mammals, IRS1 and IRS2 play key roles in regulating growth and survival, metabolism, and aging. Meanwhile, the roles of IRS1 and IRS2 in the central nervous system with respect to cognitive abilities remain to be clarified. In contrast to IRS2 in peripheral tissues, inactivation of neural IRS2 exerts beneficial effects, resulting in the reduction of amyloid ß accumulation and premature mortality in AD mouse models. On the other hand, the increased phosphorylation of IRS1 at several serine sites is observed in the brains from patients with AD and animal models of AD or cognitive impairment induced by type 2 diabetes. However, these serine sites are also activated in a mouse model of type 2 diabetes, in which the diabetes drug metformin improves memory impairment. Because IRS1 and IRS2 signaling pathways are regulated through complex mechanisms including positive and negative feedback loops, whether the elevated phosphorylation of IRS1 at specific serine sites found in AD brains is a primary response to cognitive dysfunction remains unknown. Here, we examine the associations between IRS1/IRS2-mediated signaling in the central nervous system and cognitive decline.

Metformin treatment ameliorates diabetes-associated decline in hippocampal neurogenesis and memory via phosphorylation of insulin receptor substrate 1.

Age-related reduction in adult hippocampal neurogenesis is correlated with cognitive impairment. Diabetes is a chronic systemic disease that negatively affects adult neural stem cells and memory functions in the hippocampus. Despite growing concern regarding the potential role of diabetic drugs in neural abnormalities, their effects on progressive deterioration of neurogenesis and cognitive functions remain unknown. Here, we show that the combination of aging and diabetes in mice causes a marked decrease in hippocampal neurogenesis along with memory impairment and elevated neuroinflammation. Prolonged treatment with metformin, a biguanide antidiabetic medication, promotes cell proliferation and neuronal differentiation and inhibits aging- and diabetes-associated microglial activation, which is related to homeostatic neurogenesis, leading to enhanced hippocampal neurogenesis in middle-aged diabetic mice. Although chronic therapy with metformin fails to achieve recovery from hyperglycemia, a key feature of diabetes in middle-aged diabetic mice, it improves hippocampal-dependent spatial memory functions accompanied by increased phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), atypical protein kinase C ζ (aPKC ζ), and insulin receptor substrate 1 (IRS1) at selective serine residues in the hippocampus. Our findings suggest that signaling networks acting through long-term metformin-stimulated phosphorylation of AMPK, aPKC ζ/λ, and IRS1 serine sites contribute to neuroprotective effects on hippocampal neurogenesis and cognitive function independent of a hypoglycemic effect.

Mitochondria are devoid of amyloid ß-protein (Aß)-producing secretases: Evidence for unlikely occurrence within mitochondria of Aß generation from amyloid precursor protein.

Mitochondrial dysfunction is implicated in the pathological mechanism of Alzheimer's disease (AD). Amyloid ß-protein (Aß), which plays a central role in AD pathogenesis, is reported to accumulate within mitochondria. However, a question remains as to whether Aß is generated locally from amyloid precursor protein (APP) within mitochondria. We investigated this issue by analyzing the expression patterns of APP, APP-processing secretases, and APP metabolites in mitochondria separated from human neuroblastoma SH-SY5Y cells and those expressing Swedish mutant APP. APP, BACE1, and PEN-2 protein levels were significantly lower in crude mitochondria than microsome fractions while those of ADAM10 and the other γ-secretase complex components (presenilin 1, nicastrin, and APH-1) were comparable between fractions. The crude mitochondrial fraction containing substantial levels of cathepsin D, a lysosomal marker, was further separated via iodixanol gradient centrifugation to obtain mitochondria- and lysosome-enriched fractions. Mature APP, BACE1, and all γ-secretase complex components (in particular, presenilin 1 and PEN-2) were scarcely present in the mitochondria-enriched fraction, compared to the lysosome-enriched fraction. Moreover, expression of the ß-C-terminal fragment (ß-CTF) of APP was markedly low in the mitochondria-enriched fraction. Additionally, immunocytochemical analysis showed very little co-localization between presenilin 1 and Tom20, a marker protein of mitochondria. In view of the particularly low expression levels of BACE1, γ-secretase complex proteins, and ß-CTF in mitochondria, we propose that it is unlikely that Aß generation from APP occurs locally within this organelle.

The neurotoxicity of amyloid ß-protein oligomers is reversible in a primary neuron model.

Alzheimer's disease (AD) is characterized by the accumulation of extracellular amyloid ß-protein (Aß) and intracellular hyperphosphorylated tau proteins. Recent evidence suggests that soluble Aß oligomers elicit neurotoxicity and synaptotoxicity, including tau abnormalities, and play an initiating role in the development of AD pathology. In this study, we focused on the unclarified issue of whether the neurotoxicity of Aß oligomers is a reversible process. Using a primary neuron culture model, we examined whether the neurotoxic effects induced by 2-day treatment with Aß42 oligomers (Aß-O) are reversible during a subsequent 2-day withdrawal period. Aß-O treatment resulted in activation of caspase-3 and eIF2α, effects that were considerably attenuated following Aß-O removal. Immunocytochemical analyses revealed that Aß-O induced aberrant phosphorylation and caspase-mediated cleavage of tau, both of which were mostly reversed by Aß-O removal. Furthermore, Aß-O caused intraneuronal dislocation of ß-catenin protein and a reduction in its levels, and these alterations were partially reversed upon Aß-O withdrawal. The dislocation of ß-catenin appeared to reflect synaptic disorganization. These findings indicate that removal of extracellular Aß-O can fully or partially reverse Aß-O-induced neurotoxic alterations in our neuron model. Accordingly, we propose that the induction of neurotoxicity by Aß oligomers is a reversible process, which has important implications for the development of AD therapies.

Amyloid ß-protein oligomers upregulate the ß-secretase, BACE1, through a post-translational mechanism involving its altered subcellular distribution in neurons.

BACKGROUND: ß-Site amyloid precursor protein cleaving enzyme 1 (BACE1) is a membrane-bound aspartyl protease that initiates amyloid ß-protein (Aß) generation. Aberrant elevation of BACE1 levels in brains of Alzheimer's disease (AD) patients may involve Aß. In the present study, we used a neuron culture model system to investigate the effects of Aß on BACE1 expression as well as the underlying mechanisms. RESULTS: Rat primary cortical neurons were treated with relatively low concentrations (2.5 µM) of Aß42 oligomers (Aß-O) or fibrils (Aß-F) for 2-3 days. Aß-O induced a significant increase in protein levels of BACE1, while Aß-F only had a marginal effect. Levels of amyloid precursor protein (APP) and the major α-secretase, ADAM10, remained unaltered upon treatment with both types of Aß. Aß-O treatment resulted in activation of eIF2α and caspase 3 in a time-dependent manner, with no changes in the endoplasmic reticulum (ER) stress marker, GRP78, indicating that a typical ER stress response is not induced under our experimental conditions. Furthermore, Aß-O did not affect BACE1 mRNA expression but augmented the levels of exogenous BACE1 expressed via recombinant adenoviruses, indicating regulation of BACE1 protein expression, not at the transcriptional or translational but the post-translational level. Immunocytochemical analysis revealed that Aß-O causes a significant increase in BACE1 immunoreactivity in neurites (both axons and dendrites), but not soma of neurons; this change appears relevant to the mechanism of Aß-O-induced BACE1 elevation, which may involve impairment of BACE1 trafficking and degradation. In contrast, Aß-O had no effect on APP immunoreactivity. CONCLUSION: Our results collectively suggest that Aß oligomers induce BACE1 elevation via a post-translational mechanism involving its altered subcellular distribution in neurons, which possibly triggers a vicious cycle of Aß generation, thus contributing to the pathogenetic mechanism of AD.

LRP1 Downregulates the Alzheimer's ß-Secretase BACE1 by Modulating Its Intraneuronal Trafficking

The ß-secretase called BACE1 is a membrane-associated protease that initiates the generation of amyloid ß-protein (Aß), a key event in Alzheimer's disease (AD). However, the mechanism of intraneuronal regulation of BACE1 is poorly understood. Here, we present evidence that low-density lipoprotein receptor-related protein 1 (LRP1), a multi-functional receptor, has a previously unrecognized function to regulate BACE1 in neurons. We show that deficiency of LRP1 exerts promotive effects on the protein expression and function of BACE1, whereas expression of LRP-L4, a functional LRP1 mini-receptor, specifically decreases BACE1 levels in both human embryonic kidney (HEK) 293 cells and rat primary neurons, leading to reduced Aß production. Our subsequent analyses further demonstrate that (1) both endogenous and exogenous BACE1 and LRP1 interact with each other and are colocalized in soma and neurites of primary neurons, (2) LRP1 reduces the protein stability and cell-surface expression of BACE1, and (3) LRP1 facilitates the shift in intracellular localization of BACE1 from early to late endosomes, thereby promoting lysosomal degradation. These findings establish that LRP1 specifically downregulates BACE1 by modulating its intraneuronal trafficking and stability through protein interaction and highlight LRP1 as a potential therapeutic target in AD.

Glucocorticoid suppresses dendritic spine development mediated by down-regulation of caldesmon expression.

Glucocorticoids (GCs) mediate the effects of stress to cause structural plasticity in brain regions such as the hippocampus, including simplification of dendrites and shrinkage of dendritic spines. However, the molecular mechanics linking stress and GCs to these effects remain largely unclear. Here, we demonstrated that corticosterone (CORT) reduces the expression levels of caldesmon (CaD), causing dendritic spines to become vulnerable. CaD regulates cell motility by modulating the actin-myosin system and actin filament stability. In cultured rat hippocampal neurons, CaD localized to dendritic spines by binding to filamentous actin (F-actin), and CaD expression levels increased during spine development. CaD stabilized the F-actin dynamics in spines, thereby enlarging the spine heads, whereas CaD knockdown decreased the spine-head size via destabilization of the F-actin dynamics. CaD was also required for chemical LTP-induced actin stabilization. The CaD expression levels were markedly decreased by exposure to CORT mediated by suppression of serum response factor-dependent transcription. High CORT levels reduced both the spine-head size and F-actin stability similarly to CaD knockdown, and overexpressing CaD abolished the detrimental effect of CORT on dendritic spine development. These results indicate that CaD enlarges the spine-head size by stabilizing F-actin dynamics, and that CaD is a critical target in the GC-induced detrimental effects on dendritic spine development.