Prostaglandin I2 upregulates the expression of anterior pharynx‐defective‐1α and anterior pharynx‐defective‐1β in amyloid precursor protein/presenilin 1 transgenic mice

Cyclooxygenase‐2 (COX‐2) has been recently identified to be involved in the pathogenesis of Alzheimer's disease (AD). Yet, the role of an important COX‐2 metabolic product, prostaglandin (PG) I₂, in the pathogenesis of AD remains unknown. Using human‐ and mouse‐derived neuronal cells as well as amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice as model systems, we elucidated the mechanism of anterior pharynx‐defective (APH)‐1α and pharynx‐defective‐1β induction. In particular, we found that PGI₂ production increased during the course of AD development. Then, PGI₂ accumulation in neuronal cells activates PKA/CREB and JNK/c‐Jun signaling pathways by phosphorylation, which results in APH‐1α/1β expression. As PGI₂ is an important metabolic by‐product of COX‐2, its suppression by NS398 treatment decreases the expression of APH‐1α/1β in neuronal cells and APP/PS1 mice. More importantly, β‐amyloid protein (Aβ) oligomers in the cerebrospinal fluid (CSF) of APP/PS1 mice are critical for stimulating the expression of APH‐1α/1β, which was blocked by NS398 incubation. Finally, the induction of APH‐1α/1β was confirmed in the brains of patients with AD. Thus, these findings not only provide novel insights into the mechanism of PGI₂‐induced AD progression but also are instrumental for improving clinical therapies to combat AD.     

Dyrk1 inhibition improves Alzheimer's disease‐like pathology

There is an urgent need for the development of new therapeutic strategies for Alzheimer's disease (AD). The dual‐specificity tyrosine phosphorylation‐regulated kinase‐1A (Dyrk1a) is a protein kinase that phosphorylates the amyloid precursor protein (APP) and tau and thus represents a link between two key proteins involved in AD pathogenesis. Furthermore, Dyrk1a is upregulated in postmortem human brains, and high levels of Dyrk1a are associated with mental retardation. Here, we sought to determine the effects of Dyrk1 inhibition on AD‐like pathology developed by 3xTg‐AD mice, a widely used animal model of AD. We dosed 10‐month‐old 3xTg‐AD and nontransgenic (NonTg) mice with a Dyrk1 inhibitor (Dyrk1‐inh) or vehicle for eight weeks. During the last three weeks of treatment, we tested the mice in a battery of behavioral tests. The brains were then analyzed for the pathological markers of AD. We found that chronic Dyrk1 inhibition reversed cognitive deficits in 3xTg‐AD mice. These effects were associated with a reduction in amyloid‐β (Aβ) and tau pathology. Mechanistically, Dyrk1 inhibition reduced APP and insoluble tau phosphorylation. The reduction in APP phosphorylation increased its turnover and decreased Aβ levels. These results suggest that targeting Dyrk1 could represent a new viable therapeutic approach for AD.     

Intranasal insulin alleviates cognitive deficits and amyloid pathology in young adult APPswe/PS1dE9 mice

Brain insulin signaling deficits contribute to multiple pathological features of Alzheimer's disease (AD). Although intranasal insulin has shown efficacy in patients with AD, the underlying mechanisms remain largely unillustrated. Here, we demonstrate that intranasal insulin improves cognitive deficits, ameliorates defective brain insulin signaling, and strongly reduces β‐amyloid (Aβ) production and plaque formation after 6 weeks of treatment in 4.5‐month‐old APPswe/PS1dE9 (APP/PS1) mice. Furthermore, c‐Jun N‐terminal kinase activation, which plays a pivotal role in insulin resistance and AD pathologies, is significantly inhibited. The alleviation of amyloid pathology by intranasal insulin results mainly from enhanced nonamyloidogenic processing and compromised amyloidogenic processing of amyloid precursor protein (APP), and from a reduction in apolipoprotein E protein which is involved in Aβ metabolism. In addition, intranasal insulin effectively promotes hippocampal neurogenesis in APP/PS1 mice. This study, exploring the mechanisms underlying the beneficial effects of intranasal insulin on Aβ pathologies in vivo for the first time, highlights important preclinical evidence that intranasal insulin is potentially an effective therapeutic method for the prevention and treatment of AD.     

HDAC3 negatively regulates spatial memory in a mouse model of Alzheimer's disease

The accumulation and deposition of beta‐amyloid (Aβ) is a key neuropathological hallmark of Alzheimer's disease (AD). Histone deacetylases (HDACs) are promising therapeutic targets for the treatment of AD, while the specific HDAC isoforms associated with cognitive improvement are poorly understood. In this study, we investigate the role of HDAC3 in the pathogenesis of AD. Nuclear HDAC3 is significantly increased in the hippocampus of 6‐ and 9‐month‐old APPswe/PS1dE9 (APP/PS1) mice compared with that in age‐matched wild‐type C57BL/6 (B6) mice. Lentivirus ‐mediated inhibition or overexpression of HDAC3 was used in the hippocampus of APP/PS1 mice to investigate the role of HDAC3 in spatial memory, amyloid burden, dendritic spine density, glial activation and tau phosphorylation. Inhibition of HDAC3 in the hippocampus attenuates spatial memory deficits, as indicated in the Morris water maze test, and decreases amyloid plaque load and Aβ levels in the brains of APP/PS1 mice. Dendritic spine density is increased, while microglial activation is alleviated after HDAC3 inhibition in the hippocampus of 9‐month‐old APP/PS1 mice. Furthermore, HDAC3 overexpression in the hippocampus increases Aβ levels, activates microglia, and decreases dendritic spine density in 6‐month‐old APP/PS1 mice. In conclusion, our results indicate that HDAC3 negatively regulates spatial memory in APP/PS1 mice and HDAC3 inhibition might represent a potential therapy for the treatment of AD.     

Conditional inactivation of nicastrin restricts amyloid deposition in an Alzheimer's disease mouse model

Production of Aβ by γ‐secretase is a key event in Alzheimer's disease (AD). The γ‐secretase complex consists of presenilin (PS) 1 or 2, nicastrin (ncstn), Pen‐2, and Aph‐1 and cleaves type I transmembrane proteins, including the amyloid precursor protein (APP). Although ncstn is widely accepted as an essential component of the complex required for γ‐secretase activity, recent in vitro studies have suggested that ncstn is dispensable for APP processing and Aβ production. The focus of this study was to answer this controversy and evaluate the role of ncstn in Aβ generation and the development of the amyloid‐related phenotype in the mouse brain. To eliminate ncstn expression in the mouse brain, we used a ncstn conditional knockout mouse that we mated with an established AD transgenic mouse model (5XFAD) and a neuronal Cre‐expressing transgenic mouse (CamKIIα‐iCre), to generate AD mice (5XFAD/CamKIIα‐iCre/ncstn^f/f mice) where ncstn was conditionally inactivated in the brain. 5XFAD/CamKIIα‐iCre/ncstn^f/f mice at 10 week of age developed a neurodegenerative phenotype with a significant reduction in Aβ production and formation of Aβ aggregates and the absence of amyloid plaques. Inactivation of nctsn resulted in substantial accumulation of APP‐CTFs and altered PS1 expression. These results reveal a key role for ncstn in modulating Aβ production and amyloid plaque formation in vivo and suggest ncstn as a target in AD therapeutics.     

Deferoxamine inhibits iron induced hippocampal tau phosphorylation in the Alzheimer transgenic mouse brain

Prior work has shown that iron interacts with hyperphosphorylated tau, which contributes to the formation of neurofibrillary tangles (NFTs) in Alzheimer’s disease (AD), whereas iron chelator desferrioxamine (DFO) slows down the clinical progression of the cognitive decline associated with this disease. However, the effects of DFO on tau phosphorylation in the presence or absence of iron have yet to be determined. Using amyloid precursor protein (APP) and presenilin 1 (PS1) double transgenic mouse brain as a model system, we investigated the effects and potential mechanisms of intranasal administration of DFO on iron induced abnormal tau phosphorylation. High-dose iron treatment markedly increased the levels of tau phosphorylation at the sites of Thr205, Thr231 and Ser396, whereas highly induced tau phosphorylation was abolished by intranasal administration of DFO in APP/PS1 transgenic mice. Moreover, DFO intranasal administration also decreases Fe-induced the activities of cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3β (GSK3β), which in turn suppressing tau phosphorylation. Cumulatively, our data show that intranasal DFO treatment exerts its suppressive effects on iron induced tau phosphorylation via CDK5 and GSK3β pathways. More importantly, elucidation of DFO mechanism in suppressing tau phosphorylation may provide insights for developing therapeutic strategies to combat AD.     

Mutant presenilin‐1 deregulated peripheral immunity exacerbates Alzheimer‐like pathology

Mutations in the presenilin‐1 (PS1) gene are independent causes of familial Alzheimer's disease (AD). AD patients have dysregulated immunity, and PS1 mutant mice exhibit abnormal systemic immune responses. To test whether immune function abnormality caused by a mutant human PS1 gene (mhPS1) could modify AD‐like pathology, we reconstituted immune systems of AD model mice carrying a mutant human amyloid precursor protein gene (mhAPP; Tg2576 mice) or both mhAPP and mhPS1 genes (PSAPP mice) with allo‐geneic bone marrow cells. Here, we report a marked reduction in amyloid‐β (Aβ) levels, β‐amyloid plaques and brain inflammatory responses in PSAPP mice following strain‐matched wild‐type PS1 bone marrow reconstitution. These effects occurred with immune switching from pro‐inflammatory T helper (Th) 1 to anti‐inflammatory Th2 immune responses in the periphery and in the brain, which likely instructed microglia to phagocytose and clear Aβ in an ex vivo assay. Conversely, Tg2576 mice displayed accelerated AD‐like pathology when reconstituted with mhPS1 bone marrow. These data show that haematopoietic cells bearing the mhPS1 transgene exacerbate AD‐like pathology, suggesting a novel therapeutic strategy for AD based on targeting PS1 in peripheral immune cells.     

The cytoplasmic domain of Alzheimer's amyloid precursor protein is phosphorylated at Thr654, Ser655, and Thr668 in adult rat brain and cultured cells.

BACKGROUND: The cytoplasmic domain of the Alzheimer's disease amyloid precursor protein (APP) is phosphorylated in vitro at Thr654 and Ser655, and both in vitro and in intact cells at Thr668 (numbering for APP695 isoform). MATERIALS AND METHODS: We have developed phosphorylation state-specific antibodies to each of the sites, and we have used these to analyze the phosphorylation of APP in adult rat brain and in cultured cell lines. RESULTS: We demonstrate that all three sites in APP are phosphorylated in adult rat brain. Phosphorylation at Thr654, Ser655, and Thr668 was also observed in several cultured cell lines. In PC12 cells, phosphorylation at Ser655 was increased more than 10-fold by treatment with okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, but was not affected by activators of protein kinase C. In HeLa cells, phosphorylation at Thr668 was regulated in a cell cycle-dependent manner with near-stoichiometric phosphorylation being observed at the G2/M phase of the cell cycle. In general, phosphorylation at Ser655 was found to be highest in mature APP isoforms, whereas phosphorylation of Thr668 was highest in immature APP isoforms in cultured cells. CONCLUSIONS: The results demonstrate that phosphorylation of the cytoplasmic domain of APP occurs at Thr654, Ser655, and Thr668 under physiological conditions. The further characterization of APP phosphorylation using phosphorylation-specific antibodies may help in the elucidation of the biological function of APP.     

Mitosis-specific phosphorylation of amyloid precursor protein at Threonine 668 leads to its altered processing and association with centrosomes

Background

Atypical expression of cell cycle regulatory proteins has been implicated in Alzheimer's disease (AD), but the molecular mechanisms by which they induce neurodegeneration are not well understood. We examined transgenic mice expressing human amyloid precursor protein (APP) and presenilin 1 (PS1) for changes in cell cycle regulatory proteins to determine whether there is a correlation between cell cycle activation and pathology development in AD.

Results

Our studies in the AD transgenic mice show significantly higher levels of cyclin E, cyclin D1, E2F1, and P-cdc2 in the cells in the vicinity of the plaques where maximum levels of Threonine 668 (Thr668)-phosphorylated APP accumulation was observed. This suggests that the cell cycle regulatory proteins might be influencing plaque pathology by affecting APP phosphorylation. Using neuroglioma cells overexpressing APP we demonstrate that phosphorylation of APP at Thr668 is mitosis-specific. Cells undergoing mitosis show altered cellular distribution and localization of P-APP at the centrosomes. Also, Thr668 phosphorylation in mitosis correlates with increased processing of APP to generate Aβ and the C-terminal fragment of APP, which is prevented by pharmacological inhibitors of the G1/S transition.

Conclusions

The data presented here suggests that cell cycle-dependent phosphorylation of APP may affect its normal cellular function. For example, association of P-APP with the centrosome may affect spindle assembly and cell cycle progression, further contributing to the development of pathology in AD. The experiments with G1/S inhibitors suggest that cell cycle inhibition may impede the development of Alzheimer's pathology by suppressing modification of βAPP, and thus may represent a novel approach to AD treatment. Finally, the cell cycle regulated phosphorylation and processing of APP into Aβ and the C-terminal fragment suggest that these proteins may have a normal function during mitosis.     

NGF controls APP cleavage by downregulating APP phosphorylation at Thr668: relevance for Alzheimer's disease

NGF has been implicated in forebrain neuroprotection from amyloidogenesis and Alzheimer's disease (AD). However, the underlying molecular mechanisms are still poorly understood. Here, we investigated the role of NGF signalling in the metabolism of amyloid precursor protein (APP) in forebrain neurons using primary cultures of septal neurons and acute septo‐hippocampal brain slices. In this study, we show that NGF controls the basal level of APP phosphorylation at Thr668 (T668) by downregulating the activity of the Ser/Thr kinase JNK(p54) through the Tyr kinase signalling adaptor SH2‐containing sequence C (ShcC). We also found that the specific NGF receptor, Tyr kinase A (TrkA), which is known to bind to APP, fails to interact with the fraction of APP molecules phosphorylated at T668 (APP^pT668). Accordingly, the amount of TrkA bound to APP is significantly reduced in the hippocampus of ShcC KO mice and of patients with AD in which elevated APP^pT668 levels are detected. NGF promotes TrkA binding to APP and APP trafficking to the Golgi, where APP–BACE interaction is hindered, finally resulting in reduced generation of sAPPβ, CTFβ and amyloid‐beta (1‐42). These results demonstrate that NGF signalling directly controls basal APP phosphorylation, subcellular localization and BACE cleavage, and pave the way for novel approaches specifically targeting ShcC signalling and/or the APP–TrkA interaction in AD therapy.     

Temporal and regional progression of Alzheimer’s disease‐like pathology in 3xTg‐AD mice

Accumulation of amyloid‐β (Aβ) and fibrillary tangles, as well as neuroinflammation and memory loss, are hallmarks of Alzheimer’s disease (AD). After almost 15 years from their generation, 3xTg‐AD mice are still one of the most used transgenic models of AD. Converging evidence indicates that the phenotype of 3xTg‐AD mice has shifted over the years and contradicting reports about onset of pathology or cognitive deficits are apparent in the literature. Here, we assessed Aβ and tau load, neuroinflammation, and cognitive changes in 2‐, 6‐, 12‐, and 20‐month‐old female 3xTg‐AD and nontransgenic (NonTg) mice. We found that ~80% of the mice analyzed had Aβ plaques in the caudal hippocampus at 6 months of age, while 100% of them had Aβ plaques in the hippocampus at 12 months of age. Cortical Aβ plaques were first detected at 12 months of age, including in the entorhinal cortex. Phosphorylated Tau at Ser202/Thr205 and Ser422 was apparent in the hippocampus of 100% of 6‐month‐old mice, while only 50% of mice showed tau phosphorylation at Thr212/Ser214 at this age. Neuroinflammation was first evident in 6‐month‐old mice and increased as a function of age. These neuropathological changes were clearly associated with progressive cognitive decline, which was first apparent at 6 months of age and became significantly worse as the mice aged. These data indicate a consistent and predictable progression of the AD‐like pathology in female 3xTg‐AD mice, and will facilitate the design of future studies using these mice.     

Role of phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (PICALM) in intracellular amyloid precursor protein (APP) processing and amyloid plaque pathogenesis

One of the pathological hallmarks of Alzheimer disease is the accumulation of amyloid plaques in the extracellular space in the brain. Amyloid plaques are primarily composed of aggregated amyloid β peptide (Aβ), a proteolytic fragment of the transmembrane amyloid precursor protein (APP). For APP to be proteolytically cleaved into Aβ, it must be internalized into the cell and trafficked to endosomes where specific protease complexes can cleave APP. Several recent genome-wide association studies have reported that several single nucleotide polymorphisms (SNPs) in the phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (PICALM) gene were significantly associated with Alzheimer disease, suggesting a role in APP endocytosis and Aβ generation. Here, we show that PICALM co-localizes with APP in intracellular vesicles of N2a-APP cells after endocytosis is initiated. PICALM knockdown resulted in reduced APP internalization and Aβ generation. Conversely, PICALM overexpression increased APP internalization and Aβ production. In vivo, PICALM was found to be expressed in neurons and co-localized with APP throughout the cortex and hippocampus in APP/PS1 mice. PICALM expression was altered using AAV8 gene transfer of PICALM shRNA or PICALM cDNA into the hippocampus of 6-month-old APP/PS1 mice. PICALM knockdown decreased soluble and insoluble Aβ levels and amyloid plaque load in the hippocampus. Conversely, PICALM overexpression increased Aβ levels and amyloid plaque load. These data indicate that PICALM, an adaptor protein involved in clathrin-mediated endocytosis, regulates APP internalization and subsequent Aβ generation. PICALM contributes to amyloid plaque load in brain likely via its effect on Aβ metabolism.     

Differential transgene expression patterns in Alzheimer mouse models revealed by novel human amyloid precursor protein‐specific antibodies

Alzheimer's disease (AD) is histopathologically characterized by neurodegeneration, the formation of intracellular neurofibrillary tangles and extracellular Aβ deposits that derive from proteolytic processing of the amyloid precursor protein (APP). As rodents do not normally develop Aβ pathology, various transgenic animal models of AD were designed to overexpress human APP with mutations favouring its amyloidogenic processing. However, these mouse models display tremendous differences in the spatial and temporal appearance of Aβ deposits, synaptic dysfunction, neurodegeneration and the manifestation of learning deficits which may be caused by age‐related and brain region‐specific differences in APP transgene levels. Consequentially, a comparative temporal and regional analysis of the pathological effects of Aβ in mouse brains is difficult complicating the validation of therapeutic AD treatment strategies in different mouse models. To date, no antibodies are available that properly discriminate endogenous rodent and transgenic human APP in brains of APP‐transgenic animals. Here, we developed and characterized rat monoclonal antibodies by immunohistochemistry and Western blot that detect human but not murine APP in brains of three APP‐transgenic mouse and one APP‐transgenic rat model. We observed remarkable differences in expression levels and brain region‐specific expression of human APP among the investigated transgenic mouse lines. This may explain the differences between APP‐transgenic models mentioned above. Furthermore, we provide compelling evidence that our new antibodies specifically detect endogenous human APP in immunocytochemistry, FACS and immunoprecipitation. Hence, we propose these antibodies as standard tool for monitoring expression of endogenous or transfected APP in human cells and APP expression in transgenic animals.     

Specificity of presenilin‐1‐ and presenilin‐2‐dependent γ‐secretases towards substrate processing

The two presenilin‐1 (PS1) and presenilin‐2 (PS2) homologs are the catalytic core of the γ‐secretase complex, which has a major role in cell fate decision and Alzheimer's disease (AD) progression. Understanding the precise contribution of PS1‐ and PS2‐dependent γ‐secretases to the production of β‐amyloid peptide (Aβ) from amyloid precursor protein (APP) remains an important challenge to design molecules efficiently modulating Aβ release without affecting the processing of other γ‐secretase substrates. To that end, we studied PS1‐ and PS2‐dependent substrate processing in murine cells lacking presenilins (PSs) (PS1KO, PS2KO or PS1‐PS2 double‐KO noted PSdKO) or stably re‐expressing human PS1 or PS2 in an endogenous PS‐null (PSdKO) background. We characterized the processing of APP and Notch on both endogenous and exogenous substrates, and we investigated the effect of pharmacological inhibitors targeting the PSs activity (DAPT and L‐685,458). We found that murine PS1 γ‐secretase plays a predominant role in APP and Notch processing when compared to murine PS2 γ‐secretase. The inhibitors blocked more efficiently murine PS2‐ than murine PS1‐dependent processing. Human PSs, especially human PS1, expression in a PS‐null background efficiently restored APP and Notch processing. Strikingly, and contrary to the results obtained on murine PSs, pharmacological inhibitors appear to preferentially target human PS1‐ than human PS2‐dependent γ‐secretase activity.     

Nitric oxide‐mediated regulation of ‐amyloid clearance via alterations of MMP‐9/TIMP‐1

Fibrillar amyloid plaques are largely composed of amyloid‐beta (Aβ) peptides that are metabolized into products, including Aβ1‐16, by proteases including matrix metalloproteinase 9 (MMP‐9). The balance between production and degradation of Aβ proteins is critical to amyloid accumulation and resulting disease. Regulation of MMP‐9 and its endogenous inhibitor tissue inhibitor of metalloproteinase (TIMP)‐1 by nitric oxide (NO) has been shown. We hypothesize that nitric oxide synthase (NOS2) protects against Alzheimer's disease pathology by increasing amyloid clearance through NO regulation of MMP‐9/TIMP‐1 balance. We show NO‐mediated increased MMP‐9/TIMP‐1 ratios enhanced the degradation of fibrillar Aβ in vitro, which was abolished when silenced for MMP‐9 protein translation. The in vivo relationship between MMP‐9, NO and Aβ degradation was examined by comparing an Alzheimer's disease mouse model that expresses NOS2 with a model lacking NOS2. To quantitate MMP‐9 mediated changes, we generated an antibody recognizing the Aβ1‐16 fragment, and used mass spectrometry multi‐reaction monitoring assay for detection of immunoprecipitated Aβ1‐16 peptides. Aβ1‐16 levels decreased in brain lysates lacking NOS2 when compared with strains that express human amyloid precursor protein on the NOS2 background. TIMP‐1 increased in the APPSwDI/NOS2^?/? mice with decreased MMP activity and increased amyloid burden, thereby supporting roles for NO in the regulation of MMP/TIMP balance and plaque clearance.     

Identification of new Presenilin‐1 phosphosites: implication for γ‐secretase activity and Aβ production

An important pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid‐beta (Aβ) peptides in the brain parenchyma, leading to neuronal death and impaired learning and memory. The protease γ‐secretase is responsible for the intramembrane proteolysis of the amyloid‐β precursor protein (APP), which leads to the production of the toxic Aβ peptides. Thus, an attractive therapeutic strategy to treat AD is the modulation of the γ‐secretase activity, to reduce Aβ42 production. Because phosphorylation of proteins is a post‐translational modification known to modulate the activity of many different enzymes, we used electrospray (LC‐MS/MS) mass spectrometry to identify new phosphosites on highly purified human γ‐secretase. We identified 11 new single or double phosphosites in two well‐defined domains of Presenilin‐1 (PS1), the catalytic subunit of the γ‐secretase complex. Next, mutagenesis and biochemical approaches were used to investigate the role of each phosphosite in the maturation and activity of γ‐secretase. Together, our results suggest that the newly identified phosphorylation sites in PS1 do not modulate γ‐secretase activity and the production of the Alzheimer's Aβ peptides. Individual PS1 phosphosites shall probably not be considered therapeutic targets for reducing cerebral Aβ plaque formation in AD.

     

Metabolic syndrome exacerbates amyloid pathology in a comorbid Alzheimer's mouse model

Alzheimer's disease (AD) often coexists with other aging-associated diseases including obesity, diabetes, hypertension, and cardiovascular diseases. The early stage of these comorbidities is known as metabolic syndrome (MetS) which is highly prevalent in mid-life. An important cause of MetS is the deficiency of SIRT3, a mitochondrial deacetylase which enhances the functions of critical mitochondrial proteins, including metabolic enzymes, by deacetylation. Deletion of Sirt3 gene has been reported to result in the acceleration of MetS. In a recently published study, we demonstrated in the brain of Sirt3^−/− mice, downregulation of metabolic enzymes, insulin resistance and elevation of inflammatory markers including microglial proliferation. These findings suggested a novel pathway that could link SIRT3 deficiency to neuroinflammation, an important cause of Alzheimer's pathogenesis. Therefore, we hypothesized that MetS and amyloid pathology may interact through converging pathways of insulin resistance and neuroinflammation in comorbid AD. To investigate these interactions, we crossed Sirt3^−/− mice with APP/PS1 mice and successfully generated APP/PS1/Sirt3^−/− mice with amyloid pathology and MetS. In these comorbid AD mice, we observed exacerbation of insulin resistance, glucose intolerance, amyloid plaque deposition, markers of neuroinflammation, including elevated expression of IL-1β, TNF-α and Cox-2 at 8 months of age. There was also increased microglial proliferation and activation. Our observations suggest a novel mechanism by which MetS may interact with amyloid pathology during the cellular phase of AD. Therapeutic targeting of SIRT3 in AD with comorbidities may produce beneficial effects.