Faculty Bibliography

Background: The risk of developing Alzheimer's disease (AD) is magnified in individuals with metabolic dysfunction, specifically obesity and type 2 diabetes. In cases of insulin deficiency or resistance, elevated and fluctuating levels of blood glucose lead to the production of Advanced Glycation End Products (AGEs) that bind their receptor, RAGE, with pathological consequences. AGE-RAGE ligand binding induces intracellular signaling cascades, in part via the formin signal transduction effector, diaphanous- 1 (mDia1). Activation of this cellular mechanism activates NF-KB activation, upregulates pro-inflammatory molecules, increases RAGE expression, and ignites a positive feedback loop driving chronic inflammation in the periphery. Given that AGEs are increased in the brains of both diabetic and AD patients, here we investigate AGE-RAGE binding and subsequent mDia1 signal transduction as a possible mechanism of neuroinflammation, which contributes to the pathogenesis of AD. Methods: mDia1 and RAGE expression in microglia was evaluated by Immunoflourescent IHC in temporal cortex brain slices of AD and Non-Demented Aged human tissue. CD11b+ microglia were isolated from young (<10 month) and old (>16 month) APP London mice and aged matched controls and subjected to molecular analysis. BV-2 microglial- like cells were stimulated by the prototypic RAGE ligand, Carboxy Methyl Lysine (CML-AGE) (100 mug/mL) for 24 h, harvested, and Ager (RAGE), Drf1 (mDia1), Cd36, and Cd68RNA transcript levels were analyzed by q-RT-PCR. Results: mDia1 and RAGE are highly expressed in human AD and aged brain and colocalize, at least in part, to CD68+ activated microglia. mDia1 is highly expressed in CD11b+ microglia from APP London mice vs. aged matched controls. CML-AGE stimulated BV-2 cells display a 2-fold increased expression of mDia1, RAGE, CD36, and CD68 vs. vehicle treatment. Conclusions: RAGE and mDia1 are highly expressed in AD human brain, supporting their possible role in mediating pathogenesis. Acute stimulation with RAGE ligands upregulates inflammatory and phagocytosis markers in cultured BV-2 cells. Primary isolates of murine CD11b+ microglia demonstrate high levels of expression of mDia. These data suggest that RAGE may exert its pathogenic effects in AD brain, at least in part via mDia1-mediated neuroinflammation, thereby driving the AD phenotype. (Figure presented)

Research indicates that female risk of developing Alzheimer's disease (AD) is greater than that of males. Moderate reduction of calorie intake, known as calorie restriction (CR), reduces pathology in AD mouse models and is a potentially translatable prevention measure for individuals at-risk for AD, as well as an important tool for understanding how the brain endogenously attenuates age-related pathology. Whether sex influences the response to CR remains unknown. In this study, we assessed the effect of CR on beta-amyloid peptide (Abeta) pathology and hippocampal CA1 neuron specific gene expression in the Tg2576 mouse model of cerebral amyloidosis. Relative to ad libitum (AL) feeding, CR feeding significantly reduced hippocampal Abeta burden in 15-month-old female, but not age-matched male, Tg2576 mice. Sustained CR also significantly reduced expression of presenilin enhancer 2 (Psenen) and presenilin 1, components of the gamma-secretase complex, in Tg2576 females. These results indicate that long-term CR significantly reduces age-dependent female Tg2576 Abeta pathology, which is likely to involve CR-mediated reductions in gamma-secretase-dependent amyloid precursor protein (APP) metabolism.

Objectives: Given that epidemiologic studies have shown that diabetes mellitus increases the risk of Alzheimer's disease (AD), our objective was to examine the mechanistic links between the two diseases in a non-human primate. Methods: Tissue from multiple brain regions of a vervet monkey model of streptozotocin-induced type 1 diabetes (n=10 control; n=7 diabetic) was examined by Western blot analysis, sandwich ELISA, and qPCR for biochemical changes in tau protein and Abeta peptide, as well as changes in key enzymes that contribute to their processing and posttranslational modification. Results: Regional brain analyses showed a global increase in tau phosphorylation in areas vulnerable to AD pathology as well as in spared structures such as the cerebellum. An examination of tau phosphatases and kinases showed a brain-wide increase in active ERK1/2. A diabetes-induced increase in Abeta levels, however, was specific to brain regions affected during the early stages of AD pathogenesis, with the greatest increase observed in the hippocampus. Examination of the amyloid precursor protein, its metabolites, and proteins involved in the clearance and degradation of brain Abeta indicated that a hippocampal-specific decrease in the Abeta-degrading enzyme neprilysin is a major contributor to this localized Abeta increase. Conclusions: Our study suggests protein changes in the brain that link diabetes to AD risk: decreased neprilysin expression leads to an increase in Abeta in the temporal lobe structures that are at the earliest risk in AD while increased ERK1/2 activity appears to contribute to a brain-wide increase in tau phosphorylation

The entorhinal cortex (EC) is one of the first brain areas to display neuropathology in Alzheimer's disease. A mouse model which simulates amyloid-beta (Abeta) neuropathology, the Tg2576 mouse, was used to address these early changes. Here, we show EC abnormalities occur in 2- to 4-month-old Tg2576 mice, an age before Abeta deposition and where previous studies suggest that there are few behavioral impairments. First we show, using a sandwich enzyme-linked immunosorbent assay, that soluble human Abeta40 and Abeta42 are detectable in the EC of 2-month-old Tg2576 mice before Abeta deposition. We then demonstrate that 2- to 4-month-old Tg2576 mice are impaired at object placement, an EC-dependent cognitive task. Next, we show that defects in neuronal nuclear antigen expression and myelin uptake occur in the superficial layers of the EC in 2- to 4-month-old Tg2576 mice. In slices from Tg2576 mice that contained the EC, there were repetitive field potentials evoked by a single stimulus to the underlying white matter, and a greater response to reduced extracellular magnesium ([Mg2+]o), suggesting increased excitability. However, deep layer neurons in Tg2576 mice had longer latencies to antidromic activation than wild type mice. The results show changes in the EC at early ages and suggest that altered excitability occurs before extensive plaque pathology.

gamma-Secretase plays a pivotal role in the production of neurotoxic amyloid beta-peptides (Abeta) in Alzheimer disease (AD) and consists of a heterotetrameric core complex that includes the aspartyl intramembrane protease presenilin (PS). The human genome codes for two presenilin paralogs. To understand the causes for distinct phenotypes of PS paralog-deficient mice and elucidate whether PS mutations associated with early-onset AD affect the molecular environment of mature gamma-secretase complexes, quantitative interactome comparisons were undertaken. Brains of mice engineered to express wild-type or mutant PS1, or HEK293 cells stably expressing PS paralogs with N-terminal tandem-affinity purification tags served as biological source materials. The analyses revealed novel interactions of the gamma-secretase core complex with a molecular machinery that targets and fuses synaptic vesicles to cellular membranes and with the H(+)-transporting lysosomal ATPase macrocomplex but uncovered no differences in the interactomes of wild-type and mutant PS1. The catenin/cadherin network was almost exclusively found associated with PS1. Another intramembrane protease, signal peptide peptidase, predominantly co-purified with PS2-containing gamma-secretase complexes and was observed to influence Abeta production.

The histopathological hallmarks of Alzheimer disease (AD) include intraneuronal neurofibrillary tangles composed of abnormally hyperphosphorylated tau protein. Insulin dysfunction might influence AD pathology, as population-based and cohort studies have detected higher AD incidence rates in diabetic patients. But how diabetes affects tau pathology is not fully understood. In this study, we investigated the impact of insulin dysfunction on tau phosphorylation in a genetic model of spontaneous type 1 diabetes: the nonobese diabetic (NOD) mouse. Brains of young and adult female NOD mice were examined, but young NOD mice did not display tau hyperphosphorylation. tau phosphorylation at tau-1 and pS422 epitopes was slightly increased in nondiabetic adult NOD mice. At the onset of diabetes, tau was hyperphosphorylated at the tau-1, AT8, CP13, pS262, and pS422. A subpopulation of diabetic NOD mice became hypothermic, and tau hyperphosphorylation further extended to paired helical filament-1 and TG3 epitopes. Furthermore, elevated tau phosphorylation correlated with an inhibition of protein phosphatase 2A (PP2A) activity. Our data indicate that insulin dysfunction in NOD mice leads to AD-like tau hyperphosphorylation in the brain, with molecular mechanisms likely involving a deregulation of PP2A. This model may be a useful tool to address further mechanistic association between insulin dysfunction and AD pathology.

Olfaction is often impaired in Alzheimer's disease (AD) and is also dysfunctional in mouse models of the disease. We recently demonstrated that short-term passive anti-murine-Abeta immunization can rescue olfactory behavior in the Tg2576 mouse model overexpressing a human mutation of the amyloid precursor protein (APP) after beta-amyloid deposition. Here we tested the ability to preserve normal olfactory behaviors by means of long-term passive anti-murine-Abeta immunization. Seven-month-old Tg2576 and non-transgenic littermate (NTg) mice were IP-injected biweekly with the m3.2 murine-Abeta-specific antibody until 16mo of age when mice were tested in the odor habituation test. While Tg2576 mice treated with a control antibody showed elevations in odor investigation times and impaired odor habituation compared to NTg, olfactory behavior was preserved to NTg levels in m3.2-immunized Tg2576 mice. Immunized Tg2576 mice had significantly less beta-amyloid immunolabeling in the olfactory bulb and entorhinal cortex, yet showed elevations in Thioflavin-S labeled plaques in the piriform cortex. No detectable changes in APP metabolite levels other than Abeta were found following m3.2 immunization. These results demonstrate efficacy of chronic, long-term anti-murine-Abeta m3.2 immunization in preserving normal odor-guided behaviors in a human APP Tg model. Further, these results provide mechanistic insights into olfactory dysfunction as a biomarker for AD by yielding evidence that focal reductions of Abeta may be sufficient to preserve olfaction.

Although anti-human beta-amyloid (Abeta) immunotherapy clears brain beta-amyloid plaques in Alzheimer's disease (AD), targeting additional brain plaque constituents to promote clearance has not been attempted. Endogenous murine Abeta is a minor Abeta plaque component in amyloid precursor protein (APP) transgenic AD models, which we show is approximately 3%-8% of the total accumulated Abeta in various human APP transgenic mice. Murine Abeta codeposits and colocalizes with human Abeta in amyloid plaques, and the two Abeta species coimmunoprecipitate together from brain extracts. In the human APP transgenic mouse model Tg2576, passive immunization for 8 weeks with a murine-Abeta-specific antibody reduced beta-amyloid plaque pathology, robustly decreasing both murine and human Abeta levels. The immunized mice additionally showed improvements in two behavioral assays, odor habituation and nesting behavior. We conclude that passive anti-murine Abeta immunization clears Abeta plaque pathology-including the major human Abeta component-and decreases behavioral deficits, arguing that targeting minor endogenous brain plaque constituents can be beneficial, broadening the range of plaque-associated targets for AD therapeutics.

Early endosomal changes, a prominent pathology in neurons early in Alzheimer's disease, also occur in neurons and peripheral tissues in Down syndrome. While in Down syndrome models increased amyloid-beta protein precursor (AbetaPP) expression is known to be a necessary contributor on the trisomic background to this early endosomal pathology, increased AbetaPP alone has yet to be shown to be sufficient to drive early endosomal alterations in neurons. Comparing two AbetaPP transgenic mouse models, one that contains the AbetaPP Swedish K670N/M671L double mutation at the beta-cleavage site (APP23) and one that has the AbetaPP London V717I mutation near the gamma-cleavage site (APPLd2), we show significantly altered early endosome morphology in fronto-parietal neurons as well as enlargement of early endosomes in basal forebrain cholinergic neurons of the medial septal nucleus in the APP23 model, which has the higher levels of AbetaPP beta-C-terminal fragment (betaCTF) accumulation. Early endosomal changes correlate with a marked loss of the cholinergic population, which is consistent with the known dependence of the large projection cholinergic cells on endosome-mediated retrograde neurotrophic transport. Our findings support the idea that increased expression of AbetaPP and AbetaPP metabolites in neurons is sufficient to drive early endosomal abnormalities in vivo, and that disruption of the endocytic system is likely to contribute to basal forebrain cholinergic vulnerability.

We report that neuronal overexpression of the endogenous inhibitor of calpains, calpastatin (CAST), in a mouse model of human Alzheimer's disease (AD) beta-amyloidosis, the APP23 mouse, reduces beta-amyloid (Abeta) pathology and Abeta levels when comparing aged, double transgenic (tg) APP23/CAST with APP23 mice. Concurrent with Abeta plaque deposition, aged APP23/CAST mice show a decrease in the steady-state brain levels of the amyloid precursor protein (APP) and APP C-terminal fragments (CTFs) when compared with APP23 mice. This CAST-dependent decrease in APP metabolite levels was not observed in single tg CAST mice expressing endogenous APP or in younger, Abeta plaque predepositing APP23/CAST mice. We also determined that the CAST-mediated inhibition of calpain activity in the brain is greater in the CAST mice with Abeta pathology than in non-APP tg mice, as demonstrated by a decrease in calpain-mediated cytoskeleton protein cleavage. Moreover, aged APP23/CAST mice have reduced extracellular signal-regulated kinase 1/2 (ERK1/2) activity and tau phosphorylation when compared with APP23 mice. In summary, in vivo calpain inhibition mediated by CAST transgene expression reduces Abeta pathology in APP23 mice, with our findings further suggesting that APP metabolism is modified by CAST overexpression as the mice develop Abeta pathology. Our results indicate that the calpain system in neurons is more responsive to CAST inhibition under conditions of Abeta pathology, suggesting that in the disease state neurons may be more sensitive to the therapeutic use of calpain inhibitors.