Disrupted HSF1 regulation in normal and exceptional brain aging.

Brain aging is a major risk factor for cognitive diseases such as Alzheimer's disease (AD) and vascular dementia. The rate of aging and age-related pathology are modulated by stress responses and repair pathways that gradually decline with age. However, recent reports indicate that exceptional longevity sustains and may even enhance the stress response. Whether normal and exceptional aging result in either attenuated or enhanced stress responses across all organs is unknown. This question arises from our understanding that biological age differs from chronological age and evidence that the rate of aging varies between organs. Thus, stress responses may differ between organs and depend upon regenerative capacity and ability to manage damaged proteins and proteotoxicity. To answer these questions, we assessed age-dependent changes in brain stress responses with normally aged wild type and long-lived Dwarf mice. Results from this study show that normal aging unfavorably impacts activation of the brain heat shock (HS) axis with key changes noted in the transcription factor, HSF1, and its regulation. Exceptional aging appears to preserve and strengthen many elements of HSF1 activation in the brain. These results support the possibility that reconstitution of aging brain stress responses requires a multi-factorial approach that addresses HSF1 protein levels, its DNA binding, and regulatory elements such as phosphorylation and protein interactions.

Augmentation of the heat shock axis during exceptional longevity in Ames dwarf mice.

How the heat shock axis, repair pathways, and proteostasis impact the rate of aging is not fully understood. Recent reports indicate that normal aging leads to a 50% change in several regulatory elements of the heat shock axis. Most notably is the age-dependent enhancement of inhibitory signals associated with accumulated heat shock proteins and hyper-acetylation associated with marked attenuation of heat shock factor 1 (HSF1)-DNA binding activity. Because exceptional longevity is associated with increased resistance to stress, this study evaluated regulatory check points of the heat shock axis in liver extracts from 12 months and 24 months long-lived Ames dwarf mice and compared these findings with aging wild-type mice. This analysis showed that 12M dwarf and wild-type mice have comparable stress responses, whereas old dwarf mice, unlike old wild-type mice, preserve and enhance activating elements of the heat shock axis. Old dwarf mice thwart negative regulation of the heat shock axis typically observed in usual aging such as noted in HSF1 phosphorylation at Ser307 residue, acetylation within its DNA binding domain, and reduction in proteins that attenuate HSF1-DNA binding. Unlike usual aging, dwarf HSF1 protein and mRNA levels increase with age and further enhance by stress. Together these observations suggest that exceptional longevity is associated with compensatory and enhanced HSF1 regulation as an adaptation to age-dependent forces that otherwise downregulate the heat shock axis.

IGF-1R Inhibitor Ameliorates Neuroinflammation in an Alzheimer's Disease Transgenic Mouse Model.

Aging is a major risk factor for Alzheimer's disease (AD). Insulin-like growth factor-1 receptor (IGF-1R) regulates general aging and lifespan. However, the contribution of IGF-1 to age-related AD pathology and progression is highly controversial. Based on our previous work, AßPP/PS1 double transgenic mice, which express human mutant amyloid precursor protein (APP) and presenilin-1 (PS-1), demonstrated a decrease in brain IGF-1 levels when they were crossed with IGF-1 deficient Ames dwarf mice (df/df). Subsequently, a reduction in gliosis, amyloid-ß (Aß) plaque deposition, and Aß1-40/42 concentrations were observed in this mouse model. This supported the hypothesis that IGF-1 may contribute to the progression of the disease. To assess the role of IGF-1 in AD, 9-10-month-old male littermate control wild type and AßPP/PS1 mice were randomly divided into two treatment groups including control vehicle (DMSO) and picropodophyllin (PPP), a selective, competitive, and reversible IGF-1R inhibitor. The brain penetrant inhibitor was given ip. at 1 mg/kg/day. Mice were sacrificed after 7 days of daily injection and the brains, spleens, and livers were collected to quantify histologic and biochemical changes. The PPP-treated AßPP/PS1 mice demonstrated attenuated insoluble Aß1-40/42. Additionally, an attenuation in microgliosis and protein p-tyrosine levels was observed due to drug treatment in the hippocampus. Our data suggest IGF-1R signaling is associated with disease progression in this mouse model. More importantly, modulation of the brain IGF-1R signaling pathway, even at mid-life, was enough to attenuate aspects of the disease phenotype. This suggests that small molecule therapy targeting the IGF-1R pathway may be viable for late-stage disease treatment.

Multifactorial Attenuation of the Murine Heat Shock Response With Age.

Age-dependent perturbation of the cellular stress response affects proteostasis and other key functions relevant to cellular action and survival. Central to age-related changes in the stress response is loss of heat shock factor 1 (HSF1)-DNA binding and transactivation properties. This report elucidates how age alters different checkpoints of HSF1 activation related to posttranslational modification and protein interactions. When comparing liver extracts from middle aged (12 M) and old (24 M) mice, significant differences are found in HSF1 phosphorylation and acetylation. HSF1 protein levels and messenger RNA decline with age, but its protein levels are stress-inducible and exempt from age-dependent changes. This surprising adaptive change in the stress response has additional implications for aging and chronic physiological stress that might explain an age-dependent dichotomy of HSF1 protein levels that are low in neurodegeneration and elevated in cancer.

Temporal progression of Alzheimer's disease in brains and intestines of transgenic mice.

The amyloid beta (Aß) peptide is associated with the neurodegenerative and inflammatory changes in brains affected by Alzheimer's disease (AD). We hypothesized that the enteric nervous system also produces Aß in an intestinal component of disease. To test this idea, we compared C57BL/6 wild-type (WT) male and female mice to two models of Alzheimer's disease, amyloid precursor protein (APP)/presenilin 1 (PS1) mice and amyloid precursor protein NL-G-F (AppNL-G-F) mice, at 3, 6, and 12 months of age. Brain Aß plaque deposition in AppNL-G-F mice preceded that in the APP/PS1 mice, observable by 3 months. Three-month-old female AppNL-G-F mice had decreased intestinal motility compared with WT and APP/PS1 mice. However, 3-month-old female APP/PS1 mice demonstrated increased intestinal permeability compared with WT and AppNL-G-F mice. Both sexes of APP/PS1 and AppNL-G-F mice demonstrated increased colon lipocalin 2 mRNA and insoluble Aß 1-42 levels at 3 months. These data demonstrate an unrecognized enteric aspect of disease in 2 different mouse models correlating with the earliest brain changes.

Ablation of amyloid precursor protein increases insulin-degrading enzyme levels and activity in brain and peripheral tissues.

The amyloid precursor protein (APP) is a type I transmembrane glycoprotein widely studied for its role as the source of ß-amyloid peptide, accumulation of which is causal in at least some cases of Alzheimer's disease (AD). APP is expressed ubiquitously and is involved in diverse biological processes. Growing bodies of evidence indicate connections between AD and somatic metabolic disorders related to type 2 diabetes, and App-/- mice show alterations in glycemic regulation. We find that App-/- mice have higher levels of insulin-degrading enzyme (IDE) mRNA, protein, and activity compared with wild-type controls. This regulation of IDE by APP was widespread across numerous tissues, including liver, skeletal muscle, and brain as well as cell types within neural tissue, including neurons, astrocytes, and microglia. RNA interference-mediated knockdown of APP in the SIM-A9 microglia cell line elevated IDE levels. Fasting levels of blood insulin were lower in App-/- than App+/+ mice, but the former showed a larger increase in response to glucose. These low basal levels may enhance peripheral insulin sensitivity, as App-/- mice failed to develop impairment of glucose tolerance on a high-fat, high-sucrose ("Western") diet. Insulin levels and insulin signaling were also lower in the App-/- brain; synaptosomes prepared from App-/- hippocampus showed diminished insulin receptor phosphorylation compared with App+/+ mice when stimulated ex vivo. These findings represent a new molecular link connecting APP to metabolic homeostasis and demonstrate a novel role for APP as an upstream regulator of IDE in vivo.

In utero exposure to fine particulate matter results in an altered neuroimmune phenotype in adult mice.

Environmental exposure to air pollution has been linked to a number of health problems including organ rejection, lung damage and inflammation. While the deleterious effects of air pollution in adult animals are well documented, the long-term consequences of particulate matter (PM) exposure during animal development are uncertain. In this study we tested the hypothesis that environmental exposure to PM 2.5 µm in diameter in utero promotes long term inflammation and neurodegeneration. We evaluated the behavior of PM exposed animals using several tests and observed deficits in spatial memory without robust changes in anxiety-like behavior. We then examined how this affects the brains of adult animals by examining proteins implicated in neurodegeneration, synapse formation and inflammation by western blot, ELISA and immunohistochemistry. These tests revealed significantly increased levels of COX2 protein in PM2.5 exposed animal brains in addition to changes in synaptophysin and Arg1 proteins. Exposure to PM2.5 also increased the immunoreactivity for GFAP, a marker of activated astrocytes. Cytokine concentrations in the brain and spleen were also altered by PM2.5 exposure. These findings indicate that in utero exposure to particulate matter has long term consequences which may affect the development of both the brain and the immune system in addition to promoting inflammatory change in adult animals.

NFATc2 Modulates Microglial Activation in the AßPP/PS1 Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) brains are characterized by fibrillar amyloid-ß (Aß) peptide containing plaques and associated reactive microglia. The proinflammatory phenotype of the microglia suggests that they may negatively affect disease course and contribute to behavioral decline. This hypothesis predicts that attenuating microglial activation may provide benefit against disease. Prior work from our laboratory and others has characterized a role for the transcription factor, nuclear factor of activated T cells (NFAT), in regulating microglial phenotype in response to different stimuli, including Aß peptide. We observed that the NFATc2 isoform was the most highly expressed in murine microglia cultures, and inhibition or deletion of NFATc2 was sufficient to attenuate the ability of the microglia to secrete cytokines. In order to determine whether the NFATc2 isoform, in particular, was a valid immunomodulatory target in vivo, we crossed an NFATc2-/- line to a well-known AD mouse model, an AßPP/PS1 mouse line. As expected, the AßPP/PS1 x NFATc2-/- mice had attenuated cytokine levels compared to AßPP/PS1 mice as well as reduced microgliosis and astrogliosis with no effect on plaque load. Although some species differences in relative isoform expression may exist between murine and human microglia, it appears that microglial NFAT activity is a viable target for modulating the proinflammatory changes that occur during AD.

APP Regulates Microglial Phenotype in a Mouse Model of Alzheimer's Disease.

UNLABELLED: Prior work suggests that amyloid precursor protein (APP) can function as a proinflammatory receptor on immune cells, such as monocytes and microglia. Therefore, we hypothesized that APP serves this function in microglia during Alzheimer's disease. Although fibrillar amyloid ß (Aß)-stimulated cytokine secretion from both wild-type and APP knock-out (mAPP(-/-)) microglial cultures, oligomeric Aß was unable to stimulate increased secretion from mAPP(-/-) cells. This was consistent with an ability of oligomeric Aß to bind APP. Similarly, intracerebroventricular infusions of oligomeric Aß produced less microgliosis in mAPP(-/-) mice compared with wild-type mice. The mAPP(-/-) mice crossed to an APP/PS1 transgenic mouse line demonstrated reduced microgliosis and cytokine levels and improved memory compared with wild-type mice despite robust fibrillar Aß plaque deposition. These data define a novel function for microglial APP in regulating their ability to acquire a proinflammatory phenotype during disease. SIGNIFICANCE STATEMENT: A hallmark of Alzheimer's disease (AD) brains is the accumulation of amyloid ß (Aß) peptide within plaques robustly invested with reactive microglia. This supports the notion that Aß stimulation of microglial activation is one source of brain inflammatory changes during disease. Aß is a cleavage product of the ubiquitously expressed amyloid precursor protein (APP) and is able to self-associate into a wide variety of differently sized and structurally distinct multimers. In this study, we demonstrate both in vitro and in vivo that nonfibrillar, oligomeric forms of Aß are able to interact with the parent APP protein to stimulate microglial activation. This provides a mechanism by which metabolism of APP results in possible autocrine or paracrine Aß production to drive the microgliosis associated with AD brains.

An improved approach to align and embed multiple brain samples in a gelatin-based matrix for simultaneous histological processing.

BACKGROUND: Preparation and processing of free-floating histological sections involve a series of steps. The amount of labor, particularly sectioning and mounting, quickly multiplies as the number of samples increases. Embedding tissue samples in a flexible matrix allows simultaneous handling of multiple samples and preserves the integrity of the tissue during histological processing. However, aligning multiple asymmetrical samples, for example small-animal brains, in a particular orientation requires skillful arrangement and securing of the samples by pinning onto a solid surface. Consequently, costly technical services offered by contract research organizations are often sought. NEW METHOD: An improved approach to align and embed multiple whole or half rodent brain samples into a gelatin-based matrix is described. Using a template specifically designed to form arrayed mouse brain-shaped cavities, a "receiving matrix" is prepared. Inserting brain samples directly into the cavities allows the samples to be effortlessly positioned into a uniform orientation and embedded in a block of matrix. RESULTS: Multiple mouse brains were arrayed in a uniform orientation in a gelatin matrix block with ease using the receiving matrix. The gelatin-embedded brains were simultaneously sectioned and stained, and effortlessly mounted onto glass slides. COMPARISON WITH EXISTING METHODS: The improved approach allowed multiple whole or half mouse brains to be easily arrayed without pinning the samples onto a solid surface and prevented damages or shifting of the samples during embedding. CONCLUSIONS: The new approach to array multiple brain samples provides a simple way to prepare gelatin-embedded whole or half brain arrays of commercial quality.

Characterization of Novel Src Family Kinase Inhibitors to Attenuate Microgliosis.

Microgliosis is a major hallmark of Alzheimer's disease (AD) brain pathology. Aß peptide is hypothesized to act as a stimulus for microglia leading to activation of non-receptor tyrosine kinases and subsequent secretion of pro-inflammatory cytokines. Therefore, the signaling pathways mediating microglial activation may be important therapeutic targets of anti-inflammatory therapy for AD. Four novel compounds were chosen after high throughput screening kinase activity assays determined them as potential Lyn kinase inhibitors. Their kinase inhibitory and anti-inflammatory effect on Aß-stimulated activation was assessed using the murine microglial cell line, BV2. Cells were treated with the compounds to determine effects on active, phosphorylated levels of Src family kinases, Src and Lyn, as well as MAP kinases ERK, JNK and p38. Only one compound, LDDN-0003499, produced a dose dependent decrease in basal levels of active, phosphorylated Src and Lyn in the BV2 cells. LDDN-0003499 treatment also attenuated the Aß-stimulated increase in active, phosphorylated levels of Lyn/Src and TNFα and IL-6 secretion. This study identifies a novel small molecule Src family tyrosine kinase inhibitor with anti-inflammatory effects in response to Aß stimulation of microglia. Further in vitro/in vivo characterization of LDDN-0003499 as well as structural modification may provide a new tool for attenuating microglial-mediated brain inflammatory conditions such as that occurring in AD.

Attenuation of microglial activation in a mouse model of Alzheimer's disease via NFAT inhibition.

BACKGROUND: Amyloid ß (Aß) peptide is hypothesized to stimulate microglia to acquire their characteristic proinflammatory phenotype in Alzheimer's disease (AD) brains. The specific mechanisms by which Aß leads to microglial activation remain an area of interest for identifying attractive molecular targets for intervention. Based upon the fact that microglia express the proinflammatory transcription factor, nuclear factor of activated T cells (NFAT), we hypothesized that NFAT activity is required for the Aß-stimulated microgliosis that occurs during disease. METHODS: Primary murine microglia cultures were stimulated with Aß in the absence or presence of NFAT inhibitors, FK506 and tat-VIVIT peptide, to quantify secretion of cytokines, neurotoxins, or Aß phagocytosis. A transgenic mouse model of AD, APP/PS1, was treated subcutaneously via mini-osmotic pumps with FK506 or tat-VIVIT to quantify effects on cytokines, microgliosis, plaque load, and memory. RESULTS: Expression of various NFAT isoforms was verified in primary murine microglia through Western blot analysis. Microglial cultures were stimulated with Aß fibrils in the absence or presence of the NFAT inhibitors, FK506 and tat-VIVIT, to demonstrate that NFAT activity regulated Aß phagocytosis, neurotoxin secretion, and cytokine secretion. Delivery of FK506 and tat-VIVIT to transgenic APP/PS1 mice attenuated spleen but not brain cytokine levels. However, FK506 and tat-VIVIT significantly attenuated both microgliosis and Aß plaque load in treated mice compared to controls. Surprisingly, this did not correlate with changes in memory performance via T-maze testing. CONCLUSIONS: Our findings suggest that development of specific NFAT inhibitors may offer promise as an effective strategy for attenuating the microgliosis and Aß plaque deposition that occur in AD.

Amyloid precursor protein mediated changes in intestinal epithelial phenotype in vitro.

BACKGROUND: Although APP and its proteolytic metabolites have been well examined in the central nervous system, there remains limited information of their functions outside of the brain. For example, amyloid precursor protein (APP) and amyloid beta (Aß) immunoreactivity have both been demonstrated in intestinal epithelial cells. Based upon the critical role of these cells in absorption and secretion, we sought to determine whether APP or its metabolite amyloid ß (Aß), had a definable function in these cells. METHODOLOGY/PRINCIPAL FINDINGS: The human colonic epithelial cell line, Caco-2 cells, were cultured to examine APP expression and Aß secretion, uptake, and stimulation. Similar to human colonic epithelium stains, Caco-2 cells expressed APP. They also secreted Aß 1-40 and Aß 1-42, with LPS stimulating higher concentrations of Aß 1-40 secretion. The cells also responded to Aß 1-40 stimulation by increasing IL-6 cytokine secretion and decreasing cholesterol uptake. Conversely, stimulation with a sAPP-derived peptide increased cholesterol uptake. APP was associated with CD36 but not FATP4 in co-IP pull down experiments from the Caco-2 cells. Moreover, stimulation of APP with an agonist antibody acutely decreased CD36-mediated cholesterol uptake. CONCLUSIONS/SIGNIFICANCE: APP exists as part of a multi-protein complex with CD36 in human colonic epithelial cells where its proteolytic fragments have complex, reciprocal roles in regulating cholesterol uptake. A biologically active peptide fragment from the N-terminal derived, sAPP, potentiated cholesterol uptake while the ß secretase generated product, Aß1-40, attenuated it. These data suggest that APP is important in regulating intestinal cholesterol uptake in a fashion dependent upon specific proteolytic pathways. Moreover, this biology may be applicable to cells beyond the gastrointestinal tract.

Overexpression of mutant amyloid-ß protein precursor and presenilin 1 modulates enteric nervous system.

Alzheimer's disease (AD) is a neurodegenerative disorder histologically characterized by amyloid-ß (Aß) protein accumulation and activation of associated microglia. Although these features are well described in the central nervous system, the process and consequences of Aß accumulation in the enteric nervous system have not been extensively studied. We hypothesized that Aß also may accumulate in the enteric nervous system and lead to immune cell activation and neuronal dysfunction in the digestive tract not unlike that observed in diseased brain. To test this hypothesis, ileums of the small intestine of thirteen month old AßPP/PS1 and C57BL/6 (wild type) mice were collected and analyzed using immunohistochemistry, western blot analysis, cytokine arrays, and ELISA. AßPP/PS1 mice demonstrated no differences in intestinal motility or water absorption but elevated luminal IgA levels compared to wild type mice. They also had increased protein levels of AßPP and the proteolytic enzyme, BACE, corresponding to an increase in Aß1-40 in the intestinal lysate as well as an increase in both Aß1-40 and Aß1-42 in the stool. This correlated with increased protein markers of proinflammatory and immune cell activation. Histologic analysis localized AßPP within enteric neurons but also intestinal epithelial cells with elevated Aß immunoreactivity in the AßPP/PS1 mice. The presence of AßPP, Aß, and CD68 immunoreactivity in the intestines of some patients with neuropathologically-confirmed AD are consistent with the findings in this mouse model. These data support the hypothesis that in AD the intestine, much like the brain, may develop proinflammatory and immune changes related to AßPP and Aß.