Role Of The Alzheimer\u27s Amyloid Precursor Protein In High Fat Diet Induced Obesity And Regulating Macrophage Phenotype

Amyloid precursor protein (APP) derived amyloid beta peptides have been extensively investigated in Alzheimer\u27s disease pathology of the brain. However, the function of full length APP in the central nervous system remains unclear. Even less is known about the behavior of this ubiquitously expressed protein and it metabolites outside of the central nervous system. Therefore, we sought to broaden our understanding of the expression and function of APP and its proteolytic fragments in specific non-neuronal tissues. Although the majority of research effort is currently focused on neuronal amyloid beta production and its effects on cells, prior work in our lab demonstrated a novel role for APP in regulating the phenotype of monocytic lineage cells. Therefore, we hypothesized that APP can behave as a proinflammatory receptor on these cells involved in modulating their tissue infiltration and differentiation. Based upon the fact that midlife obesity is a risk factor for Alzheimer\u27s disease and both obese adipose tissue and Alzheimer\u27s disease brains share a common presence of increased, reactive macrophage and microglia, respectively, we hypothesized that APP may have a common role in both diseases regulating the infiltration or proinflammatory activation of microglia and macrophage characterizing both diseases. Indeed, recent data has demonstrated that APP levels are increased in adipose tissue from obese versus control individuals. To test this idea we utilized a high fat diet feeding paradigm on both C57BL6 wild type and APP-/- mice to examine any role for APP and high fat diet dependent changes in adipose tissue, brain, and intestine. In vivo changes were compared to those obtained using primary cells isolated from the murine models. Collectively, these data suggest that APP does regulate microglia and macrophage phenotype in a manner responsible for altering their behavior in tissue specific fashion. This suggests that immune-related functions of APP may be a common type of pathophysiology linking the complex diseases of obesity and Alzheimer\u27s disease

NFATc2 Modulates Microglial Activation in the AβPP/PS1 Mouse Model of Alzheimer\u27s Disease

Alzheimer’s disease (AD) brains are characterized by fibrillar amyloid-β (Aβ) 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 Aβ 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 AβPP/PS1 mouse line. As expected, the AβPP/PS1 x NFATc2–/– mice had attenuated cytokine levels compared to AβPP/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\u27s Disease

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\u27s disease. Although fibrillar amyloid β (Aβ)-stimulated cytokine secretion from both wild-type and APP knock-out (mAPP−/−) microglial cultures, oligomeric Aβ was unable to stimulate increased secretion from mAPP−/− cells. This was consistent with an ability of oligomeric Aβ to bind APP. Similarly, intracerebroventricular infusions of oligomeric Aβ 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 Aβ 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\u27s disease (AD) brains is the accumulation of amyloid β (Aβ) peptide within plaques robustly invested with reactive microglia. This supports the notion that Aβ stimulation of microglial activation is one source of brain inflammatory changes during disease. Aβ 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 Aβ 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 Aβ production to drive the microgliosis associated with AD brains

Characterization of Novel Src Family Kinase Inhibitors to Attenuate Microgliosis

Microgliosis is a major hallmark of Alzheimer’s disease (AD) brain pathology. Aβ 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 Aβ-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 Aβ-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 Aβ 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

Amyloid Precursor Protein and Proinflammatory Changes Are Regulated in Brain and Adipose Tissue in a Murine Model of High Fat Diet-Induced Obesity

Background: Middle age obesity is recognized as a risk factor for Alzheimer’s disease (AD) although a mechanistic linkage remains unclear. Based upon the fact that obese adipose tissue and AD brains are both areas of proinflammatory change, a possible common event is chronic inflammation. Since an autosomal dominant form of AD is associated with mutations in the gene coding for the ubiquitously expressed transmembrane protein, amyloid precursor protein (APP) and recent evidence demonstrates increased APP levels in adipose tissue during obesity it is feasible that APP serves some function in both disease conditions. Methodology/Principal Findings: To determine whether diet-induced obesity produced proinflammatory changes and altered APP expression in brain versus adipose tissue, 6 week old C57BL6/J mice were maintained on a control or high fat diet for 22 weeks. Protein levels and cell-specific APP expression along with markers of inflammation and immune cell activation were compared between hippocampus, abdominal subcutaneous fat and visceral pericardial fat. APP stimulation-dependent changes in macrophage and adipocyte culture phenotype were examined for comparison to the in vivo changes. Conclusions/Significance: Adipose tissue and brain from high fat diet fed animals demonstrated increased TNF-a and microglial and macrophage activation. Both brains and adipose tissue also had elevated APP levels localizing to neurons and macrophage/adipocytes, respectively. APP agonist antibody stimulation of macrophage cultures increased specific cytokin

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

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 (Aβ) 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 β (Aβ), had a definable function in these cells.The human colonic epithelial cell line, Caco-2 cells, were cultured to examine APP expression and Aβ secretion, uptake, and stimulation. Similar to human colonic epithelium stains, Caco-2 cells expressed APP. They also secreted Aβ 1-40 and Aβ 1-42, with LPS stimulating higher concentrations of Aβ 1-40 secretion. The cells also responded to Aβ 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.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 β secretase generated product, Aβ1-40, 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

Aβ, APP, and CD36 stimulations selectively altered protein levels in Caco-2 cells.

<p>To assess changes in phenotype Caco-2 cells were stimulated overnight with or without 1μM Aβ 1–40, 1μg/mL IgG₁ (isotype control), 1μg/mL 22C11 (APP agonist), 1μg/mL SMΦ (CD36 agonist), or both 22C11 and CD36 and then lysed for western blotting. Data are from 3 samples in each condition and are displayed as mean (+/-SD), *p<0.05.</p

Aβ and 22C11 agonist decreased cholesterol uptake whereas the sAPP-derived peptide and SM-ϕ stimulated increased cholesterol uptake by Caco-2 cells.

<p>To demonstrate effects on lipid uptake a cell-based cholesterol uptake assay was performed. Cells were stimulated with or without 10ng/mL LPS, 100nM Aβ 1–40, 1μM Aβ 1–40, 5μM Aβ1–40, 1μg/mL IgG₁ (isotype control), 1μg/mL 22C11 (APP agonist), 1μg/mL SMΦ (CD36 agonist), 10pM, 100pM, and 1nM sAPP peptide, or both 22C11 and SMΦ for 40 min and NBD Cholesterol uptake was quantified using a fluorescent plate reader (480 nm excitation and 520 nm emission). Data are from 8 samples in each condition and are displayed as mean (+/-SD), *p<0.05. Representative data from 3 independent experiments are shown.</p

Caco-2 cells secreted increased IL-6 levels upon Aβ stimulation.

<p>To demonstrate treatment effects on cytokine secretion cells were stimulated with or without 10ng/mL LPS, 100nM Aβ 1–40, 1μM Aβ 1–40, or 5μM Aβ1–40 overnight and media was analyzed via IL-8, MCP-1, MDC, IL-6 and TNFα ELISAs. Cytokine concentrations are normalized to their respective well protein concentrations (pg/mL cytokine/μg protein) from 6 samples in each condition and are displayed as mean (+/-SD), *p<0.05. Representative data from 3 independent experiments are shown.</p

Caco-2 cells secreted and took up Aβ.

<p>To demonstrate Caco-2 cell ability to secrete Aβ they were stimulated with or without 10ng/mL LPS, 1μg/mL IgG₁, or 1μg/mL 22C11 overnight, and Aβ secretion was measured via Aβ 1–40 and Aβ 1–42 ELISA (<b>A</b>). To demonstrate Caco-2 cell ability to take up Aβ, they were incubated with or without 500nM FITC conjugated Aβ 1–40 in the absence or presence of 10 ng/mL LPS stimulation for 4 hr. The cells were rinsed with trypan blue to quench extracellular signal from FITC-Aβ on the cell surface or surface of the tissue culture dish and intracellular Aβ fluorescence was measured with a fluorescent plate reader (480 nm excitation and 520 nm emission) (<b>B</b>). Data are from 3 experiments in a replicate of 8 each displayed as mean (+/-SD), *p<0.05.</p