Gut bacteria regulate brain pathology of Alzheimer's disease (AD) patients and animal models; however, the underlying mechanism remains unclear. In this study, 3-month-old APP-transgenic female mice with and without knock-out of Il-17a gene were treated with antibiotics-supplemented or normal drinking water for 2 months. The antibiotic treatment eradicated almost all intestinal bacteria, which led to a reduction in Il-17a-expressing CD4-positive T lymphocytes in the spleen and gut, and to a decrease in bacterial DNA in brain tissue. Depletion of gut bacteria inhibited inflammatory activation in both brain tissue and microglia, lowered cerebral A beta levels, and promoted transcription of Arc gene in the brain of APP-transgenic mice, all of which effects were abolished by deficiency of Il-17a. As possible mechanisms regulating A beta pathology, depletion of gut bacteria inhibited beta-secretase activity and increased the expression of Abcb1 and Lrp1 in the brain or at the blood-brain barrier, which were also reversed by the absence of Il-17a. Interestingly, a crossbreeding experiment between APP-transgenic mice and Il-17a knockout mice further showed that deficiency of Il-17a had already increased Abcb1 and Lrp1 expression at the blood-brain barrier. Thus, depletion of gut bacteria attenuates inflammatory activation and amyloid pathology in APP-transgenic mice via Il-17a-involved signaling pathways. Our study contributes to a better understanding of the gut-brain axis in AD pathophysiology and highlights the therapeutic potential of Il-17a inhibition or specific depletion of gut bacteria that stimulate the development of Il-17a-expressing T cells.

Gut bacteria regulate brain pathology of Alzheimer’s disease (AD) patients and animal models; however, the underlying mechanism remains unclear. In this study, 3-month-old APP-transgenic female mice with and without knock-out of Il-17a gene, or haploinsufficiency of MyD88 in microglia were treated with antibiotics-supplemented or normal drinking water for 2 months. Antibiotic treatment eradicated gut bacteria, particularly in the phyla Bacteroidetes and Firmicutes , and reduced Il-17a-expressing CD4-positive T lymphocytes. Deletion of gut bacteria inhibited inflammatory activation in the brain and microglia, and reduced cerebral Aβ levels in APP-transgenic mice, which was abolished by deficiency of Il-17a or haploinsufficiency of MyD88 in microglia. As possible mechanisms regulating Aβ pathology, deletion of gut bacteria inhibited β-secretase activity and increased the expression of Abcb1 and Lrp1 in the brain or at the blood-brain barrier, which were also reversed by the absence of Il-17a. Interestingly, a crossbreeding experiment between APP-transgenic mice and Il-17a knockout mice further showed that deficiency of Il-17a had already increased Abcb1 and Lrp1 expression at the blood-brain barrier. Thus, deletion of gut bacteria attenuates inflammatory activation and amyloid pathology in APP-transgenic mice via Il-17a and microglial MyD88-involved signalling pathways. Our study contributes to a better understanding of the gut-brain axis in AD pathophysiology.

In Alzheimer's disease (AD) brain, inflammatory activation regulates protein levels of amyloid-beta-peptide (A beta) and phosphorylated tau (p-tau), as well as neurodegeneration; however, the regulatory mechanisms remain unclear. We constructed APP- and tau-transgenic AD mice with deletion of IKK beta specifically in neurons, and observed that IKK beta deficiency reduced cerebral A beta and p-tau, and modified inflammatory activation in both AD mice. However, neuronal deficiency of IKK beta decreased apoptosis and maintained synaptic proteins (e.g., PSD-95 and Munc18-1) in the brain and improved cognitive function only in APP-transgenic mice, but not in tau-transgenic mice. Additionally, IKK beta deficiency decreased BACE1 protein and activity in APP-transgenic mouse brain and cultured SH-SY5Y cells. IKK beta deficiency increased expression of PP2A catalytic subunit isoform A, an enzyme dephosphorylating cerebral p-tau, in the brain of tau-transgenic mice. Interestingly, deficiency of IKK beta in neurons enhanced autophagy as indicated by the increased ratio of LC3B-II/I in brains of both APP- and tau-transgenic mice. Thus, IKK beta deficiency in neurons ameliorates AD-associated pathology in APP- and tau-transgenic mice, perhaps by decreasing A beta production, increasing p-tau dephosphorylation, and promoting autophagy-mediated degradation of BACE1 and p-tau aggregates in the brain. However, IKK beta deficiency differently protects neurons in APP- and tau-transgenic mice. Further studies are needed, particularly in the context of interaction between A beta and p-tau, before IKK beta/NF-kappa B can be targeted for AD therapies.

Abstract Background: Alzheimer’s disease (AD) is pathologically characterized by extracellular deposition of amyloid β peptide (Aβ), intracellular neurofibrillary tangles (composed mainly of phosphorylated tau [p-tau]) and neuroinflammation. The pathogenic role of inflammatory activation in AD has been extensively studied; however, the underlying mechanisms remain unclear. In this project, we investigated how neuronal IKKβ/NF-kB regulates AD-associated pathologies in APP- and tau-transgenic AD mice. Methods: APP- and tau-transgenic mice were cross-bred with ikbkb-floxed and nex-cre knock-in mice to generate AD models with deletion of IKKβ specifically in neurons. After assessing cognitive function with the Morris water maze test, mice were analyzed for Aβ and p-tau levels, microglial numbers, transcription of inflammatory genes, apoptosis and synaptic protein levels in the brain by histological, biochemical and molecular biology methods. To explore pathogenic mechanisms, we analyzed the activity and/or protein levels of: 1) β- and γ-secretases, 2) tau-phosphorylating (e.g., GSK3β and p38α-MAPK) and dephosphorylating enzymes (e.g., PP2A and PP2B), and 3) autophagy-related proteins (e.g., LC3B, beclin1 and SQSTM1/p62). In addition, IKKβ-knockdown and wild-type SH-SY5Y cell lines were created to verify the in vivo results.Results: In APP-transgenic mice, neuronal deficiency of IKKβ decreased Aβ load, inflammatory activation, and apoptosis in the brain, and improved cognitive function and maintenance of synaptic proteins (e.g., PSD-95). IKKβ deficiency decreased BACE1 activity and protein in the brain and cultured neuronal cells. In tau-transgenic mice, neuronal deficiency of IKKβ decreased p-tau, shifted pro- to anti-inflammatory activation and inhibited autophagy in the brain. IKKβ deficiency increased expression of PP2A catalytic subunit isoform A, an enzyme dephosphorylating cerebral p-tau. However, deficiency of IKKβ in neurons did not alter the cognitive function and even increased apoptosis in the brain.Conclusions: Deficiency of IKKβ in neurons attenuates Aβ and p-tau loads in the brains of AD mice. As possible molecular mechanisms, IKKβ deficiency decreases BACE1 activity, thereby reducing Aβ production, and increases PP2A expression, promoting p-tau dephosphorylation. However, IKKβ deficiency protects neurons only in APP-transgenic mice, but not in tau-transgenic AD mice. Further studies are needed before IKKβ/NF-kB can be targeted for AD therapies.

Alzheimer's disease (AD), the most common cause of dementia in the elderly, is pathologically characterized by extracellular deposition of amyloid-β peptides (Aβ) and microglia-dominated inflammatory activation in the brain. p38α-MAPK is activated in both neurons and microglia. How p38α-MAPK in microglia contributes to AD pathogenesis remains unclear. In this study, we conditionally knocked out p38α-MAPK in all myeloid cells or specifically in microglia of APP-transgenic mice, and examined animals for AD-associated pathologies (i.e., cognitive deficits, Aβ pathology, and neuroinflammation) and individual microglia for their inflammatory activation and Aβ internalization at different disease stages (e.g., at 4 and 9 months of age). Our experiments showed that p38α-MAPK-deficient myeloid cells were more effective than p38α-MAPK-deficient microglia in reducing cerebral Aβ and neuronal impairment in APP-transgenic mice. Deficiency of p38α-MAPK in myeloid cells inhibited inflammatory activation of individual microglia at 4 months but enhanced it at 9 months. Inflammatory activation promoted microglial internalization of Aβ. Interestingly, p38α-MAPK-deficient myeloid cells reduced IL-17a-expressing CD4-positive lymphocytes in 9 but not 4-month-old APP-transgenic mice. By cross-breeding APP-transgenic mice with Il-17a-knockout mice, we observed that IL-17a deficiency potentially activated microglia and reduced Aβ deposition in the brain as shown in 9-month-old myeloid p38α-MAPK-deficient AD mice. Thus, p38α-MAPK deficiency in all myeloid cells, but not only in microglia, prevents AD progression. IL-17a-expressing lymphocytes may partially mediate the pathogenic role of p38α-MAPK in peripheral myeloid cells. Our study supports p38α-MAPK as a therapeutic target for AD patients.

Amyloid beta peptide (A beta) is the major pathogenic molecule in Alzheimer's disease (AD). BACE1 enzyme is essential for the generation of A beta. Deficiency of p38 alpha-MAPK in neurons increases lysosomal degradation of BACE1 and decreases A beta deposition in the brain of APP-transgenic mice. However, the mechanisms mediating effects of p38 alpha-MAPK are largely unknown. In this study, we used APP-transgenic mice and cultured neurons and observed that deletion of p38 alpha-MAPK specifically in neurons decreased phosphorylation of Snapin at serine, increased retrograde transportation of BACE1 in axons and reduced BACE1 at synaptic terminals, which suggests that p38 alpha-MAPK deficiency promotes axonal transportation of BACE1 from its predominant locations, axonal terminals, to lysosomes in the cell body. In vitro kinase assay revealed that p38 alpha-MAPK directly phosphorylates Snapin. By further performing mass spectrometry analysis and site-directed mutagenic experiments in SH-SY5Y cell lines, we identified serine residue 112 as a p38 alpha-MAPK-phosphorylating site on Snapin. Replacement of serine 112 with alanine did abolish p38 alpha-MAPK knockdown-induced reduction of BACE1 activity and protein level, and transportation to lysosomes in SH-SY5Y cells. Taken together, our study suggests that activation of p38 alpha-MAPK phosphorylates Snapin and inhibits the retrograde transportation of BACE1 in axons, which might exaggerate amyloid pathology in AD brain.

Growing evidence indicates that innate immune molecules regulate microglial activation in Alzheimer's disease (AD); however, their effects on amyloid pathology and neurodegeneration remain inconclusive. Here, we conditionally deleted one allele of myd88 gene specifically in microglia in APP/PS1-transgenic mice by 6 months and analyzed AD-associated pathologies by 9 months. We observed that heterozygous deletion of myd88 gene in microglia decreased cerebral amyloid β (Aβ) load and improved cognitive function of AD mice, which was correlated with reduced number of microglia in the brain and inhibited transcription of inflammatory genes, for example, tnf-α and il-1β, in both brain tissues and individual microglia. To investigate mechanisms underlying the pathological improvement, we observed that haploinsufficiency of MyD88 increased microglial recruitment toward Aβ deposits, which might facilitate Aβ clearance. Microglia with haploinsufficient expression of MyD88 also increased vasculature in the brain of APP/PS1-transgenic mice, which was associated with up-regulated transcription of osteopontin and insulin-like growth factor genes in microglia. Moreover, MyD88-haploinsufficient microglia elevated protein levels of LRP1 in cerebral capillaries of APP/PS1-transgenic mice. Cell culture experiments further showed that treatments with interleukin-1β decreased LRP1 expression in pericytes. In summary, haploinsufficiency of MyD88 in microglia at a late disease stage attenuates pro-inflammatory activation and amyloid pathology, prevents the impairment of microvasculature and perhaps also protects LRP1-mediated Aβ clearance in the brain of APP/PS1-transgenic mice, all of which improves neuronal function of AD mice.

Microglial activation is a hall marker of Alzheimer’s disease (AD); its pathogenic role and regulating mechanisms are unclear. p38α-MAPK, a stress-responding kinase, is activated in AD brain in early disease stages. In APP-transgenic mice, we deleted p38α-MAPK in whole myeloid cells from birth or specifically in microglia from 9 months, and analysed AD pathology at the age of 4, 9 and 12 months. In both experimental settings, p38α-MAPK deficiency decreased cerebral Aβ and improved cognitive function of AD mice; however, p38α-MAPK-deficient myeloid cells were more effective than p38α-MAPK-deficient microglia in preventing AD pathogenesis. Deficiency of p38α-MAPK in myeloid cells inhibited the inflammatory activation of individual microglia by 4 months, but enhanced it by 9 months. Inflammatory activation was essential for p38α-MAPK deficiency to promote microglial internalization of Aβ. Interestingly, p38α-MAPK deficiency in peripheral myeloid cells reduced il-17a transcription in CD4-positive spleen cells. By cross-breeding APP-transgenic mice and IL-17a knockout mice, we further observed that IL-17a deficiency activated microglia and decreased Aβ deposits in AD mouse brain. Thus, p38α-MAPK deficiency in myeloid cells prevents AD pathogenesis, perhaps through reducing IL-17a-expressing T lymphocytes, and promoting Aβ clearance in the brain. Our study supports p38α-MAPK as a novel target for AD therapy.

Alzheimer's disease (AD) is the leading cause of dementia with very limited therapeutic options. Amyloid β (Aβ) and phosphorylated Tau (p‐Tau) are key pathogenic molecules in AD. P38α‐MAPK is specifically activated in AD lesion sites. However, its effects on AD pathogenesis, especially on p‐Tau‐associated brain pathology, and the underlying molecular mechanisms remain unclear. We mated human APP‐transgenic mice and human P301S Tau‐transgenic mice with mapk14‐floxed and neuron‐specific Cre‐knock‐in mice. We observed that deletion of p38α‐MAPK specifically in neurons improves the cognitive function of both 9‐month‐old APP and Tau‐transgenic AD mice, which is associated with decreased Aβ and p‐Tau load in the brain. We further used next‐generation sequencing to analyze the gene transcription in brains of p38α‐MAPK deficient and wild‐type APP‐transgenic mice, which indicated that deletion of p38α‐MAPK regulates the transcription of calcium homeostasis‐related genes, especially downregulates the expression of grin2a, a gene encoding NMDAR subunit NR2A. Cell culture experiments further verified that deletion of p38α‐MAPK inhibits NMDA‐triggered calcium influx and neuronal apoptosis. Our systemic studies of AD pathogenic mechanisms using both APP‐ and Tau‐transgenic mice suggested that deletion of neuronal p38α‐MAPK attenuates AD‐associated brain pathology and protects neurons in AD pathogenesis. This study supports p38α‐MAPK as a novel target for AD therapy.

Alzheimer's disease (AD) is a neurodegenerative disease pathologically characterized by extracellular amyloid-β (Aβ) deposits and intracellular neurofibrillary tangles (NFT) in many brain regions. NFT are primarily composed of hyperphosphorylated tau protein (p-Tau). Aβ and p-Tau are two major path ogenic molecules with tau acting downstream to Aβ to induce neuronal degeneration. In this study, we investigated whether Ginkgo biloba extract EGb 761 reduces cerebral p-Tau level and prevents AD pathogenesis. Human P301S tau mutant-transgenic mice were fed with EGb 761, added to the regular diet for 2 or 5 months. We observed that treatment with EGb 761 for 5 months significantly improved the cognitive function of mice, attenuated the loss of synaptophysin and recovered the phosphorylation of CREB in the mouse brain. Treatment with EGb 761 for 5 but not 2 months also decreased p-Tau protein amount and shifted microglial pro-inflammatory to anti-inflammatory activation in the brain. As potential therapeutic mechanisms, we demonstrated that treatment with EGb 761, especially the components of ginkgolide A, bilobalide, and flavonoids, but not with purified ginkgolide B or C, increased autophagic activity and degradation of p-Tau in lysosomes of neurons. Inhibiting ATG5 function or treating cells with Bafilomycin B1 abolished EGb 761-enhanced degradation of p-Tau in cultured neurons. Additionally, we observed that 5- instead of 2-month-treatment with EGb 761 inhibited the activity of p38-MAPK and GSK-3β. Therefore, long-term treatment with Ginkgo biloba extract EGb 761, a clinically available and well-tolerated herbal medication, ameliorates AD pathology through mechanisms against multiple AD pathogenic processes.

Alzheimer's disease (AD) is characterized by intracellular neurofibrillary tangles. The primary component, hyperphosphorylated Tau (p-Tau), contributes to neuronal death. Recent studies have shown that autophagy efficiently degrades p-Tau, but the mechanisms modulating autophagy and subsequent p-Tau clearance in AD remain unclear. In our study, we first analyzed the relationship between the inflammatory activation and autophagy in brains derived from aged mice and LPS-injected inflammatory mouse models. We found that inflammatory activation was essential for activation of autophagy in the brain, which was neuronal ATG5-dependent. Next, we found that autophagy in cultured neurons was enhanced by LPS treatment of cocultured macrophages. In further experiments designed to provoke chronic mild stimulation of TLR4 without inducing obvious neuroinflammation, we gave repeated LPS injections (i.p., 0.15 mg/kg, weekly for 3 mo) to transgenic mice overexpressing human Tau mutant (P301S) in neurons. We observed significant enhancement of neuronal autophagy, which was associated with a reduction of cerebral p-Tau proteins and improved cognitive function. In summary, these results show that neuroinflammation promotes neuronal autophagy and that chronic mild TLR4 stimulation attenuates AD-related tauopathy, likely by activating neuronal autophagy. Our study displays the beneficial face of neuroinflammation and suggests a possible role in the treatment of AD patients.

Amyloid β (Aβ) damages neurons and triggers microglial inflammatory activation in the Alzheimer disease (AD) brain. BACE1 is the primary enzyme in Aβ generation. Neuroinflammation potentially up-regulates BACE1 expression and increases Aβ production. In Alzheimer amyloid precursor protein-transgenic mice and SH-SY5Y cell models, we specifically knocked out or knocked down gene expression of mapk14, which encodes p38α MAPK, a kinase sensitive to inflammatory and oxidative stimuli. Using immunological and biochemical methods, we observed that reduction of p38α MAPK expression facilitated the lysosomal degradation of BACE1, decreased BACE1 protein and activity, and subsequently attenuated Aβ generation in the AD mouse brain. Inhibition of p38α MAPK also enhanced autophagy. Blocking autophagy by treating cells with 3-methyladenine or overexpressing dominant-negative ATG5 abolished the deficiency of the p38α MAPK-induced BACE1 protein reduction in cultured cells. Thus, our study demonstrates that p38α MAPK plays a critical role in the regulation of BACE1 degradation and Aβ generation in AD pathogenesis.

Neuroleptic antipychotic medications are powerful agents for treatment of mental illness. However they have been associated with increased stroke incidence in dementia. Their postulated co-factor role in the neurodegeneration of schizophrenia is noted, albeit less robust. In vitro research suggests an array of effects upon amyloid metabolism between antipsychotic drug families, and in vivo schizophrenic tau neurofibrillary pathology may relate more to use of these drugs than the underlying diagnosis. The adult patients with early onset depression may have a more robust neuropil. There is some conflicting in vivo evidence that antipsychotic medications alter cerebrospinal fluid (CSF) tau in depression, but a paucity of information on amyloid. Human ventricular (V)CSF was obtained at stereotactic psychosurgery for intractable depression. VCSF Aβ40 and Aβ42 was determined by quantitative immunoprecipitation and Western blotting (mab G2–10, G2–11) with densitometric scanning (MACBAS 2) . Total Tau and Tau 181P determination utlised Innogenetics colorimetric ELISAs. Concentrations of Aβ40 and tau were approximately1.6-fold higher in patients who were receiving antipsychotic medication at surgery compared with those not receiving such treatment. No similar alterations were observed in concentrations of Aβ42, phosphotau or Aβ40/42 or phosphotau/tau ratios. Among those patients receiving antipsychotic medication there was no correlation between the approximate dose equivalent to chlorpromazine and any of the CSF proteins (p> 0.05). There was no significant differences between patients with unipolar and unipolar psychotic diagnoses for any of the CSF proteins (Student's t-test, p>0.05). We demonstrate a novel effect of neuroleptic (antipsychotic) drugs upon Aβ, that may deleteriously affect vulnerable individuals exposed to these treatments. These findings have three important implications: 1) Conditions other than AD are associated with profound alterations in Aβ, 2) Neuroleptics appear to be modulators of Aβ1–40, 3) Alterations in Aβ1–40 production or clearance may underlie the deleterious effect of anti-psychotic drugs in dementia, and perhaps schizophrenia and depression.

The major molecular risk factor for Alzheimer disease so far identified is the amyloidogenic peptide Aβ42. In addition, growing evidence suggests a role of cholesterol in Alzheimer disease pathology and Aβ generation. However, the cellular mechanism of lipid-dependent Aβ production remains unclear. Here we describe that the two enzymatic activities responsible for Aβ production, β-secretase and γ-secretase, are inhibited in parallel by cholesterol reduction. Importantly, our data indicate that cholesterol depletion within the cellular context inhibits both secretases additively and independently from each other. This is unexpected because the β-secretase β-site amyloid precursor protein cleaving enzyme and the presenilin-containing γ-secretase complex are structurally different from each other, and these enzymes are apparently located in different subcellular compartments. The parallel and additive inhibition has obvious consequences for therapeutic research and may indicate an intrinsic cross-talk between Alzheimer disease-related amyloid precursor protein processing, amyloid precursor protein function, and lipid biology.

Generation of amyloid-beta (Abeta) from the amyloid precursor protein (APP) requires proteolytic cleavage by two proteases, beta- and gamma-secretase. Several lines of evidence suggest a role for cholesterol on secretase activities, although the responsible cellular mechanisms remain unclear. Here we show that alterations in cholesterol transport from late endocytic organelles to the endoplasmic reticulum have important consequences for both APP processing and the localization of gamma-secretase-associated presenilins (PS). Exposure of neuronal cells to cholesterol transport-inhibiting agents resulted in a marked decrease in beta-cleavage of full-length APP. In contrast, gamma-secretase activity on APP C-terminal fragments was enhanced, increasing the production of both Abeta40 and Abeta42. Remarkably, retention of cholesterol in endosomal/lysosomal compartments induced PS1 and PS2 to accumulate in Rab7-positive vesicular organelles implicated in cholesterol sorting. Accumulation of PS in vesicular compartments was prominent in both Chinese hamster ovary cells deficient in Niemann-Pick C1 protein as well as in neuronal cells exposed to the cholesterol transport-inhibiting agent U18666A. Because Abeta42 also localized to PS1-containing vesicular compartments, organelles involved in cholesterol transport might represent an important site for gamma-secretase activity. Our results suggest that the subcellular distribution of cholesterol may be an important factor in how cholesterol alters Abeta production and the risk of Alzheimer's disease.

We examined the effect of cerebrospinal fluid (CSF) from 23 Alzheimer's disease (AD) patients and 22 age-matched non-demented controls with apolipoprotein E4/4, 3/3, or 3/2 phenotypes on in vitro aggregation of amyloid beta-protein (Aβ) 1–40 by Thioflavin T fluorescence spectroscopy. CSF from both AD and control groups inhibited Aβ aggregation, as compared to that of phosphate buffered saline, in agreement with an earlier report (Wisniewski et al., 1993). However, there was significantly less aggregation of Aβ in presence of CSF from AD than that from non-demented controls. The presence of CSF from controls with apoE3/3 phenotype resulted in higher Aβ aggregation as compared to other phenotypes. There was a positive correlation between CSF apoE concentrations and Aβ aggregation; whereas age, CSF soluble Aβ levels or severity of dementia did not correlate with Aβ aggregation. These results suggest that mechanism of sequestration of Aβ in CSF may not be defective in AD. Amyloid formation in AD may be impact of altered balance of other factors such as amyloid-associated proteins/extracellular matrix components that can immobilize Aβ in the brain, and promote its fibrillogenesis in AD.