Modulation of Alzheimer's disease brain pathology in mice by gut bacterial depletion: the role of IL-17a.

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 Aß 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 Aß pathology, depletion of gut bacteria inhibited ß-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.

Risk factors and management of gastrointestinal bleeding in patients with or without antiplatelet and anticoagulation therapy: a multicenter real-world prospective study.

BACKGROUND: Antiplatelet and anticoagulation drugs complicate acute gastrointestinal bleeding (GIB) patients. Limited data about the risk factors and patient management has been presented. This study explored the association between previous antiplatelet or anticoagulant drug usage and clinical outcomes in GIB patients to improve awareness further and optimize treatment. METHODS: We conducted a multicenter, non-interventional, real-world prospective study in 106 hospitals in 23 provinces in China. GIB patients confirmed in the emergency department were included and were grouped according to previous drug histories. Univariate analysis, multivariate logistic regression, and multivariate stratification models were performed separately to investigate the associations. RESULTS: A total of 2299 patients (57.23 ± 17.21 years old, 68.3% male) were included, of whom 20.1% and 2.9% received antiplatelet and anticoagulation therapy, respectively. The all-cause 28-day mortality rates in patients without antiplatelet or anticoagulants, patients undergoing antiplatelet treatment, and patients with anticoagulation therapy were 2.8%, 4.6%, and 10.5%, respectively. After adjusting for confounding factors, both antiplatelet [odd ratio (OR), 2.92; 95% confidence interval (CI), 1.48-5.76; p = 0.002] and anticoagulation therapy (OR, 8.87; 95% CI, 3.02-26.02; p < 0.001) were associated with higher 28-day mortality. In the subgroup analysis, blood transfusion, especially red blood cell transfusion, in patients undergoing antiplatelet and anticoagulation therapy was associated with a decreased death risk. CONCLUSION: We confirmed an association between concurrent antiplatelet or anticoagulation therapy in GIB patients and elevated 28-day mortality. Blood transfusions could improve poor outcomes in such patients.

[Treatment and factors associated with prognosis of hyperkalemia in the emergency department].

OBJECTIVE: To survey treatment and prognosis of hyperkalemia patients in the emergency department and to analyze factors associated with all-cause in-hospital mortality. METHODS: We implemented electronic hospital information system, extracted demographic characteristics, underlying diseases, laboratory findings, potassium lowering therapy and prognosis of hyperkalemia patients [age ≥ 18 years, serum potassium (K+) concentration ≥ 5.5 mmol/L] in the emergency department of Peking Union hospital in Beijing between June 1st 2019 to May 31st 2020. The enrolled subjects were divided into the non-survival group and the survival group according to their prognosis. Univariate analysis and Cox regression model were adopted to analyze factors affecting all-cause in-hospital mortality of hyperkalemia patients. RESULTS: A total of 579 patients [median age 64 (22) years; 310 men (53.5%) and 269 women (46.5%)] with hyperkalemia were enrolled, among which, 317 (54.7%), 143 (24.7%) and 119 (20.6%) were mild, moderate, and severe hyperkalemia, respectively. 499 (86.20%) patients received potassium-lowering therapy, forty-four treatment regimens were administered. Insulin and glucose (I+G, 61.3%), diuretics (Diu, 57.2%), sodium bicarbonate (SB, 41.9%) and calcium gluconate/chloride (CA, 44.4%) were commonly used for the treatment of hyperkalemiain the emergency department. The combination of insulin and glucose, calcium gluconate/chloride, diuretics and sodium bicarbonate (I+G+CA+Diu+SB) was the most favored combined treatment regimen of hyperkalemia in the emergency department. The higher serum potassium concentration, the higher proportion of administrating combined treatment regimen and/or hemodialysis (HD) (the proportion of administrating combined treatment regimen in mild, moderate, and severe hyperkalemia patients were 58.4%, 82.5% and 94.8%; the proportion of administrating HD in mild, moderate, and severe hyperkalemia patients were 9.7%, 13.3% and 16.0%, respectively). The proportion of achievement of normokalaemia elevated as the kinds of potassium lowering treatment included in the combined treatment regimen increased. The proportion of achievement of normokalaemia was 100% in the combined treatment regimen including 6 kinds of potassium lowering therapy. Among various potassium lowering treatments, HD contributed to the highest rate of achievement of normokalaemia (93.8%). 111 of 579 (19.20%) hyperkalemia patients died in hospital. Cox regression model revealed that complicated with cardiac dysfunction predicted higher mortality [hazard ratio (HR) = 1.757, 95% confidence interval (95%CI) was 1.155-2.672, P = 0.009]. Achievement of normokalaemia and administration of diuretics attributed to lower mortality (HR = 0.248, 95%CI was 0.155-0.398, P = 0.000; HR = 0.335, 95%CI was 0.211-0.531, P = 0.000, respectively). CONCLUSIONS: Treatment of hyperkalemia in the emergency department were various. Complicated with cardiac dysfunction were associated with higher mortality. Achieving normokalaemia was associated with decreased mortality.

Deficiency of IKKß in neurons ameliorates Alzheimer's disease pathology in APP- and tau-transgenic mice.

In Alzheimer's disease (AD) brain, inflammatory activation regulates protein levels of amyloid-ß-peptide (Aß) 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 IKKß specifically in neurons, and observed that IKKß deficiency reduced cerebral Aß and p-tau, and modified inflammatory activation in both AD mice. However, neuronal deficiency of IKKß 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, IKKß deficiency decreased BACE1 protein and activity in APP-transgenic mouse brain and cultured SH-SY5Y cells. IKKß 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 IKKß in neurons enhanced autophagy as indicated by the increased ratio of LC3B-II/I in brains of both APP- and tau-transgenic mice. Thus, IKKß deficiency in neurons ameliorates AD-associated pathology in APP- and tau-transgenic mice, perhaps by decreasing Aß production, increasing p-tau dephosphorylation, and promoting autophagy-mediated degradation of BACE1 and p-tau aggregates in the brain. However, IKKß deficiency differently protects neurons in APP- and tau-transgenic mice. Further studies are needed, particularly in the context of interaction between Aß and p-tau, before IKKß/NF-κB can be targeted for AD therapies.

p38α-MAPK-deficient myeloid cells ameliorate symptoms and pathology of APP-transgenic Alzheimer's disease mice.

Alzheimer's disease (AD), the most common cause of dementia in the elderly, is pathologically characterized by extracellular deposition of amyloid-ß peptides (Aß) 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, Aß pathology, and neuroinflammation) and individual microglia for their inflammatory activation and Aß 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 Aß 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 Aß. 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 Aß 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.

Haploinsufficiency of microglial MyD88 ameliorates Alzheimer's pathology and vascular disorders in APP/PS1-transgenic mice.

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 ß (Aß) 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-1ß, in both brain tissues and individual microglia. To investigate mechanisms underlying the pathological improvement, we observed that haploinsufficiency of MyD88 increased microglial recruitment toward Aß deposits, which might facilitate Aß 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-1ß 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 Aß clearance in the brain of APP/PS1-transgenic mice, all of which improves neuronal function of AD mice.

Treatment With CD52 Antibody Protects Neurons in Experimental Autoimmune Encephalomyelitis Mice During the Recovering Phase.

Multiple sclerosis (MS) is a chronic autoimmune disease driven by T and B lymphocytes. The remyelination failure and neurodegeneration results in permanent clinical disability in MS patients. A desirable therapy should not only modulate the immune system, but also promote neuroprotection and remyelination. To investigate the neuroprotective effect of CD52 antibody in MS, both C57BL/6J and SJL mice with experimental autoimmune encephalomyelitis (EAE) were treated with CD52 antibody at the peak of disease. Treatment with CD52 antibody depleted T but not B lymphocytes in the blood, reduced the infiltration of T lymphocytes and microglia/macrophages in the spinal cord. Anti-CD52 therapy attenuated EAE scores during the recovery phase. It protected neurons immediately after treatment (within 4 days) as shown by reducing the accumulation of amyloid precursor proteins. It potentially promoted remyelination as it increased the number of olig2/CC-1-positive mature oligodendrocytes and prevented myelin loss in the following days (e.g., 14 days post treatment). In further experiments, EAE mice with a conditional knockout of BDNF in neurons were administered with CD52 antibodies. Neuronal deficiency of BDNF attenuated the effect of anti-CD52 treatment on reducing EAE scores and inflammatory infiltration but did not affect anti-CD52 treatment-induced improvement of myelin coverage in the spinal cord. In summary, anti-CD52 therapy depletes CD4-positive T lymphocytes, prevents myelin loss and protects neurons in EAE mice. Neuronal BDNF regulates neuroprotective and anti-inflammatory effect of CD52 antibody in EAE mice.

Ginkgo biloba Extract EGb 761 and Its Specific Components Elicit Protective Protein Clearance Through the Autophagy-Lysosomal Pathway in Tau-Transgenic Mice and Cultured Neurons.

Alzheimer's disease (AD) is a neurodegenerative disease pathologically characterized by extracellular amyloid-ß (Aß) deposits and intracellular neurofibrillary tangles (NFT) in many brain regions. NFT are primarily composed of hyperphosphorylated tau protein (p-Tau). Aß and p-Tau are two major pathogenic molecules with tau acting downstream to Aß 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-3ß. 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.

Stimulation of TLR4 Attenuates Alzheimer's Disease-Related Symptoms and Pathology in Tau-Transgenic Mice.

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.

Deficiency of IκB Kinase ß in Myeloid Cells Reduces Severity of Experimental Autoimmune Encephalomyelitis.

In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), peripherally developed myelin-reactive T lymphocytes stimulate myeloid cells (ie, microglia and infiltrated macrophages) to trigger an inflammatory reaction in the central nervous system, resulting in demyelination and neurodegeneration. IκB kinase ß (IKKß) is a kinase that modulates transcription of inflammatory genes. To investigate the pathogenic role of IKKß in MS, we developed strains in which IKKß was conditionally ablated in myeloid cells and established active or passive EAE in these animals. Deficiency of IKKß in myeloid cells ameliorated EAE symptoms and suppressed neuroinflammation, as shown by decreased infiltration of T lymphocytes and macrophages and reduced inflammatory gene transcription in the spinal cord at the peak or end stage of EAE. Myeloid deficiency of IKKß also reduced the transcription of Rorc or Il17 genes in T lymphocytes isolated from lymph nodes, spleen, and spinal cord of EAE mice. Moreover, cultured splenocytes isolated from myeloid IKKß-deficient EAE mice released less IL-17, interferon-γ, and granulocyte-macrophage colony-stimulating factor after treatment with myelin peptide than splenocytes from IKKß wild-type EAE mice. Thus, deficiency of myeloid IKKß attenuates the severity of EAE by inhibiting both the neuroinflammatory activity and the activation of encephalitogenic T lymphocytes. These results suggest IKKß may be a potential target for MS patients, especially when neuroinflammation is the primary problem.

Deficiency of Neuronal p38α MAPK Attenuates Amyloid Pathology in Alzheimer Disease Mouse and Cell Models through Facilitating Lysosomal Degradation of BACE1.

Amyloid ß (Aß) damages neurons and triggers microglial inflammatory activation in the Alzheimer disease (AD) brain. BACE1 is the primary enzyme in Aß generation. Neuroinflammation potentially up-regulates BACE1 expression and increases Aß 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 Aß 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 Aß generation in AD pathogenesis.

Long-term treatment with Ginkgo biloba extract EGb 761 improves symptoms and pathology in a transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by extracellular deposits of amyloid ß peptide (Aß) and microglia-dominated neuroinflammation. The therapeutic options for AD are currently limited. In this study, we investigated the antiinflammatory effects and the underlying molecular mechanisms of Ginkgo biloba extract EGb 761 when administered to TgCRND8 AD mice, which overexpress human Alzheimer's amyloid precursor protein (APP) specifically in neurons. We gave APP-transgenic mice EGb 761 as a dietary supplement for 2 or 5months. Plasma concentrations of EGb 761 components in mice were in the same range as such concentrations in humans taking EGb 761 at the recommended dose (240mg daily). Treatment with EGb 761 for 5months significantly improved the cognitive function of the mice as measured by the Barnes Maze test. It also attenuated the loss of synaptic structure proteins, such as PSD-95, Munc18-1, and SNAP25. Treatment with EGb 761 for 5months inhibited microglial inflammatory activation in the brain. The effects of treatment with EGb 761 for 2months were weak and not statistically significant. Moreover, EGb 761 activated autophagy in microglia. Treatment with EGb 761 decreased Aß-induced microglial secretion of TNF-α and IL-1ß and activation of caspase-1, both of which were abolished by the inhibition of autophagy. Treatment with EGb 761 also reduced the concentrations of NLRP3 protein that colocalized with LC3-positive autophagosomes or autolysosomes in microglia. Additionally, long-term treatment with EGb 761 may reduce cerebral Aß pathology by inhibiting ß-secretase activity and Aß aggregation. Therefore, long-term treatment with G. biloba extract EGb 761, a clinically available and well-tolerated herbal medication, ameliorates AD pathology by antiinflammatory and Aß-directed mechanisms.

IKKß deficiency in myeloid cells ameliorates Alzheimer's disease-related symptoms and pathology.

Alzheimer's disease (AD) is characterized by extracellular amyloid-ß (Aß) deposits and microglia-dominated inflammatory activation. Innate immune signaling controls microglial inflammatory activities and Aß clearance. However, studies examining innate immunity in Aß pathology and neuronal degeneration have produced conflicting results. In this study, we investigated the pathogenic role of innate immunity in AD by ablating a key signaling molecule, IKKß, specifically in the myeloid cells of TgCRND8 APP-transgenic mice. Deficiency of IKKß in myeloid cells, especially microglia, simultaneously reduced inflammatory activation and Aß load in the brain and these effects were associated with reduction of cognitive deficits and preservation of synaptic structure proteins. IKKß deficiency enhanced microglial recruitment to Aß deposits and facilitated Aß internalization, perhaps by inhibiting TGF-ß-SMAD2/3 signaling, but did not affect Aß production and efflux. Therefore, inhibition of IKKß signaling in myeloid cells improves cognitive functions in AD mice by reducing inflammatory activation and enhancing Aß clearance. These results contribute to a better understanding of AD pathogenesis and could offer a new therapeutic option for delaying AD progression.

Tenascin-C deficiency ameliorates Alzheimer's disease-related pathology in mice.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by deposits of amyloid ß peptide (Aß) and microglia-driven inflammatory activation. Tenascin-C (tnc) is an extracellular matrix protein that is upregulated in inflammation and induces further inflammatory responses. We hypothesized that tnc contributes to the inflammatory pathology in AD. Using real-time polymerase chain reaction, we observed that tnc gene transcription was upregulated in cultured microglia after Aß challenge and in the brain of an AD mouse model that overexpresses mutated amyloid precursor protein (APP) in neural cells. By cross-breeding APP-transgenic mice and tenascin-C-deficient mice, we demonstrated using real-time polymerase chain reaction, Western blot analysis, enzyme-linked immunosorbent assay, and immunohistochemistry that tnc deficiency reduces pro- but enhances anti-inflammatory activation in the mutated APP-transgenic mouse brain, associated with a reduced cerebral Aß load and higher levels of the postsynaptic density protein 95. Thus, our study indicates that functional inhibition of tnc exerts beneficial effects on AD pathogenesis, suggesting a potential for tnc as a new therapeutic target in AD.

Matrix metalloproteinase-12 contributes to neuroinflammation in the aged brain.

During aging the brain displays an increased proinflammatory status, which is associated with the pathogenesis of aging-related diseases such as Alzheimer's and Parkinson diseases. Matrix metalloproteinases (MMPs) facilitate the migration of inflammatory cells in tissues and modulate their inflammatory activity. In this study, we screened expression of MMPs in 3-, 10-, and 18-month-old mice and observed that cerebral MMP-12 expression was strongly upregulated during aging. We compared the neuroinflammation of 3-, 10-, and 18-month-old MMP-12-deficient versus wild type mice by counting microglia and measuring inflammatory gene transcripts in the brain and observed that MMP-12 deficiency reduced neuroinflammation during aging. In order to identify potential mechanisms, we analyzed the inflammatory activity of microglia directly isolated from adult mouse brains or cultured from newborn mice. We observed that MMP-12 deficiency increased the inflammatory activity of adult brain-derived microglia, but did not affect cultured microglia. We found greater numbers of CD11b/CD45(high) cells in the parenchyma of MMP-12 wild type than in the parenchyma of MMP-12-deficient mouse brains. Thus, our study suggested that the upregulated cerebral MMP-12 during aging enhances aging-associated neuroinflammation by facilitating recruitment of bone marrow-derived microglia into the brain.

TLR2 is a primary receptor for Alzheimer's amyloid ß peptide to trigger neuroinflammatory activation.

Microglia activated by extracellularly deposited amyloid ß peptide (Aß) act as a two-edged sword in Alzheimer's disease pathogenesis: on the one hand, they damage neurons by releasing neurotoxic proinflammatory mediators (M1 activation); on the other hand, they protect neurons by triggering anti-inflammatory/neurotrophic M2 activation and by clearing Aß via phagocytosis. TLRs are associated with Aß-induced microglial inflammatory activation and Aß internalization, but the mechanisms remain unclear. In this study, we used real-time surface plasmon resonance spectroscopy and conventional biochemical pull-down assays to demonstrate a direct interaction between TLR2 and the aggregated 42-aa form of human Aß (Aß42). TLR2 deficiency reduced Aß42-triggered inflammatory activation but enhanced Aß phagocytosis in cultured microglia and macrophages. By expressing TLR2 in HEK293 cells that do not endogenously express TLR2, we observed that TLR2 expression enabled HEK293 cells to respond to Aß42. Through site-directed mutagenesis of tlr2 gene, we identified the amino acids EKKA (741-744) as a critical cytoplasmic domain for transduction of inflammatory signals. By coexpressing TLR1 or TLR6 in TLR2-transgenic HEK293 cells or silencing tlrs genes in RAW264.7 macrophages, we observed that TLR2-mediated Aß42-triggered inflammatory activation was enhanced by TLR1 and suppressed by TLR6. Using bone marrow chimeric Alzheimer's amyloid precursor transgenic mice, we observed that TLR2 deficiency in microglia shifts M1- to M2-inflammatory activation in vivo, which was associated with improved neuronal function. Our study demonstrated that TLR2 is a primary receptor for Aß to trigger neuroinflammatory activation and suggested that inhibition of TLR2 in microglia could be beneficial in Alzheimer's disease pathogenesis.

Myeloid differentiation factor 88-deficient bone marrow cells improve Alzheimer's disease-related symptoms and pathology.

Alzheimer's disease is characterized by extracellular deposits of amyloid ß peptide in the brain. Increasing evidence suggests that amyloid ß peptide injures neurons both directly and indirectly by triggering neurotoxic innate immune responses. Myeloid differentiation factor 88 is the key signalling molecule downstream to most innate immune receptors crucial in inflammatory activation. For this reason, we investigated the effects of myeloid differentiation factor 88-deficient bone marrow cells on Alzheimer's disease-related symptoms and pathology by establishing bone marrow chimeric amyloid ß peptide precursor transgenic mice, in which bone marrow cells differentiate into microglia and are recruited to amyloid ß peptide deposits. We observed that myeloid differentiation factor 88-deficient bone marrow reconstruction reduced both inflammatory activation and amyloid ß peptide burden in the brain. In addition, synaptophysin, a marker of neuronal integrity, was preserved and the expression of neuronal plasticity-related genes, ARC and NMDA-R1, was increased. Thus, myeloid differentiation factor 88-deficient microglia significantly improved the cognitive function of amyloid ß peptide precursor protein transgenic mice. Myeloid differentiation factor 88-deficiency enhanced amyloid ß peptide phagocytosis by microglia/macrophages and blunted toxic inflammatory activation. Both the expression of amyloid ß peptide precursor protein and amyloid ß peptide degrading enzymes and also the efflux of amyloid ß peptide from brain parenchyma were unaffected by myeloid differentiation factor 88-deficient microglia. By contrast, the activity of ß-secretase was increased. ß-Secretase is expressed primarily in neurons, with relatively little expression in astrocytes and microglia. Therefore, microglial replenishment with myeloid differentiation factor 88-deficient bone marrow cells might improve cognitive functions in Alzheimer's disease mouse models by enhancing amyloid ß peptide phagocytosis and reducing inflammatory activation. These results could offer a new therapeutic option that might delay the progression of Alzheimer's disease.

Screening of innate immune receptors in neurodegenerative diseases: a similar pattern.

In Alzheimer's disease (AD), Parkinson's disease (PD), dementia with Lewy bodies (DLB) and amyotrophic lateral sclerosis (ALS), neuroinflammatory responses are considered to contribute to neuronal injury. Recently, the innate immune receptors, toll-like receptors (TLRs) and the LPS receptor (CD14) have been related to neurodegeneration. In this study, we systematically assessed the expression of most TLRs and CD14 in AD, PD/DLB and ALS using murine models of these diseases and human post-mortem brain tissues. A common upregulation of TLR2 and CD14 was found in all three animal models. While these two receptors could also be detected in AD patient tissues, they were absent from DLB and ALS tissues. This uniform pattern of innate immune response in animal models of neurodegenerative diseases clearly indicates that this response is part of a non-specific neuroinflammatory effector phase rather than a disease-specific event. The less dynamic disease progression in humans and the location (extracellular versus intracellular) of the aggregated proteins deposits might explain the divergent results seen between animal models and human tissues.

Expression of amyotrophic lateral sclerosis-linked SOD1 mutant increases the neurotoxic potential of microglia via TLR2.

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease, in which activated microglia overexpressing ALS-linked SOD1 mutants (mSOD1) are known to contribute to neuronal death. However, it is unclear how mSOD1 expression affects micoglial activation and subsequently damages neurons. In this study, we created mSOD1-overexpressing BV-2 microglial cell lines. Following TLR2, but not TLR4 stimulation, we observed that overexpression of human SOD1 G93A, L8Q, or G10V mutant, as compared with the wild-type SOD1 or a mock control, significantly enhanced microglial secretion of a neurotoxic cytokine, tumor necrosis factor-alpha (TNF-alpha), which was dependent on the NADPH-oxidase-mediated increased generation of reactive oxygen species (ROS). In further experiments, we demonstrated that mSOD1 expression regulated TNF-alpha secretion at a post-transcriptional level and involved ROS-sensitive TNF-alpha-converting enzymes, e.g. ADAM10 and -17, which shed TNF-alpha from its membrane-anchored precursor. Together with a recent report that the function of SOD1, as a self-regulating redox sensor in NADPH oxidase-dependent ROS production, is lost due to its genetic mutations, we conclude that mSOD1 expression in ALS facilitates microglial neurotoxic inflammatory responses via TLR2, which is mediated by an uncontrolled ROS generation. The link, between mSOD1, innate immunity and NADPH oxidase, offers new opportunities in ALS therapies.

Hematopoietic stem cells reduce postischemic inflammation and ameliorate ischemic brain injury.

BACKGROUND AND PURPOSE: Systemic injection of hematopoietic stem cells after ischemic cardiac or neural lesions is one approach to promote tissue repair. However, mechanisms of possible protective or reparative effects are poorly understood. In this study we analyzed the effect of lineage-negative bone marrow-derived hematopoietic stem and precursor cells (Lin(-)-HSCs) on ischemic brain injury in mice. METHODS: Lin(-)-HSCs were injected intravenously at 24 hours after onset of a 45-minute transient cerebral ischemia. Effects of Lin(-)-HSCs injection on infarct size, apoptotic cell death, postischemic inflammation and cytokine gene transcription were analyzed. RESULTS: Green fluorescent protein (GFP)-marked Lin(-)-HSCs were detected at 24 hours after injection in the spleen and later in ischemic brain parenchyma, expressing microglial but no neural marker proteins. Tissue injury assessment showed significantly smaller infarct volumes and less apoptotic neuronal cell death in peri-infarct areas of Lin(-)-HSC-treated animals. Analysis of immune cell infiltration in ischemic hemispheres revealed a reduction of invading T cells and macrophages in treated mice. Moreover, Lin(-)-HSC therapy counter-regulated proinflammatory cytokine and chemokine receptor gene transcription within the spleen. CONCLUSIONS: Our data demonstrate that systemically applied Lin(-)-HSCs reduce cerebral postischemic inflammation, attenuate peripheral immune activation and mediate neuroprotection after ischemic stroke.