Aß efflux impairment and inflammation linked to cerebrovascular accumulation of amyloid-forming amylin secreted from pancreas.

Impairment of vascular pathways of cerebral ß-amyloid (Aß) elimination contributes to Alzheimer disease (AD). Vascular damage is commonly associated with diabetes. Here we show in human tissues and AD-model rats that bloodborne islet amyloid polypeptide (amylin) secreted from the pancreas perturbs cerebral Aß clearance. Blood amylin concentrations are higher in AD than in cognitively unaffected persons. Amyloid-forming amylin accumulates in circulating monocytes and co-deposits with Aß within the brain microvasculature, possibly involving inflammation. In rats, pancreatic expression of amyloid-forming human amylin indeed induces cerebrovascular inflammation and amylin-Aß co-deposits. LRP1-mediated Aß transport across the blood-brain barrier and Aß clearance through interstitial fluid drainage along vascular walls are impaired, as indicated by Aß deposition in perivascular spaces. At the molecular level, cerebrovascular amylin deposits alter immune and hypoxia-related brain gene expression. These converging data from humans and laboratory animals suggest that altering bloodborne amylin could potentially reduce cerebrovascular amylin deposits and Aß pathology.

Microglial-associated responses to comorbid amyloid pathology and hyperhomocysteinemia in an aged knock-in mouse model of Alzheimer's disease.

BACKGROUND: Elevated blood homocysteine levels, termed hyperhomocysteinemia (HHcy), is a prevalent risk factor for Alzheimer's disease (AD) in elderly populations. While dietary supplementation of B-vitamins is a generally effective method to lower homocysteine levels, there is little if any benefit to cognition. In the context of amyloid pathology, dietary-induced HHcy is known to enhance amyloid deposition and certain inflammatory responses. Little is known, however, about whether there is a more specific effect on microglia resulting from combined amyloid and HHcy pathologies. METHODS: The present study used a knock-in mouse model of amyloidosis, aged to 12 months, given 8 weeks of B-vitamin deficiency-induced HHcy to better understand how microglia are affected in this comorbidity context. RESULTS: We found that HHcy-inducing diet increased amyloid plaque burden, altered the neuroinflammatory milieu, and upregulated the expression of multiple damage-associated and "homeostatic" microglial genes. CONCLUSIONS: Taken together, these data indicate complex effects of comorbid pathologies on microglial function that are not driven solely by increased amyloid burden. Given the highly dynamic nature of microglia, their central role in AD pathology, and the frequent occurrence of various comorbidities in AD patients, it is increasingly important to understand how microglia respond to mixed pathological processes.

Genetic knockout of myosin light chain kinase (MLCK210) prevents cerebral microhemorrhages and attenuates neuroinflammation in a mouse model of vascular cognitive impairment and dementia.

The blood-brain barrier (BBB) is critical in maintenance of brain homeostasis, and loss of its functional integrity is a key feature across a broad range of neurological insults. This includes both acute injuries such as traumatic brain injury and stroke, as well as more chronic pathologies associated with aging, such as vascular cognitive impairment and dementia (VCID). A specific form of myosin light chain kinase (MLCK210) is a major regulator of barrier integrity in general, including the BBB. Studies have demonstrated the potential of MLCK210 as a therapeutic target for peripheral disorders involving tissue barrier dysfunction, but less is known about its potential as a target for chronic neurologic disorders. We report here that genetic knockout (KO) of MLCK210 protects against cerebral microhemorrhages and neuroinflammation induced by chronic dietary hyperhomocysteinemia. Overall, the results are consistent with an accumulating body of evidence supporting MLCK210 as a potential therapeutic target for tissue barrier dysfunction and specifically implicate it in BBB dysfunction and neuroinflammation in a model of VCID.

Diffuse traumatic brain injury induces prolonged immune dysregulation and potentiates hyperalgesia following a peripheral immune challenge.

BACKGROUND: Nociceptive and neuropathic pain occurs as part of the disease process after traumatic brain injury (TBI) in humans. Central and peripheral inflammation, a major secondary injury process initiated by the traumatic brain injury event, has been implicated in the potentiation of peripheral nociceptive pain. We hypothesized that the inflammatory response to diffuse traumatic brain injury potentiates persistent pain through prolonged immune dysregulation. RESULTS: To test this, adult, male C57BL/6 mice were subjected to midline fluid percussion brain injury or to sham procedure. One cohort of mice was analyzed for inflammation-related cytokine levels in cortical biopsies and serum along an acute time course. In a second cohort, peripheral inflammation was induced seven days after surgery/injury with an intraplantar injection of carrageenan. This was followed by measurement of mechanical hyperalgesia, glial fibrillary acidic protein and Iba1 immunohistochemical analysis of neuroinflammation in the brain, and flow cytometric analysis of T-cell differentiation in mucosal lymph. Traumatic brain injury increased interleukin-6 and chemokine ligand 1 levels in the cortex and serum that peaked within 1-9 h and then resolved. Intraplantar carrageenan produced mechanical hyperalgesia that was potentiated by traumatic brain injury. Further, mucosal T cells from brain-injured mice showed a distinct deficiency in the ability to differentiate into inflammation-suppressing regulatory T cells (Tregs). CONCLUSIONS: We conclude that traumatic brain injury increased the inflammatory pain associated with cutaneous inflammation by contributing to systemic immune dysregulation. Regulatory T cells are immune suppressors and failure of T cells to differentiate into regulatory T cells leads to unregulated cytokine production which may contribute to the potentiation of peripheral pain through the excitation of peripheral sensory neurons. In addition, regulatory T cells are identified as a potential target for therapeutic rebalancing of peripheral immune homeostasis to improve functional outcome and decrease the incidence of peripheral inflammatory pain following traumatic brain injury.

Targeting human central nervous system protein kinases: An isoform selective p38αMAPK inhibitor that attenuates disease progression in Alzheimer's disease mouse models.

The first kinase inhibitor drug approval in 2001 initiated a remarkable decade of tyrosine kinase inhibitor drugs for oncology indications, but a void exists for serine/threonine protein kinase inhibitor drugs and central nervous system indications. Stress kinases are of special interest in neurological and neuropsychiatric disorders due to their involvement in synaptic dysfunction and complex disease susceptibility. Clinical and preclinical evidence implicates the stress related kinase p38αMAPK as a potential neurotherapeutic target, but isoform selective p38αMAPK inhibitor candidates are lacking and the mixed kinase inhibitor drugs that are promising in peripheral tissue disease indications have limitations for neurologic indications. Therefore, pursuit of the neurotherapeutic hypothesis requires kinase isoform selective inhibitors with appropriate neuropharmacology features. Synaptic dysfunction disorders offer a potential for enhanced pharmacological efficacy due to stress-induced activation of p38αMAPK in both neurons and glia, the interacting cellular components of the synaptic pathophysiological axis, to be modulated. We report a novel isoform selective p38αMAPK inhibitor, MW01-18-150SRM (=MW150), that is efficacious in suppression of hippocampal-dependent associative and spatial memory deficits in two distinct synaptic dysfunction mouse models. A synthetic scheme for biocompatible product and positive outcomes from pharmacological screens are presented. The high-resolution crystallographic structure of the p38αMAPK/MW150 complex documents active site binding, reveals a potential low energy conformation of the bound inhibitor, and suggests a structural explanation for MW150's exquisite target selectivity. As far as we are aware, MW150 is without precedent as an isoform selective p38MAPK inhibitor or as a kinase inhibitor capable of modulating in vivo stress related behavior.

The p38alpha mitogen-activated protein kinase limits the CNS proinflammatory cytokine response to systemic lipopolysaccharide, potentially through an IL-10 dependent mechanism.

BACKGROUND: The p38α mitogen-activated protein kinase (MAPK) is a well-characterized intracellular kinase involved in the overproduction of proinflammatory cytokines from glia. As such, p38α appears to be a promising therapeutic target for neurodegenerative diseases associated with neuroinflammation. However, the in vivo role of p38α in cytokine production in the CNS is poorly defined, and prior work suggests that p38α may be affecting a yet to be identified negative feedback mechanism that limits the acute, injury-induced proinflammatory cytokine surge in the CNS. METHODS: To attempt to define this negative feedback mechanism, we used two in vitro and two in vivo models of neuroinflammation in a mouse where p38α is deficient in cells of the myeloid lineage. RESULTS: We found that p38α in myeloid cells has an important role in limiting amplitude of the acute proinflammatory cytokine response to a systemic inflammatory challenge. Moreover, we identified IL-10 as a potential negative feedback mechanism regulated by p38α. CONCLUSIONS: Our data suggest that p38α regulates a proper balance between the pro- and anti-inflammatory cytokine responses to systemic inflammation, and that if circulating IL-10 levels are not elevated to counter-balance the increased systemic proinflammatory responses, the spread of the inflammatory response from the periphery to the CNS is exaggerated.

Neuroinflammation and neurologic deficits in diabetes linked to brain accumulation of amylin.

BACKGROUND: We recently found that brain tissue from patients with type-2 diabetes (T2D) and cognitive impairment contains deposits of amylin, an amyloidogenic hormone synthesized and co-secreted with insulin by pancreatic ß-cells. Amylin deposition is promoted by chronic hypersecretion of amylin (hyperamylinemia), which is common in humans with obesity or pre-diabetic insulin resistance. Human amylin oligomerizes quickly when oversecreted, which is toxic, induces inflammation in pancreatic islets and contributes to the development of T2D. Here, we tested the hypothesis that accumulation of oligomerized amylin affects brain function. METHODS: In contrast to amylin from humans, rodent amylin is neither amyloidogenic nor cytotoxic. We exploited this fact by comparing rats overexpressing human amylin in the pancreas (HIP rats) with their littermate rats which express only wild-type (WT) non-amyloidogenic rodent amylin. Cage activity, rotarod and novel object recognition tests were performed on animals nine months of age or older. Amylin deposition in the brain was documented by immunohistochemistry, and western blot. We also measured neuroinflammation by immunohistochemistry, quantitative real-time PCR and cytokine protein levels. RESULTS: Compared to WT rats, HIP rats show i) reduced exploratory drive, ii) impaired recognition memory and iii) no ability to improve the performance on the rotarod. The development of neurological deficits is associated with amylin accumulation in the brain. The level of oligomerized amylin in supernatant fractions and pellets from brain homogenates is almost double in HIP rats compared with WT littermates (P < 0.05). Large amylin deposits (>50 µm diameter) were also occasionally seen in HIP rat brains. Accumulation of oligomerized amylin alters the brain structure at the molecular level. Immunohistochemistry analysis with an ED1 antibody indicates possible activated microglia/macrophages which are clustering in areas positive for amylin infiltration. Multiple inflammatory markers are expressed in HIP rat brains as opposed to WT rats, confirming that amylin deposition in the brain induces a neuroinflammatory response. CONCLUSIONS: Hyperamylinemia promotes accumulation of oligomerized amylin in the brain leading to neurological deficits through an oligomerized amylin-mediated inflammatory response. Additional studies are needed to determine whether brain amylin accumulation may predispose to diabetic brain injury and cognitive decline.

The p38α MAPK regulates microglial responsiveness to diffuse traumatic brain injury.

Neuropathology after traumatic brain injury (TBI) is the result of both the immediate impact injury and secondary injury mechanisms. Unresolved post-traumatic glial activation is a secondary injury mechanism that contributes to a chronic state of neuroinflammation in both animal models of TBI and human head injury patients. We recently demonstrated, using in vitro models, that p38α MAPK signaling in microglia is a key event in promoting cytokine production in response to diverse disease-relevant stressors and subsequent inflammatory neuronal dysfunction. From these findings, we hypothesized that the p38α signaling pathway in microglia could be contributing to the secondary neuropathologic sequelae after a diffuse TBI. Mice where microglia were p38α-deficient (p38α KO) were protected against TBI-induced motor deficits and synaptic protein loss. In wild-type (WT) mice, diffuse TBI produced microglia morphological activation that lasted for at least 7 d; however, p38α KO mice failed to activate this response. Unexpectedly, we found that the peak of the early, acute phase cytokine and chemokine levels was increased in injured p38α KO mice compared with injured WT mice. The increased cytokine levels in the p38α KO mice could not be accounted for by more infiltration of macrophages or neutrophils, or increased astrogliosis. By 7 d after injury, the cytokine and chemokine levels remained elevated in injured WT mice but not in p38α KO mice. Together, these data suggest that p38α balances the inflammatory response by acutely attenuating the early proinflammatory cytokine surge while perpetuating the chronic microglia activation after TBI.

Adjusting for mortality when identifying risk factors for transitions to mild cognitive impairment and dementia.

Risk factors for mild cognitive impairment (MCI) and dementia are often investigated without accounting for the competing risk of mortality, which can bias results and lead to spurious conclusions, particularly regarding protective factors. Here, we apply a semi-Markov modeling approach to 531 participants in the University of Kentucky Biologically Resilient Adults in Neurological Studies (BRAiNS) longitudinal cohort, over one-third of whom died without transitioning to a cognitively impaired clinical state. A semi-Markov approach enables a statistical study of clinical state transitions while accounting for the competing risk of death and facilitates insights into both the odds that a risk factor will affect clinical transitions as well as the age at which the transition to MCI or dementia will occur. Risk factors assessed in the current study were identified by matching those reported in the literature with the data elements collected on participants. The presence of Type II diabetes at baseline shortens the time it takes cognitively intact individuals to transition to MCI by seven years on average while use of estrogen replacement therapy at enrollment (baseline) decreases the time required to convert from MCI to dementia by 1.5 years. Finally, smoking and being overweight do not promote transitions to impaired states but instead hasten death without a dementia. In contrast, conventional statistical analyses based on Cox proportional hazards models fail to recognize diabetes as a risk, show that being overweight increases the risk of clinical MCI, and that high blood pressure at baseline increases the risk of a dementia.

Targeting astrocytes ameliorates neurologic changes in a mouse model of Alzheimer's disease.

Astrocytes are the most abundant cell type in the brain and play a critical role in maintaining healthy nervous tissue. In Alzheimer's disease (AD) and most other neurodegenerative disorders, many astrocytes convert to a chronically "activated" phenotype characterized by morphologic and biochemical changes that appear to compromise protective properties and/or promote harmful neuroinflammatory processes. Activated astrocytes emerge early in the course of AD and become increasingly prominent as clinical and pathological symptoms progress, but few studies have tested the potential of astrocyte-targeted therapeutics in an intact animal model of AD. Here, we used adeno-associated virus (AAV) vectors containing the astrocyte-specific Gfa2 promoter to target hippocampal astrocytes in APP/PS1 mice. AAV-Gfa2 vectors drove the expression of VIVIT, a peptide that interferes with the immune/inflammatory calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway, shown by our laboratory and others to orchestrate biochemical cascades leading to astrocyte activation. After several months of treatment with Gfa2-VIVIT, APP/PS1 mice exhibited improved cognitive and synaptic function, reduced glial activation, and lower amyloid levels. The results confirm a deleterious role for activated astrocytes in AD and lay the groundwork for exploration of other novel astrocyte-based therapies.

Microglial p38α MAPK is critical for LPS-induced neuron degeneration, through a mechanism involving TNFα.

BACKGROUND: The p38α MAPK isoform is a well-established therapeutic target in peripheral inflammatory diseases, but the importance of this kinase in pathological microglial activation and detrimental inflammation in CNS disorders is less well understood. To test the role of the p38α MAPK isoform in microglia-dependent neuron damage, we used primary microglia from wild-type (WT) or p38α MAPK conditional knockout (KO) mice in co-culture with WT cortical neurons, and measured neuron damage after LPS insult. RESULTS: We found that neurons in co-culture with p38α-deficient microglia were protected against LPS-induced synaptic loss, neurite degeneration, and neuronal death. The involvement of the proinflammatory cytokine TNFα was demonstrated by the findings that p38α KO microglia produced much less TNFα in response to LPS compared to WT microglia, that adding back TNFα to KO microglia/neuron co-cultures increased the LPS-induced neuron damage, and that neutralization of TNFα in WT microglia/neuron co-cultures prevented the neuron damage. These results using cell-selective, isoform-specific KO mice demonstrate that the p38α MAPK isoform in microglia is a key mediator of LPS-induced neuronal and synaptic dysfunction. The findings also provide evidence that a major mechanism by which LPS activation of microglia p38α MAPK signaling leads to neuron damage is through up-regulation of the proinflammatory cytokine TNFα. CONCLUSIONS: The data suggest that selective targeting of p38α MAPK signaling should be explored as a potential therapeutic strategy for CNS disorders where overproduction of proinflammatory cytokines is implicated in disease progression.

Microglial p38α MAPK is a key regulator of proinflammatory cytokine up-regulation induced by toll-like receptor (TLR) ligands or beta-amyloid (Aß).

BACKGROUND: Overproduction of proinflammatory cytokines from activated microglia has been implicated as an important contributor to pathophysiology progression in both acute and chronic neurodegenerative diseases. Therefore, it is critical to elucidate intracellular signaling pathways that are significant contributors to cytokine overproduction in microglia exposed to specific stressors, especially pathways amenable to drug interventions. The serine/threonine protein kinase p38α MAPK is a key enzyme in the parallel and convergent intracellular signaling pathways involved in stressor-induced production of IL-1ß and TNFα in peripheral tissues, and is a drug development target for peripheral inflammatory diseases. However, much less is known about the quantitative importance of microglial p38α MAPK in stressor-induced cytokine overproduction, or the potential of microglial p38α MAPK to be a druggable target for CNS disorders. Therefore, we examined the contribution of microglial p38αMAPK to cytokine up-regulation, with a focus on the potential to suppress the cytokine increase by inhibition of the kinase with pharmacological or genetic approaches. METHODS: The microglial cytokine response to TLR ligands 2/3/4/7/8/9 or to Aß1-42 was tested in the presence of a CNS-penetrant p38α MAPK inhibitor, MW01-2-069A-SRM. Primary microglia from mice genetically deficient in p38α MAPK were used to further establish a linkage between microglia p38α MAPK and cytokine overproduction. The in vivo significance was determined by p38α MAPK inhibitor treatment in a LPS-induced model of acute neuroinflammation. RESULTS: Increased IL-1ß and TNFα production by the BV-2 microglial cell line and by primary microglia cultures was inhibited in a concentration-dependent manner by the p38α MAPK-targeted inhibitor. Cellular target engagement was demonstrated by the accompanying decrease in the phosphorylation state of two p38α MAPK protein substrates, MK2 and MSK1. Consistent with the pharmacological findings, microglia from p38α-deficient mice showed a diminished cytokine response to LPS. Further, oral administration of the inhibitor blocked the increase of IL-1ß in the cerebral cortex of mice stressed by intraperitoneal injection of LPS. CONCLUSION: The p38α MAPK pathway is an important contributor to the increased microglial production of proinflammatory cytokines induced by diverse stressors. The results also indicate the feasibility of targeting p38α MAPK to modulate CNS proinflammatory cytokine overproduction.

Association between male gender and cortical Lewy body pathology in large autopsy series.

Sex-linked factors may alter risk for neurodegenerative diseases. Definitive diagnoses are not established until autopsy, so neuropathological studies are critical. There have not been reported gender-related differences in neocortical Lewy bodies (LBs) using large multi-center autopsy series. We evaluated the associations between gender and pathologically characterized neurodegenerative diseases. Cases with Alzheimer's disease (AD), neocortical LBs, AD + neocortical LBs, or neither pathology were evaluated as separate groups. Results were corrected for possible confounders including age at death, smoking history, and education. The settings were the University of Kentucky Alzheimer's Disease Center and the National Alzheimer's Coordinating Center (NACC) Registry autopsy series; 3,830 subjects met inclusion criteria. Patients with neocortical ("diffuse") or intermediate ("limbic") LB pathologies tended to be male (male:female odds ratios ~2.9 with 95% CI 2.02-4.18). The preponderance of males dying with neocortical LB pathology was seen consistently across age groups and was not due to the potential confounders evaluated. By contrast, individuals dying with AD pathology were more likely to be female if dying over 80 (male:female odds ratio 0.66, 95% CI 0.50-0.88), but that tendency was not seen in individuals dying with AD pathology prior to age 80. Increased understanding of the male predominance in neocortical LB pathology may help guide clinicians, because males are more likely to be "undercalled" for neocortical LBs clinically, and females are more likely to be "overcalled" (P < 0.05 for both). Males are far more likely than females to die with neocortical LB pathology. This phenomenon may help guide medical practice including clinical trial study design.

Enhanced microglial activation and proinflammatory cytokine upregulation are linked to increased susceptibility to seizures and neurologic injury in a 'two-hit' seizure model.

Early-life seizures result in increased susceptibility to seizures and greater neurologic injury with a second insult in adulthood. The mechanisms which link seizures in early-life to increased susceptibility to neurologic injury following a 'second hit' are not known. We examined the contribution of microglial activation and increased proinflammatory cytokine production to the subsequent increase in susceptibility to neurologic injury using a kainic acid (KA)-induced, established 'two-hit' seizure model in rats. Postnatal day (P)15 rats were administered intraperitoneal KA (early-life seizures) or saline, followed on P45 with either a 'second hit' of KA, a first exposure to KA (adult seizures), or saline. We measured the levels of proinflammatory cytokines (IL-1 beta, TNF-alpha, and S100B), the chemokine CCL2, microglial activation, seizure susceptibility and neuronal outcomes in adult rats 12 h and 10 days after the second hit on P45. The 'two-hit' group exposed to KA on both P15 and P45 had higher levels of cytokines, greater microglial activation, and increased susceptibility to seizures and neurologic injury compared to the adult seizures group. Treatment after early-life seizures with Minozac, a small molecule experimental therapeutic that targets upregulated proinflammatory cytokine production, attenuated the enhanced microglial and cytokine responses, the increased susceptibility to seizures, and the greater neuronal injury in the 'two-hit' group. These results implicate microglial activation as one mechanism by which early-life seizures contribute to increased vulnerability to neurologic insults in adulthood, and indicate the potential longer term benefits of early-life intervention with therapies that target up-regulation of proinflammatory cytokines.

MCP-1-deficient mice show reduced neuroinflammatory responses and increased peripheral inflammatory responses to peripheral endotoxin insult.

BACKGROUND: An endotoxin insult mimics a severe peripheral infection and recent evidence suggests that a single exposure can cause long-term cognitive deficits. A peripheral injection of LPS results in production of pro-inflammatory cytokines, such as IL-1beta and TNF-alpha, in the brain and periphery and these cytokines mediate many effects of the acute phase response including activation of the HPA axis. The chemokine MCP-1 is highly expressed during endotoxemia and although much is known about the importance of MCP-1 in peripheral inflammatory responses to LPS, information about MCP-1 and CNS responses to peripheral LPS is lacking. METHODS: C57Bl/6 mice were administered LPS by intraperitoneal (i.p.) injection, serum and brains were collected at several time points, and the time course of MCP-1 protein up-regulation was measured. To examine the role of MCP-1 in activation of the brain during acute systemic inflammation, we injected MCP-1 knockout (MCP-1-/-) or control C57Bl/6 (MCP-1+/+) mice with LPS i.p. and measured the levels of selected cytokines and chemokines in serum and brain extracts 6 hours later. Activated microglia were examined by CD45 immunohistochemistry, and serum corticosterone and ACTH levels were measured by enzyme immunoassay. RESULTS: We report that LPS injection induces a robust increase in MCP-1 protein levels in serum and brain, with peak brain levels reached at 6 hrs after LPS administration. MCP-1-/- mice injected with LPS showed higher levels of serum IL-1beta and TNF-alpha compared to LPS-treated MCP-1+/+ mice. In contrast, these MCP-1-/- mice showed significantly lower inductions of brain pro-inflammatory cytokines and chemokines, fewer activated microglia, and a reduction in serum corticosterone levels. CONCLUSION: MCP-1-/- mice have decreased brain inflammation after a peripheral LPS insult, despite an exaggerated peripheral response. These data demonstrate an important role for MCP-1 in regulation of brain inflammation after peripheral endotoxemia.

MFG-E8 regulates microglial phagocytosis of apoptotic neurons.

Phagocytosis is an essential mechanism for clearance of pathogens, dying cells, and other unwanted debris in order to maintain tissue health in the body. Macrophages execute this process in the peripheral immune system but in the brain microglia act as resident macrophages to accomplish this function. In the peripheral immune system, macrophages secrete Milk Fat Globule Factor-E8 (MFG-E8) that recognizes phosphatidylserine "eat me" signals expressed on the surface of apoptotic cells. MFG-E8 then acts as a tether to attach the apoptotic cell to the macrophage and trigger a signaling cascade that stimulates the phagocyte development, allowing the macrophage to engulf the dying cell. When this process becomes disrupted, inflammation and autoimmunity can result. MFG-E8 resides in the brain as well as in the periphery, and microglia express MFG-E8. However, the function of MFG-E8 in the brain has not been elucidated. We measured MFG-E8 production in the BV-2 microglial cell line and the role of this protein in the recognition and engulfment of apoptotic SY5Y neuroblastoma cells. BV-2 cells produced and released MFG-E8, which apoptotic SY5Y cells and the chemokine fractalkine further stimulated. Furthermore, MFG-E8 increased phagocytosis of apoptotic SY5Y cells, and a dominant negative form of MFG-E8 inhibited phagocytosis by BV-2 cells. Finally, brain MFG-E8 levels were altered in a mouse model of Alzheimer's disease. Our data suggest that MFG-E8 acts in the brain via microglia to aid in clearance of apoptotic neurons, and we hypothesize that a dysregulation of this process may be involved in neurodegenerative disease.

Inhibition of experimental autoimmune encephalomyelitis by a novel small molecular weight proinflammatory cytokine suppressing drug.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated, autoimmune disease of the central nervous system (CNS) that serves as a model for various cellular and molecular aspects of the human disease, multiple sclerosis (MS). Although EAE has long been considered a T cell-mediated disease, macrophages play a role in disease pathogenesis and are known to accumulate in the CNS under the control of chemokines. In the present report we demonstrate that mice induced to develop EAE were treated with a small molecular weight molecule that suppresses proinflammatory cytokine production which resulted in significantly decreased clinical EAE, CNS CCL2 expression and CNS macrophage accumulation. These results demonstrate the efficacy of a novel class of therapeutic molecules for CNS demyelinating disease.

Suppression of acute proinflammatory cytokine and chemokine upregulation by post-injury administration of a novel small molecule improves long-term neurologic outcome in a mouse model of traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) with its associated morbidity is a major area of unmet medical need that lacks effective therapies. TBI initiates a neuroinflammatory cascade characterized by activation of astrocytes and microglia, and increased production of immune mediators including proinflammatory cytokines and chemokines. This inflammatory response contributes both to the acute pathologic processes following TBI including cerebral edema, in addition to longer-term neuronal damage and cognitive impairment. However, activated glia also play a neuroprotective and reparative role in recovery from injury. Thus, potential therapeutic strategies targeting the neuroinflammatory cascade must use careful dosing considerations, such as amount of drug and timing of administration post injury, in order not to interfere with the reparative contribution of activated glia. METHODS: We tested the hypothesis that attenuation of the acute increase in proinflammatory cytokines and chemokines following TBI would decrease neurologic injury and improve functional neurologic outcome. We used the small molecule experimental therapeutic, Minozac (Mzc), to suppress TBI-induced up-regulation of glial activation and proinflammatory cytokines back towards basal levels. Mzc was administered in a clinically relevant time window post-injury in a murine closed-skull, cortical impact model of TBI. Mzc effects on the acute increase in brain cytokine and chemokine levels were measured as well as the effect on neuronal injury and neurobehavioral function. RESULTS: Administration of Mzc (5 mg/kg) at 3 h and 9 h post-TBI attenuates the acute increase in proinflammatory cytokine and chemokine levels, reduces astrocyte activation, and the longer term neurologic injury, and neurobehavioral deficits measured by Y maze performance over a 28-day recovery period. Mzc-treated animals also have no significant increase in brain water content (edema), a major cause of the neurologic morbidity associated with TBI. CONCLUSION: These results support the hypothesis that proinflammatory cytokines contribute to a glial activation cycle that produces neuronal dysfunction or injury following TBI. The improvement in long-term functional neurologic outcome following suppression of cytokine upregulation in a clinically relevant therapeutic window indicates that selective targeting of neuroinflammation may lead to novel therapies for the major neurologic morbidities resulting from head injury, and indicates the potential of Mzc as a future therapeutic for TBI.

Glia as a therapeutic target: selective suppression of human amyloid-beta-induced upregulation of brain proinflammatory cytokine production attenuates neurodegeneration.

A corollary of the neuroinflammation hypothesis is that selective suppression of neurotoxic products produced by excessive glial activation will result in neuroprotection. We report here that daily oral administration to mice of the brain-penetrant compound 4,6-diphenyl-3-(4-(pyrimidin-2-yl)piperazin-1-yl)pyridazine (MW01-5-188WH), a selective inhibitor of proinflammatory cytokine production by activated glia, suppressed the human amyloid-beta (Abeta) 1-42-induced upregulation of interleukin-1beta, tumor necrosis factor-alpha, and S100B in the hippocampus. Suppression of neuroinflammation was accompanied by restoration of hippocampal synaptic dysfunction markers synaptophysin and postsynaptic density-95 back toward control levels. Consistent with the neuropathophysiological improvements, MW01-5-188WH therapy attenuated deficits in Y maze behavior, a hippocampal-linked task. Oral MW01-5-188WH therapy begun 3 weeks after initiation of intracerebroventricular infusion of human Abeta decreased the numbers of activated astrocytes and microglia and the cytokine levels in the hippocampus without modifying amyloid plaque burden or altering peripheral tissue cytokine upregulation in response to an in vivo inflammatory challenge. The results provide a novel integrative chemical biology proof in support of the neuroinflammation hypothesis of disease progression, demonstrate that neurodegeneration can be attenuated independently of plaque modulation by targeting innate brain proinflammatory cytokine responses, and indicate the feasibility of developing efficacious, safe, and selective therapies for neurodegenerative disorders by targeting key glial activation pathways.

Human amyloid beta-induced neuroinflammation is an early event in neurodegeneration.

Using a human amyloid beta (Abeta) intracerebroventricular infusion mouse model of Alzheimer's disease-related injury, we previously demonstrated that systemic administration of a glial activation inhibitor could suppress neuroinflammation, prevent synaptic damage, and attenuate hippocampal-dependent behavioral deficits. We report that Abeta-induced neuroinflammation is an early event associated with onset and progression of pathophysiology, can be suppressed by the glial inhibitor over a range of intervention start times, and is amenable to suppression without inhibiting peripheral tissue inflammatory responses. Specifically, hippocampal neuroinflammation and neurodegeneration occur in close time proximity at 4-6 weeks after the start of infusion. Intraperitoneal administration of inhibitor for 2-week intervals starting at various times after initiation of Abeta infusion suppresses progression of pathophysiology. The glial inhibitor is a selective suppressor of neuroinflammation, in that it does not block peripheral tissue production of proinflammatory cytokines or markers of B- and T-cell activation after a systemic lipopolysaccharide challenge. These results support a causal link between neuroinflammation and neurodegeneration, have important implications for future therapeutic development, and provide insight into the relative time window for targeting neuroinflammation with positive neurological outcomes.