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Abstract 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-1β 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 Aβ1-42 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-1β 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-1β 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.

Abstract 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-1β and TNF-α, 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-1β and TNF-α 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.

Abstract 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.

Abstract Background An accumulating body of evidence is consistent with the hypothesis that excessive or prolonged increases in proinflammatory cytokine production by activated glia is a contributor to the progression of pathophysiology that is causally linked to synaptic dysfunction and hippocampal behavior deficits in neurodegenerative diseases such as Alzheimer's disease (AD). This raises the opportunity for the development of new classes of potentially disease-modifying therapeutics. A logical candidate CNS target is p38α MAPK, a well-established drug discovery molecular target for altering proinflammatory cytokine cascades in peripheral tissue disorders. Activated p38 MAPK is seen in human AD brain tissue and in AD-relevant animal models, and cell culture studies strongly implicate p38 MAPK in the increased production of proinflammatory cytokines by glia activated with human amyloid-beta (Aβ) and other disease-relevant stressors. However, the vast majority of small molecule drugs do not have sufficient penetrance of the blood-brain barrier to allow their use as in vivo research tools or as therapeutics for neurodegenerative disorders. The goal of this study was to test the hypothesis that brain p38α MAPK is a potential in vivo target for orally bioavailable, small molecules capable of suppressing excessive cytokine production by activated glia back towards homeostasis, allowing an improvement in neurologic outcomes. Methods A novel synthetic small molecule based on a molecular scaffold used previously was designed, synthesized, and subjected to analyses to demonstrate its potential in vivo bioavailability, metabolic stability, safety and brain uptake. Testing for in vivo efficacy used an AD-relevant mouse model. Results A novel, CNS-penetrant, non-toxic, orally bioavailable, small molecule inhibitor of p38α MAPK (MW01-2-069A-SRM) was developed. Oral administration of the compound at a low dose (2.5 mg/kg) resulted in attenuation of excessive proinflammatory cytokine production in the hippocampus back towards normal in the animal model. Animals with attenuated cytokine production had reductions in synaptic dysfunction and hippocampus-dependent behavioral deficits. Conclusion The p38α MAPK pathway is quantitatively important in the Aβ-induced production of proinflammatory cytokines in hippocampus, and brain p38α MAPK is a viable molecular target for future development of potential disease-modifying therapeutics in AD and related neurodegenerative disorders.

Abstract Background Interleukin 1 (IL-1) is a key mediator of immune responses in health and disease. Although classically the function of IL-1 has been studied in the systemic immune system, research in the past decade has revealed analogous roles in the CNS where the cytokine can contribute to the neuroinflammation and neuropathology seen in a number of neurodegenerative diseases. In Alzheimer's disease (AD), for example, pre-clinical and clinical studies have implicated IL-1 in the progression of a pathologic, glia-mediated pro-inflammatory state in the CNS. The glia-driven neuroinflammation can lead to neuronal damage, which, in turn, stimulates further glia activation, potentially propagating a detrimental cycle that contributes to progression of pathology. A prediction of this neuroinflammation hypothesis is that increased IL-1 signaling in vivo would correlate with increased severity of AD-relevant neuroinflammation and neuronal damage. Methods To test the hypothesis that increased IL-1 signaling predisposes animals to beta-amyloid (Aβ)-induced damage, we used IL-1 receptor antagonist Knock-Out (IL1raKO) and wild-type (WT) littermate mice in a model that involves intracerebroventricular infusion of human oligomeric Aβ1–42. This model mimics many features of AD, including robust neuroinflammation, Aβ plaques, synaptic damage and neuronal loss in the hippocampus. IL1raKO and WT mice were infused with Aβ for 28 days, sacrificed at 42 days, and hippocampal endpoints analyzed. Results IL1raKO mice showed increased vulnerability to Aβ-induced neuropathology relative to their WT counterparts. Specifically, IL1raKO mice exhibited increased mortality, enhanced microglial activation and neuroinflammation, and more pronounced loss of synaptic markers. Interestingly, Aβ-induced astrocyte responses were not significantly different between WT and IL1raKO mice, suggesting that enhanced IL-1 signaling predominately affects microglia. Conclusion Our data are consistent with the neuroinflammation hypothesis whereby increased IL-1 signaling in AD enhances glia activation and leads to an augmented neuroinflammatory process that increases the severity of neuropathologic sequelae.

Background: Brain p38α mitogen-activated protein kinase (MAPK), a potential therapeutic target for cognitive dysfunction based on the neuroinflammation-synaptic dysfunction cycle of pathophysiology progression, offers an innovative pharmacological strategy via inhibiting the same activated target in both glia and neurons, thereby enhancing the possibility for efficacy. The highly selective, brain-penetrant p38αMAPK inhibitor MW150 attenuates cognitive dysfunction in two distinct Alzheimer's disease (AD)-relevant models and avoids the problems encountered with previous mixed-kinase inhibitor drug candidates. Therefore, it is essential that the glial effects of this CNS-active kinase inhibitor be addressed in order to anticipate future use in clinical investigations.Methods: We explored the effects of MW150 on glial biology in the AD-relevant APP/PS1 knock-in (KI) mouse model where we previously showed efficacy in suppression of hippocampal-dependent associative and spatial memory deficits. MW150 (2.5 mg/kg/day) was administered daily to 11-12-month-old KI mice for 14 days, and levels of proinflammatory cytokines IL-1β, TNFα, and IL-6 measured in homogenates of mouse cortex using ELISA. Glial markers IBA1, CD45, CD68, and GFAP were assessed by immunohistochemistry. Microglia and amyloid plaques were quantified by immunofluorescence staining followed by confocal imaging. Levels of soluble and insoluble of Aβ40 and Aβ42 were measured by ELISA. The studies of in vivo pharmacodynamic effects on markers of neuroinflammation were complemented by mechanistic studies in the murine microglia BV2 cell line, using live cell imaging techniques to monitor proliferation, migration, and phagocytosis activities.Results: Intervention with MW150 in KI mice during the established therapeutic time window attenuated the increased levels of IL-1β and TNFα but not IL-6. MW150 treatment also increased the IBA1+ microglia within a 15 μm radius of the amyloid plaques, without significantly affecting overall microglia or plaque volume. Levels of IBA1, CD45, CD68, GFAP, and Aβ40 and Aβ42 were not affected by MW150 treatment. MW150 did not significantly alter microglial migration, proliferation, or phagocytosis in BV2 cells.Conclusions: Our results demonstrate that MW150 at an efficacious dose can selectively modulate neuroinflammatory responses associated with pathology progression without pan-suppression of normal physiological functions of microglia. [ABSTRACT FROM AUTHOR]

Background: An accumulating body of evidence is consistent with the hypothesis that excessive or prolonged increases in proinflammatory cytokine production by activated glia is a contributor to the progression of pathophysiology that is causally linked to synaptic dysfunction and hippocampal behavior deficits in neurodegenerative diseases such as Alzheimer's disease (AD). This raises the opportunity for the development of new classes of potentially disease-modifying therapeutics. A logical candidate CNS target is p38 alpha MAPK, a well-established drug discovery molecular target for altering proinflammatory cytokine cascades in peripheral tissue disorders. Activated p38 MAPK is seen in human AD brain tissue and in AD-relevant animal models, and cell culture studies strongly implicate p38 MAPK in the increased production of proinflammatory cytokines by glia activated with human amyloid-beta (A beta) and other disease-relevant stressors. However, the vast majority of small molecule drugs do not have sufficient penetrance of the blood-brain barrier to allow their use as in vivo research tools or as therapeutics for neurodegenerative disorders. The goal of this study was to test the hypothesis that brain p38 alpha MAPK is a potential in vivo target for orally bioavailable, small molecules capable of suppressing excessive cytokine production by activated glia back towards homeostasis, allowing an improvement in neurologic outcomes., Methods: A novel synthetic small molecule based on a molecular scaffold used previously was designed, synthesized, and subjected to analyses to demonstrate its potential in vivo bioavailability, metabolic stability, safety and brain uptake. Testing for in vivo efficacy used an AD-relevant mouse model., Results: A novel, CNS-penetrant, non-toxic, orally bioavailable, small molecule inhibitor of p38 alpha MAPK (MW01-2-069A-SRM) was developed. Oral administration of the compound at a low dose (2.5 mg/kg) resulted in attenuation of excessive proinflammatory cytokine production in the hippocampus back towards normal in the animal model. Animals with attenuated cytokine production had reductions in synaptic dysfunction and hippocampus-dependent behavioral deficits., Conclusion: The p38 alpha MAPK pathway is quantitatively important in the A beta-induced production of proinflammatory cytokines in hippocampus, and brain p38 alpha MAPK is a viable molecular target for future development of potential disease-modifying therapeutics in AD and related neurodegenerative disorders.

Background: Interleukin 1 (IL-1) is a key mediator of immune responses in health and disease. Although classically the function of IL-1 has been studied in the systemic immune system, research in the past decade has revealed analogous roles in the CNS where the cytokine can contribute to the neuroinflammation and neuropathology seen in a number of neurodegenerative diseases. In Alzheimer's disease (AD), for example, pre-clinical and clinical studies have implicated IL-1 in the progression of a pathologic, glia-mediated pro-inflammatory state in the CNS. The glia-driven neuroinflammation can lead to neuronal damage, which, in turn, stimulates further glia activation, potentially propagating a detrimental cycle that contributes to progression of pathology. A prediction of this neuroinflammation hypothesis is that increased IL-1 signaling in vivo would correlate with increased severity of AD-relevant neuroinflammation and neuronal damage., Methods: To test the hypothesis that increased IL-1 signaling predisposes animals to beta-amyloid (Abeta)-induced damage, we used IL-1 receptor antagonist Knock-Out (IL1raKO) and wild-type (WT) littermate mice in a model that involves intracerebroventricular infusion of human oligomeric Abeta1-42. This model mimics many features of AD, including robust neuroinflammation, Abeta plaques, synaptic damage and neuronal loss in the hippocampus. IL1raKO and WT mice were infused with Abeta for 28 days, sacrificed at 42 days, and hippocampal endpoints analyzed., Results: IL1raKO mice showed increased vulnerability to Abeta-induced neuropathology relative to their WT counterparts. Specifically, IL1raKO mice exhibited increased mortality, enhanced microglial activation and neuroinflammation, and more pronounced loss of synaptic markers. Interestingly, Abeta-induced astrocyte responses were not significantly different between WT and IL1raKO mice, suggesting that enhanced IL-1 signaling predominately affects microglia., Conclusion: Our data are consistent with the neuroinflammation hypothesis whereby increased IL-1 signaling in AD enhances glia activation and leads to an augmented neuroinflammatory process that increases the severity of neuropathologic sequelae.