BackgroundOverproduction 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.MethodsThe 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.ResultsIncreased 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.ConclusionThe 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.

Serine-threonine protein kinases are critical to CNS function, yet there is a dearth of highly selective, CNS-active kinase inhibitors for in vivo investigations. Further, prevailing assumptions raise concerns about whether single kinase inhibitors can show in vivo efficacy for CNS pathologies, and debates over viable approaches to the development of safe and efficacious kinase inhibitors are unsettled. It is critical, therefore, that these scientific challenges be addressed in order to test hypotheses about protein kinases in neuropathology progression and the potential for in vivo modulation of their catalytic activity. Identification of molecular targets whose in vivo modulation can attenuate synaptic dysfunction would provide a foundation for future disease-modifying therapeutic development as well as insight into cellular mechanisms. Clinical and preclinical studies suggest a critical link between synaptic dysfunction in neurodegenerative disorders and the activation of p38αMAPK mediated signaling cascades. Activation in both neurons and glia also offers the unusual potential to generate enhanced responses through targeting a single kinase in two distinct cell types involved in pathology progression. However, target validation has been limited by lack of highly selective inhibitors amenable to in vivo use in the CNS. Therefore, we employed high-resolution co-crystallography and pharmacoinformatics to design and develop a novel synthetic, active site targeted, CNS-active, p38αMAPK inhibitor (MW108). Selectivity was demonstrated by large-scale kinome screens, functional GPCR agonist and antagonist analyses of off-target potential, and evaluation of cellular target engagement. In vitro and in vivo assays demonstrated that MW108 ameliorates beta-amyloid induced synaptic and cognitive dysfunction. A serendipitous discovery during co-crystallographic analyses revised prevailing models about active site targeting of inhibitors, providing insights that will facilitate future kinase inhibitor design. Overall, our studies deliver highly selective in vivo probes appropriate for CNS investigations and demonstrate that modulation of p38αMAPK activity can attenuate synaptic dysfunction.

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Inhibition of proteasome activity occurs in normal aging and in a wide variety of neurodegenerative conditions including Alzheimer's disease and Parkinson's disease. Although each of these conditions is also associated with mitochondrial dysfunction potentially mediated by proteasome inhibition, the relationship between proteasome inhibition and the loss of mitochondrial homeostasis in each of these conditions has not been fully elucidated. In this study, we conducted experimentation in order to begin to develop a more complete understanding of the effects proteasome inhibition has on neural mitochondrial homeostasis. Mitochondria within neural SH-SY5Y cells exposed to low level proteasome inhibition possessed similar morphological features and similar rates of electron transport chain activity under basal conditions as compared with untreated neural cultures of equal passage number. Despite such similarities, maximal complex I and complex II activities were dramatically reduced in neural cells subject to proteasome inhibition. Proteasome inhibition also increased mitochondrial reactive oxygen species production, reduced intramitochondrial protein translation, and increased cellular dependence on glycolysis. Finally, whereas proteasome inhibition generated cells that consistently possessed mitochondria located in close proximity to lysosomes with mitochondria present in the cellular debris located within autophagosomes, increased levels of lipofuscin suggest that impairments in mitochondrial turnover may occur following proteasome inhibition. Taken together, these data demonstrate that proteasome inhibition dramatically alters specific aspects of neural mitochondrial homeostasis and alters lysosomal-mediated degradation of mitochondria with both of these alterations potentially contributing to aging and age-related disease in the nervous system.

Increasing evidence suggests that proteasome inhibition plays a causal role in promoting the neurodegeneration and neuron death observed in multiple disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD). The ability of severe and acute inhibition of proteasome function to induce neuron death and neuropathology similar to that observed in AD and PD is well documented. However, at present the effects of chronic low-level proteasome inhibition on neural homeostasis has not been elucidated. In order to determine the effects of chronic low-level proteasome inhibition on neural homeostasis, we conducted studies in individual colonies of neural SH-SY5Y cells that were isolated following continual exposure to low concentrations (100 nM) of the proteasome inhibitor MG115. Clonal cell lines appeared morphologically similar to control cultures but exhibited significantly different rates of both proliferation and differentiation. Elevated levels of protein oxidation and protein insolubility were observed in clonal cell lines, with all clonal cell lines being more resistant to neural death induced by serum withdrawal and oxidative stress. Interestingly, clonal cell lines demonstrated evidence for increased macroautophagy, suggesting that chronic low-level proteasome inhibition may cause an excessive activation of the lysosomal system. Taken together, these data indicate that chronic lowlevel proteasome inhibition has multiple effects on neural homeostasis, and suggests that studying the effects of chronic low-level proteasome inhibition may be useful in understanding the relationship between protein oxidation, protein insolubility, proteasome function, macroautophagy and neural viability in AD and PD.