Deletion of p38α MAPK in microglia blunts trauma-induced inflammatory responses in mice.

Traumatic brain injury (TBI) is a significant cause of morbidity and mortality in the USA and other developed countries worldwide. Following the initial mechanical insult, the brain's primary innate immune effector, microglia, initiate inflammatory signaling cascades and pathophysiological responses that can lead to chronic neuroinflammation and neurodegenerative sequelae. The p38α MAPK signaling pathway in microglia is a key contributor to inflammatory responses to diverse disease-relevant stressors and injury conditions. Therefore, we tested here whether microglia p38α contributes to acute and persistent inflammatory responses induced by a focal TBI. We generated conditional cell-specific knockout of p38α in microglia using a CX3CR1 Cre-lox system, subjected the p38α knockout and wild-type mice to a controlled cortical impact TBI, and measured inflammatory responses at acute (1-day) and subacute (7-day) post-injury time points. We found that deletion of p38α in microglia only was sufficient to attenuate multiple pro-inflammatory responses following TBI, notably reducing pro-inflammatory cytokine/chemokine production and recruitment of inflammatory monocytes into the brain and preventing the persistent microglial morphological activation. These data provide strong evidence supporting a role for microglial p38α in propagation of a chronic and potentially neurotoxic pro-inflammatory environment in the brain following TBI.

Attenuation of traumatic brain injury-induced cognitive impairment in mice by targeting increased cytokine levels with a small molecule experimental therapeutic.

BACKGROUND: Evidence from clinical studies and preclinical animal models suggests that proinflammatory cytokine overproduction is a potential driving force for pathology progression in traumatic brain injury (TBI). This raises the possibility that selective targeting of the overactive cytokine response, a component of the neuroinflammation that contributes to neuronal dysfunction, may be a useful therapeutic approach. MW151 is a CNS-penetrant, small molecule experimental therapeutic that selectively restores injury- or disease-induced overproduction of proinflammatory cytokines towards homeostasis. We previously reported that MW151 administered post-injury (p.i.) is efficacious in a closed head injury (CHI) model of diffuse TBI in mice. Here we test dose dependence of MW151 to suppress the target mechanism (proinflammatory cytokine up-regulation), and explore the therapeutic window for MW151 efficacy. METHODS: We examined suppression of the acute cytokine surge when MW151 was administered at different times post-injury and the dose-dependence of cytokine suppression. We also tested a more prolonged treatment with MW151 over the first 7 days post-injury and measured the effects on cognitive impairment and glial activation. RESULTS: MW151 administered up to 6 h post-injury suppressed the acute cytokine surge, in a dose-dependent manner. Administration of MW151 over the first 7 days post-injury rescues the CHI-induced cognitive impairment and reduces glial activation in the focus area of the CHI. CONCLUSIONS: Our results identify a clinically relevant time window post-CHI during which MW151 effectively restores cytokine production back towards normal, with a resultant attenuation of downstream cognitive impairment.

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.

Early stage drug treatment that normalizes proinflammatory cytokine production attenuates synaptic dysfunction in a mouse model that exhibits age-dependent progression of Alzheimer's disease-related pathology.

Overproduction of proinflammatory cytokines in the CNS has been implicated as a key contributor to pathophysiology progression in Alzheimer's disease (AD), and extensive studies with animal models have shown that selective suppression of excessive glial proinflammatory cytokines can improve neurologic outcomes. The prior art, therefore, raises the logical postulation that intervention with drugs targeting dysregulated glial proinflammatory cytokine production might be effective disease-modifying therapeutics if used in the appropriate biological time window. To test the hypothesis that early stage intervention with such drugs might be therapeutically beneficial, we examined the impact of intervention with MW01-2-151SRM (MW-151), an experimental therapeutic that selectively attenuates proinflammatory cytokine production at low doses. MW-151 was tested in an APP/PS1 knock-in mouse model that exhibits increases in AD-relevant pathology progression with age, including increases in proinflammatory cytokine levels. Drug was administered during two distinct but overlapping therapeutic time windows of early stage pathology development. MW-151 treatment attenuated the increase in microglial and astrocyte activation and proinflammatory cytokine production in the cortex and yielded improvement in neurologic outcomes, such as protection against synaptic protein loss and synaptic plasticity impairment. The results also demonstrate that the therapeutic time window is an important consideration in efficacy studies of drugs that modulate glia biological responses involved in pathology progression and suggest that such paradigms should be considered in the development of new therapeutic regimens that seek to delay the onset or slow the progression of AD.

APOE4 drives transcriptional heterogeneity and maladaptive immunometabolic responses of astrocytes.

Apolipoprotein E4 (APOE4) is the strongest risk allele associated with the development of late onset Alzheimer's disease (AD). Across the CNS, astrocytes are the predominant expressor of APOE while also being critical mediators of neuroinflammation and cerebral metabolism. APOE4 has been consistently linked with dysfunctional inflammation and metabolic processes, yet insights into the molecular constituents driving these responses remain unclear. Utilizing complementary approaches across humanized APOE mice and isogenic human iPSC astrocytes, we demonstrate that ApoE4 alters the astrocyte immunometabolic response to pro-inflammatory stimuli. Our findings show that ApoE4-expressing astrocytes acquire distinct transcriptional repertoires at single-cell and spatially-resolved domains, which are driven in-part by preferential utilization of the cRel transcription factor. Further, inhibiting cRel translocation in ApoE4 astrocytes abrogates inflammatory-induced glycolytic shifts and in tandem mitigates production of multiple pro-inflammatory cytokines. Altogether, our findings elucidate novel cellular underpinnings by which ApoE4 drives maladaptive immunometabolic responses of astrocytes.

APOE modulates microglial immunometabolism in response to age, amyloid pathology, and inflammatory challenge.

The E4 allele of Apolipoprotein E (APOE) is associated with both metabolic dysfunction and a heightened pro-inflammatory response: two findings that may be intrinsically linked through the concept of immunometabolism. Here, we combined bulk, single-cell, and spatial transcriptomics with cell-specific and spatially resolved metabolic analyses in mice expressing human APOE to systematically address the role of APOE across age, neuroinflammation, and AD pathology. RNA sequencing (RNA-seq) highlighted immunometabolic changes across the APOE4 glial transcriptome, specifically in subsets of metabolically distinct microglia enriched in the E4 brain during aging or following an inflammatory challenge. E4 microglia display increased Hif1α expression and a disrupted tricarboxylic acid (TCA) cycle and are inherently pro-glycolytic, while spatial transcriptomics and mass spectrometry imaging highlight an E4-specific response to amyloid that is characterized by widespread alterations in lipid metabolism. Taken together, our findings emphasize a central role for APOE in regulating microglial immunometabolism and provide valuable, interactive resources for discovery and validation research.

Acute brain inflammation, white matter oxidative stress, and myelin deficiency in a model of neonatal intraventricular hemorrhage.

OBJECTIVE: Neonatal intraventricular hemorrhage (IVH) leads to posthemorrhagic hydrocephalus (PHH), brain injury, and long-term disability. Current therapy for IVH is based on treating PHH but does not address the underlying brain injury. In order to develop pharmacological treatment for IVH, there must be a better understanding of the underlying pathology of this disease. This study was designed to determine the time course of the acute inflammation and oxidative stress that may underlie the progressive pathology of IVH. The authors sought to understand the temporal relationships among inflammation, oxidative stress, and white matter pathology in a rat model of IVH. METHODS: A rat model of IVH consisting of hemoglobin injection into the lateral ventricle was used. Tissue was analyzed via biochemical and histological methods to map the spatiotemporal distribution of innate immune activation and oxidative stress. White matter was quantified using both immunohistochemistry and Western blot for myelin basic protein (MBP) in the corpus callosum. RESULTS: IVH led to acute induction of inflammatory cytokines, followed by oxidative stress. Oxidative stress was concentrated in white matter, adjacent to the lateral ventricles. Animals with IVH initially gained weight at a lower rate than control animals and had larger ventricles and less MBP than control animals. CONCLUSIONS: Experimental IVH induces global inflammation throughout the brain and oxidative stress concentrated in the white matter. Both of these phenomena occur early after IVH. This has implications for human neonates with immature white matter that is exquisitely sensitive to inflammation and oxidative stress. Antiinflammatory or antioxidant therapy for IVH may need to be initiated early in order to protect developing white matter.

Neonatal hydrocephalus leads to white matter neuroinflammation and injury in the corpus callosum of Ccdc39 hydrocephalic mice.

OBJECTIVE: The authors sought to determine if hydrocephalus caused a proinflammatory state within white matter as is seen in many other forms of neonatal brain injury. Common causes of hydrocephalus (such as trauma, infection, and hemorrhage) are inflammatory insults themselves and therefore confound understanding of how hydrocephalus itself affects neuroinflammation. Recently, a novel animal model of hydrocephalus due to a genetic mutation in the Ccdc39 gene has been developed in mice. In this model, ciliary dysfunction leads to early-onset ventriculomegaly, astrogliosis, and reduced myelination. Because this model of hydrocephalus is not caused by an antecedent proinflammatory insult, it was utilized to study the effect of hydrocephalus on inflammation within the white matter of the corpus callosum. METHODS: A Meso Scale Discovery assay was used to measure levels of proinflammatory cytokines in whole brain from animals with and without hydrocephalus. Immunohistochemistry was used to measure macrophage activation and NG2 expression within the white matter of the corpus callosum in animals with and without hydrocephalus. RESULTS: In this model of hydrocephalus, levels of cytokines throughout the brain revealed a more robust increase in classic proinflammatory cytokines (interleukin [IL]-1ß, CXCL1) than in immunomodulatory cytokines (IL-10). Increased numbers of macrophages were found within the corpus callosum. These macrophages were polarized toward a proinflammatory phenotype as assessed by higher levels of CD86, a marker of proinflammatory macrophages, compared to CD206, a marker for antiinflammatory macrophages. There was extensive structural damage to the corpus callosum of animals with hydrocephalus, and an increase in NG2-positive cells. CONCLUSIONS: Hydrocephalus without an antecedent proinflammatory insult induces inflammation and tissue injury in white matter. Future studies with this model will be useful to better understand the effects of hydrocephalus on neuroinflammation and progenitor cell development. Antiinflammatory therapy for diseases that cause hydrocephalus may be a powerful strategy to reduce tissue damage.

The effects of mild closed head injuries on tauopathy and cognitive deficits in rodents: Primary results in wild type and rTg4510 mice, and a systematic review.

In humans, the majority of sustained traumatic brain injuries (TBIs) are classified as 'mild' and most often a result of a closed head injury (CHI). The effects of a non-penetrating CHI are not benign and may lead to chronic pathology and behavioral dysfunction, which could be worsened by repeated head injury. Clinical-neuropathological correlation studies provide evidence that conversion of tau into abnormally phosphorylated proteotoxic intermediates (p-tau) could be part of the pathophysiology triggered by a single TBI and enhanced by repeated TBIs. However, the link between p-tau and CHI in rodents remains controversial. To address this question experimentally, we induced a single CHI or two CHIs to WT or rTg4510 mice. We found that 2× CHI increased tau phosphorylation in WT mice and rTg4510 mice. Behavioral characterization in WT mice found chronic deficits in the radial arm water maze in 2× CHI mice that had partially resolved in the 1× CHI mice. Moreover, using Manganese-Enhanced Magnetic Resonance Imaging with R1 mapping - a novel functional neuroimaging technique - we found greater deficits in the rTg4510 mice following 2× CHI compared to 1× CHI. To integrate our findings with prior work in the field, we conducted a systematic review of rodent mild repetitive CHI studies. Following Prisma guidelines, we identified 25 original peer-reviewed papers. Results from our experiments, as well as our systematic review, provide compelling evidence that tau phosphorylation is modified by experimental mild TBI studies; however, changes in p-tau levels are not universally reported. Together, our results provide evidence that repetitive TBIs can result in worse and more persistent neurological deficits compared to a single TBI, but the direct link between the worsened outcome and elevated p-tau could not be established.

MW151 Inhibited IL-1ß Levels after Traumatic Brain Injury with No Effect on Microglia Physiological Responses.

A prevailing neuroinflammation hypothesis is that increased production of proinflammatory cytokines contributes to progressive neuropathology, secondary to the primary damage caused by a traumatic brain injury (TBI). In support of the hypothesis, post-injury interventions that inhibit the proinflammatory cytokine surge can attenuate the progressive pathology. However, other post-injury neuroinflammatory responses are key to endogenous recovery responses. Therefore, it is critical that pharmacological attenuation of detrimental or dysregulated neuroinflammatory processes avoid pan-suppression of inflammation. MW151 is a CNS-penetrant, small molecule experimental therapeutic that restores injury- or disease-induced overproduction of proinflammatory cytokines towards homeostasis without immunosuppression. Post-injury administration of MW151 in a closed head injury model of mild TBI suppressed acute cytokine up-regulation and downstream cognitive impairment. Here, we report results from a diffuse brain injury model in mice using midline fluid percussion. Low dose (0.5-5.0 mg/kg) administration of MW151 suppresses interleukin-1 beta (IL-1ß) levels in the cortex while sparing reactive microglia and astrocyte responses. To probe molecular mechanisms, we used live cell imaging of the BV-2 microglia cell line to demonstrate that MW151 does not affect proliferation, migration, or phagocytosis of the cells. Our results provide insight into the roles of glial responses to brain injury and indicate the feasibility of using appropriate dosing for selective therapeutic modulation of injurious IL-1ß increases while sparing other glial responses to injury.

Generation and behavior characterization of CaMKIIß knockout mice.

The calcium/calmodulin-dependent protein kinase II (CaMKII) is abundant in the brain, where it makes important contributions to synaptic organization and homeostasis, including playing an essential role in synaptic plasticity and memory. Four genes encode isoforms of CaMKII (α, ß, δ, γ), with CaMKIIα and CaMKIIß highly expressed in the brain. Decades of molecular and cellular research, as well as the use of a large number of CaMKIIα mutant mouse lines, have provided insight into the pivotal roles of CaMKIIα in brain plasticity and cognition. However, less is known about the CaMKIIß isoform. We report the development and extensive behavioral and phenotypic characterization of a CaMKIIß knockout (KO) mouse. The CaMKIIß KO mouse was found to be smaller at weaning, with an altered body mass composition. The CaMKIIß KO mouse showed ataxia, impaired forelimb grip strength, and deficits in the rotorod, balance beam and running wheel tasks. Interestingly, the CaMKIIß KO mouse exhibited reduced anxiety in the elevated plus maze and open field tests. The CaMKIIß KO mouse also showed cognitive impairment in the novel object recognition task. Our results provide a comprehensive behavioral characterization of mice deficient in the ß isoform of CaMKII. The neurologic phenotypes and the construction of the genotype suggest the utility of this KO mouse strain for future studies of CaMKIIß in brain structure, function and development.