Neural stem and progenitor cells (NSCs/NPCs) are distinct groups of cells found in the mammalian central nervous system (CNS). Previously we determined that members of the High Mobility Group (HMG) B family of chromatin structural proteins modulate NSC proliferation and self-renewal. Among them HMGB2 was found to be dynamically expressed in proliferating and differentiating NSCs, suggesting that it may regulate NSC maintenance. We report now that Hmgb2(-/-) mice exhibit SVZ hyperproliferation, increased numbers of SVZ NSCs, and a trend towards aberrant increases in newly born neurons in the olfactory bulb (OB) granule cell layer. Increases in the levels of the transcription factor p21 and the Neural cell adhesion molecule (NCAM), along with down-regulation of the transcription/pluripotency factor Oct4 in the Hmgb2-/- SVZ point to a possible pathway for this increased proliferation/differentiation. Our findings suggest that HMGB2 functions as a modulator of neurogenesis in young adult mice through regulation of NSC proliferation, and identify a potential target via which CNS repair could be amplified following trauma or disease-based neuronal degeneration.

Multiple sclerosis (MS) is a demyelinating autoimmune disease mediated by infiltration of T cells into the central nervous system after compromise of the blood-brain barrier. We have previously shown that administration of tuftsin, a macrophage/microglial activator, dramatically improves the clinical course of experimental autoimmune encephalomyelitis (EAE), a well-established animal model for MS. Tuftsin administration correlates with upregulation of the immunosuppressive Helper-2 T cell (Th2) cytokine transcription factor GATA-3. We now show that tuftsin-mediated microglial activation results in shifting microglia to an anti-inflammatory phenotype. Moreover, the T cell phenotype is shifted towards immunoprotection after exposure to tuftsin-treated activated microglia; specifically, downregulation of pro-inflammatory Th1 responses is triggered in conjunction with upregulation of Th2-specific responses and expansion of immunosuppressive regulatory T cells (Tregs). Finally, tuftsin-shifted T cells, delivered into animals via adoptive transfer, reverse the pathology observed in mice with established EAE. Taken together, our findings demonstrate that tuftsin decreases the proinflammatory environment of EAE and may represent a therapeutic opportunity for treatment of MS.

Spinal cord injury (SCI) induces an immune response during which microglia, the resident immunocompetent cells of the central nervous system, become activated and migrate to the site of damage. Depending on their state of activation, microglia secrete neurotoxic or neurotrophic factors that influence the surrounding environment and have a detrimental or restorative effect following SCI, including causing or protecting bystander damage to nearby undamaged tissue. Subsequent infiltration of macrophages contributes to the SCI outcome. We show here that suppressing microglia/macrophage activation using the tripeptide macrophage/microglia inhibitory factor (MIF/TKP) reduced secondary injury around the lesion epicenter in the murine dorsal hemisection model of SCI; it decreased the hypertrophic change of astrocytes and caused an increase in the number of axons present within the lesion epicenter. Moreover, timely inhibition of microglial/macrophage activation prevented demyelination and axonal dieback by modulating oligodendrocyte survival and oligodendrocyte precursor maturation. Microglia/macrophages located within or proximal to the lesion produced neurotoxic factors, such as tumor necrosis factor alpha (TNF-α). These results suggest that microglia/macrophages within the epicenter at early time points post injury are neurotoxic, contributing to demyelination and axonal degeneration and that MIF/TKP could be used in combination with other therapies to promote functional recovery.

Microglia are reported to have significant roles in regulating normal mammalian adult neurogenesis. There are two neurogenic niches in the adult mammal brain: the subgranular zone (SGZ) in the hippocampus, and the subventricular zone (SVZ), which makes up the lining of the lateral ventricles. While the microglia interactions on adult neurogenesis in the hippocampus have been characterized, the SVZ niche is not as well investigated. The SVZ niche is unique in that the newborn neurons migrate a much longer distance through multiple brain structures compared to newborn neurons in the hippocampus, making it more difficult to fully characterize how microglia influence this process. To examine the SVZ niche and migration pathway, we used the colony stimulating factor 1 receptor (CSF1R) antagonist PLX5622, which promotes brain wide microglia ablation. Microglia ablation resulted in no changes in the numbers of neural stem cells (NSCs), transient amplifying cells, and neuroblasts. Microglia ablation in the olfactory bulb (OB) was decreased compared to the SVZ. CSF1R inhibition had no effect on the ability of microglia to proliferate. Thus, our data suggest that microglia are not required for normal functioning SVZ adult neurogenesis.

Axonal regeneration in the mature CNS is limited by extracellular inhibitory factors. Triple knockout mice lacking the major myelin-associated inhibitors do not display spontaneous regeneration after injury, indicating the presence of other inhibitors. Searching for such inhibitors, we have detected elevated levels of histone H3 in human CSF 24 h after spinal cord injury. Following dorsal column lesions in mice and optic nerve crushes in rats, elevated levels of extracellular histone H3 were detected at the injury site. Similar to myelin-associated inhibitors, these extracellular histones induced growth cone collapse and inhibited neurite outgrowth. Histones mediate inhibition through the transcription factor Y-box-binding protein 1 and Toll-like receptor 2, and these effects are independent of the Nogo receptor. Histone-mediated inhibition can be reversed by the addition of activated protein C in vitro, and activated protein C treatment promotes axonal regeneration in the crushed optic nerve in vivo. These findings identify extracellular histones as a new class of nerve regeneration-inhibiting molecules within the injured CNS.

Microglia, the dynamic innate immune cells of the central nervous system, become activated in epilepsy. The process of microglial activation in epilepsy results in the creation of an inflammatory environment around the site of seizure onset, which contributes to the epileptogenic process and epilepsy progression. Cannabidiol (CBD) has been effective for use as an adjunctive treatment for two severe pediatric seizure disorders. Newly recognized as an Food and Drug Administration (FDA)-approved drug treatment in epilepsy, it has gained in popularity primarily for pain management. Although CBD is readily available in stores and online retailers, its mechanism of action and specifically its effects on microglia and their functions are yet fully understood. In this study, we examine the effects of commercially available CBD on microglia inflammatory activation and neurogenic response, in the presence and absence of seizures. We use systemic administration of kainate to elicit seizures in mice, which are assessed behaviorally. Artisanal CBD is given in different modes of administration and timing to dissect its effect on seizure intensity, microglial activation and aberrant seizure-related neurogenesis. CBD significantly dampens microglial migration and accumulation to the hippocampus. While long term artisanal CBD use does not prevent or lessen seizure severity, CBD is a promising adjunctive partner for its ability to depress epileptogenic processes. These studies indicate that artisanal CBD is beneficial as it both decreases inflammation in the CNS and reduces the number of ectopic neurons deposited in the hippocampal area post seizure.

Gliomas are highly aggressive and accompanied by numerous microglia/macrophages (MG/MP) in and about the tumor. Little is known about what MG/MP do in this setting, or whether modulating MG/MP activation might affect glioma progression. Here, we used a glioma-microglia in culture system to establish the effects the tumor and microglia have on each other. We assessed glioma progression in vivo after MG/MP ablation or in the setting of exaggerated MG/MP activation. We show that glioma cells activate microglia but inhibit their phagocytic activities. Local ablation of MG/MP in vivo decreased tumor size and improved survival curves. Conversely, pharmacological activation of MG/MP increased glioma size through stimulating tumor proliferation and inhibiting apoptosis. In agreement with recent reports, expression of the chemokine CCL21 is enhanced after MG/MP activation and correlates with tumor growth. Taken together, our findings demonstrate that inhibition of MG/MP activation may constitute a new and effective contribution towards suppressing glioma proliferation.

Microglia are the immunocompetent cells of the central nervous system. In the physiological setting, their highly motile processes continually survey the local brain parenchyma and transiently contact synaptic elements. Although recent work has shown that the interaction of microglia with synapses contributes to synaptic remodeling during development, the role of microglia in synaptic physiology is just starting to get explored. To assess this question, we employed an electrophysiological approach using two methods to manipulate microglia in culture: organotypic hippocampal brain slices in which microglia were depleted using clodronate liposomes, and cultured hippocampal neurons to which microglia were added. We show here that the frequency of excitatory postsynaptic current increases in microglia-depleted brain slices, consistent with a higher synaptic density, and that this enhancement ensures from the loss of microglia since it is reversed when the microglia are replenished. Conversely, the addition of microglia to neuronal cultures decreases synaptic activity and reduces the density of synapses, spine numbers, surface expression of AMPA receptor (GluA1), and levels of synaptic adhesion molecules. Taken together, our findings demonstrate that non-activated microglia acutely modulate synaptic activity by regulating the number of functional synapses in the central nervous system.

Medically refractory seizures cause inflammation and neurodegeneration. Seizure initiation thresholds have been linked in mice to the serine protease tissue plasminogen activator (tPA); mice lacking tPA exhibit resistance to seizure induction, and the ensuing inflammation and neurodegeneration are similarly suppressed. Seizure foci in humans can be examined using PET employing 2-deoxy-2[(18)F]fluoro-d-glucose ((18)FDG) as a tracer to visualize metabolic dysfunction. However, there currently exist no such methods in mice to correlate measures of brain activation with behavior. Using a novel method for small animal PET data analysis, we examine patterns of (18)FDG uptake in wild-type and tPA(-/-) mice and find that they correlate with the severity of drug-induced seizure initiation. Furthermore, we report unexpected activations that may underlie the tPA modulation of seizure susceptibility. The methods described here should be applicable to other mouse models of human neurological disease.

We have synthesized several morphologies and crystal structures of MgWO4 using a one-pot hydrothermal method, producing not only monoclinic stars and large nanoparticles but also triclinic wool balls and sub-10 nm nanoparticles. Herein we describe the importance of reaction parameters in demonstrating morphology control of as-prepared MgWO4. Moreover, we correlate structure and composition with the resulting photoluminescence and radioluminescence properties. Specifically, triclinic-phase samples yielded a photoluminescence emission of 421 nm, whereas monoclinic-phase materials gave rise to an emission maximum of 515 nm. The corresponding radioluminescence data were characterized by a broad emission peak, located at 500 nm for all samples. Annealing the wool balls and sub-10 nm particles to transform the crystal structure from a triclinic to a monoclinic phase yielded a radioluminescence (RL) emission signal that was two orders of magnitude greater than that of their unannealed counterparts. Finally, to confirm the practical utility of these materials for biomedical applications, a series of sub-10 nm particles, including as-prepared and annealed samples, were functionalized with biocompatible PEG molecules, and subsequently were found to be readily taken up by various cell lines as well as primary cultured hippocampal neurons with low levels of toxicity, thereby highlighting for the first time the potential of this particular class of metal oxides as viable and readily generated platforms for a range of biomedical applications.

Neural Stem Cells (NSCs) are a distinct group of cells present in the embryonic and adult mammalian central nervous system (CNS) that are able to differentiate into neurons, astrocytes and oligodendrocytes. As NSC proliferation declines with age, factors that regulate this process need to be defined. To search for NSC regulatory factors, we performed a quantitative shotgun proteomics study that revealed that members of the High Mobility Group B (HMGB) family are highly expressed in NSCs. Using a neurosphere assay, we report the differential expression of HMGB 1, 2, 3, and 4 mRNAs in proliferating NSCs isolated from various time points during embryonic development, as well as the dynamic expression of HMGB1 and B2 mRNAs and proteins in differentiating embryonic NSCs. Expression of HMGB2 underwent the most dramatic changes during the developmental ages examined; as a result, we assessed its role in NSC proliferation and differentiation. We report the predominance of small diameter HMGB2-/- neurospheres in comparison to wild-type, which correlated with increased proliferation in these smaller HMGB2-/- neurospheres. Our data suggest that HMGB2 plays a regulatory role in NSC cell proliferation and maintenance pathways.

Reactive oxygen species (ROS) are vital regulators of many cellular functions in the body. The intracellular ROS concentration is highly regulated by a balance between pro-oxidants and anti-oxidants. A chronic excess of pro-oxidants leads to elevated ROS concentrations and inflammation, possibly initiating or enhancing disease onset. Mineral-induced generation of ROS, the role of minerals in upregulating cellular ROS, and their role in the development of several occupational diseases are now widely recognized. However, there is no standard protocol to determine changes in ROS production in cells after exposure to mineral dust or earth materials in general. In this study, a new method for determining the degree of cellular toxicity (i.e., cytotoxicity) of particles is described that will help bridge the gap in knowledge.

Epidemiological studies have reported that cigarette smoking increases the risk of developing multiple sclerosis (MS) and accelerates its progression. However, the molecular mechanisms underlying these effects remain unsettled. We have investigated here the effects of the nicotine and the non-nicotine components in cigarette smoke on MS using the experimental autoimmune encephalomyelitis (EAE) model, and have explored their underlying mechanism of action. Our results show that nicotine ameliorates the severity of EAE, as shown by reduced demyelination, increased body weight, and attenuated microglial activation. Nicotine administration after the development of EAE symptoms prevented further disease exacerbation, suggesting that it might be useful as an EAE/MS therapeutic. In contrast, the remaining components of cigarette smoke, delivered as cigarette smoke condensate (CSC), accelerated and increased adverse clinical symptoms during the early stages of EAE, and we identify a particular cigarette smoke compound, acrolein, as one of the potential mediators. We also show that the mechanisms underlying the opposing effects of nicotine and CSC on EAE are likely due to distinct effects on microglial viability, activation, and function.

Monocyte chemoattractant protein 1 (MCP1) is an important chemoattractant for microglia. Rodent MCP1 carries a heavily glycosylated C-terminus, which has been predicted to increase local MCP1 concentration, promote MCP1 dimerization/oligomerization, and facilitate receptor engagement. Previous studies have shown that MCP1 mutant lacking the glycosylated C-terminus cannot dimerize/oligomerize, but has higher chemotactic potency than the wild-type (full-length) MCP1, suggesting that rodent MCP1 may function as a monomer. Although many groups support this hypothesis, there is no direct evidence on whether rodent MCP1 dimer is functional. In this paper, using forced recombinant dimeric MCP1 proteins we show that mouse MCP1 dimer is unable to activate Rac1, promote protrusion of lamellipodia, or induce microglial migration, although it can bind to CCR2 and mediate its internalization. These results support the idea that signaling events mediated by MCP1 require the presence of the monomeric form of this chemokine.

The efficacy of combining radiation therapy with immune checkpoint inhibitor blockade to treat brain tumors is currently the subject of multiple investigations and holds significant therapeutic promise. However, the long-term effects of this combination therapy on the normal brain tissue are unknown. Here, we examined mice that were intracranially implanted with murine glioma cell line and became long-term survivors after treatment with a combination of 10 Gy cranial irradiation (RT) and anti-PD-1 checkpoint blockade (aPD-1). Post-mortem analysis of the cerebral hemisphere contralateral to tumor implantation showed complete abolishment of hippocampal neurogenesis, but neural stem cells were well preserved in subventricular zone. In addition, we observed a drastic reduction in the number of mature oligodendrocytes in the subcortical white matter. Importantly, this observation was evident specifically in the combined (RT + aPD-1) treatment group but not in the single treatment arm of either RT alone or aPD-1 alone. Elimination of microglia with a small molecule inhibitor of colony stimulated factor-1 receptor (PLX5622) prevented the loss of mature oligodendrocytes. These results identify for the first time a unique pattern of normal tissue changes in the brain secondary to combination treatment with radiotherapy and immunotherapy. The results also suggest a role for microglia as key mediators of the adverse treatment effect.

Early invasion of the central nervous system (CNS) by human immunodeficiency virus (HIV) (Gray et al. in Brain Pathol 6:1-15, 1996; An et al. in Ann Neurol 40:611-6172, 1996), results in neuroinflammation, potentially through extracellular vesicles (EVs) and their micro RNAs (miRNA) cargoes (Sharma et al. in FASEB J 32:5174-5185, 2018; Hu et al. in Cell Death Dis 3:e381, 2012). Although the basal ganglia (BG) is a major target and reservoir of HIV in the CNS (Chaganti et al. in Aids 33:1843-1852, 2019; Mintzopoulos et al. in Magn Reson Med 81:2896-2904, 2019), whether BG produces EVs and the effect of HIV and/or the phytocannabinoid-delta-9-tetrahydrocannabinol (THC) on BG-EVs and HIV neuropathogenesis remain unknown.

Microglia are important for brain homeostasis and immunity, but their role in regulating vigilance remains unclear. We employed genetic, physiological, and metabolomic methods to examine microglial involvement in the regulation of wakefulness and sleep. Microglial depletion decreased stable nighttime wakefulness in mice by increasing transitions between wakefulness and non-rapid eye movement (NREM) sleep. Metabolomic analysis revealed that the sleep-wake behavior closely correlated with diurnal variation of the brain ceramide, which disappeared in microglia-depleted mice. Ceramide preferentially influenced microglia in the thalamic reticular nucleus (TRN), and local depletion of TRN microglia produced similar impaired wakefulness. Chemogenetic manipulations of anterior TRN neurons showed that they regulated transitions between wakefulness and NREM sleep. Their firing capacity was suppressed by both microglial depletion and added ceramide. In microglia-depleted mice, activating anterior TRN neurons or inhibiting ceramide production both restored stable wakefulness. These findings demonstrate that microglia can modulate stable wakefulness through anterior TRN neurons via ceramide signaling.

Though promoting remyelination in multiple sclerosis (MS) has emerged as a promising therapeutic strategy, it does not address inflammatory signals that continue to induce neuronal damage and inhibit effectiveness of repair mechanisms. Our lab has previously characterized the immunomodulatory tetrapeptide, tuftsin, which induces an anti-inflammatory shift in microglia and macrophages. This targeted anti-inflammatory agent improves physical deficits in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Here, we sought to determine whether tuftsin is also effective in combination with benztropine, an FDA-approved drug that stimulates remyelination, in both EAE and in the cuprizone model of demyelination. We show that combining these two agents to promote anti-inflammatory and remyelinating mechanisms alleviates symptoms in EAE and lessens pathological hallmarks in both MS models. Importantly, tuftsin is required to transform the inflammatory CNS environment normally present in EAE/MS into one of an anti-inflammatory nature, and benztropine is required in the cuprizone model to improve remyelination. Our data further support tuftsin's beneficial immunomodulatory activity in the context of EAE, and show that when studying remyelination in the absence of an autoimmune insult, tuftsin still activated microglia toward an anti-inflammatory fate, but benztropine was necessary for significant repair of the damaged myelin. Overall, tuftsin effectively combined with benztropine to significantly improve MS-like pathologies in both models.

Mouse cortical development relies heavily on a delicate balance between neurogenesis and gliogenesis. The lateral ventricular zone produces different classes of excitatory pyramidal cells until just before birth, when the production of astroglia begins to prevail. Epigenetic control of this fate shift is of critical importance and chromatin regulatory elements driving neuronal or astroglial development play an vital role. Different classes of chromatin binding proteins orchestrate the transcriptional repression of neuronal-specific genes, while allowing for the activation of astrocyte-specific genes. Through proteomic analysis of embryonic neural progenitor cells (NPCs) our group had previously identified high mobility group B2 (HMGB2), a chromatin protein dynamically expressed throughout embryonic development. In the current study using cultures of perinatal NPCs from HMGB2+/+ and HMGB2-/- mice we discovered that vital elements of the polycomb group (PcG) epigenetic complexes polycomb repressive complexes 1 and 2 (PRC1/2) were downregulated during the differentiation process of HMGB2-null NPCs. These epigenetic changes led to downstream changes in specific histone modification levels, specifically the trimethylation of H3K27, and a subsequent shift in the perinatal neurogenesis to gliogenesis fate transition. Collectively these results demonstrate that chromatin binding proteins, such as HMGB2, can have significant effects on the epigenetic landscape of perinatal neural stem/progenitor cells.

Comorbid post-traumatic stress disorder with traumatic brain injury (TBI) produce more severe affective and cognitive deficits than PTSD or TBI alone. Both PTSD and TBI produce long-lasting neuroinflammation, which may be a key underlying mechanism of the deficits observed in co-morbid TBI/PTSD. We developed a model of co-morbid TBI/PTSD by combining the closed head (CHI) model of TBI with the chronic variable stress (CVS) model of PTSD and examined multiple behavioral and neuroinflammatory outcomes. Male C57/Bl6 mice received sham treatment, CHI, CVS, CHI then CVS (CHI → CVS) or CVS then CHI (CVS → CHI). The CVS → CHI group had deficits in Barnes maze or active place avoidance not seen in the other groups. The CVS → CHI, CVS and CHI → CVS groups displayed increased basal anxiety level, based on performance on elevated plus maze. The CVS → CHI had impaired performance on Barnes Maze, and Active Place Avoidance. These performance deficits were strongly correlated with increased hippocampal Iba-1 level an indication of activated MP/MG. These data suggest that greater cognitive deficits in the CVS → CHI group were due to increased inflammation. The increased deficits and neuroinflammation in the CVS → CHI group suggest that the order by which a subject experiences TBI and PTSD is a major determinant of the outcome of brain injury in co-morbid TBI/PTSD.