Proto-oncogene tyrosine-protein kinase SRC (Src) inhibition in microglia relieves neuroinflammation in neuropathic pain mouse models.

Chronic neuroinflammation is an important factor in the development of neuropathic pain (NP). Excess microglia activation releases a mass of pro-inflammatory cytokines during neuroinflammation process, leading to a constant painful irritation of the sensory nerve. Src belongs to a non-receptor tyrosine kinase associated with sarcoma, whereas the role of Src in neuropathic pain is controversial. We designed to testify the inflammation-regulatory role of Src in the lipopolysaccharide (LPS)-induced BV2 microglia line and the mouse model of neuropathic pain by partial sciatic nerve ligation (PNL). In BV2 microglia, Src expression was inhibited using a Src family kinase inhibitor PP2 after LPS induced inflammatory response. In vivo, the neuropathic pain in mice was induced by PNL surgery and then treated with PP2. The neuroinflammation level in vitro was detected by enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF), trans-well and Western blotting (WB) assays, in vivo was examined in PNL mice using immunohistochemistry (IHC) and IF. Finally, mechanical allodynia and thermal hyperalgesia assays were used to access the functional evaluation. Inhibition of Src was decreased microglial inflammation and migration after LPS stimuli. Mechanistically, the expression of nuclear factor kappa B (NF-κB) pathway decreased after Src inhibition. The data in vivo showed that the decrease expression of Src reduced neuroinflammation and the amount of microglia in spinal dorsal horn (SDH), the mechanical allodynia of mice thereby attenuated after Src inhibition. These results indicated that the inhibition of Src took a protective effect in neuropathic pain mouse models via reducing microglia-induced neuroinflammation.

Bavachin exerted anti-neuroinflammatory effects by regulation of A20 ubiquitin-editing complex.

Neuroinflammation is a major pathophysiological contributor to the progression of the central nervous system disorders. Bavachin is a natural product belonging to the flavonoid class. The anti-neuroinflammatory effect and the molecular mechanisms are not well understood. In this study, we found bavachin can exert anti-neuroinflammatory effect via inhibition of nuclear factor-kappa B (NF-κB) signaling. We found that bavachin can obviously upregulate the expression of A20 (TNFAIP3) in microglial cells. Further studies suggested siRNA-A20 knockdown treatment can attenuate the inhibitory effects of bavachin on neuroinflammation. We further found bavachin can increase the interaction of ubiquitin-editing enzyme A20 complex including A20, Tax1-binding protein 1 (TAX1BP1) and Itch, the subsequently downregulated the K63-ubiquitination of TNF receptor associated factor 6 (TRAF6) and NF-κB signaling pathway. Altogether, our results indicated that bavachin exerted anti-neuroinflammatory effects through inhibition of NF-κB signaling mediated by regulation of ubiquitin-editing enzyme A20 complex. Our finding has important clinical significance for the potential application of bavachin in the treatment of neurological disorders.

GCH1-regulated miRNAs are potential targets for microglial activation in neuropathic pain.

Neuropathic pain (NP) is a chronic pain directly caused by injury or disease of the somatosensory nervous system. Previous studies suggest that GTP cyclohydrolase I (GCH1) may play a pivotal role in microglial activation, which has been shown to be essential for NP. However, its underlying mechanisms in microglial activation remain unclear. A wide range of microRNAs (miRNAs) have been found to be involved in microglial activation-induced NP. To identify the miRNAs regulated by GCH1 and predict their functions in the progression of microglial activation, we analyzed the miRNA expression profiles of GCH1-knockdown (KD) BV2 microglial cells. Small RNA-sequencing analysis revealed 13 differentially expressed (DE) miRNAs in GCH1-KD cells. The target genes of DE miRNAs mainly participate in PI3K-Akt signaling pathway, peroxisome and ferroptosis. The miRNA-mRNA regulatory network analysis showed that GCH1, MAP4K5 and YWHAB acted as hub genes. qRT-PCR results further verified the expression levels of mmu-miR-1a-3p, mmu-miR-133a-3p, mmu-miR-7a-5p and mmu-miR-10a-5p in GCH1-KD cells, which were consistent with the sequencing data. In addition, our data indicated that overexpression of mmu-miR-133a-3p alleviated the pro-inflammatory cytokines IL-1ß and IL-6 production induced by lipopolysaccharide (LPS), indicating that mmu-miR-133a-3p has a negative effect on microglial activation. Taken together, our findings suggest that many miRNAs regulated by GCH1 may be involved in microglial activation, which may provide new potential targets for GCH1 in the pathogenesis of NP.

Myeloid Arginase 1 Insufficiency Exacerbates Amyloid-ß Associated Neurodegenerative Pathways and Glial Signatures in a Mouse Model of Alzheimer's Disease: A Targeted Transcriptome Analysis.

Brain myeloid cells, include infiltrating macrophages and resident microglia, play an essential role in responding to and inducing neurodegenerative diseases, such as Alzheimer's disease (AD). Genome-wide association studies (GWAS) implicate many AD casual and risk genes enriched in brain myeloid cells. Coordinated arginine metabolism through arginase 1 (Arg1) is critical for brain myeloid cells to perform biological functions, whereas dysregulated arginine metabolism disrupts them. Altered arginine metabolism is proposed as a new biomarker pathway for AD. We previously reported Arg1 deficiency in myeloid biased cells using lysozyme M (LysM) promoter-driven deletion worsened amyloidosis-related neuropathology and behavioral impairment. However, it remains unclear how Arg1 deficiency in these cells impacts the whole brain to promote amyloidosis. Herein, we aim to determine how Arg1 deficiency driven by LysM restriction during amyloidosis affects fundamental neurodegenerative pathways at the transcriptome level. By applying several bioinformatic tools and analyses, we found that amyloid-ß (Aß) stimulated transcriptomic signatures in autophagy-related pathways and myeloid cells' inflammatory response. At the same time, myeloid Arg1 deficiency during amyloidosis promoted gene signatures of lipid metabolism, myelination, and migration of myeloid cells. Focusing on Aß associated glial transcriptomic signatures, we found myeloid Arg1 deficiency up-regulated glial gene transcripts that positively correlated with Aß plaque burden. We also observed that Aß preferentially activated disease-associated microglial signatures to increase phagocytic response, whereas myeloid Arg1 deficiency selectively promoted homeostatic microglial signature that is non-phagocytic. These transcriptomic findings suggest a critical role for proper Arg1 function during normal and pathological challenges associated with amyloidosis. Furthermore, understanding pathways that govern Arg1 metabolism may provide new therapeutic opportunities to rebalance immune function and improve microglia/macrophage fitness.

A RIPK1-regulated inflammatory microglial state in amyotrophic lateral sclerosis.

Microglial-derived inflammation has been linked to a broad range of neurodegenerative and neuropsychiatric conditions, including amyotrophic lateral sclerosis (ALS). Using single-cell RNA sequencing, a class of Disease-Associated Microglia (DAMs) have been characterized in neurodegeneration. However, the DAM phenotype alone is insufficient to explain the functional complexity of microglia, particularly with regard to regulating inflammation that is a hallmark of many neurodegenerative diseases. Here, we identify a subclass of microglia in mouse models of ALS which we term RIPK1-Regulated Inflammatory Microglia (RRIMs). RRIMs show significant up-regulation of classical proinflammatory pathways, including increased levels of Tnf and Il1b RNA and protein. We find that RRIMs are highly regulated by TNFα signaling and that the prevalence of these microglia can be suppressed by inhibiting receptor-interacting protein kinase 1 (RIPK1) activity downstream of the TNF receptor 1. These findings help to elucidate a mechanism by which RIPK1 kinase inhibition has been shown to provide therapeutic benefit in mouse models of ALS and may provide an additional biomarker for analysis in ongoing phase 2 clinical trials of RIPK1 inhibitors in ALS.

Dexmedetomidine Alleviates Hypoxia-Induced Synaptic Loss and Cognitive Impairment via Inhibition of Microglial NOX2 Activation in the Hippocampus of Neonatal Rats.

BACKGROUND: Perinatal hypoxia is a universal cause of death and neurological deficits in neonates worldwide. Activation of microglial NADPH oxidase 2 (NOX2) leads to oxidative stress and neuroinflammation, which may contribute to hypoxic damage in the developing brain. Dexmedetomidine has been reported to exert potent neuroprotection in several neurological diseases, but the mechanism remains unclear. We investigated whether dexmedetomidine acts through microglial NOX2 to reduce neonatal hypoxic brain damage. METHODS: The potential role of microglial NOX2 in dexmedetomidine-mediated alleviation of hypoxic damage was evaluated in cultured BV2 microglia and neonatal rats subjected to hypoxia. In vivo, neonatal rats received dexmedetomidine (25 µg/kg, i.p.) 30 min before or immediately after hypoxia (5% O2, 2 h). Apocynin-mediated NOX inhibition and lentivirus-mediated NOX2 overexpression were applied to further assess the involvement of microglial NOX2 activation. RESULTS: Pre- or posttreatment with dexmedetomidine alleviated hypoxia-induced cognitive impairment, restored damaged synapses, and increased postsynaptic density-95 and synaptophysin protein expression following neonatal hypoxia. Importantly, dexmedetomidine treatment suppressed hypoxia-induced microglial NOX2 activation and subsequent oxidative stress and the neuroinflammatory response, as reflected by reduced 4-hydroxynonenal and ROS accumulation, and decreased nuclear NF-κB p65 and proinflammatory cytokine levels in cultured BV2 microglia and the developing hippocampus. In addition, treating primary hippocampal neurons with conditioned medium (CM) from hypoxia-activated BV2 microglia resulted in neuronal damage, which was alleviated by CM from dexmedetomidine-treated microglia. Moreover, the neuroprotective effect of dexmedetomidine was reversed in NOX2-overexpressing BV2 microglia and diminished in apocynin-pretreated neonatal rats. CONCLUSION: Dexmedetomidine targets microglial NOX2 to reduce oxidative stress and neuroinflammation and subsequently protects against hippocampal synaptic loss following neonatal hypoxia.

Moringa oleifera modulates cholinergic and purinergic enzymes activity in BV-2 microglial cells.

Microglia are immune cells that are resident in central nervous system. Activation of microglial cells are detrimental to the survival of neurons. Thus, prevention of microglia activation and/or protection against microglia activation could be potential therapeutic strategy towards the management of inflammation-mediated neurodegenerative diseases. Moringa oleifera is widely consumed as food and used in folklore medicine for treating several diseases. This study was convened to investigate the effect of aqueous extract of Moringa oleifera on cell viability, cholinergic and purinergic enzymes in BV-2 microglial cultured cell. Aqueous extract of Moringa oleifera was prepared, lyophilized and reconstituted in 0.5% dimethylsulphoxide (DMSO). Cells were treated with Moringa oleifera extracts (0.1-100 µg/mL) and assessed for cell viability and nitric oxide production. Furthermore, the effect of Moringa oleifera on enzymes of cholinergic (acetylcholinesterase) and purinergic (nucleoside triphosphate diphosphohydrolase; NTPDase, 5' nucleotidase and adenosine deaminase; ADA) systems in BV-2 microglial cells were determined. Incubation of BV-2 microglia cell with M. oleifera extract maintained cell viability, modulated cholinergic and purinergic enzymes activity. The phenolic compounds found in M. oleifera extracts, include chlorogenic acid, rutin; quercetin pentoside, kaempferol derivative and quercetin derivative. Thus, this study suggest that the potential therapeutic effect of the phenolic compounds found in M. oleifera may have been responsible for the maintenance of cell viability in BV-2 microglia cells and modulation of cholinergic as well as purinergic enzymes activity.

Mesenchymal stem cell-derived extracellular vesicles promote microglial M2 polarization after subarachnoid hemorrhage in rats and involve the AMPK/NF-κB signaling pathway.

Subarachnoid hemorrhage (SAH) is an acute and severe disease with high disability and mortality. Inflammatory reactions have been proven to occur throughout SAH. Extracellular vesicles derived from mesenchymal stem cells (MSCs-EVs) have shown broad potential for the treatment of brain dysfunction and neuroprotective effects through neurogenesis and angiogenesis after stroke. However, the mechanisms of EVs in neuroinflammation during the acute phase of SAH are not well known. Our present study was designed to investigate the effects of MSCs-EVs on neuroinflammation and the polarization regulation of microglia to the M2 phenotype and related signaling pathways after SAH in rats. The SAH model was induced by an improved method of intravascular perforation, and MSCs-EVs were injected via the tail vein. Post-SAH assessments included neurobehavioral tests as well as brain water content, immunohistochemistry, PCR and Western blot analyses. Our results showed that MSCs-EVs alleviated the expression of inflammatory cytokines in the parietal cortex and hippocampus 24 h and 48 h after SAH and that MSCs-EVs inhibited NF-κB and activated AMPK to reduce inflammation after SAH. Furthermore, MSC-EVs regulated the polarization of microglia toward the M2 phenotype by downregulating interleukin-1ß, cluster of differentiation 16, cluster of differentiation 11b, and inducible nitric oxide synthase and upregulating the expression of cluster of differentiation 206 and arginase-1. Additionally, MSCs-EVs inhibited the neuroinflammatory response and had neuroprotective effects in the brain tissues of rats after SAH. This study may support their use as a potential treatment strategy for early SAH in the future.

N-Acylethanolamine Acid Amidase contributes to disease progression in a mouse model of multiple sclerosis.

N-Acylethanolamine acid amidase (NAAA) deactivates the endogenous peroxisome proliferator-activated receptor-α (PPAR-α) agonist palmitoylethanolamide (PEA). NAAA-regulated PEA signaling participates in the control of peripheral inflammation, but evidence suggests also a role in the modulation of neuroinflammatory pathologies such as multiple sclerosis (MS). Here we show that disease progression in the mouse experimental autoimmune encephalomyelitis (EAE) model of MS is accompanied by induction of NAAA expression in spinal cord, which in presymptomatic animals is confined to motor neurons and oligodendrocytes but, as EAE progresses, extends to microglia/macrophages and other cell types. As previously reported for NAAA inhibition, genetic NAAA deletion delayed disease onset and attenuated symptom intensity in female EAE mice, suggesting that accrued NAAA expression may contribute to pathology. To further delineate the role of NAAA in EAE, we generated a mouse line that selectively overexpresses the enzyme in macrophages, microglia and other monocyte-derived cells. Non-stimulated alveolar macrophages from these NaaaCD11b+ mice contain higher-than-normal levels of inducible nitric oxide synthase and display an activated morphology. Furthermore, intranasal lipopolysaccharide injections cause greater alveolar leukocyte accumulation in NaaaCD11b+ than in control mice. NaaaCD11b+ mice also display a more aggressive clinical response to EAE induction, compared to their wild-type littermates. The results identify NAAA as a critical control step in EAE pathogenesis, and point to this enzyme as a possible target for the treatment of MS.

6-Gingerol attenuates microglia-mediated neuroinflammation and ischemic brain injuries through Akt-mTOR-STAT3 signaling pathway.

Neuroinflammation is critical for the pathogenesis of ischemia brain damage. Over-activated microglia-mediated inflammation plays a very important role in ischemia cerebral injuries. 6-Gingerol, obtained from edible ginger (Zingiber Officinale) exhibits protective effects against inflammation. In this study, we found that 6-Gingerol could reduce the size of infarction (P = 0.0184) and improve neurological functions (P = 0.04) at the third day after ischemic brain injury in vivo. Since 6-Gingerol has the anti-inflammatory effects, we further investigated its impacts on neuroinflammation mediated by microglia both in vivo and in vitro. We found that the levels of pro-inflammatory cytokines Interleukin-1 beta (IL-1ß, P = 0.0213), Interleukin-6 (IL-6, P = 0.0316), and inducible NO synthase (iNOS, P = 0.0229) in the infarct penumbra were lower in 6-Gingerol treated groups. Furthermore, microglia induced pro-inflammatory cytokines, such as IL-6, IL-1ß, incremental intercellular nitric oxide (NO), as well as iNOS were blocked by the treatment of 6-Gingerol in lipopolysaccharide (LPS) stimulated microglia. In terms of mechanism, 6-Gingerol potently suppressed phosphorylation of serine-threonine protein kinase (Akt) - mammalian target of rapamycin (mTOR) - signal transducer and activator of transcription 3 (STAT3) in LPS-treated microglia. Taken together, the present study suggested that 6-Gingerol improved cerebral ischemia injury by suppressing microglia-mediated neuroinflammation by down-regulating Akt-mTOR-STAT3 pathway.

Inhibition of lncRNA DILC attenuates neuropathic pain via the SOCS3/JAK2/STAT3 pathway.

Long noncoding RNAs (lncRNAs) have been involved in the development of multiple pathological processes including neuropathic pain. The aim of the present study is to investigate the role of lncRNA down-regulated in liver cancer stem cells (DILC) in the progression of neuropathic pain and its underlying mechanism. Neuropathic pain rat model was established with the bilateral chronic constriction injury (bCCI) method. The results from quantitative PCR analysis in the spinal cord showed that DILC was significantly up-regulated in rats with bCCI compared with the sham group. DILC down-regulation mediated by intrathecal administration of DILC siRNA significantly increased the mechanical shrinkage threshold (MWT) and paw withdrawal threshold latency (PWTL), decreased the positive frequency for nerve sensitivity to cold and suppressed the expression of inflammatory genes in bCCI rats. Down-regulation of DILC induced suppressor of cytokine signaling (SOCS3) expression and inhibited the phosphorylation of signal transducer and activator of transcription 3 (p-STAT3) in spinal cord tissues. Western blotting showed that down-regulation of DILC by DILC siRNA transfection induced SOCS3 expression and inhibited the expression of p-Janus kinase 2 (p-JAK2) and p-STAT3 and their downstream genes in primary microglia. Furthermore, down-regulation of DILC increased the viability of primary microglia, suppressed apoptosis, and inhibited the production of interleukin (IL)-6 and IL-1ß in microglia. In contrast, overexpression of DILC showed the opposite functions to those of DILC knockdown. In conclusion, silence of lncRNA DILC attenuates neuropathic pain via SOCS3-induced suppression of the JAK2/STAT3 pathway.

Induction of inducible nitric oxide synthase expression in activated microglia and astrocytes following pre- and postnatal immune challenge in an animal model of schizophrenia.

In the central nervous system, activated microglia and astrocytes produce proinflammatory mediators such as inducible nitric oxide (iNOS) and cytokines. Uncontrolled release of these mediators induced by immune challenge can lead to increased vulnerability to complex brain disorders such as schizophrenia. In this study, BALB/c mice were injected intraperitoneally (i.p) with the viral mimetic polyriboinosinic-polyribocytidilic acid (poly(I:C)) or saline. At postnatal day 30 (PND0), the animals were sacrificed and the hippocampus, corpus callosum, striatum, cortex, fimbria and ventricle were immunostained for Iba-1, a microglial marker, glial fibrillary acidic protein (GFAP), an astrocyte marker, and iNOS, an activation marker for NO. Additionally, serum cytokine profiling (Interleukin-2 (IL-2), IL- 4, IL-6, interferon gamma (IFN-γ), tumour necrosis factor (TNF), IL-17A and IL-10) was determined using serum samples from poly(I:C)-treated and control mice. Our results demonstrated that poly(I:C) induced overactivation of differential proinflammatory responses in microglia and astrocytes, which could be strongly enhanced by a postnatal poly(I:C) administration before PND 30 in one part of the animals investigated. Specifically, there was significant iNOS upregulation in hippocampus, cortex and corpus callosum of poly(I:C)-affected off-springs. These inflammatory alterations were accompanied by increased circulating levels of the proinflammatory cytokines tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). This study provides insight into the role of microglia and astrocytes in an animal model of schizophrenia and an understanding of the regulation of iNOS expression in glial cells and cytokine networks. This knowledge could help identify novel targets for anti-oxidative and anti-inflammatory therapeutic schizophrenia intervention.

Triad3A displays a critical role in suppression of cerebral ischemic/reperfusion (I/R) injury by regulating necroptosis.

Ischemic stroke is a major cause of death and disability worldwide. Necroptosis is known as a form of cell death, playing an essential role in regulating ischemia-induced brain injury. Triad3A is a ubiquitin ligase of the RING-in-between-RING family, and regulates necroptotic cell death under different pathological conditions, including neurodegenerative disorders. In the present study, the effects of Triad3A on experimental stroke were explored on a mouse model with middle cerebral artery occlusion (MCAO). The results indicated that Triad3A expression was markedly induced in the ischemic brain after MCAO operation. The neurons and microglia cells were the major cellular sources for Triad3A induction. Triad3A knockdown enhanced the infarction area, cell death, microglia activity, and the expression levels of pro-inflammatory markers including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, inducible nitric oxide synthase (iNOS), CD32 and CD68 in MCAO mice. Triad3A and necroptosis were triggered in mouse microglia cells treated with oxygen and glucose deprivation (OGD), and in TNFα-incubated mouse hippocampal neuronal cells treated with Z-VAD-fmk, known as a pan-caspase inhibitor. Moreover, Triad3A knockdown accelerated cell death in microglial cells and neurons under these stresses. Furthermore, pre-treatment with necroptosis inhibitor markedly inhibited the cell death promoted by Triad3A silence in brain of mice with MCAO operation, demonstrating that Triad3A could regulate necroptosis to meditate the progression of cerebral I/R injury. Collectively, these finding illustrated that Triad3A could be served as a potential target for stroke therapy.

The Parkinson's disease-associated kinase LRRK2 regulates genes required for cell adhesion, polarization, and chemotaxis in activated murine macrophages.

Leucine-rich repeat kinase 2 (LRRK2) encodes a complex protein that includes kinase and GTPase domains. Genome-wide association studies have identified dominant LRRK2 alleles that predispose their carriers to late-onset idiotypic Parkinson's disease (PD) and also to autoimmune disorders such as Crohn's disease. Considerable evidence indicates that PD initiation and progression involve activation of innate immune functions in microglia, which are brain-resident macrophages. Here we asked whether LRRK2 modifies inflammatory signaling and how this modification might contribute to PD and Crohn's disease. We used RNA-Seq-based high-resolution transcriptomics to compare gene expression in activated primary macrophages derived from WT and Lrrk2 knockout mice. Remarkably, expression of a single gene, Rap guanine nucleotide exchange factor 3 (Rapgef3), was strongly up-regulated in the absence of LRRK2 and down-regulated in its presence. We observed similar regulation of Rapgef3 expression in cells treated with a highly specific inhibitor of LRRK2 protein kinase activity. Rapgef3 encodes an exchange protein, activated by cAMP 1 (EPAC-1), a guanine nucleotide exchange factor that activates the small GTPase Rap-1. Rap-1 mediates cell adhesion, polarization, and directional motility, and our results indicate that LRRK2 modulates chemotaxis of microglia and macrophages. Dominant PD-associated LRRK2 alleles may suppress EPAC-1 activity, further restricting motility and preventing efficient migration of microglia to sites of neuronal damage. Functional analysis in vivo in a subclinical infection model also indicated that Lrrk2 subtly modifies the inflammatory response. These results indicate that LRRK2 modulates the expression of genes involved in murine immune cell chemotaxis.

Genetic Ablation of Hematopoietic Cell Kinase Accelerates Alzheimer's Disease-Like Neuropathology in Tg2576 Mice.

Microglial dysregulation, pertaining to impairment in phagocytosis, clearance and containment of amyloid-ß (Aß), and activation of neuroinflammation, has been posited to contribute to the pathogenesis of Alzheimer's disease (AD). Detailed cellular mechanisms that are disrupted during the disease course to display such impairment in microglia, however, remain largely undetermined. We hypothesize that loss of hematopoietic cell kinase (HCK), a phagocytosis-regulating member of the Src family tyrosine kinases that mediate signals from triggering receptor expressed on myeloid cells 2 and other immunoreceptors, impairs microglial homeostasis and Aß clearance, leading to the accelerated buildup of Aß pathology and cognitive decline during the early stage of neuropathological development. To elucidate the pivotal role of HCK in AD, we generated a constitutive knockout of HCK in the Tg2576 mouse model of AD. We found that HCK deficiency accelerated cognitive decline along with elevated Aß level and plaque burden, attenuated microglial Aß phagocytosis, induced iNOS expression in microglial clusters, and reduced pre-synaptic protein at the hippocampal regions. Our findings substantiate that HCK plays a prominent role in regulating microglial neuroprotective functions and attenuating early AD neuropathology.

Stearoyl-CoA desaturase-1 impairs the reparative properties of macrophages and microglia in the brain.

Failure of remyelination underlies the progressive nature of demyelinating diseases such as multiple sclerosis. Macrophages and microglia are crucially involved in the formation and repair of demyelinated lesions. Here we show that myelin uptake temporarily skewed these phagocytes toward a disease-resolving phenotype, while sustained intracellular accumulation of myelin induced a lesion-promoting phenotype. This phenotypic shift was controlled by stearoyl-CoA desaturase-1 (SCD1), an enzyme responsible for the desaturation of saturated fatty acids. Monounsaturated fatty acids generated by SCD1 reduced the surface abundance of the cholesterol efflux transporter ABCA1, which in turn promoted lipid accumulation and induced an inflammatory phagocyte phenotype. Pharmacological inhibition or phagocyte-specific deficiency of Scd1 accelerated remyelination ex vivo and in vivo. These findings identify SCD1 as a novel therapeutic target to promote remyelination.

CD38 in Neurodegeneration and Neuroinflammation.

Neurodegenerative diseases are characterized by neuronal degeneration as well as neuroinflammation. While CD38 is strongly expressed in brain cells including neurons, astrocytes as well as microglial cells, the role played by CD38 in neurodegeneration and neuroinflammation remains elusive. Yet, CD38 expression increases as a consequence of aging which is otherwise the primary risk associated with neurodegenerative diseases, and several experimental data demonstrated that CD38 knockout mice are protected from neurodegenerative and neuroinflammatory insults. Moreover, nicotinamide adenine dinucleotide, whose levels are tightly controlled by CD38, is a recognized and potent neuroprotective agent, and NAD supplementation was found to be beneficial against neurodegenerative diseases. The aims of this review are to summarize the physiological role played by CD38 in the brain, present the arguments indicating the involvement of CD38 in neurodegeneration and neuroinflammation, and to discuss these observations in light of CD38 complex biology.

Targeting aurora kinase B alleviates spinal microgliosis and neuropathic pain in a rat model of peripheral nerve injury.

Peripheral nerve injury elicits spinal microgliosis, contributing to neuropathic pain. The aurora kinases A (AURKA), B (AURKB), and C (AURKC) are potential therapeutic targets in proliferating cells. However, their role has not been clarified in microglia. The aim of this study was to examine the regulation of aurora kinases and their roles and druggability in spinal microgliosis and neuropathic pain. Sprague-Dawley rats received chronic constriction injury (CCI). Gene expression of aurora kinases A-C was evaluated by quantitative RT-PCR and western blot, respectively, in spinal cords at 1, 3, 7, and 14 days after CCI. AURKB gene and protein expression was up-regulated concomitantly with the development of spinal microgliosis and neuropathic pain. Using lentiviral over-expression and adeno-associated viral knockdown approaches, the function of AURKB was further investigated by western blot, immunohistochemistry, RNA sequencing, and pain behavior tests. We found that AURKB over-expression in naive rats caused spinal microgliosis and pain hypersensitivity, whereas AURKB knockdown reduced microgliosis and alleviated CCI-induced neuropathic pain. Accordingly, RNA sequencing data revealed down-regulation of genes critically involved in signaling pathways associated with spinal microgliosis and neuropathic pain after AURKB knockdown in CCI rats. To examine its therapeutic potential for treatment of neuropathic pain, animals were treated intrathecally with the pharmacological AURKB inhibitor AZD1152-HQPA resulting in the alleviation of CCI-induced pain. Taken together, our findings indicated that AURKB plays a critical role in spinal microgliosis and neuropathic pain. Targeting AURKB may be an efficient method for treatment of neuropathic pain subsequent to peripheral nerve injury.

Signaling pathways involved in anti-inflammatory effects of Pulsed Electromagnetic Field in microglial cells.

Literature studies suggest important protective effects of low-frequency, low-energy pulsed electromagnetic fields (PEMFs) on inflammatory pathways affecting joint and cerebral diseases. However, it is not clear on which bases they affect neuroprotection and the mechanism responsible is yet unknown. Therefore the aim of this study was to identify the molecular targets of PEMFs anti-neuroinflammatory action. The effects of PEMF exposure in cytokine production by lipopolysaccharide (LPS)-activated N9 microglial cells as well as the pathways involved, including adenylyl cyclase (AC), phospholipase C (PLC), protein kinase C epsilon (PKC-ε) and delta (PKC-δ), p38, ERK1/2, JNK1/2 mitogen activated protein kinases (MAPK), Akt and caspase 1, were investigated. In addition, the ability of PEMFs to modulate ROS generation, cell invasion and phagocytosis, was addressed. PEMFs reduced the LPS-increased production of TNF-α and IL-1ß in N9 cells, through a pathway involving JNK1/2. Furthermore, they decreased the LPS-induced release of IL-6, by a mechanism not dependent on AC, PLC, PKC-ε, PKC-δ, p38, ERK1/2, JNK1/2, Akt and caspase 1. Importantly, a significant effect of PEMFs in the reduction of crucial cell functions specific of microglia like ROS generation, cell invasion and phagocytosis was found. PEMFs inhibit neuroinflammation in N9 cells through a mechanism involving, at least in part, the activation of JNK MAPK signalling pathway and may be relevant to treat a variety of diseases characterized by neuroinflammation.

Inhibition of the Dead Box RNA Helicase 3 Prevents HIV-1 Tat and Cocaine-Induced Neurotoxicity by Targeting Microglia Activation.

HIV-1 Associated Neurocognitive Disorder (HAND) is a common and clinically detrimental complication of HIV infection. Viral proteins, including Tat, released from infected cells, cause neuronal toxicity. Substance abuse in HIV-infected patients greatly influences the severity of neuronal damage. To repurpose small molecule inhibitors for anti-HAND therapy, we employed MOLIERE, an AI-based literature mining system that we developed. All human genes were analyzed and prioritized by MOLIERE to find previously unknown targets connected to HAND. From the identified high priority genes, we narrowed the list to those with known small molecule ligands developed for other applications and lacking systemic toxicity in animal models. To validate the AI-based process, the selective small molecule inhibitor of DDX3 helicase activity, RK-33, was chosen and tested for neuroprotective activity. The compound, previously developed for cancer treatment, was tested for the prevention of combined neurotoxicity of HIV Tat and cocaine. Rodent cortical cultures were treated with 6 or 60 ng/ml of HIV Tat and 10 or 25 µM of cocaine, which caused substantial toxicity. RK-33 at doses as low as 1 µM greatly reduced the neurotoxicity of Tat and cocaine. Transcriptome analysis showed that most Tat-activated transcripts are microglia-specific genes and that RK-33 blocks their activation. Treatment with RK-33 inhibits the Tat and cocaine-dependent increase in the number and size of microglia and the proinflammatory cytokines IL-6, TNF-α, MCP-1/CCL2, MIP-2, IL-1α and IL-1ß. These findings reveal that inhibition of DDX3 may have the potential to treat not only HAND but other neurodegenerative diseases. Graphical Abstract RK-33, selective inhibitor of Dead Box RNA helicase 3 (DDX3) protects neurons from combined Tat and cocaine neurotoxicity by inhibition of microglia activation and production of proinflammatory cytokines.