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People with alcohol use disorders (AUD) may be at higher risk for COVID-19. Angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) are required for cellular entry of SARS-CoV-2, but information on their expression in specific brain regions after alcohol exposure is limited. We sought to clarify how chronic alcohol exposure affects ACE2 expression in monoaminergic brainstem circuits and other putative SARS-CoV-2 entry points.Brains were examined for ACE2 using immunofluorescence after 4 weeks of chronic intermittent ethanol (CIE) vapor inhalation. Additionally, we examined TMPRSS2, Cathepsin L, and ADAM17 by Western blot and RAS pathway mediators and pro-inflammatory markers via RT-qPCR.ACE2 increased in most brain regions following CIE including olfactory bulb (OB), hypothalamus (HT), raphe magnus (RMG), raphe obscurus (ROB), locus coeruleus (LC), and periaqueductal gray (PAG). We also observed increased colocalization of ACE2 with monoaminergic neurons in brainstem nuclei. Moreover, soluble ACE2 (sACE2) was elevated in OB, HT, and LC. This increase in sACE2 in OB and HT was accompanied by upregulation of ADAM17, an ACE2 sheddase, while TMPRSS2 increased in HT and LC. Cathepsin L, an endosomal receptor involved in viral entry, was also increased in OB. Alcohol can increase Angiotensin II, which triggers a pro-inflammatory response that may upregulate ACE2 via activation of RAS pathway receptors AT1R/AT2R. ACE2 then metabolizes Angiotensin II to Angiotensin (1-7) and provokes an anti-inflammatory response via MAS1. Accordingly, we report that AT1R/AT2R mRNA decreased in OB and increased in the LC, while MAS1 mRNA increased in both OB and LC. Other mRNAs for pro-inflammatory markers were also dysregulated in OB, HT, raphe, and LC.Our results suggest that alcohol triggers a compensatory upregulation of ACE2 in the brain due to disturbed RAS and may increase the risk or severity of SARS-CoV-2 infection.

Alzheimer’s disease (AD) poses an ever-increasing public health concern as the population ages, affecting more than 6 million Americans. AD patients present with mood and sleep changes in the prodromal stages that may be partly driven by loss of monoaminergic neurons in brainstem, but a causal relationship has not been firmly established. The goal of the present study was to evaluate depressive and anxiety-like behaviors in a mouse model of human tauopathy (htau mice) at 4 and 6 months of age prior to the onset of cognitive impairments and correlate these behavior changes with tau pathology, neuroinflammation, and monoaminergic dysregulation in the DRN and LC. We observed depressive-like behaviors at 4 months of age in male and female htau mice and hyperlocomotion in male htau mice. At 6 months, male htau mice developed anxiety-like behavior in the EZM, whereas hyperlocomotion had resolved by this time point. Depressive-like behaviors in the social interaction test persisted at 6 months but were resolved in the sucrose preference test. There was also a significant reduction in number and density of 5-HT-immunoreactive neurons in the rostral DRN in htau mice at 4 months and 5-HT neuronal density was negatively correlated with the intensity of phosphorylated tau staining in this subregion. Additionally, we found evidence of microglial activation in the mid and caudal DRN and astrocytic activation in the rostral DRN. 5-HT neuronal activity was reduced in the DRN and accompanied by downregulation of Tph2 and Sert, whereas genes that promote neuroinflammation and tau phosphorylation were upregulated. Finally, there was enhanced ptau202/205 staining and microglial activity in the LC of htau mice and reduced TH optical density, although the number and density of TH+ neurons were not altered. In total, these results suggest that tau pathology in the DRN and the resulting loss of serotonergic neurotransmission may drive depressive-like behaviors in the early stages of AD, whereas anxiety-like behaviors develop later and may result from neurodegeneration in other regions.

Neuroinflammation leads to neurodegeneration, cognitive defects, and neurodegenerative disorders. Neurotrauma/traumatic brain injury (TBI) can cause activation of glial cells, neurons, and neuroimmune cells in the brain to release neuroinflammatory mediators. Neurotrauma leads to immediate primary brain damage (direct damage), neuroinflammatory responses, neuroinflammation, and late secondary brain damage (indirect) through neuroinflammatory mechanism. Secondary brain damage leads to chronic inflammation and the onset and progression of neurodegenerative diseases. Currently, there are no effective and specific therapeutic options to treat these brain damages or neurodegenerative diseases. Flavone luteolin is an important natural polyphenol present in several plants that show anti-inflammatory, antioxidant, anticancer, cytoprotective, and macrophage polarization effects. In this short review article, we have reviewed the neuroprotective effects of luteolin in neurotrauma and neurodegenerative disorders and pathways involved in this mechanism. We have collected data for this study from publications in the PubMed using the keywords luteolin and mast cells, neuroinflammation, neurodegenerative diseases, and TBI. Recent reports suggest that luteolin suppresses systemic and neuroinflammatory responses in Coronavirus disease 2019 (COVID-19). Studies have shown that luteolin exhibits neuroprotective effects through various mechanisms, including suppressing immune cell activation, such as mast cells, and inflammatory mediators released from these cells. In addition, luteolin can suppress neuroinflammatory response, activation of microglia and astrocytes, oxidative stress, neuroinflammation, and the severity of neuroinflammatory diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, and TBI pathogenesis. In conclusion, luteolin can improve cognitive decline and enhance neuroprotection in neurodegenerative diseases, TBI, and stroke.

Neurotrauma especially traumatic brain injury (TBI) is the leading cause of death and disability worldwide. To improve upon the early diagnosis and develop precision-targeted therapies for TBI, it is critical to understand the underlying molecular mechanisms and signaling pathways. The transcription factor, nuclear factor kappa B (NFκB), which is ubiquitously expressed, plays a crucial role in the normal cell survival, proliferation, differentiation, function, as well as in disease states like neuroinflammation and neurodegeneration. Here, we hypothesized that real-time noninvasive bioluminescence molecular imaging allows rapid and precise monitoring of TBI-induced immediate and rapid spatio-temporal activation of NFκB signaling pathway in response to Glia maturation factor (GMF) upregulation which in turn leads to neuroinflammation and neurodegeneration post-TBI. To test and validate our hypothesis and to gain novel mechanistic insights, we subjected NFκB-RE-Luc transgenic male and female mice to TBI and performed real-time noninvasive bioluminescence imaging (BLI) as well as photoacoustic and ultrasound imaging (PAI). Our BLI data revealed that TBI leads to an immediate and sustained activation of NFκB signaling. Further, our BLI data suggest that especially in male NFκB-RE-Luc transgenic mice subjected to TBI, in addition to brain, there is widespread activation of NFκB signaling in multiple organs. However, in the case of the female NFκB-RE-Luc transgenic mice, TBI induces a very specific and localized activation of NFκB signaling in the brain. Further, our microRNA data suggest that TBI induces significant upregulation of mir-9-5p, mir-21a-5p, mir-34a-5p, mir-16-3p, as well as mir-155-5p within 24 h and these microRNAs can be successfully used as TBI-specific biomarkers. To the best of our knowledge, this is one of the first and unique study of its kind to report immediate and sustained activation of NFκB signaling post-TBI in a gender-specific manner by utilizing real-time non-invasive BLI and PAI in NFκB-RE-Luc transgenic mice. Our study will prove immensely beneficial to gain novel mechanistic insights underlying TBI, unravel novel therapeutic targets, as well as enable us to monitor in real-time the response to innovative TBI-specific precision-targeted gene and stem cell-based precision medicine.

Traumatic brain injury (TBI) induces inflammatory responses through microglial activation and polarization towards a more inflammatory state that contributes to the deleterious secondary brain injury. Glia maturation factor (GMF) is a pro-inflammatory protein that is responsible for neuroinflammation following insult to the brain, such as in TBI. We hypothesized that the absence of GMF in GMF-knockout (GMF-KO) mice would regulate microglial activation state and the M1/M2 phenotypes following TBI. We used the weight drop model of TBI in C57BL/6 mice wild-type (WT) and GMF-KO mice. Immunofluorescence staining, Western blot, and ELISA assays were performed to confirm TBI-induced histopathological and neuroinflammatory changes. Behavioral analysis was done to check motor coordination ability and cognitive function. We demonstrated that the deletion of GMF in GMF-KO mice significantly limited lesion volume, attenuated neuronal loss, inhibited gliosis, and activated microglia adopted predominantly anti-inflammatory (M2) phenotypes. Using an ELISA method, we found a gradual decrease in pro-inflammatory cytokines (TNF-α and IL-6) and upregulation of anti-inflammatory cytokines (IL-4 and IL-10) in GMF-KO mice compared with WT mice, thus, promoting the transition of microglia towards a more predominantly anti-inflammatory (M2) phenotype. GMF-KO mice showed significant improvement in motor ability, memory, and cognition. Overall, our results demonstrate that GMF deficiency regulates microglial polarization, which ameliorates neuronal injury and behavioral impairments following TBI in mice and concludes that GMF is a regulator of neuroinflammation and an ideal therapeutic target for the treatment of TBI.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new pandemic infectious disease that originated in China. COVID-19 is a global public health emergency of international concern. COVID-19 causes mild to severe illness with high morbidity and mortality, especially in preexisting risk groups. Therapeutic options are now limited to COVID-19. The hallmark of COVID-19 pathogenesis is the cytokine storm with elevated levels of interleukin-6 (IL-6), IL-1β, tumor necrosis factor-alpha (TNF-α), chemokine (C-C-motif) ligand 2 (CCL2), and granulocyte-macrophage colony-stimulating factor (GM-CSF). COVID-19 can cause severe pneumonia, and neurological disorders, including stroke, the damage to the neurovascular unit, blood-brain barrier disruption, high intracranial proinflammatory cytokines, and endothelial cell damage in the brain. Mast cells are innate immune cells and also implicated in adaptive immune response, systemic inflammatory diseases, neuroinflammatory diseases, traumatic brain injury and stroke, and stress disorders. SARS-CoV-2 can activate monocytes/macrophages, dendritic cells, T cells, mast cells, neutrophils, and induce cytokine storm in the lung. COVID-19 can activate mast cells, neurons, glial cells, and endothelial cells. SARS-CoV-2 infection can cause psychological stress and neuroinflammation. In conclusion, COVID-19 can induce mast cell activation, psychological stress, cytokine storm, and neuroinflammation.

Traumatic brain injury (TBI) is the primary cause of death and disability affecting over 10 million people in the industrialized world. TBI causes a wide spectrum of secondary molecular and cellula...

Traumatic brain injury (TBI) causes disability and death, accelerating the progression towards Alzheimer's disease and Parkinson's disease (PD). TBI causes serious motor and cognitive impairments, as seen in PD that arise during the period of the initial insult. However, this has been understudied relative to TBI induced neuroinflammation, motor and cognitive decline that progress towards PD. Neuronal ubiquitin-C-terminal hydrolase- L1 (UCHL1) is a thiol protease that breaks down ubiquitinated proteins and its level represents the severity of TBI. Previously, we demonstrated the molecular action of glia maturation factor (GMF); a proinflammatory protein in mediating neuroinflammation and neuronal loss. Here, we show that the weight drop method induced TBI neuropathology using behavioral tests, western blotting, and immunofluorescence techniques on sections from wild type (WT) and GMF-deficient (GMF-KO) mice. Results reveal a significant improvement in substantia nigral tyrosine hydroxylase and dopamine transporter expression with motor behavioral performance in GMF-KO mice following TBI. In addition, a significant reduction in neuroinflammation was manifested, as shown by activation of nuclear factor-kB, reduced levels of inducible nitric oxide synthase, and cyclooxygenase- 2 expressions. Likewise, neurotrophins including brain-derived neurotrophic factor and glial-derived neurotrophic factor were significantly improved in GMF-KO mice than WT 72 h post-TBI. Consistently, we found that TBI enhances GFAP and UCHL-1 expression and reduces the number of dopaminergic TH-positive neurons in WT compared to GMF-KO mice 72 h post-TBI. Interestingly, we observed a reduction of THpositive tanycytes in the median eminence of WT than GMF-KO mice. Overall, we found that absence of GMF significantly reversed these neuropathological events and improved behavioral outcome. This study provides evidence that PD-associated pathology progression can be initiated upon induction of TBI.

Purpose Psychological stress is a significant health problem in veterans and their family members. Traumatic brain injury (TBI) and stress lead to the onset, progression, and worsening of several inflammatory and neurodegenerative diseases in veterans and civilians. Alzheimer's disease (AD) is a progressive, irreversible neuroinflammatory disease that causes problems with memory, thinking, and behavior. TBIs and chronic psychological stress cause and accelerate the pathology of neuroinflammatory diseases such as AD. However, the precise molecular and cellular mechanisms governing neuroinflammation and neurodegeneration are currently unknown, especially in veterans. The purpose of this review article was to advance the hypothesis that stress and TBI-mediated immune response substantially contribute and accelerate the pathogenesis of AD in veterans and their close family members and civilians. Methods The information in this article was collected and interpreted from published articles in PubMed between 1985 and 2020 using the key words stress, psychological stress, Afghanistan war, Operation Enduring Freedom (OEF), Iraq War, Operation Iraqi Freedom (OIF), Operation New Dawn (OND), traumatic brain injury, mast cell and stress, stress and neuroimmune response, stress and Alzheimer's disease, traumatic brain injury, and Alzheimer's disease. Findings Chronic psychological stress and brain injury induce the generation and accumulation of beta-amyloid peptide, amyloid plaques, neurofibrillary tangles, and phosphorylation of tau in the brain, thereby contributing to AD pathogenesis. Active military personnel and veterans are under enormous psychological stress due to various war-related activities, including TBIs, disabilities, fear, new environmental conditions, lack of normal life activities, insufficient communications, explosions, military-related noise, and health hazards. Brain injury, stress, mast cell, and other immune cell activation can induce headache, migraine, dementia, and upregulate neuroinflammation and neurodegeneration in veterans of Operation Enduring Freedom, Operation Iraqi Freedom, and Operation New Dawn. TBIs, posttraumatic stress disorder, psychological stress, pain, glial activation, and dementia in active military personnel, veterans, or their family members can cause AD several years later in their lives. We suggest that there are increasing numbers of veterans with TBIs and stress and that these veterans may develop AD late in life if no appropriate therapeutic intervention is available. Implications Per these published reports, the fact that TBIs and psychological stress can accelerate the pathogenesis of AD should be recognized. Active military personnel, veterans, and their close family members should be evaluated regularly for stress symptoms to prevent the pathogenesis of neurodegenerative diseases, including AD.

Traumatic brain injury (TBI) is a major health problem in the United States, which affects about 1.7 million people each year. Glial cells, T-cells, and mast cells perform specific protective functions in different regions of the brain for the recovery of cognitive and motor functions after central nervous system (CNS) injuries including TBI. Chronic neuroinflammatory responses resulting in neuronal death and the accompanying stress following brain injury predisposes or accelerates the onset and progression of Alzheimer’s disease (AD) in high-risk individuals. About 5.7 million Americans are currently living with AD. Immediately following brain injury, mast cells respond by releasing prestored and preactivated mediators and recruit immune cells to the CNS. Blood-brain barrier (BBB), tight junction and adherens junction proteins, neurovascular and gliovascular microstructural rearrangements, and dysfunction associated with increased trafficking of inflammatory mediators and inflammatory cells from the periphery across the BBB leads to increase in the chronic neuroinflammatory reactions following brain injury. In this review, we advance the hypothesis that neuroinflammatory responses resulting from mast cell activation along with the accompanying risk factors such as age, gender, food habits, emotional status, stress, allergic tendency, chronic inflammatory diseases, and certain drugs can accelerate brain injury-associated neuroinflammation, neurodegeneration, and AD pathogenesis.

Despite significant advancements in the field of molecular neurobiology especially neuroinflammation and neurodegeneration, the highly complex molecular mechanisms underlying neurodegenerative diseases remain elusive. As a result, the development of the next generation neurotherapeutics has experienced a considerable lag phase. Recent advancements in the field of genome editing offer a new template for dissecting the precise molecular pathways underlying the complex neurodegenerative disorders. We believe that the innovative genome and transcriptome editing strategies offer an excellent opportunity to decipher novel therapeutic targets, develop novel neurodegenerative disease models, develop neuroimaging modalities, develop next-generation diagnostics as well as develop patient-specific precision-targeted personalized therapies to effectively treat neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic lateral sclerosis, Frontotemporal dementia etc. Here, we review the latest developments in the field of CRISPR-mediated genome editing and provide unbiased futuristic insights regarding its translational potential to improve the treatment outcomes and minimize financial burden. However, despite significant advancements, we would caution the scientific community that since the CRISPR field is still evolving, currently we do not know the full spectrum of CRISPR-mediated side effects. In the wake of the recent news regarding CRISPR-edited human babies being born in China, we urge the scientific community to maintain high scientific and ethical standards and utilize CRISPR for developing in vitro disease in a dish model, in vivo testing in nonhuman primates and lower vertebrates and for the development of neurotherapeutics for the currently incurable neurodegenerative disorders. Graphical Abstract.

The pathological form of amyloid beta (Aβ) peptide is shown to be toxic to the mitochondria and implicates this organelle in the progression and pathogenesis of Alzheimer's disease (AD). Mitochondria are dynamic structures constantly undergoing fission and fusion, and altering their shape and size while traveling through neurons. Mitochondrial fission (Drp1, Fis1) and fusion (OPA1, Mfn1, and Mfn2) proteins are balanced in healthy neuronal cells. Glia maturation factor (GMF), a neuroinflammatory protein isolated and cloned in our laboratory plays an important role in the pathogenesis of AD. We hypothesized that GMF, a brain-localized inflammatory protein, promotes oxidative stress-mediated disruption of mitochondrial dynamics by alterations in mitochondrial fission and fusion proteins which eventually leads to apoptosis in the Aβ (1-42)-treated human neuroblastoma (SH-SY5Y) cells. The SH-SY5Y cells were incubated with GMF and Aβ (1-42) peptide, and mitochondrial fission and fusion proteins were analyzed by immunofluorescence, western blotting, and co-immunoprecipitation. We report that SH-SY5Y cells incubated with GMF and Aβ (1-42) promote mitochondrial fragmentation, by potentiating oxidative stress, mitophagy and shifts in the Bax/Bcl2 expression and release of cytochrome-c, and eventual apoptosis. In the present study, we show that GMF and Aβ treatments significantly upregulate fission proteins and downregulate fusion proteins. The study shows that extracellular GMF is an important inflammatory mediator that mediates mitochondrial dynamics by altering the balance in fission and fusion proteins and amplifies similar effects promoted by Aβ. Upregulated GMF in the presence of Aβ could be an additional risk factor for AD, and their synergistic actions need to be explored as a potential therapeutic target to suppress the progression of AD.

Acute traumatic brain injury (TBI) leads to neuroinflammation, neurodegeneration, cognitive decline, psychological disorders, increased blood-brain barrier (BBB) permeability, and microvascular damage in the brain. Inflammatory mediators secreted from activated glial cells, neurons, and mast cells are implicated in the pathogenesis of TBI through secondary brain damage. Abnormalities or damage to the neurovascular unit is the indication of secondary injuries in the brain after TBI. However, the precise mechanisms of molecular and ultrastructural neurovascular alterations involved in the pathogenesis of acute TBI are not yet clearly understood. Moreover, currently, there are no precision-targeted effective treatment options to prevent the sequelae of TBI. In this study, mice were subjected to closed head weight-drop-induced acute TBI and evaluated neuroinflammatory and neurovascular alterations in the brain by immunofluorescence staining or quantitation by enzyme-linked immunosorbent assay (ELISA) procedure. Mast cell stabilizer drug cromolyn was administered to inhibit the neuroinflammatory response of TBI. Results indicate decreased level of pericyte marker platelet-derived growth factor receptor-beta (PDGFR-β) and BBB-associated tight junction proteins junctional adhesion molecule-A (JAM-A) and zonula occludens-1 (ZO-1) in the brains 7 days after weight-drop-induced acute TBI as compared with the brains from sham control mice indicating acute TBI-associated BBB/tight junction protein disruption. Further, the administration of cromolyn drug significantly inhibited acute TBI-associated decrease of PDGFR-β, JAM-A, and ZO-1 in the brain. These findings suggest that acute TBI causes BBB/tight junction damage and that cromolyn administration could protect this acute TBI-induced brain damage as well as its long-time consequences.

Neurotrauma, especially Traumatic Brain Injury (TBI) is a major health concern not only for the civilian population but also for the military personnel. Currently there are no precision and regenerative therapies available for the successful treatment of TBI patients. Hence, early detection and treatment options may prevent the severity and untoward harmful effects of TBI. However, currently there are no effective biomarkers available for the rapid and robust diagnosis as well as prognosis of TBI. Several biomarkers in blood, cerebrospinal fluid (CSF), saliva and urine have been explored to assess the onset, progression, severity and prognosis of TBI recently. Present knowledge on the blood biomarkers including cytokines and chemokines and in vivo imaging modalities are useful to some extent to detect and treat TBI patients. Here, we review S100B, Glial Fibrillary Acidic Protein (GFAP), Neuron Specific Enolase (NSE), Myelin Basic Protein (MBP), Ubiquitin C-terminal Hydrolase L1 (UCHL1), tau protein, and alpha spectrin II break down products regarding their usefulness as a set of reliable biomarkers for the robust diagnosis of TBI. We suggest that these biomarkers may prove very useful for the diagnosis and prognosis of TBI.

Alzheimer’s disease (AD) is a neurodegenerative and neuroinflammatory disease characterized by the presence of extracellular amyloid plaques (APs) and intracellular neurofibrillary tangles (NFTs) in the brain. There is no disease modifying therapeutic options currently available for this disease. Hippocampus, entorhinal cortex (Broadmann area 28), perirhinal cortex (Broadmann area 35) and insular cortices are areas within the brain that are first ones to be severely affected in AD. Neuroinflammation is an important factor that induces neurodegeneration in AD. Glia maturation factor (GMF), a proinflammatory factor plays a crucial role in AD through activation of microglia and astrocytes to release proinflammatory mediators in the brain. Through immunohistochemical studies, we have previously shown that GMF is highly expressed in the vicinity of APs and NFTs in AD brains. Glial fibrillary acidic protein (GFAP), reactive astrocytes, ionized calcium binding adaptor molecule-1 (Iba-1) labelled activated microglia and GMF immunoreactive glial cells are increased in the entorhinal cortical layers especially at the sites of APs and Tau containing NFTs indicating a role for GMF. Overexpression of GMF in glial cells leads to neuroinflammation and neurodegeneration. Inhibition of GMF expression reduces neurodegeneration. Therefore, we suggest that GMF is a novel therapeutic target not only for AD but also for various other neurodegenerative diseases.

Traumatic brain injury (TBI) is the primary cause of death and disability affecting over 10 million people in the industrialized world. TBI causes a wide spectrum of secondary molecular and cellular complications in the brain. However, the pathological events are still not yet fully understood. Previously, we have shown that the glia maturation factor (GMF) is a mediator of neuroinflammation in neurodegenerative diseases. To identify the potential molecular pathways accompanying TBI, we used an in vitro cell culture model of TBI. A standardized injury was induced by scalpel cut through a mixed primary cell culture of astrocytes, microglia and neurons obtained from both wild type (WT) and GMF-deficient (GMF-KO) mice. Cell culture medium and whole–cell lysates were collected at 24, 48, and 72 h after the scalpel cuts injury and probed for oxidative stress using immunofluorescence analysis. Results showed that oxidative stress markers such as glutathione and glutathione peroxidase were significantly reduced, while release of cytosolic enzyme lactate dehydrogenase along with nitric oxide and prostaglandin E2 were significantly increased in injured WT cells compared with injured GMF-KO cells. In addition, injured WT cells showed increased levels of oxidation product 4-hydroxynonenal and 8-oxo-2′-deoxyguanosine compared with injured GMF-KO cells. Further, we found that injured WT cells showed a significantly increased expression of glial fibrillary acidic protein, ionized calcium binding adaptor molecule 1, and phosphorylated ezrin/radixin/moesin proteins, and reduced microtubule associated protein expression compared with injured GMF-KO cells after injury. Collectively, our results demonstrate that GMF exacerbates the oxidative stress–mediated neuroinflammation that could be brought about by TBI-induced astroglial activation.

Alzheimer's disease (AD) is an irreversible progressive neurodegenerative disorder recognized by accumulation of amyloid-plaques (APs) and neurofibrillary tangles (NFTs) and eventually loss of memory. Glia maturation factor (GMF), a neuroinflammatory protein first time isolated and cloned in our laboratory plays an important role in the pathogenesis of AD. However, no studies have been reported on whether anti-GMF antibody administration could downregulate neuroinflammation and attenuate amyloid pathology in AD brain. We investigated the potential effect of single dose of (2 mg/kg b.wt/mouse) intravenously (iv) injected with anti-GMF antibodyon cognitive function, neuroprotection, neuroinflammation and Aβ load in the brain of 9-month-old 5XFAD mice. Following 4 weeks of anti-GMF antibody delivery in mice, we found reduced expression of GMF, astrocytic glial fibrillary acidic protein (GFAP) and microglial ionizing calcium binding adaptor molecule 1 (Iba1) as well as improvement inneuroinflammatory response via inhibition of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) production and amyloid pathology in the cerebral cortex and hippocampal CA1 region of 5XFAD mice. Correspondingly, blockade of GMF function with anti-GMF antibody improved spatial learning, memory, and long-term recognition memory in 5XFAD mice. The present study demonstrates that the immune checkpoint blockade of GMF function with anti-GMF antibody coordinates anti-inflammatory effects to attenuate neurodegeneration in the cortex and hippocampal CA1 region of 5XFAD mouse brain. Further, our data suggest, that pharmacological immune neutralization of GMF is a promising neuroprotective strategy totherapeutically target neuroinflammation and neurodegeneration in AD. Graphical Abstract 5XFAD mice Polyclonal anti-GMF antibody.

Traumatic brain injury (TBI) is one of the major health problems worldwide that causes death or permanent disability through primary and secondary damages in the brain. TBI causes primary brain damage and activates glial cells and immune and inflammatory cells, including mast cells in the brain associated with neuroinflammatory responses that cause secondary brain damage. Though the survival rate and the neurological deficiencies have shown significant improvement in many TBI patients with newer therapeutic options, the underlying pathophysiology of TBI-mediated neuroinflammation, neurodegeneration, and cognitive dysfunctions is understudied. In this study, we analyzed mast cells and neuroinflammation in weight drop-induced TBI. We analyzed mast cell activation by toluidine blue staining, serum chemokine C-C motif ligand 2 (CCL2) level by enzyme-linked immunosorbent assay (ELISA), and proteinase-activated receptor-2 (PAR-2), a mast cell and inflammation-associated protein, vascular endothelial growth factor receptor 2 (VEGFR2), and blood-brain barrier tight junction-associated claudin 5 and Zonula occludens-1 (ZO-1) protein expression in the brains of TBI mice. Mast cell activation and its numbers increased in the brains of 24 h and 72 h TBI when compared with sham control brains without TBI. Mouse brains after TBI show increased CCL2, PAR-2, and VEGFR2 expression and derangement of claudin 5 and ZO-1 expression as compared with sham control brains. TBI can cause mast cell activation, neuroinflammation, and derangement of tight junction proteins associated with increased BBB permeability. We suggest that inhibition of mast cell activation can suppress neuroimmune responses and glial cell activation-associated neuroinflammation and neurodegeneration in TBI.

Alzheimer's disease (AD) is a devastating, progressive neurodegenerative disorder that leads to severe cognitive impairment in elderly patients. Chronic neuroinflammation plays an important role in the AD pathogenesis. Glia maturation factor (GMF), a proinflammatory molecule discovered in our laboratory, is significantly upregulated in various regions of AD brains. We have previously reported that GMF is predominantly expressed in the reactive glial cells surrounding the amyloid plaques (APs) in the mouse and human AD brain. Microglia are the major source of proinflammatory cytokines and chemokines including GMF. Recently clustered regularly interspaced short palindromic repeats (CRISPR) based genome editing has been recognized to study the functions of genes that are implicated in various diseases. Here, we investigated if CRISPR-Cas9-mediated GMF gene editing leads to inhibition of GMF expression and suppression of microglial activation. Confocal microscopy of murine BV2 microglial cell line transduced with an adeno-associated virus (AAV) coexpressing Staphylococcus aureus (Sa) Cas9 and a GMF-specific guide RNA (GMF-sgRNA) revealed few cells expressing SaCas9 while lacking GMF expression, thereby confirming successful GMF gene editing. To further improve GMF gene editing efficiency, we developed lentiviral vectors (LVs) expressing either Streptococcus pyogenes (Sp) Cas9 or GMF-sgRNAs. BV2 cells cotransduced with LVs expressing SpCas9 and GMF-sgRNAs revealed reduced GMF expression and the presence of indels in the exons 2 and 3 of the GMF coding sequence. Lipopolysaccharide (LPS) treatment of GMF-edited cells led to reduced microglial activation as shown by reduced p38 MAPK phosphorylation. We believe that targeted in vivo GMF gene editing has a significant potential for developing a unique and novel AD therapy.

Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by the presence of intracellular neurofibrillary tangles (NFTs) containing hyper-phosphorylated tau, and the extracellular deposition of amyloid plaques (APs) with misfolded amyloid–β (Aβ) peptide. Glia maturation factor (GMF), a highly conserved pro-inflammatory protein, isolated and cloned in our laboratory has been shown to activate glial cells leading to neuroinflammation and neurodegeneration in AD. We hypothesized that inflammatory reactions promoted by NLRP3-Caspase-1inflammasome pathway trigger dysfunction in autophagy and accumulation of Aβ which is amplified and regulated by GMF in AD. In this study, using immunohistochemical techniques we analyzed components of the NLRP3 inflammasome and autophagy-lysosomal markers in relation to Aβ, p-tau and GMF in human post-mortem AD and age-matched non-AD brains. Tissue sections were prepared from the temporal cortex of human post-mortem brains. Here, we demonstrate an increased expression of the inflammasome components NLRP3 and Caspase-1 and the products of inflammasome activation IL-1β and IL-18 along with GMF in the temporal cortex of AD brains. These inflammasome components and the pro-inflammatory cytokines co-localized with GMF in the vicinity and periphery of the amyloid plaques and NFTs. Moreover, using double immunofluorescence staining, AD brain displayed an increase in the autophagy SQSTM1/p62 and LC3 positive vesicles and the lysosomal marker LAMP1 that also co-localized with GMF, amyloid beta and hyper-phosphorylated p-tau. Our results indicate that in AD, the neuroinflammation promoted by the NLRP3 inflammasome may be amplified and regulated by GMF, which further impairs clearance of protein aggregates mediated by the autophagosomal pathway.

The molecular mechanism mediating degeneration of nigrostriatal dopaminergic neurons in Parkinson's disease (PD) is not yet fully understood. Previously, we have shown the contribution of glia maturation factor (GMF), a proinflammatory protein in dopaminergic neurodegeneration mediated by activation of mast cells (MCs). In this study, methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigrostriatal neurodegeneration and astro-glial activations were determined by western blot and immunofluorescence techniques in wild type (WT) mice, MC-deficient (MC-KO) mice and GMF-deficient (GMF-KO) mice, with or without MC reconstitution before MPTP administration. We show that GMF-KO in the MCs reduces the synergistic effects of MC and Calpain1 (calcium-activated cysteine protease enzyme)-dependent dopaminergic neuronal loss that reduces motor behavioral impairments in MPTP-treated mouse. Administration of MPTP increase in calpain-mediated proteolysis in nigral dopaminergic neurons further resulting in motor decline in mice. We found that MPTP administered WT mice exhibits oxidative stress due to significant increases in the levels of malondialdehyde, superoxide dismutase and reduction in the levels of reduced glutathione and glutathione peroxidase activity as compared with both MC-KO and GMF-KO mice. The number of TH-positive neurons in the ventral tegmental area, substantia nigra and the fibers in the striatum were significantly reduced while granulocyte macrophage colony-stimulating factor (GM-CSF), MC-Tryptase, GFAP, IBA1, Calpain1 and intracellular adhesion molecule 1 expression were significantly increased in WT mice. Similarly, tyrosine hydroxylase, dopamine transporters and vesicular monoamine transporters 2 proteins expression were significantly reduced in the SN of MPTP treated WT mice. The motor behavior as analyzed by rotarod and hang test was significantly reduced in WT mice as compared with both the MC-KO and GMF-KO mice. We conclude that GMF-dependent MC activation enhances the detrimental effect of astro-glial activation-mediated oxidative stress and neuroinflammation in the midbrain, and its inhibition may slowdown the progression of PD.

Microglial cells are brain specific professional phagocytic immune cells that play a crucial role in the inflammation-mediated neurodegeneration especially in Parkinson’s disease (PD) and Alzheimer’s disease. Glia maturation factor (GMF) is a neuroinflammatory protein abundantly expressed in the brain. We have previously shown that GMF expression is significantly upregulated in the substantia nigra (SN) of PD brains. However, its possible role in PD progression is still not fully understood. The Clustered-Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR Associated (Cas) protein9 (CRISPR/Cas9) system is a simple, rapid and often extremely efficient gene editing tool at desired loci, enabling complete gene knockout or homology directed repair. In this study, we examined the effect of GMF editing by using the CRISPR/Cas9 technique in BV2 microglial cells (hereafter referred to as BV2-G) on oxidative stress and nuclear factor erythroid 2-related factor 2 (NRF2)/Hemeoxygenase1 (HO-1)-dependent ferritin activation after treatment with 1-methyl-4-phenylpyridinium (MPP(+)). Knockout of GMF in BV2-G cells significantly attenuated oxidative stress via reduced ROS production and calcium flux. Furthermore, deficiency of GMF significantly reduced nuclear translocation of NRF2, which modulates HO-1 and ferritin activation, cyclooxygenase 2 (COX2) and nitric oxide synthase 2 (NOS2) expression in BV2 microglial cells. Lack of GMF significantly improved CD11b and CD68 positive microglial cells as compared with untreated cells. Our results also suggest that pharmacological and genetic intervention targeting GMF may represent a promising and a novel therapeutic strategy in controlling Parkinsonism by regulating microglial functions. Targeted regulation of GMF possibly mediates protein aggregation in microglial homeostasis associated with PD progression through regulation of iron metabolism by modulating NRF2-HO1 and ferritin expression.

Parkinson’s disease (PD) is one of the several neurodegenerative diseases where accumulation of aggregated proteins like α-synuclein occurs. Dysfunction in autophagy leading to this protein build-up and subsequent dopaminergic neurodegeneration may be one of the causes of PD. The mechanisms that impair autophagy remain poorly understood. 1-Methyl-4-phenylpiridium ion (MPP+) is a neurotoxin that induces experimental PD in vitro. Our studies have shown that glia maturation factor (GMF), a brain-localized inflammatory protein, induces dopaminergic neurodegeneration in PD and that suppression of GMF prevents MPP+-induced loss of dopaminergic neurons. In the present study, we demonstrate a molecular action of GMF on the autophagic machinery resulting in dopaminergic neuronal loss and propose GMF-mediated autophagic dysfunction as one of the contributing factors in PD progression. Using dopaminergic N27 neurons, primary neurons from wild type (WT), and GMF-deficient (GMF-KO) mice, we show that GMF and MPP+ enhanced expression of MAPKs increased the mammalian target of rapamycin (mTOR) activation and endoplasmic reticulum stress markers such as phospho-eukaryotic translation initiation factor 2 alpha kinase 3 (p-PERK) and inositol-requiring enzyme 1α (IRE1α). Further, GMF and MPP+ reduced Beclin 1, focal adhesion kinase (FAK) family-interacting protein of 200 kD (FIP200), and autophagy-related proteins (ATGs) 3, 5, 7, 16L, and 12. The combined results demonstrate that GMF affects autophagy through autophagosome formation with significantly reduced lysosomal-associated membrane protein 1/2, and the number of autophagic acidic vesicles. Using primary neurons, we show that MPP+ treatment leads to differential expression and localization of p62/sequestosome and in GMF-KO neurons, there was a marked increase in p62 staining implying autophagy deficiency with very little co-localization of α-synuclein and p62 as compared with WT neurons. Collectively, this study provides a bidirectional role for GMF in executing dopaminergic neuronal death mediated by autophagy that is relevant to PD.

Tanycytes are highly specialized bipolar ependymal cells that line the ventrolateral wall and the floor of the third ventricle in the brain and form a blood-cerebrospinal fluid barrier at the level of the median eminence. They play a pivotal role in regulating metabolic networks that control body weight and energy homeostasis. Due to the glucosensing function of tanycytes, they could be considered as a critical player in the pathogenesis of type 2 diabetes. Genetic fate mapping studies have established the role of tanycytes for the newly detected adult hypothalamic neurogenesis with important implications for metabolism as well as pathophysiology of various neurodegenerative diseases. We believe that a comprehensive understanding of the physiological mechanisms underlying their neuroplasticity, glucosensing, and cross talk with endothelial cells will enable us to achieve metabolic homeostasis in type 2 diabetes patients and possibly delay the progression of Alzheimer's disease and hopefully improve cognitive function.

Apolipoprotein E4 (ApoE4) is a major genetic risk factor for Alzheimer’s disease (AD). The E4 allele of ApoE plays a crucial role in the inflammatory and neurodegenerative processes associated with AD. This is evident from the multiple effects of the ApoE isoforms in beta amyloid (Aβ) aggregation. Glia maturation factor (GMF) is a brain-specific neuroinflammatory protein, that we have previously demonstrated to be significantly upregulated in various regions of AD brains compared to non-AD control brains and that it induces neurodegeneration. We have previously reported that GMF is predominantly expressed in the reactive astrocytes surrounding APs in AD brain. In the present study, using immunohistochemical staining methods we show the expression of GMF and ApoE4 in AD brains. Double immunofluorescence labelling of GMF and ApoE4 showed their co-localization in the amyloid plaques (APs) of AD brain. We show the co-localization of GMF and ApoE4 in APs of AD brain by double immunohistochemical and dual immunofluorescence methods. Our results show that ApoE4 is present in APs of AD brain. Double immunofluorescence labelling method shows the relationship between ApoE4 and GMF expression in the APs in the AD brain. We found that GMF and ApoE4 were strongly expressed and co-associated in APs and in the reactive astrocytes surrounding APs in AD. The increased expression of GMF in APs and neurofibrillary tangles (NFTs) in the AD brain, and the co-localization of GMF and ApoE4 in APs suggest that GMF and ApoE4 together should be contributing to the pathogenesis of AD.

Parkinson's disease (PD) is a progressive neurodegenerative disease affecting over five million individuals worldwide. The exact molecular events underlying PD pathogenesis are still not clearly known. Glia maturation factor (GMF), a neuroinflammatory protein in the brain plays an important role in the pathogenesis of PD. Mitochondrial dysfunctions and oxidative stress trigger apoptosis leading to dopaminergic neuronal degeneration in PD. Peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α or PPARGC-α) acts as a transcriptional co-regulator of mitochondrial biogenesis and energy metabolism by controlling oxidative phosphorylation, antioxidant activity, and autophagy. In this study, we found that incubation of immortalized rat dopaminergic (N27) neurons with GMF influences the expression of peroxisome PGC-1α and increases oxidative stress, mitochondrial dysfunction, and apoptotic cell death. We show that incubation with GMF reduces the expression of PGC-1α with concomitant decreases in the mitochondrial complexes. Besides, there is increased oxidative stress and depolarization of mitochondrial membrane potential (MMP) in these cells. Further, GMF reduces tyrosine hydroxylase (TH) expression and shifts Bax/Bcl-2 expression resulting in release of cytochrome-c and increased activations of effector caspase expressions. Transmission electron microscopy analyses revealed alteration in the mitochondrial architecture. Our results show that GMF acts as an important upstream regulator of PGC-1α in promoting dopaminergic neuronal death through its effect on oxidative stress-mediated apoptosis. Our current data suggest that GMF is a critical risk factor for PD and suggest that it could be explored as a potential therapeutic target to inhibit PD progression.

Parkinson’s disease (PD) is a progressive movement disorder characterized by neuroinflammation and dopaminergic neurodegeneration in the brain. 1-methyl-4-phenylpyridinium (MPP+), a metabolite of the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces the release of inflammatory mediators from glial cells and neurons. Glia maturation factor (GMF), a brain proinflammatory protein, MPP+, and mast cell-derived inflammatory mediators induce neurodegeneration which eventually leads to PD. However, the precise mechanisms underlying interaction between glial cells, neurons and mast cells in PD still remain elusive. In the present study, mouse bone marrow-derived mast cells (BMMCs) and mouse fetal brain-derived mixed glia/neurons, astrocytes and neurons were incubated with MPP+, GMF and mast cell-derived inflammatory mediators mouse mast cell protease-6 (MMCP-6), MMCP-7 or tryptase/brain-specific serine protease-4 (tryptase/BSSP-4). Inflammatory mediators released from these cells in the culture medium were quantitated by enzyme-linked immunosorbent assay. Neurodegeneration was quantified by measuring total neurite outgrowth following microtubule-associated protein-2 immunocytochemistry. MPP+− induced significant neurodegeneration with reduced total neurite outgrowth. MPP+− induced the release of tryptase/BSSP-4 from the mouse mast cells, and tryptase/BSSP-4 induced chemokine (C-C motif) ligand 2 (CCL2) release from astrocytes and glia/neurons. Overall our results suggest that MPP+, GMF, MMCP-6 or MMCP-7 stimulate glia/neurons, astrocytes or neurons to release CCL2 and matrix metalloproteinase-3. Additionally, CD40L expression is increased in BMMCs after incubation with MPP+ in a co-culture system consisting of BMMCs and glia/neurons. We propose that mast cell interaction with glial cells and neurons during neuroinflammation can be explored as a new therapeutic target for PD.

Neuroinflammatory response is primarily a protective mechanism in the brain. However, excessive and chronic inflammatory responses can lead to deleterious effects involving immune cells, brain cells and signaling molecules. Neuroinflammation induces and accelerates pathogenesis of Parkinson's disease (PD), Alzheimer's disease (AD) and Multiple sclerosis (MS). Neuroinflammatory pathways are indicated as novel therapeutic targets for these diseases. Mast cells are immune cells of hematopoietic origin that regulate inflammation and upon activation release many proinflammatory mediators in systemic and central nervous system (CNS) inflammatory conditions. In addition, inflammatory mediators released from activated glial cells induce neurodegeneration in the brain. Systemic inflammation-derived proinflammatory cytokines/chemokines and other factors cause a breach in the blood brain-barrier (BBB) thereby allowing for the entry of immune/inflammatory cells including mast cell progenitors, mast cells and proinflammatory cytokines and chemokines into the brain. These peripheral-derived factors and intrinsically generated cytokines/chemokines, α-synuclein, corticotropin-releasing hormone (CRH), substance P (SP), beta amyloid 1-42 (Aβ1-42) peptide and amyloid precursor proteins can activate glial cells, T-cells and mast cells in the brain can induce additional release of inflammatory and neurotoxic molecules contributing to chronic neuroinflammation and neuronal death. The glia maturation factor (GMF), a proinflammatory protein discovered in our laboratory released from glia, activates mast cells to release inflammatory cytokines and chemokines. Chronic increase in the proinflammatory mediators induces neurotoxic Aβ and plaque formation in AD brains and neurodegeneration in PD brains. Glial cells, mast cells and T-cells can reactivate each other in neuroinflammatory conditions in the brain and augment neuroinflammation. Further, inflammatory mediators from the brain can also enter into the peripheral system through defective BBB, recruit immune cells into the brain, and exacerbate neuroinflammation. We suggest that mast cell-associated inflammatory mediators from systemic inflammation and brain could augment neuroinflammation and neurodegeneration in the brain. This review article addresses the role of some atypical inflammatory mediators that are associated with mast cell inflammation and their activation of glial cells to induce neurodegeneration.