Low-dose lipopolysaccharide as an immune regulator for homeostasis maintenance in the central nervous system through transformation to neuroprotective microglia

Microglia,which are tissue-resident macrophages in the brain,play a central role in the brain innate immunity and contribute to the maintenance of brain homeostasis.Lipopolysaccharide is a component of the outer membrane of gram-negative bacteria,and activates immune cells including microglia via Toll-like receptor 4 signaling.Lipopolysaccharide is generally known as an endotoxin,as administration of highdose lipopolysaccharide induces potent systemic inflammation.Also,it has long been recognized that lipopolysaccharide exacerbates neuroinflammation.In contrast,our study revealed that oral administration of lipopolysaccharide ameliorates Alzheimer’s disease pathology and suggested that neuroprotective microglia are involved in this phenomenon.Additionally,other recent studies have accumulated evidence demonstrating that controlled immune training with low-dose lipopolysaccharide prevents neuronal damage by transforming the microglia into a neuroprotective phenotype.Therefore,lipopolysaccharide may not a mere inflammatory inducer,but an immunomodulator that can lead to neuroprotective effects in the brain.In this review,we summarized current studies regarding neuroprotective microglia transformed by immune training with lipopolysaccharide.We state that microglia transformed by lipopolysaccharide preconditioning cannot simply be characterized by their general suppression of proinflammatory mediators and general promotion of anti-inflammatory mediators,but instead must be described by their complex profile comprising various molecules related to inflammatory regulation,phagocytosis,neuroprotection,anti-apoptosis,and antioxidation.In addition,microglial transformation seems to depend on the dose of lipopolysaccharide used during immune training.Immune training of neuroprotective microglia using lowdose lipopolysaccharide,especially through oral lipopolysaccharide administration,may represent an innovative prevention or treatment for neurological diseases;however more vigorous studies are still required to properly modulate these treatments.     

Shifting equilibriums in Alzheimer's disease: the complex roles of microglia in neuroinflammation,neuronal survival and neurogenesis

Alzheimer's disease is the leading cause of dementia.Its increased prevalence in developed countries,due to the sharp rise in ageing populations,presents one of the costliest challenges to modern medicine.In order to find disease-modifying therapies to confront this challenge,a more complete understanding of the pathogenesis of Alzheimer's disease is necessary.Recent studies have revealed increasing evidence for the roles played by microglia,the resident innate immune system cells of the brain.Reflecting the well-established roles of microglia in reacting to pathogens and inflammatory stimuli,there is now a growing literature describing both protective and detrimental effects for individual cytokines and chemokines produced by microglia in Alzheimer's disease.A smaller but increasing number of studies have also addressed the divergent roles played by microglial neurotrophic and neurogenic factors,and how their perturbation may play a key role in the pathogenesis of Alzheimer's disease.Here we review recent findings on the roles played by microglia in neuroinflammation,neuronal survival and neurogenesis in Alzheimer's disease.In each case,landmark studies have provided evidence for the divergent ways in which microglia can either promote neuronal function and survival,or perturb neuronal function,leading to cell death.In many cases,the secreted molecules of microglia can lead to divergent effects depending on the magnitude and context of microglial activation.This suggests that microglial functions must be maintained in a fine equilibrium,in order to support healthy neuronal function,and that the cellular microenvironment in the Alzheimer's disease brain disrupts this fine balance,leading to neurodegeneration.Thus,an understanding of microglial homeostasis,both in health and across the trajectory of the disease state,will improve our understanding of the pathogenic mechanisms underlying Alzheimer's disease,and will hopefully lead to the development of microglial-based therapeutic strategies to restore equilibrium in the Alzheimer's disease brain.     

Targeting the P2X7 receptor in microglial cells to prevent brain inflammation

Microglial cells are the key innate immune cells in the brain and they are crucial in maintaining brain parenchyma homeostasis.Under physiological conditions,microglial cells assume a ramified morphology with a small cell body and an extensive network of fine processes,which secrete neurotrophic factors and patrol the surroundings in search for pathogens and eliminate cellular debris via phagocytosis.Microglial cells express a repertoire of pattern recognition receptors(PRRs)that enable them to detect diverse danger-associated molecular patterns(DAMPs)released from damaged cells or cells under stress,or pathogen-associated molecular patterns generated by pathogens during infection.     

CD93 and GIPC expression and localization during central nervous system inlfammation

CD93 and GAIP-interacting protein, C termius （GIPC） have been shown to interactively alter phagocytic processes of immune cells. CD93 and GIPC expression and localization during cen-tral nervous system inflammation have not yet been reported. In this study, we established a rat model of brain inlfammation by lipopolysaccharide injection to the lateral ventricle. In the brain of rats with inlfammation, western blots showed increased CD93 expression that decreased over time. GIPC expression was unaltered. Immunohistochemistry demonstrated extensive distribution of CD93 expression mainly in cell membranes in the cerebral cortex. After lipopoly-saccharide stimulation, CD93 expression increased and then reduced, with distinct staining in the cytoplasm and nucleus. Double immunolfuorescence staining in cerebral cortex of normal rats showed that CD93 and GIPC widely expressed in resting microglia and neurons. CD93 was mainly expressed in microglial and neuronal cell membranes, while GIPC was expressed in both cell membrane and cytoplasm. In the cerebral cortex at 9 hours after model establishment, CD93-immunoreactive signal diminished in microglial membrane, with cytoplasmic transloca-tion and aggregation detected. GIPC localization was unaltered in neurons and microglia. These results are the ifrst to demonstrate CD93 participation in pathophysiological processes of central nervous system inlfammation.     

Non-coding RNAs and other determinants of neuroinflammation and endothelial dysfunction: regulation of gene expression in the acute phase of ischemic stroke and possible therapeutic applications

Ischemic stroke occurs under a variety of clinical conditions and has different pathogeneses,resulting in necrosis of brain parenchyma.Stroke pathogenesis is characterized by neuroinflammation and endothelial dysfunction.Some of the main processes triggered in the early stages of ischemic damage are the rapid activation of resident inflammatory cells(microglia,astrocytes and endothelial cells),inflammatory cytokines,and translocation of intercellular nuclear factors.Inflammation in stroke includes all the processes mentioned above,and it consists of either protective or detrimental effects concerning the“polarization”of these processes.This polarization comes out from the interaction of all the molecular pathways that regulate genome expression:the epigenetic factors.In recent years,new regulation mechanisms have been cleared,and these include non-coding RNAs,adenosine receptors,and the activity of mesenchymal stem/stromal cells and microglia.We reviewed how long non-coding RNA and microRNA have emerged as an essential mediator of some neurological diseases.We also clarified that their roles in cerebral ischemic injury may provide novel targets for the treatment of ischemic stroke.To date,we do not have adequate tools to control pathophysiological processes associated with stroke.Our goal is to review the role of non-coding RNAs and innate immune cells(such as microglia and mesenchymal stem/stromal cells)and the possible therapeutic effects of their modulation in patients with acute ischemic stroke.A better understanding of the mechanisms that influence the“polarization”of the inflammatory response after the acute event seems to be the way to change the natural history of the disease.     

Astrocyte in prion disease: a double-edged sword

Prion diseases are infectious protein misfolding disorders of the central nervous system that result from misfolding of the cellular prion protein(PrPC)into the pathologic isoform PrPSc.Pathologic hallmarks of prion disease are depositions of pathological prion protein PrPSc,neuronal loss,spongiform degeneration and astrogliosis in the brain.Prion diseases affect human and animals,there is no effective therapy,and they invariably remain fatal.For a long time,neuronal loss was considered the sole reason for neurodegeneration in prion pathogenesis,and the contribution of non-neuronal cells like microglia and astrocytes was considered less important.Recent evidence suggests that neurodegeneration during prion pathogenesis is a consequence of a complex interplay between neuronal and non-neuronal cells in the brain,but the exact role of these non-neuronal cells during prion pathology is still elusive.Astrocytes are non-neuronal cells that regulate brain homeostasis under physiological conditions.However,astrocytes can deposit PrPSc aggregates and propagate prions in prion-infected brains.Additionally,sub-populations of reactive astrocytes that include neurotrophic and neurotoxic species have been identified,differentially expressed in the brain during prion infection.Revealing the exact role of astrocytes in prion disease is hampered by the lack of in vitro models of prion-infected astrocytes.Recently,we established a murine astrocyte cell line persistently infected with mouse-adapted prions,and showed how such astrocytes differentially process various prion strains.Considering the complexity of the role of astrocytes in prion pathogenesis,we need more in vitro and in vivo models for exploring the contribution of sub-populations of reactive astrocytes,their differential regulation of signaling cascades,and the interaction with neurons and microglia during prion pathogenesis.This will help to establish novel in vivo models and define new therapeutic targets against prion diseases.In this review,we will discuss the complex role of astrocytes in prion disease,the existing experimental resources,the challenges to analyze the contribution of astrocytes in prion disease pathogenesis,and future strategies to improve the understanding of their role in prion disease.     

Do phagocytotic mechanisms regulate soluble factor secretion in microglia?

正Microglia are responsible for phagocytosis in the brain:Phagocytosis, one of the major mechanisms of innate immune defense, is the process by which several types of cells in the immune system recognize, engulf, and digest large particles, such as pathogens and cell debris. In the brain, microglia play phagocytotic ro les to regulate the micro-enviro nment of brains under both physiological and pathological conditions. For example, during development, microglia help develop functional synaptic connections by pruning excessively produced synapses.     

Expression of NG2 and platelet-derived growth facto receptor alpha in the developing neonatal rat brain

Platelet-derived growth factor receptor alpha(PDGFRα) is a marker of oligodendrocyte precursor cells in the central nervous system. NG2 is also considered a marker of oligodendrocyte precursor cells. However, whether there are differences in the distribution and morphology of oligodendrocyte precursor cells labeled by NG2 or PDGFRα in the developing neonatal rat brain remains unclear. In this study, by immunohistochemical staining, NG2 positive(NG2~+) cells were ubiquitous in the molecular layer, external pyramidal layer, internal pyramidal layer, and polymorphic layer of the cerebral cortex, and corpus callosum, external capsule, piriform cortex, and medial septal nucleus. NG2~+ cells were stellate or fusiform in shape with long processes that were progressively decreased and shortened over the course of brain development. The distribution and morphology of PDGFRα positive(PDGFRα~+) cells were coincident with NG2~+ cells. The colocalization of NG2 and PDGFRα in the cell bodies and processes of some cells was confirmed by double immunofluorescence labeling. Moreover, cells double-labeled for NG2 and PDGFRα were predominantly in the early postnatal stage of development. The numbers of NG2~+/PDGFRα~+ cells and PDGFRα~+ cells decreased, but the number of NG2~+ cells increased from postnatal days 3 to 14 in the developing brain. In addition, amoeboid microglial cells of the corpus callosum, newborn brain macrophages in the normal developing brain, did not express NG2 or PDGFRα, but NG2 expression was detected in amoeboid microglia after hypoxia. The present results suggest that NG2 and PDGFRα are specific markers of oligodendrocyte precursor cells at different stages during early development. Additionally, the NG2 protein is involved in inflammatory and pathological processes of amoeboid microglial cells.     

Modulation of microglial functions by methyl jasmonate

Neuroinflammation contributes to the neurodegenerative processes in Alzheimer's disease(AD);therefore,characterization of novel drug candidates aimed at combatting inflammation in the central nervous system is one of the potential avenues for the development of effective AD treatment and prevention strategies.Non-neuronal microglial cells orchestrate neuroinflammatory reactions,and their adverse activation has been linked to AD pathogenesis.Methyl jasmonate(MJ) has anti-cancer properties and has also been shown to reduce peripheral inflammation in pre-clinical models.Recently,anti-neuroinflammatory activity of MJ was demonstrated in mice,but the exact cellular and molecular mechanisms responsible for this beneficial effect are unknown.We hypothesized that MJ can regulate select microglial functions,and used two different in vitro models of microglia to test this hypothesis.MJ inhibited the production of damaging reactive oxygen species by differentiated human HL-60 promyelocytic leukemia cells without reducing their viability.MJ also selectively upregulated phagocytic activity of murine BV-2 microglia,but had no effect on nitric oxide secretion by these cells.Since microglial phagocytosis can be beneficial for clearance of amyloid β aggregates in AD,the observed upregulation of phagocytic activity by MJ,combined with its inhibitory effect on reactive oxygen species production,supports continued studies of MJ as a candidate drug for managing adverse neuroinflammation in AD.     

Targeting chronic and evolving neuroinflammation following traumatic brain injury to improve long-term outcomes:insights from microglial-depletion models

正Microglia, the resident innate immune cells of the central nervous system (CNS), play important roles in brain development, maintenance, and disease. As brain sentinels, microglia adopt a surveillant state in healthy tissue characterized by a ramified scanning morphology that maintains CNS homeostasis and contributes to learningassociated synaptic plasticity. Following acute CNS injury or during chronic disease,     

Don’t know what you got till it’s gone:microglial depletion and neurodegeneration

In the central nervous system,immunologic surveillance and response are carried out,in large part,by microglia.These resident macrophages derive from myeloid precursors in the embryonic yolk sac,migrating to the brain and eventually populating local tissue prior to blood-brain barrier formation.Preserved for the duration of lifespan,microglia serve the host as more than just a central arm of innate immunity,also contributing significantly to the development and maintenance of neurons and neural networks,as well as neuroregeneration.The critical nature of these varied functions makes the characterization of key roles played by microglia in neurodegenerative disorders,especially Alzheimer’s disease,of paramount importance.While genetic models and rudimentary pharmacologic approaches for microglial manipulation have greatly improved our understanding of central nervous system health and disease,significant advances in the selective and near complete in vitro and in vivo depletion of microglia for neuroscience application continue to push the boundaries of research.Here we discuss the research efficacy and utility of various microglial depletion strategies,including the highly effective CSF1R inhibitor models,noteworthy insights into the relationship between microglia and neurodegeneration,and the potential for therapeutic repurposing of microglial depletion and repopulation.     

Microglial voltage-gated proton channel Hv1 in spinal cord injury

After spinal cord injury,microglia as the first responders to the lesion display both beneficial and detrimental characteristics.Activated microglia phagocyte and eliminate cell debris,release cytokines to recruit peripheral immune cells to the injury site.Excessively activated microglia can aggravate the secondary damage by producing extravagant reactive oxygen species and pro-inflammatory cytokines.Recent studies demonstrated that the voltage-gated proton channel Hv1 is selectively expressed in microglia and regulates microglial activation upon injury.In mouse models of spinal cord injury,Hv1 deficiency ameliorates microglia activation,resulting in alleviated production of reactive oxygen species and pro-inflammatory cytokines.The reduced secondary damage subsequently decreases neuronal loss and correlates with improved locomotor recovery.This review provides a brief historical perspective of advances in investigating voltage-gated proton channel Hv1 and home in on microglial Hv1.We discuss recent studies on the roles of Hv1 activation in pathophysiological activities of microglia,such as production of NOX-dependent reactive oxygen species,microglia polarization,and tissue acidosis,particularly in the context of spinal cord injury.Further,we highlight the rationale for targeting Hv1 for the treatment of spinal cord injury and related disorders.     

Microinjection of a growth factor cocktail affects activated microglia in the neocortex of adult rats

Microglia, as the resident immune cells in the central nervous system, play important roles in regulating neuronal processes, such as neural excitability, synaptic activity, and apoptotic cell clearance. Growth factors can activate multiple signaling pathways in central nervous system microglia and can regulate their immune effects, but whether growth factors can affect the morphological characteristics and ultrastructure of microglia has not been reported. After microinjecting 300 nL of a growth factor cocktail, including 10 μg/mL epidermal growth factor, 10 μg/mL basic fibroblast growth factor, 10 μg/mL hepatocyte growth factor and 10 μg/mL insulin-like growth factor into adult rat cortex, we found that the number of IBA1-positive microglia around the injection area increased significantly, indicating local activation of microglia. All CD68-positive labeling co-localized with IBA1 in microglia. Cell bodies and protrusions of CD68-positive cells were strongly attached to or were engulfing neurons. Characteristic huge phagosomes were observed in activated phagocytes by electron microscopy. The phagosomes generally included non-degraded neuronal protrusions and mitochondria, yet they contained no myelin membrane or remnants, which might indicate selective phagocytosis by the phagocytes. The remnant myelin sheath after phagocytosis still had regenerative ability and formed "myelin-like" structures around phagocytes. These results show that microinjection of a growth factor cocktail into the cerebral cortex of rodents can locally activate microglia and induce selective phagocytosis of neural structures by phagocytes. The study was approved by the Institute of Laboratory Animal Science, Beijing Institute of Basic Medical Sciences(approval No. IACUC-AMMS-2014-501) on June 30, 2014.     

Using induced pluripotent stem cells derived neurons to model brain diseases

The ability to use induced pluripotent stem cells(i PSC)to model brain diseases is a powerful tool for unraveling mechanistic alterations in these disorders.Rodent models of brain diseases have spurred understanding of pathology but the concern arises that they may not recapitulate the full spectrum of neuron disruptions associated with human neuropathology.iPSC derived neurons,or other neural cell types,provide the ability to access pathology in cells derived directly from a patient’s blood sample or skin biopsy where availability of brain tissue is limiting.Thus,utilization of iPSC to study brain diseases provides an unlimited resource for disease modelling but may also be used for drug screening for effective therapies and may potentially be used to regenerate aged or damaged cells in the future.Many brain diseases across the spectrum of neurodevelopment,neurodegenerative and neuropsychiatric are being approached by iPSC models.The goal of an iPSC based disease model is to identify a cellular phenotype that discriminates the disease-bearing cells from the control cells.In this mini-review,the importance of iPSC cell models validated for pluripotency,germline competency and function assessments is discussed.Selected examples for the variety of brain diseases that are being approached by iPSC technology to discover or establish the molecular basis of the neuropathology are discussed.     

Cell proliferation and apoptosis in optic nerve and brain integration centers of adult troutOncorhynchus mykiss after optic nerve injury

Fishes have remarkable ability to effectively rebuild the structure of nerve cells and nerve fibers after central nervous system injury.However,the underlying mechanism is poorly understood.In order to address this issue,we investigated the proliferation and apoptosis of cells in contralateral and ipsilateral optic nerves,after stab wound injury to the eye of an adult trout Oncorhynchus mykiss.Heterogenous population of proliferating cells was investigated at 1 week after injury.TUNEL labeling gave a qualitative and quantitative assessment of apoptosis in the cells of optic nerve of trout 2 days after injury.After optic nerve injury,apoptotic response was investigated,and mass patterns of cell migration were found.The maximal concentration of apoptotic bodies was detected in the areas of mass clumps of cells.It is probably indicative of massive cell death in the area of high phagocytic activity of macrophages/microglia.At 1 week after optic nerve injury,we observed nerve cell proliferation in the trout brain integration centers:the cerebellum and the optic tectum.In the optic tectum,proliferating cell nuclear antigen(PCNA)-immunopositive radial glia-like cells were identified.Proliferative activity of nerve cells was detected in the dorsal proliferative(matrix) area of the cerebellum and in parenchymal cells of the molecular and granular layers whereas local clusters of undifferentiated cells which formed neurogenic niches were observed in both the optic tectum and cerebellum after optic nerve injury.In vitro analysis of brain cells of trout showed that suspension cells compared with monolayer cells retain higher proliferative activity,as evidenced by PCNA immunolabeling.Phase contrast observation showed mitosis in individual cells and the formation of neurospheres which gradually increased during 1–4 days of culture.The present findings suggest that trout can be used as a novel model for studying neuronal regeneration.     

Nigral dopaminergic neuron replenishment in adult mice through VE-cadherin-expressing neural progenitor cells

The function of dopaminergic neurons in the substantia nigra is of central importance to the coordination of movement by the brain’s basal ganglia circuitry.This is evidenced by the loss of these neurons,resulting in the cardinal motor deficits associated with Parkinson’s disease.In order to fully understand the physiology of these key neurons and develop potential therapies for their loss,it is essential to determine if and how dopaminergic neurons are replenished in the adult brain.Recent work has presented evidence for adult neurogenesis of these neurons by Nestin~+/Sox2~–neural progenitor cells.We sought to further validate this finding and explore a potential atypical origin for these progenitor cells.Since neural progenitor cells have a proximal association with the vasculature of the brain and subsets of endothelial cells are Nestin~+,we hypothesized that dopaminergic neural progenitors might share a common cell lineage.Therefore,we employed a VE-cadherin promoter-driven CRE~(ERT2):TH~(lox)/TH~(lox) transgenic mouse line to ablate the tyrosine hydroxylase gene from endothelial cells in adult animals.After 26 weeks,but not 13 weeks,following the genetic blockade of tyrosine hydroxylase expression in VE-cadherin~+cells,we observed a significant reduction in tyrosine hydroxylase~+neurons in the substantia nigra.The results from this genetic lineage tracing study suggest that dopaminergic neurons are replenished in adult mice by a VE-cadherin~+progenitor cell population potentially arising from an endothelial lineage.