Multiple system atrophy (MSA) exhibits widespread astrogliosis together with α-synuclein (α-syn) glial cytoplasmic inclusions (GCIs) in mature oligodendrocytes. We quantified astrocyte activation by morphometric analysis of MSA cases, and investigated the correlation to GCI proximity. Using Imaris software, we obtained "skinned" three-dimensional models of GFAP-positive astrocytes in MSA and control tissue (n=75) from confocal z-stacks and measured the astrocyte process length and thickness and radial distance to the GCI. Astrocytes proximal to GCI-containing oligodendrocytes (r<25μm) had significantly (p, 0.05) longer and thicker processes characteristic of activation than distal astrocytes (r>25μm), with a reciprocal linear correlation (m, 90μm(2)) between mean process length and radial distance to the nearest GCI (R(2), 0.7). In primary cell culture studies, α-syn addition caused ERK-dependent activation of rat astrocytes and perinuclear α-syn inclusions in mature (MOSP-positive) rat oligodendrocytes. Activated astrocytes were also observed in close proximity to α-syn deposits in a unilateral rotenone-lesion mouse model. Moreover, unilateral injection of MSA tissue-derived α-syn into the mouse medial forebrain bundle resulted in widespread neuroinflammation in the α-syn-injected, but not sham-injected hemisphere. Taken together, our data suggests that the action of localized concentrations of α-syn may underlie both astrocyte and oligodendrocyte MSA pathological features.
Brain oedema is thought to form and to clear through the use of water-protein channels, aquaporin-4 (AQP4), which are found in the astrocyte endfeet. The model developed here is used to study the function of AQP4 in the formation and elimination of oedema fluid in ischaemia-reperfusion injury. The cerebral space is assumed to be made of four fluid compartments: astrocyte, neuron, ECS and blood microvessels, and a solid matrix for the tissue, and this is modelled using multiple-network poroelastic theory. AQP4 allows the movement of water between astrocyte and the ECS and the microvessels. It is found that the presence of AQP4 may help in reducing vasogenic oedema shown by a decrease in brain tissue extracellular pressure. However, the astrocyte pressure will increase to compensate for this decrease, which may lead to cytotoxic oedema. In addition, the swelling will also depend on the ionic concentrations in the astrocyte and extracellular space, which may change after ischaemic stroke. Understanding the role of AQP4 in oedema may thus help the development of a treatment plan in reducing brain swelling after ischaemia-reperfusion.
The exact etiology of Parkinson's disease (PD) remains obscure, lacking effective diagnostic and prognostic biomarkers. In search of novel molecular factors that may contribute to PD pathogenesis, emerging evidence highlights the multifunctional role of the calcium-binding protein S100B that is widely expressed in the brain and predominantly in astrocytes. Preclinical evidence points towards the possible time-specific contributing role of S100B in the pathogenesis of neurodegenerative disorders including PD, mainly by regulating neuroinflammation and dopamine metabolism. Although existing clinical evidence presents some contradictions, estimation of S100B in the serum and cerebrospinal fluid seems to hold a great promise as a potential PD biomarker, particularly regarding the severity of motor and non-motor PD symptoms. Furthermore, given the recent development of S100B inhibitors that are able to cross the blood brain barrier, novel opportunities are arising in the research field of PD therapeutics. In this review, we provide an update on recent advances in the implication of S100B protein in the pathogenesis of PD and discuss relevant studies investigating the biomarker potential of S100B in PD, aiming to shed more light on clinical targeting approaches related to this incurable disorder.
Cannabidiol (CBD) and cannabigerol (CBG) are Cannabis sativa terpenophenols. Although CBD's effectiveness against neurological diseases has already been demonstrated, nothing is known about CBG. Therefore, a comparison of the effects of these compounds was performed in two experimental models mimicking the oxidative stress and neurotoxicity occurring in neurological diseases. Rat astrocytes were exposed to hydrogen peroxide and cell viability, reactive oxygen species production and apoptosis occurrence were investigated. Cortexes were exposed to K+ 60 mM depolarizing stimulus and serotonin (5-HT) turnover, 3-hydroxykinurenine and kynurenic acid levels were measured. A proteomic analysis and bioinformatics and docking studies were performed. Both compounds exerted antioxidant effects in astrocytes and restored the cortex level of 5-HT depleted by neurotoxic stimuli, whereas sole CBD restored the basal levels of 3-hydroxykinurenine and kynurenic acid. CBG was less effective than CBD in restoring the levels of proteins involved in neurotransmitter exocytosis. Docking analyses predicted the inhibitory effects of these compounds towards the neurokinin B receptor. Conclusion: The results in the in vitro system suggest brain non-neuronal cells as a target in the treatment of oxidative conditions, whereas findings in the ex vivo system and docking analyses imply the potential roles of CBD and CBG as neuroprotective agents.
The past 20 years have resulted in unprecedented progress in understanding brain energy metabolism and its role in health and disease. In this review, which was initiated at the 14th International Society for Neurochemistry Advanced School, we address the basic concepts of brain energy metabolism and approach the question of why the brain has high energy expenditure. Our review illustrates that the vertebrate brain has a high need for energy because of the high number of neurons and the need to maintain a delicate interplay between energy metabolism, neurotransmission, and plasticity. Disturbances to the energetic balance, to mitochondria quality control or to glia-neuron metabolic interaction may lead to brain circuit malfunction or even severe disorders of the CNS. We cover neuronal energy consumption in neural transmission and basic ('housekeeping') cellular processes. Additionally, we describe the most common (glucose) and alternative sources of energy namely glutamate, lactate, ketone bodies, and medium chain fatty acids. We discuss the multifaceted role of non-neuronal cells in the transport of energy substrates from circulation (pericytes and astrocytes) and in the supply (astrocytes and microglia) and usage of different energy fuels. Finally, we address pathological consequences of disrupted energy homeostasis in the CNS.
Chlorpyrifos (CPF), an organophosphate pesticide inhibits acetylcholinesterase (AChE) and causes neuromuscular incoordination among children and elderly. The objectives of the present study were to compare the neurotoxic effects of dermal application of CPF on the cerebellum in the parameters of glial fibrillary acidic protein (GFAP) expression in young and adult mice and to correlate with the changes in acetylcholinesterase levels. Male Balb/c mice, 150 days old (adult) and 18 days old (young) were dermally applied with ½ LD(50) of CPF over the tails for 14 days. Serum AChE concentration was estimated and GFAP immunostaining was performed on sagittal paraffin sections through the vermis of cerebellum. Although reduced in both age-groups exposed to CPF, percentage of reduction in serum AChE was more in adult compared to the young. Under GFAP immunostaining, brown colour fibres and glial cells were observed in cerebellar cortex and medulla in both the experimental groups. The mean GFAP-positive glial cell count in cerebellar medulla per mm(2) of section was significantly (p
The blood-brain barrier (BBB) consists of endothelial cells, astrocytes, and pericytes embedded in basal lamina (BL). Most in vitro models use nonhuman, monolayer cultures for therapeutic-delivery studies, relying on transendothelial electrical resistance (TEER) measurements without other tight-junction (TJ) formation parameters. We aimed to develop reliable, reproducible, in vitro 3-dimensional (3D) models incorporating relevant human, in vivo cell types and BL proteins. The 3D BBB models were constructed with human brain endothelial cells, human astrocytes, and human brain pericytes in mono-, co-, and tricultures. TEER was measured in 3D models using a volt/ohmmeter and cellZscope. Influence of BL proteins-laminin, fibronectin, collagen type IV, agrin, and perlecan-on adhesion and TEER was assessed using an electric cell-substrate impedance-sensing system. TJ protein expression was assessed by Western blotting (WB) and immunocytochemistry (ICC). Perlecan (10 µg/ml) evoked unreportedly high, in vitro TEER values (1200 Ω) and the strongest adhesion. Coculturing endothelial cells with astrocytes yielded the greatest resistance over time. ICC and WB results correlated with resistance levels, with evidence of prominent occludin expression in cocultures. BL proteins exerted differential effects on TEER, whereas astrocytes in contact yielded higher TEER values and TJ expression.-Maherally, Z., Fillmore, H. L., Tan, S. L., Tan, S. F., Jassam, S. A., Quack, F. I., Hatherell, K. E., Pilkington, G. J. Real-time acquisition of transendothelial electrical resistance in an all-human, in vitro, 3-dimensional, blood-brain barrier model exemplifies tight-junction integrity.
Neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease, are becoming more prevalent and an increasing burden on society. Neurodegenerative diseases often arise in the milieu of neuro-inflammation of the brain. Reactive astrocytes are key regulators in the development of neuro-inflammation. This study describes the effects of Palm Fruit Bioactives (PFB) on the behavior of human astrocytes which have been activated by IL-1β. When activated, the astrocytes proliferate, release numerous cytokines/chemokines including TNFα, RANTES (CCL5), IP-10 (CXCL10), generate reactive oxygen species (ROS), and express specific cell surface biomarkers such as the Intercellular Adhesion Molecule (ICAM), Vascular Cellular Adhesion Molecule (VCAM) and the Neuronal Cellular Adhesion Molecule (NCAM). Interleukin 1-beta (IL-1β) causes activation of human astrocytes with marked upregulation of pro-inflammatory genes. We show significant inhibition of these pro-inflammatory processes when IL-1β-activated astrocytes are exposed to PFB. PFB causes a dose-dependent and time-dependent reduction in specific cytokines: TNFα, RANTES, and IP-10. We also show that PFB significantly reduces ROS production by IL-1β-activated astrocytes. Furthermore, PFB also reduces the expression of ICAM and VCAM, both in activated and naïve human astrocytes in vitro. Since reactive astrocytes play an essential role in the neuroinflammatory state preceding neurodegenerative diseases, this study suggests that PFB may have a potential role in their prevention and/or treatment.
Tocotrienol rich fraction (TRF) is an extract of palm oil, which consists of 25% alpha tocopherol (α-TCP) and 75% tocotrienols. TRF has been shown to possess potent antioxidant, anti-inflammatory, anticancer, neuroprotection, and cholesterol lowering activities. Glutamate is the main excitatory amino acid neurotransmitter in the central nervous system of mammalian, which can be excitotoxic, and it has been suggested to play a key role in neurodegenerative disorders like Parkinson's and Alzheimer's diseases. In this present study, the effects of vitamin E (TRF and α-TCP) in protecting astrocytes against glutamate injury were elucidated. Astrocytes induced with 180 mM of glutamate lead to significant cell death. However, glutamate mediated cytotoxicity was diminished via pre and post supplementation of TRF and α-TCP. Hence, vitamin E acted as a potent antioxidant agent in recovering mitochondrial injury due to elevated oxidative stress, and enhanced better survivability upon glutamate toxicity.
Cerebral hypoperfusion involved a reduction in cerebral blood flow, leading to neuronal dysfunction, microglial activation and white matter degeneration. The effects on the blood-brain barrier (BBB) however, have not been well-documented. Here, two-vessel occlusion model was adopted to mimic the condition of cerebral hypoperfusion in Sprague-Dawley rats. The BBB permeability to high and low molecular weight exogenous tracers i.e. Evans blue dye and sodium fluorescein respectively, showed marked extravasation of the Evans blue dye in the frontal cortex, posterior cortex and thalamus-midbrain at day 1 following induction of cerebral hypoperfusion. Transmission electron microscopy revealed brain endothelial cell and astrocyte damages including increased pinocytotic vesicles and formation of membrane invaginations in the endothelial cells, and swelling of the astrocytes' end-feet. Investigation on brain microvessel protein expressions using two-dimensional (2D) gel electrophoresis coupled with LC-MS/MS showed that proteins involved in mitochondrial energy metabolism, transcription regulation, cytoskeleton maintenance and signaling pathways were differently expressed. The expression of aconitate hydratase, heterogeneous nuclear ribonucleoprotein, enoyl Co-A hydratase and beta-synuclein were downregulated, while the opposite observed for calreticulin and enhancer of rudimentary homolog. These findings provide insights into the BBB molecular responses to cerebral hypoperfusion, which may assist development of future therapeutic strategies.
The present study aimed to investigate the involvement of the angiogenic marker vascular endothelia growth factor (VEGF) and apoptotic markers of Bcl-2 and Bax in the neurons and astrocytes in the brain infected by Mycobacterium tuberculosis. The immunohistochemistry staining was performed to analyze the expression of the VEGF, Bcl-2 and Bax in the astrocytes and neurons. The expression of VEGF was high in neurons and astrocytes in both the infected brain and control tissues with no difference of angiogenic activity (p = 0.40). Higher Bcl-2 expression was seen in astrocytes of infected brain tissues compared to the control tissues (p = 0.004) promoted a higher anti-apoptotic activity in astrocytes. The neurons expressed strong Bax expression in the infected brain tissues compared to the control tissues (p
Neurological diseases particularly Alzheimer's disease (AD), Parkinson's disease (PD), stroke, and epilepsy are on the rise all around the world causing morbidity and mortality globally with a common symptom of gradual loss or impairment of motor behaviour. Striatum, which is a component of the basal ganglia, is involved in facilitating voluntary movement while the cerebellum is involved in the maintenance of balance and coordination of voluntary movements. Dopamine, serotonin, gamma-aminobutyric acid (GABA), and glutamate, to name a few, interact in regulating the excitation and inhibition of motor neurons. In another hand, interestingly, the motor loss associated with neurological diseases is possibly resulted from neuroinflammation induced by the neuroimmune system. Toll-like receptors (TLRs) are present in the central nervous system (CNS), specifically and primarily expressed in microglia and are also found on neurons and astrocytes, functioning mainly in the regulation of proinflammatory cytokine production. TLRs are always found to be associated or involved in the induction of neuroinflammation in neurodegenerative diseases. Activation of toll-like receptor 4 (TLR4) through TLR4 agonist, lipopolysaccharide (LPS), stimulation initiate a signaling cascade whereby the TLR4-LPS interaction has been found to result in physiological and behavioural changes including retardation of motor activity in the mouse model. TLR4 inhibitor TAK-242 was reflected in the reduction of the spinal cord pathology along with the motor improvement in ALS mouse. There is cross talk with neuroinflammation and neurochemicals. For example, TLR4 activation by LPS is noted to release proinflammatory cytokines, IL-1β, from microglia that subsequently suppresses GABA receptor activities at the postsynaptic site and reduces GABA synthesis at the presynaptic site. Glial glutamate transporter activities are also found to be suppressed, showing the association between TLR4 activation and the related neurotransmitters and corresponding receptors and transporters in the event of neuroinflammation. This review is helpful to understand the connection between neurotransmitter and neuroinflammation in striatum- and cerebellum-mediated motor behaviour.
The malignant primary brain tumor, glioblastoma (GBM) is generally incurable. New approaches are desperately needed. Adeno-associated virus (AAV) vector-mediated delivery of anti-tumor transgenes is a promising strategy, however direct injection leads to focal transgene spread in tumor and rapid tumor division dilutes out the extra-chromosomal AAV genome, limiting duration of transgene expression. Intravenous (IV) injection gives widespread distribution of AAV in normal brain, however poor transgene expression in tumor, and high expression in non-target cells which may lead to ineffective therapy and high toxicity, respectively. Delivery of transgenes encoding secreted, anti-tumor proteins to tumor stromal cells may provide a more stable and localized reservoir of therapy as they are more differentiated than fast-dividing tumor cells. Reactive astrocytes and tumor-associated macrophage/microglia (TAMs) are stromal cells that comprise a large portion of the tumor mass and are associated with tumorigenesis. In mouse models of GBM, we used IV delivery of exosome-associated AAV vectors driving green fluorescent protein expression by specific promoters (NF-κB-responsive promoter and a truncated glial fibrillary acidic protein promoter), to obtain targeted transduction of TAMs and reactive astrocytes, respectively, while avoiding transgene expression in the periphery. We used our approach to express the potent, yet toxic anti-tumor cytokine, interferon beta, in tumor stroma of a mouse model of GBM, and achieved a modest, yet significant enhancement in survival compared to controls. Noninvasive genetic modification of tumor microenvironment represents a promising approach for therapy against cancers. Additionally, the vectors described here may facilitate basic research in the study of tumor stromal cells in situ.
Excessive exposure to toxic substances or chemicals in the environment and various pathogens, including viruses and bacteria, is associated with the onset of numerous brain abnormalities. Among them, pathogens, specifically viruses, elicit persistent inflammation that plays a major role in Alzheimer's disease (AD) as well as dementia. AD is the most common brain disorder that affects thought, speech, memory and ability to execute daily routines. It is also manifested by progressive synaptic impairment and neurodegeneration, which eventually leads to dementia following the accumulation of Aβ and hyperphosphorylated Tau. Numerous factors contribute to the pathogenesis of AD, including neuroinflammation associated with pathogens, and specifically viruses. The human immunodeficiency virus (HIV) is often linked with HIV-associated neurocognitive disorders (HAND) following permeation through the blood-brain barrier (BBB) and induction of persistent neuroinflammation. Further, HIV infections also exhibited the ability to modulate numerous AD-associated factors such as BBB regulators, members of stress-related pathways as well as the amyloid and Tau pathways that lead to the formation of amyloid plaques or neurofibrillary tangles accumulation. Studies regarding the role of HIV in HAND and AD are still in infancy, and potential link or mechanism between both is not yet established. Thus, in the present article, we attempt to discuss various molecular mechanisms that contribute to the basic understanding of the role of HIV-associated neuroinflammation in AD and HAND. Further, using numerous growth factors and drugs, we also present possible therapeutic strategies to curb the neuroinflammatory changes and its associated sequels.
Hyperhomocysteinemia (HHcy) is a well-known risk factor for stroke; however, its underlying molecular mechanism remains unclear. Using both mouse and cell culture models, we have provided evidence that impairment of autophagy has a central role in HHcy-induced cellular injury in the mouse brain. We observed accumulation of LC3B-II and p62 that was associated with increased MTOR signaling in human and mouse primary astrocyte cell cultures as well as a diet-induced mouse model of HHcy, HHcy decreased lysosomal membrane protein LAMP2, vacuolar ATPase (ATP6V0A2), and protease cathepsin D, suggesting that lysosomal dysfunction also contributed to the autophagic defect. Moreover, HHcy increased unfolded protein response. Interestingly, Vitamin B supplementation restored autophagic flux, alleviated ER stress, and reversed lysosomal dysfunction due to HHCy. Furthermore, the autophagy inducer, rapamycin was able to relieve ER stress and reverse lysosomal dysfunction caused by HHcy in vitro. Inhibition of autophagy by HHcy exacerbated cellular injury during oxygen and glucose deprivation and reperfusion (OGD/R), and oxidative stress. These effects were prevented by Vitamin B co-treatment, suggesting that it may be helpful in relieving detrimental effects of HHcy in ischemia/reperfusion or oxidative stress. Collectively, these findings show that Vitamin B therapy can reverse defects in cellular autophagy and ER stress due to HHcy; and thus may be a potential treatment to reduce ischemic damage caused by stroke in patients with HHcy.
Hand, foot and mouth disease (HFMD) caused by Human Enterovirus A71 (HEVA71) infection is typically a benign infection. However, in minority of cases, children can develop severe neuropathology that culminate in fatality. Approximately 36.9% of HEVA71-related hospitalizations develop neurological complications, of which 10.5% are fatal. Yet, the mechanism by which HEVA71 induces these neurological deficits remain unclear. Here, we show that HEVA71-infected astrocytes release CXCL1 which supports viral replication in neurons by activating the CXCR2 receptor-associated ERK1/2 signaling pathway. Elevated CXCL1 levels correlates with disease severity in a HEVA71-infected mice model. In humans infected with HEVA71, high CXCL1 levels are only present in patients presenting neurological complications. CXCL1 release is specifically triggered by VP4 synthesis in HEVA71-infected astrocytes, which then acts via its receptor CXCR2 to enhance viral replication in neurons. Perturbing CXCL1 signaling or VP4 myristylation strongly attenuates viral replication. Treatment with AZD5069, a CXCL1-specific competitor, improves survival and lessens disease severity in infected animals. Collectively, these results highlight the CXCL1-CXCR2 signaling pathway as a potential target against HFMD neuropathogenesis.
Ruxolitinib is the first janus kinase 1 (JAK1) and JAK2 inhibitor that was approved by the United States Food and Drug Administration (FDA) agency for the treatment of myeloproliferative neoplasms. The drug targets the JAK/STAT signalling pathway, which is critical in regulating the gliogenesis process during nervous system development. In the study, we assessed the effect of non-maternal toxic dosages of ruxolitinib (0-30 mg/kg/day between E7.5-E20.5) on the brain of the developing mouse embryos. While the pregnant mice did not show any apparent adverse effects, the Gfap protein marker for glial cells and S100β mRNA marker for astrocytes were reduced in the postnatal day (P) 1.5 pups' brains. Gfap expression and Gfap+ cells were also suppressed in the differentiating neurospheres culture treated with ruxolitinib. Compared to the control group, adult mice treated with ruxolitinib prenatally showed no changes in motor coordination, locomotor function, and recognition memory. However, increased explorative behaviour within an open field and improved spatial learning and long-term memory retention were observed in the treated group. We demonstrated transplacental effects of ruxolitinib on astrogenesis, suggesting the potential use of ruxolitinib to revert pathological conditions caused by gliogenic-shift in early brain development such as Down and Noonan syndromes.
Oxidative stress is thought to be one of the factors that cause neurodegeneration and that this can be inhibited by antioxidants. Since astrocytes support the survival of central nervous system (CNS) neurons, we compared the effect of alpha-tocopherol and gamma-tocotrienol in minimizing the cytotoxic damage induced by H(2)O(2), a pro-oxidant. Primary astrocyte cultures were pretreated with either alpha-tocopherol or gamma-tocotrienol for 1 h before incubation with 100 microM H(2)O(2) for 24 h. Cell viability was then assessed using the MTS assay while apoptosis was determined using a commercial ELISA kit as well as by fluorescent staining of live and apoptotic cells. The uptake of alpha-tocopherol and gamma-tocotrienol by astrocytes were also determined using HPLC. Results showed that gamma-tocotrienol is toxic at concentrations >200 microM but protects against H(2)O(2) induced cell loss and apoptosis in a dose dependent manner up to 100 microM. alpha-Tocopherol was not cytotoxic in the concentration range tested (up to 750 microM), reduced apoptosis to the same degree as that of gamma-tocotrienol but was less effective in maintaining the viable cell number. Since the uptake of alpha-tocopherol and gamma-tocotrienol by astrocytes is similar, this may reflect the roles of these 2 vitamin E subfamilies in inhibiting apoptosis and stimulating proliferation in astrocytes.