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.
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.
Biomaterials provide novel platforms to deliver stem cell and growth factor therapies for central nervous system (CNS) repair. The majority of these approaches have focused on the promotion of neural progenitor cells and neurogenesis. However, it is now increasingly recognized that glial responses are critical for recovery in the entire neurovascular unit. In this study, we investigated the cellular effects of epidermal growth factor (EGF) containing hydrogels on primary astrocyte cultures. Both EGF alone and EGF-hydrogel equally promoted astrocyte proliferation, but EGF-hydrogels further enhanced astrocyte activation, as evidenced by a significantly elevated Glial fibrillary acidic protein (GFAP) gene expression. Thereafter, conditioned media from astrocytes activated by EGF-hydrogel protected neurons against injury and promoted synaptic plasticity after oxygen-glucose deprivation. Taken together, these findings suggest that EGF-hydrogels can shift astrocytes into neuro-supportive phenotypes. Consistent with this idea, quantitative-polymerase chain reaction (qPCR) demonstrated that EGF-hydrogels shifted astrocytes in part by downregulating potentially negative A1-like genes (Fbln5 and Rt1-S3) and upregulating potentially beneficial A2-like genes (Clcf1, Tgm1, and Ptgs2). Further studies are warranted to explore the idea of using biomaterials to modify astrocyte behavior and thus indirectly augment neuroprotection and neuroplasticity in the context of stem cell and growth factor therapies for the CNS. Stem Cells Translational Medicine 2019;8:1242&1248.
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
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.
Increased Connexin43 hemichannel opening is associated with inflammasome pathway activation and inflammation in a range of pathologies including ocular disorders, such as age-related macular degeneration (AMD) and diabetic retinopathy (DR). In this study, the effect on retinal function and morphology of clinically safe doses of orally delivered tonabersat, a small molecule connexin hemichannel blocker, was investigated in the light-damaged retina animal model of dry AMD and in a spontaneous rat model of DR. Clinical parameters (fundus imaging, optical coherence tomography (OCT), and electroretinography) and inflammatory markers (immunohistochemistry for Iba-1 microglial marker, astrocyte marker glial fibrillary acidic protein, and Connexin43 protein expression) were assessed. Tonabersat treatment reduced inflammation in the retina in parallel with preservation of retinal photoreceptor function when assessed up to 3 months post light damage in the dry AMD model. In the DR model, clinical signs, including the presence of aneurysms confirmed using Evans blue dye perfusion, were reduced after daily tonabersat treatment for 2 weeks. Inflammation was also reduced and retinal electrical function restored. Tonabersat regulates assembly of the inflammasome (NLRP3) through Connexin43 hemichannel block, with the potential to reduce inflammation, restore vascular integrity and improve anatomical along with some functional outcomes in retinal disease.
Oleocanthal is a minor constituent of olive oil with strong anti-inflammatory activities. Since the pathogenesis of many chronic diseases involves inflammatory and oxidative components, oleocanthal is a promising agent to prevent these conditions. This review aimed to summarise the current beneficial health effects of oleocanthal and the molecular basis of its biological actions. The anti-inflammatory, antioxidative, antimicrobial, anticancer and neuroprotective activities of oleocanthal have been examined by previous studies. Of these, studies on the anticancer effects have been the most extensive. Oleocanthal was reported to suppress melanoma, breast, liver, and colon cancer cells. Neurological studies focused on the effects of oleocanthal against Alzheimer’s disease. Oleocanthal improved clearance of the amyloid beta protein from neurons and reduced the inflammation of astrocytes. Despite the positive results, validation of the biological effects of oleocanthal in animal disease models is limited and should be emphasized in the future. As a conclusion, oleocanthal may act together with other bioactive compounds in olive oil to achieve its therapeutic potential. The use of oleocanthal alone as a single therapeutic measure awaits validation from future studies.
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.
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.