MAB360 Sigma-AldrichAnti-Glial Fibrillary Acidic Protein Antibody, clone GA5

Radial glial cells (RGCs) are abundant stem-like non-neuronal progenitors that are important for adult neurogenesis and brain repair, yet little is known about their regulation by neurotransmitters. Here we provide evidence for neuronal-glial interactions via a novel role for dopamine to stimulate RGC function. Goldfish were chosen as the model organism due to the abundance of RGCs and regenerative abilities of the adult central nervous system. A close anatomical relationship was observed between tyrosine hydroxylase-positive catecholaminergic cell bodies and axons and dopamine-D1 receptor expressing RGCs along the ventricular surface of telencephalon, a site of active neurogenesis. A primary cell culture model was established and immunofluorescence analysis indicates that in vitro RGCs from female goldfish retain their major characteristics in vivo, including expression of glial fibrillary acidic protein and brain lipid binding protein. The estrogen synthesis enzyme aromatase B is exclusively found in RGCs, but this is lost as cells differentiate to neurons and other glial types in adult teleost brain. Pharmacological experiments using the cultured RGCs established that specific activation of dopamine D1 receptors up-regulates aromatase B mRNA through a cyclic adenosine monophosphate-dependent molecular mechanism. These data indicate that dopamine enhances the steroidogenic function of this neuronal progenitor cell.

Neuropathic pain is a very troublesome and difficult pain to treat. Although opioids are the best analgesics for cancer and surgical pain in clinic, only oxycodone among opioids shows better efficacy to alleviate neuropathic pain. However, many side effects associated with the use of oxycodone render the continued use of it in neuropathic pain treatment undesirable. Hence, we explored whether dextromethorphan (DM, a known N-methyl-D-aspartate receptor antagonist with neuroprotective properties) could potentiate the anti-allodynic effect of oxycodone and underlying mechanisms regarding to glial cells (astrocytes and microglia) activation and proinflammatory cytokines release in a spinal nerve injury (SNL) mice model.Oxycodone produced a dose-dependent anti-allodynic effect. Co-administration of DM at a dose of 10 mg/kg (i.p.) (DM10) which had no anti-allodynic effect by itself enhanced the acute oxycodone (1 mg/kg, s.c.) effect. When the chronic anti-allodynic effects were examined, co-administration of DM10 also significantly enhanced the oxycodone effect at 3 mg/kg. Furthermore, oxycodone decreased SNL-induced activation of glial cells (astrocytes and microglia) and plasma levels of proinflammatory cytokines (IL-6, IL-1β and TNF-α). Co-administration of DM10 potentiated these effects of oxycodone.The combined use of DM with oxycodone may have therapeutic potential for decreasing the effective dose of oxycodone on the treatment of neuropathic pain. Attenuation of the glial activation and proinflammatory cytokines in the spinal cord may be important mechanisms for these effects of DM.

The neuroprotective role of Buyang Huanwu decoction (BYHWD) in focal ischemia is associated with decreasing glutamate concentration. However, the mechanisms are not fully understood. The present study aimed to explore whether glutamate transporter-1 (GLT-1) and glutamine synthetase (GS) participated in the decreased level of glutamate and whether pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38) was involved in this process. BYHWD was found to significantly upregulate the expression of GLT-1 and GS in the hippocampal CA1 area compared to the ischemia group, with the difference on day 3 being most significant. BYHWD increased the level of PACAP-38, and PACAP-(6-38) (PACAP receptor antagonist) significantly attenuated the effect of BYHWD on GLT-1 and GS, suggesting that PACAP-38 was involved in the upregulation of GLT-1 and GS induced by BYHWD. In addition, as GLT-1 and GS are mainly located in astrocytes, the changes of astrocytes were detected by glial fibrillary acidic protein (GFAP; an astrocytic marker) immunostaining. The results showed that BYHWD inhibited the expression of GFAP compared with the ischemia group, however, co-administration with PACAP-(6-38), which inhibited the effect of BYHWD on GLT-1 and GS in astrocytes, attenuated this effect, indicating that astrocytes participated in the protective role of BYHWD following focal ischemia. These results provided the evidence for the first time that not only neurons but also astrocytes contribute to the protective role of BYHWD, which opposes previous studies and may be a starting point for traditional medicine.

Protein carbonylation is a well-documented and quantifiable consequence of oxidative stress in several neuropathologies, including multiple sclerosis, Alzheimer׳s disease, and Parkinson׳s disease. Although oxidative stress is a hallmark of traumatic brain injury (TBI), little work has explored the specific neural regions and cell types in which protein carbonylation occurs. Furthermore, the effect of gender on protein carbonylation after TBI has not been studied. The present investigation was designed to determine the regional and cell specificity of TBI-induced protein carbonylation and how this response to injury is affected by gender. Immunohistochemistry was used to visualize protein carbonylation in the brains of adult male and female Sprague-Dawley rats subjected to controlled cortical impact (CCI) as an injury model of TBI. Cell-specific markers were used to colocalize the presence of carbonylated proteins in specific cell types, including astrocytes, neurons, microglia, and oligodendrocytes. Results also indicated that the injury lesion site, ventral portion of the dorsal third ventricle, and ventricular lining above the median eminence showed dramatic increases in protein carbonylation after injury. Specifically, astrocytes and limited regions of ependymal cells adjacent to the dorsal third ventricle and the median eminence were most susceptible to postinjury protein carbonylation. However, these patterns of differential susceptibility to protein carbonylation were gender dependent, with males showing significantly greater protein carbonylation at sites distant from the lesion. Proteomic analyses were also conducted and determined that the proteins most affected by carbonylation in response to TBI include glial fibrillary acidic protein, dihydropyrimidase-related protein 2, fructose-bisphosphate aldolase C, and fructose-bisphosphate aldolase A. Many other proteins, however, were not carbonylated by CCI. These findings indicate that there is both regional and protein specificity in protein carbonylation after TBI. The marked increase in carbonylation seen in ependymal layers distant from the lesion suggests a mechanism involving the transmission of a cerebral spinal fluid-borne factor to these sites. Furthermore, this process is affected by gender, suggesting that hormonal mechanisms may serve a protective role against oxidative stress.

Hutchinson-Gilford progeria syndrome (HGPS) is a segmental progeroid syndrome with multiple features suggestive of premature accelerated aging. Accumulation of progerin is thought to underlie the pathophysiology of HGPS. However, despite ubiquitous expression of lamin A in all differentiated cells, the HGPS mutation results in organ-specific defects. For example, bone and skin are strongly affected by HGPS, while the brain appears to be unaffected. There are no definite explanations as to the variable sensitivity to progeria disease among different organs. In addition, low levels of progerin have also been found in several tissues from normal individuals, but it is not clear if low levels of progerin contribute to the aging of the brain. In an attempt to clarify the origin of this phenomenon, we have developed an inducible transgenic mouse model with expression of the most common HGPS mutation in brain, skin, bone and heart to investigate how the mutation affects these organs. Ultrastructural analysis of neuronal nuclei after 70 weeks of expression of the LMNA c.1824Cgreater than T mutation showed severe distortion with multiple lobulations and irregular extensions. Despite severe distortions in the nuclei of hippocampal neurons of HGPS animals, there were only negligible changes in gene expression after 63 weeks of transgenic expression. Behavioral analysis and neurogenesis assays, following long-term expression of the HGPS mutation, did not reveal significant pathology. Our results suggest that certain tissues are protected from functional deleterious effects of progerin.

Autophagy is a homeostatic degradative process essential for basal turnover of long-lived proteins and organelles as well as for removal of dysfunctional cellular components. Dysregulation of the autophagic machinery has been recently associated to several conditions including neurodegenerative diseases and cancer, but only very few studies have investigated its role in pain processing.We previously described autophagy impairment at the spinal cord in the experimental model of neuropathic pain induced by spinal nerve ligation (SNL). In this study, we characterized the main autophagic markers in two other common experimental models of neuropathic pain, the chronic constriction injury (CCI) and the spared nerve injury (SNI). The different modulation of LC3-I, Beclin 1 and p62 suggested that autophagy is differentially affected in the spinal dorsal horn depending on the type of peripheral injury. Confocal analysis of p62 distribution in the spinal dorsal horn indicated its presence mainly in NeuN-positive cell bodies and occasionally in glial processes, thus suggesting a predominant expression in the neuronal compartment. Finally, we investigated the consequences of autophagy impairment on pain behaviour by using the autophagy blocker cloroquine. Intrathecal chloroquine injection in naïve mice induced spinal accumulation of LC3 and p62 paralleled by significant mechanical hypersensitivity thus confirming the block in autophagosome clearance and suggesting the participation of the autophagic process in spinal mechanisms of pain processing. Altogether, our data indicate that spinal autophagy is differentially altered in different experimental pain models of neuropathic pain and that this process may be relevant for pain control.

Conventional MRI is frequently used during the diagnosis of multiple sclerosis but provides only little additional pathological information. Proton MRS ((1) H-MRS), however, provides biochemical information on the lesion pathology by visualization of a spectrum of metabolites. In this study we aimed to better understand the changes in metabolite concentrations following demyelination of the white matter. Therefore, we used the cuprizone model, a well-established mouse model to mimic type III human multiple sclerosis demyelinating lesions. First, we identified CX3 CL1/CX3 CR1 signaling as a major regulator of microglial activity in the cuprizone mouse model. Compared with control groups (heterozygous CX3 CR1(+/-) C57BL/6 mice and wild type CX3 CR1(+/+) C57BL/6 mice), microgliosis, astrogliosis, oligodendrocyte cell death and demyelination were shown to be highly reduced or absent in CX3 CR1(-/-) C57BL/6 mice. Second, we show that (1) H-MRS metabolite spectra are different when comparing cuprizone-treated CX3 CR1(-/-) mice showing mild demyelination with cuprizone-treated CX3 CR1(+/+) mice showing severe demyelination and demyelination-associated inflammation. Following cuprizone treatment, CX3 CR1(+/+) mice show a decrease in the Glu, tCho and tNAA concentrations as well as an increased Tau concentration. In contrast, following cuprizone treatment CX3 CR1(-/-) mice only showed a decrease in tCho and tNAA concentrations. Therefore, (1) H-MRS might possibly allow us to discriminate demyelination from demyelination-associated inflammation via changes in Tau and Glu concentration. In addition, the observed decrease in tCho concentration in cuprizone-induced demyelinating lesions should be further explored as a possible diagnostic tool for the early identification of human MS type III lesions.

Primary coenzyme Q10 (CoQ10) deficiency is due to mutations in genes involved in CoQ biosynthesis. The disease has been associated with five major phenotypes, but a genotype-phenotype correlation is unclear. Here, we compare two mouse models with a genetic modification in Coq9 gene (Coq9(Q95X) and Coq9(R239X)), and their responses to 2,4-dihydroxybenzoic acid (2,4-diHB). Coq9(R239X) mice manifest severe widespread CoQ deficiency associated with fatal encephalomyopathy and respond to 2,4-diHB increasing CoQ levels. In contrast, Coq9(Q95X) mice exhibit mild CoQ deficiency manifesting with reduction in CI+III activity and mitochondrial respiration in skeletal muscle, and late-onset mild mitochondrial myopathy, which does not respond to 2,4-diHB. We show that these differences are due to the levels of COQ biosynthetic proteins, suggesting that the presence of a truncated version of COQ9 protein in Coq9(R239X) mice destabilizes the CoQ multiprotein complex. Our study points out the importance of the multiprotein complex for CoQ biosynthesis in mammals, which may provide new insights to understand the genotype-phenotype heterogeneity associated with human CoQ deficiency and may have a potential impact on the treatment of this mitochondrial disorder.

Connexin 43 (Cx43) is a member of connexin family mainly expressed in astrocytes, which forms gap junctions and hemichannels and maintains the normal shape and function of astrocytes. In this study we investigated the role of Cx43 in astrocytes in facilitating neuronal recovery during ischemic stroke.Primary culture of astrocytes or a mixed culture of astrocytes and cortical neurons was subjected to oxygen glucose deprivation and reperfusion (OGD/R). The expression of Cx43 and Ephrin-A4 in astrocytes was detected using immunocytochemical staining and Western blot assays. Intercellular Ca(2+) concentration was determined with Fluo-4 AM fluorescent staining. Middle cerebral artery occlusion (MCAO) model rats were used for in vivo studies.OGD/R treatment of cultured astrocytes caused a decrement of Cx43 expression and translocation of Cx43 from cell membrane to cytoplasm, accompanied by cell retraction. Furthermore, OGD/R increased intracellular Ca(2+) concentration, activated CaMKII/CREB pathways and upregulated expression of Ephrin-A4 in the astrocytes. All these changes in OGD/R-treated astrocytes were alleviated by overexpression of Cx43. In the cortical neurons cultured with astrocytes, OGD/R inhibited the neurite growth, whereas overexpression of Cx43 or knockdown of Ephrin-A4 in astrocytes restored the neurite growth. In MCAO model rats, neuronal recovery was found to be correlated with the recuperation of Cx43 and Ephrin-A4 in astrocytes.Cx43 can stabilize astrocytes and facilitate the resistance to the deleterious effects of a stroke-like milieu and promote neuronal recovery.

Astrocytes expressing the aquaporin-4 water channel are a primary target of pathogenic, disease-specific immunoglobulins (IgG) found in patients with neuromyelitis optica (NMO). Immunopathological analyses of active NMO lesions highlight a unique inflammatory phenotype marked by infiltration of granulocytes. Previous studies characterized this granulocytic infiltrate as a response to vasculocentric complement activation and localized tissue destruction. In contrast, we observe that granulocytic infiltration in NMO lesions occurs independently of complement-mediated tissue destruction or active demyelination. These immunopathological findings led to the hypothesis that NMO IgG stimulates astrocyte signaling that is responsible for granulocytic recruitment in NMO.Histopathology was performed on archival formalin-fixed paraffin-embedded autopsy-derived CNS tissue from 23 patients clinically and pathologically diagnosed with NMO or NMO spectrum disorder. Primary murine astroglial cultures were stimulated with IgG isolated from NMO patients or control IgG from healthy donors. Transcriptional responses were assessed by microarray, and translational responses were measured by ELISA. Signaling through the NFκB pathway was measured by western blotting and immunostaining.Stimulation of primary murine astroglial cultures with NMO IgG elicited a reactive and inflammatory transcriptional response that involved signaling through the canonical NFκB pathway. This signaling resulted in the release of pro-granulocytic chemokines and was inhibited by the clinically relevant proteasome inhibitors bortezomib and PR-957.We propose that the astrocytic NFκB-dependent inflammatory response to stimulation by NMO IgG represents one of the earliest events in NMO pathogenesis, providing a target for therapeutic intervention upstream of irreversible cell death and tissue damage.