Globoid cell leukodystrophy (GLD) or Krabbe disease, is a fatal demyelinating disease attributed to mutations in the galactocerebrosidase (GALC) gene. Loss of function mutations in GALC result in accumulation of the glycolipid intermediate, galactosylsphingosine (psychosine). Due to the cytotoxicity of psychosine, it has been hypothesized that accumulated psychosine underlie the pathophysiology of GLD. However, the cellular mechanisms of GLD pathophysiology remain unclear. Globoid cells, multinucleated microglia/macrophages in the central nervous system (CNS), are a defining characteristic of GLD. Here we report that exposure of primary glial cultures to psychosine induces the expression and the production of matrix metalloproteinase (MMP)-3 that mediated a morphological transformation of microglia into a multinucleated globoid cell type. Additionally, psychosine-induced globoid cell formation from microglia was prevented by either genetic ablation or chemical inhibition of MMP-3. These effects are microglia-specific as peripheral macrophages exposed to psychosine did not become activated or express increased levels of MMP-3. In the brain from twitcher mice, a murine model of human GLD, elevated MMP-3 expression relative to wild-type littermates was contemporaneous with disease onset and further increased with disease progression. Further, bone marrow transplantation (BMT), currently the only therapeutically beneficial treatment for GLD, did not mitigate the elevated expression of MMP-3 in twitcher mice. Hence, elevated expression of MMP-3 in GLD may promote microglial responses to psychosine that may represent an important pathophysiological process in this disease and its treatment.
microglia; macrophage; astrocyte; multinucleated cell; psychosine; twitcher mice
During development, progenitors that are committed to differentiate into oligodendrocytes, the myelinating cells of the central nervous system (CNS), are generated within discrete regions of the neuroepithelium. More specifically, within the developing spinal cord and hindbrain ventrally located progenitor cells that are characterized by the expression of the transcription factor olig2 give temporally rise to first motor neurons and then oligodendrocyte progenitors. The regulation of this temporal neuron-glial switch has been found complex and little is known about the extrinsic factors regulating it. Our studies described here identified a zebrafish ortholog to mammalian atx, which displays evolutionarily conserved expression pattern characteristics. Most interestingly, atx was found to be expressed by cells of the cephalic floor plate during a time period when ventrally-derived oligodendrocyte progenitors arise in the developing hindbrain of the zebrafish. Knock-down of atx expression resulted in a delay and/or inhibition of the timely appearance of oligodendrocyte progenitors and subsequent developmental stages of the oligodendrocyte lineage. This effect of atx knock-down was not accompanied by changes in the number of olig2-positive progenitor cells, the overall morphology of the axonal network or the number of somatic abducens motor neurons. Thus, our studies identified Atx as an extrinsic factor that is likely secreted by cells from the floor plate and that is involved in regulating specifically the progression of olig2-positive progenitor cells into lineage committed oligodendrocyte progenitors.
myelination; glia differentiation; CNS development; floor plate; zebrafish
Müller glia are normally mitotically quiescent cells, but in certain pathological states they can reenter the mitotic cell cycle. While several cell cycle regulators have been shown to be important in this process, a role for the tumor suppressor, p53, has not been demonstrated. Here, we investigated a role for p53 in limiting the ability of Müller glia to proliferate in the mature mouse retina. Our data demonstrate that müller glia undergo a developmental restriction in their potential to proliferate. Retinal explants or dissociated cultures treated with EGF become mitotically quiescent by the end of the second postnatal week. In contrast, Müller glia from adult trp53−/+ or trp53−/− mice displayed a greater ability to proliferate in response to EGF stimulation in vitro. The enhanced proliferative ability of trp53 deficient mice correlates with a decreased expression of the mitotic inhibitor Cdkn1a/p21cip and an increase in c-myc, a transcription factor that promotes cell cycle progression. These data show that p53 plays an essential role in limiting the potential of Müller glia to re-enter the mitotic cycle as the retina matures during postnatal development.
cell cycle; proliferation; retina
Type I interferons (IFNα/β) provide a primary defense against infection. Nevertheless, the dynamics of IFNα/β induction and responsiveness by central nervous system (CNS) resident cells in vivo in response to viral infections are poorly understood. Mice were infected with a neurotropic coronavirus with tropism for oligodendroglia and microglia to probe innate antiviral responses during acute encephalomyelitis. Expression of genes associated with the IFNα/β pathways were monitored in microglia and oligodendroglia purified from naïve and infected mice by fluorescent activated cell sorting. Compared to microglia, oligodendroglia were characterized by low basal expression of mRNA encoding viral RNA sensing pattern recognition receptors (PRRs), IFNα/β receptor chains, interferon sensitive genes (ISG), as well as kinases and transcription factors critical in IFNα/β signaling. Although PRRs and ISGs were upregulated by infection in both cell types, the repertoire and absolute mRNA levels were more limited in oligodendroglia. Furthermore, although oligodendroglia harbored higher levels of viral RNA compared to microglia, Ifnα/β was only induced in microglia. Stimulation with the double stranded RNA analogue poly I:C also failed to induce Ifnα/β in oligodendroglia, and resulted in reduced and delayed induction of ISGs compared to microglia. The limited antiviral response by oligodendroglia was associated with a high threshold for upregulation of Ikkε and Irf7 transcripts, both central to amplifying IFNα/β responses. Overall, these data reveal that oligodendroglia from the adult CNS are poor sensors of viral infection and suggest they require exogenous IFNα/β to establish an antiviral state.
coronavirus; central nervous system; innate immunity; IFNα/β; oligodendroglia
As brain-resident immune cells, microglia (MG) survey the brain parenchyma to maintain homeostasis during development and following injury. Recent work in perinatal stroke, a leading cause of lifelong disability, has implicated MG as targets for therapeutic intervention during stroke progression. Although MG responses are complex, work in developing rodents suggests that MG limit brain damage and promote recovery after stroke. However, little is known about how energy-limiting conditions affect MG survival and mobility in developing brain tissues. Here, we used confocal time-lapse imaging to monitor MG viability and motility during hypoxia or oxygen-glucose deprivation (OGD) in neonatal hippocampal tissue slices derived from GFP-reporter mice (CX3CR1GFP/+). We found that MG in P5-P7 neonatal tissues remain viable for at least 6hr of hypoxia but begin to die after 2hr of OGD. Both hypoxia and OGD reduced MG motility. Unexpectedly, some MG retain or recover motility during OGD, and these active MG can contact and engulf dead cells. MG from younger neonates (P2-P3) are more resistant to OGD than those from older ones, indicating increasing vulnerability with developmental age. Finally, we show that transient (2hr) OGD reduces MG motility, migration, and viability. Although MG motility is rapidly restored after transient OGD, it remains below control levels for many hours. Together, these results show that MG in neonatal mouse brain tissues are vulnerable to both transient and sustained OGD, and many MG die within hours after onset of OGD. Preventing MG death may, therefore, provide a strategy for promoting tissue restoration after stroke.
microglia; stroke; cell death; development
Toll like receptors 7 (TLR7) and 9 (TLR9) are important mediators of innate immune responses. Both receptors are located in endosomal compartments, recognize nucleic acids and signal via Myeloid differentiation factor 88 (MyD88). In the current study, we analyzed TLR7 and TLR9 induced activation of astrocytes and microglia, two cell types that contribute to innate immune responses in the CNS. TLR7 and TLR9 agonists induced similar cytokine profiles in each cell types. However, there were notable differences in the cytokine profile between astrocytes and microglia, including the production of the anti-inflammatory cytokine IL-10 and anti-apoptotic cytokines G-CSF and IL-9 by microglia but not astrocytes. Costimulation studies demonstrated that the TLR7 agonist, imiquimod, could inhibit TLR9 agonist-induced innate immune responses, in both cell types, in a concentration dependent manner. Surprisingly, this inhibition was not mediated by TLR7, as deficiency in TLR7 did not alter suppression of the TLR9 agonist-induced responses. The suppression of innate immune responses was also not due to an inhibition of TLR9 agonist uptake. This suggested that imiquimod suppression may be a direct effect, possibly by blocking CpG-ODN binding and/or signaling with TLR9, thus limiting cell activation. An antagonistic relationship was also observed between the two receptors in microglia, with TLR7 deficiency resulting in enhanced cytokine responses to CpG-ODN stimulation. Thus, both TLR7 and its agonist can have inhibitory effects on TLR9-induced cytokine responses in glial cells.
brain; cytokines; costimulation; imiquimod; CpG-ODN
Extracellular signals play essential roles in controlling the proliferation and differentiation of oligodendrocyte progenitor cells in the developing central nervous system. However, the intracellular pathways that transduce these extrinsic signals remain to be elucidated. In this study, we showed that conditional ablation of the nonreceptor tyrosine phosphatase Shp2 in Olig1-expressing oligodendrocyte lineage resulted in dramatic reduction in the generation and proliferation of oligodendrocyte progenitor cells in the spinal cord. Maturation and myelination of oligodendrocytes were also compromised in the Shp2 mutants. The deficits in oligodendrocyte development in Shp2 mutants nearly phenocopied those observed in PDGF-A mutants, suggesting that Shp2 is a crucial component in transducing PDGFRα signals in the control of oligodendrocyte proliferation and maturation.
Shp2; oligodendrocyte; spinal cord; proliferation; differentiation
Activation of the neuronal-glial network in the spinal cord dorsal horn (SCDH) mediates various chronic painful conditions. Here, we studied spinal neuronal-astrocyte signaling interactions involved in the maintenance of painful diabetic neuropathy (PDN) in type 2 diabetes. We used the db/db mouse, an animal model for PDN of type 2 diabetes, which develops mechanical allodynia from 6-12 wk of age. In the current study, enhanced substance P (SP) expression was detected in the presynaptic sensory fibers innervating lamina I-III in the lumbar spinal cord (LSC) dorsal horn (LSCDH) of the db/db mouse at 10 wk of age. This phenomenon is associated with enhanced spinal ERK1/2 phosphorylation in projection sensory neurons and regional astrocyte activation. In addition, peak phosphorylation of the NR1 subunit of NMDAR, along with upregulation of neuronal and inducible nitric oxide synthase (nNOS and iNOS) expression were detected in diabetic mice. Expression of nNOS and iNOS was detected in both interneurons and astrocytes in lamina I-III of the LSCDH. Treatment with MK801, a NMDAR inhibitor, inhibited mechanical allodynia, ERK1/2 phosphorylation, as well as n- and iNOS upregulation in diabetic mice. MK801 also reduced astrocytosis and GFAP upregulation in db/db mice. In addition, L-NAME, a nonspecific NOS inhibitor, had similar effects on NMDAR signaling and NOS expression. These results suggest that NO from surrounding interneurons and astrocytes interacts with NMDAR-dependent signaling in the projection neurons of the SCDH during the maintenance of PDN.
N-methyl-D-aspartate receptor; nitric oxide synthase; extracellular signal-regulated kinase; neuropathic pain; neuron-glial interactions
It is generally accepted that the endocannabinoid system plays important roles in spinal pain processing. Although it is documented that cannabinoid-1 receptors are strongly expressed in the superficial spinal dorsal horn, the cellular distribution of enzymes that can synthesize endocannabinoid ligands is less well studied. Thus, using immunocytochemical methods at the light and electron microscopic levels, we investigated the distribution of diacylglycerol lipase-alpha (DGLα) and N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD), enzymes synthesizing the endocannabinoid ligands, 2-arachidonoylglycerol (2-AG) and anandamide, respectively. Positive labeling was revealed only occasionally in axon terminals, but dendrites displayed strong immunoreactivity for both enzymes. However, the dendritic localization of DGLα and NAPE-PLD showed a remarkably different distribution. DGLα immunolabeling in dentrites was always revealed at membrane compartments in close vicinity to synapses. In contrast to this, dendritic NAPE-PLD labeling was never observed in association with synaptic contacts. In addition to dendrites, a substantial proportion of astrocytic (immunoreactive for GFAP) and microglial (immunoreactive for CD11b) profiles were also immunolabeled for both DGLα and NAPE-PLD. Glial processes immunostained for DGLα were frequently found near to synapses in which the postsynaptic dendrite was immunoreactive for DGLα, whereas NAPE-PLD immunoreactivity on glial profiles at the vicinity of synapses was only occasionally observed. Our results suggest that both neurons and glial cells can synthesize and release 2-AG and anandamide in the superficial spinal dorsal horn. 2-AG can primarily be released by postsynaptic dendrites and glial processes adjacent to synapses, whereas anandamide can predominantly be released from non-synaptic dendritic and glial compartments.
DGLα; NAPE-PLD; nociceptive primary afferents; interneurons; glial cells
Reactive astrogliosis is one of the pathological hallmarks of neurodegenerative diseases. Inflammatory cytokines, such as TNF-α and IL-1β, have been shown to mediate the reactive astrogliosis in neurodegenerative diseases; however, the molecular mechanism remains unclear. In this study, we investigated the role of transcription factor FOXO3a on astrocyte proliferation, one primary aspect of severe reactive astrogliosis. Our results confirmed that TNF-α and IL-1β increased astrocyte proliferation, as determined by Ki67 and BrdU immunostaining. Furthermore, we found that cytokine-mediated astrocyte proliferation was accompanied by an increase of the phosphorylation and reduced nuclear expression of FOXO3a. Intracranial injection of TNF-α and IL-1β induced astrocyte proliferation and hypertrophy, which was associated with reduced nuclear expression of Foxo3a in astrocytes. To determine the function of FOXO3a in astrocyte proliferation, wild type FOXO3a was overexpressed with adenovirus, which subsequently upregulated p27Kip1 and Gadd45α, and significantly inhibited cytokine-induced astrocyte proliferation. In contrast, overexpression of dominant negative FOXO3a decreased p27Kip1, upregulated cyclin D1 and promoted astrocyte proliferation. Along the same line, astrocytes isolated from Foxo3a-null mice have higher proliferative potential. In response to intracranial injection of cytokines, Foxo3a-null mice manifested severe astrogliosis in vivo. In conclusion, FOXO3a is important in restraining astrocyte proliferation during proinflammatory cytokine stimulation and loss of function of FOXO3a may be responsible for the proliferation of astrocytes in the severe form of reactive astrogliosis. Understanding the key regulatory role of FOXO3a in reactive astrogliosis may provide a novel therapeutic target during neuroinflammation.
Astrogliosis; Proinflammatory cytokine; FOXO3a; Akt-1; Phosphorylation; Cyclin D1
Astrocytes play a critical role in regulation of extracellular neurotransmitter levels in the central nervous system. This function is particularly prominent for the excitatory amino acid glutamate, with estimates that 80–90% of extracellular glutamate uptake in brain is through astrocytic glutamate transporters. This uptake has significance both in regulation of the potential toxic accumulation of extracellular glutamate, and in normal resupply of inhibitory and excitatory synapses with neurotransmitter. This resupply of neurotransmitter is accomplished by astroglial uptake of glutamate, transformation of glutamate to glutamine by the astrocytic enzyme glutamine synthetase, and shuttling of glutamine back to excitatory and inhibitory neurons via specialized transporters. Once in neurons, glutamine is enzymatically converted back to glutamate, which is utilized for synaptic transmission, either directly, or following decarboxylation to GABA. Many neurologic and psychiatric conditions, particularly epilepsy, are accompanied by the development of reactive gliosis, a pathology characterized by anatomical and biochemical plasticity in astrocytes, accompanied by proliferation of these cells. Among the biochemical changes evident in reactive astrocytes is a downregulation of several of the important regulators of the glutamine-glutamate cycle, including glutamine synthetase, and possibly also glutamate transporters. This downregulation may have significance in contributing both to the aberrant excitability and to the altered neuropathology characterizing epilepsy. In the present review, we provide an overview of the normal function of astrocytes in regulating extracellular glutamate homeostasis, neurotransmitter supply, and excitotoxicity. We further discuss the potential role reactive gliosis may play in the pathophysiology of epilepsy.
Extracellular levels of the brain’s endogenous anticonvulsant and neuroprotectant adenosine largely depend on an astrocyte-based adenosine cycle, comprised of ATP release, rapid degradation of ATP into adenosine, and metabolic reuptake of adenosine through equilibrative nucleoside transporters and phosphorylation by adenosine kinase (ADK). Changes in ADK expression and activity therefore rapidly translate into changes of extracellular adenosine, which exerts its potent anticonvulsive and neuroprotective effects by activation of pre- and postsynaptic adenosine A1 receptors. Increases in ADK increase neuronal excitability, whereas decreases in ADK render the brain resistant to seizures and injury. Importantly, ADK was found to be overexpressed and associated with astrogliosis and spontaneous seizures in rodent models of epilepsy, as well as in human specimen resected from patients with hippocampal sclerosis and temporal lobe epilepsy. Several lines of evidence indicate that overexpression of astroglial ADK and adenosine deficiency are pathological hallmarks of the epileptic brain. Consequently, adenosine augmentation therapies constitute a powerful approach for seizure prevention, which is effective in models of epilepsy that are resistant to conventional antiepileptic drugs. The adenosine kinase hypothesis of epileptogenesis suggests that adenosine dysfunction in epilepsy undergoes a biphasic response: An acute surge of adenosine that can be triggered by any type of injury might contribute to the development of astrogliosis via adenosine receptor –dependent and –independent mechanisms. Astrogliosis in turn is associated with overexpression of ADK, which was shown to be sufficient to trigger spontaneous recurrent electrographic seizures. Thus, ADK emerges as a promising target for the prediction and prevention of epilepsy.
The neuronal doctrine, developed a century ago regards neuronal networks as the sole substrate of higher brain function. Recent advances in glial physiology have promoted an alternative hypothesis, which places information processing in the brain into integrated neuronal-glial networks utilizing both binary (neuronal action potentials) and analogue (diffusional propagation of second messengers/metabolites through gap junctions or transmitters through the interstitial space) signal encoding. It has been proposed that the feed-forward and feed-back communication between these two types of neural cells, which underlies information transfer and processing, is accomplished by the release of neurotransmitters from neuronal terminals as well as from astroglial processes. Understanding of this subject, however, remains incomplete and important questions and controversies require resolution. Here we propose that the primary function of peri-synaptic glial processes is to create an “astroglial cradle” that shields the synapse from a multitude of extrasynaptic signaling events and provides for multifaceted support and long-term plasticity of synaptic contacts through variety of mechanisms, which may not necessarily involve the release of “glio”transmitters.
Pannexins (Panx1, 2 and 3) comprise a group of proteins expressed in vertebrates that share weak yet significant sequence homology with the invertebrate gap junction proteins, the innexins. In contrast to the other vertebrate gap junction protein family (connexin), pannexins do not form intercellular channels, but at least Panx1 forms non-junctional plasma membrane channels. Panx1 is ubiquitously expressed and has been shown to form large conductance (500pS) channels that are voltage-dependent, mechanosensitive and permeable to relatively large molecules, such as ATP. Pharmacological and knockdown approaches have indicated that Panx1 is the molecular substrate for the so-called “hemichannel” originally attributed to connexin43 (Cx43) and that Panx1 is the pore forming unit of the P2X7 receptor. Here, we describe, for the first time, conductance and permeability properties of Panx1-null astrocytes. The electrophysiological and fluorescence imaging analysis performed on these cells fully support our previous pharmacological and Panx1 knockdown studies that showed profoundly lower dye uptake and ATP release than wild-type untreated astrocytes. As a consequence of decreased ATP paracrine signaling, intercellular calcium wave spread is altered in Panx1-null astrocytes. Moreover, we found that in astrocytes as in Panx1 expressing oocytes, elevated extracellular K+ activates Panx1 channels independently of membrane potential. Thus, based on the present findings and our previous report, we propose that Panx1 channels serve as K+ sensors for changes in the extracellular milieu such as those occurring under pathological conditions.
Gap junction; P2X receptor; calcium waves; glia; connexin; pannexin
Glutamate transporter-1 (GLT-1) plays a central role in preventing excitotoxicity by removing excess glutamate from the synaptic clefts. 17β-estradiol (E2) and tamoxifen (TX), a selective estrogen receptor modulator (SERM), afford neuroprotection in a range of experimental models. However, the mechanisms that mediate E2 and TX neuroprotection have yet to be elucidated. We tested the hypothesis that E2 and TX enhance GLT-1 function by increasing transforming growth factor (TGF)-α expression and thus, attenuate manganese (Mn)-induced impairment in astrocytic GLT-1 expression and glutamate uptake in rat neonatal primary astrocytes. The results showed that E2 (10 nM) and TX (1 μM) increased GLT-1 expression and reversed the Mn-induced reduction in GLT-1, both at the mRNA and protein levels. E2/TX also concomitantly reversed the Mn-induced inhibition of astrocytic glutamate uptake. E2/TX activated the GLT-1 promoter and attenuated the Mn-induced repression of the GLT-1 promoter in astrocytes. TGF-α knock-down (siRNA) abolished the E2/TX effect on GLT-1 expression, and inhibition of epidermal growth factor receptor (TGF-α receptor) suppressed the effect of E2/TX on GLT-1 expression and GLT-1 promoter activity. E2/TX also increased TGF-α mRNA and protein levels with a concomitant increase in astrocytic glutamate uptake. All estrogen receptors (ERs: ER-α ER-β and GPR30) were involved in mediating E2 effects on the regulation of TGF-α, GLT-1, and glutamate uptake. These results indicate that E2/TX increase GLT-1 expression in astrocytes via TGF-α signaling, thus offering an important putative target for the development of novel therapeutics for neurological disorders.
GLT-1; TGF-α; glutamate uptake; glutamate transporter; manganese; tamoxifen; 17β-estradiol; astrocytes
Emerging evidence points to monocarboxylates as key players in the pathophysiology of temporal lobe epilepsy (TLE) with hippocampal sclerosis (mesial temporal lobe epilepsy, MTLE). Monocarboxylate transporters (MCT) 1 and 2, which are abundantly present on brain endothelial cells and perivascular astrocyte endfeet, respectively, facilitate the transport of monocarboxylates and protons across cell membranes. Recently, we reported that the density of MCT1 protein is reduced on endothelial cells and increased on astrocyte plasma membranes in the hippocampal formation in patients with MTLE and in several animal models of the disorder. Because the perivascular astrocyte endfeet comprise an important part of the neurovascular unit we now assessed the distribution of the MCT2 in hippocampal formations in TLE patients with (MTLE) or without hippocampal sclerosis (non-MTLE). Light microscopic immunohistochemistry revealed significantly less perivascular MCT2 immunoreactivity in the hippocampal formation in MTLE (n=6) than in non-MTLE (n=6) patients, and to a lesser degree in non-MTLE than in non-epilepsy patients (n=4). Immunogold electron microscopy indicated that the loss of MCT2 protein occurred on perivascular astrocyte endfeet. Interestingly, the loss of MCT2 on astrocyte endfeet in MTLE (n=3) was accompanied by an upregulation of the protein on astrocyte membranes facing synapses in the neuropil, when compared with non-MTLE (n=3). We propose that the altered distribution of MCT1 and MCT2 in TLE (especially MTLE) limits the flux of monocarboxylates across the blood brain barrier and enhances the exchange of monocarboxylates within the brain parenchyma.
Blood-brain barrier; astrocyte endfoot; hippocampal sclerosis; ketone bodies; ketongenic diet
Despite decades of research, primary brain tumors, gliomas, lack effective treatment options and present a huge clinical challenge. Particularly, the most malignant subtype, Glioblastoma multiforme, proliferates extensively and cells often undergo incomplete cell divisions, resulting in multinucleated cells. We now present evidence that multinucleated glioma cells result from the functional loss of transient receptor potential canonical 1 (TRPC1) channels, plasma membrane proteins involved in agonist-induced calcium entry and reloading of intracellular Ca2+ stores. Pharmacological inhibition or shRNA mediated suppression of TRPC1 causes loss of functional channels and store-operated calcium entry in D54MG glioma cells. This is associated with reduced cell proliferation and, frequently, with incomplete cell division. The resulting multinucleated cells are reminiscent of those found in patient biopsies. In a flank tumor model, tumor size was significantly decreased when TRPC1 expression was disrupted using a doxycycline inducible shRNA knockdown approach. These results suggest that TRPC1 channels play an important role in glioma cell division most likely by regulating calcium signaling during cytokinesis.
TRPC channels; glioma; proliferation; multinucleation; calcium
After stroke and brain injury, cortical gray matter recovery involves mechanisms of neurovascular matrix remodeling. In white matter however, the mechanisms of recovery remain unclear. In this present study, we demonstrate that oligodendrocytes secrete matrix metalloproteinase-9 (MMP-9), which accelerates the angiogenic response after white matter injury. In primary oligodendrocyte cultures, treatment with the pro-inflammatory cytokine interleukin-1β (IL-1β) induced an upregulation and secretion of MMP-9. Conditioned media from IL-1β-stimulated oligodendrocytes significantly amplified matrigel tube formation in brain endothelial cells, indicating that MMP-9 from oligodendrocytes can promote angiogenesis in vitro. Next we asked whether similar signals and substrates operate after white matter injury in vivo. Focal white matter injury and demyelination was induced in mice via stereotactic injection of lysophosphatidylcholine (LPC) into corpus callosum. Western blot analysis showed that IL-1β expression was increased in damaged white matter. Immunostaining demonstrated MMP-9 signals in MOBP (myelin-associated oligodendrocytic basic protein)-positive oligodendrocytes. Treatment with an IL-1β-neutralizing antibody suppressed the MMP-9 response in oligodendrocytes. Finally, we confirmed that the broad spectrum MMP inhibitor GM6001 inhibited angiogenesis around the injury area in this white matter injury model. In gray matter, a neurovascular niche promotes cortical recovery after brain injury. Our study suggests that an analogous oligovascular niche may mediate recovery in white matter.
oligodendrocyte; white matter injury; matrix metalloproteinase-9; vascular remodeling; cerebral endothelial cell
Intracerebral hemorrhage (ICH) has been associated with inflammation and apoptosis. The CCL2-CCR2 chemotactic system is one of the major signaling pathways that induce inflammation and apoptosis. However, its role on ICH has not been investigated. We subjected wild-type, CCL2−/− and CCR2−/− mice to collagenase-induced ICH, and assessed histological and behavioral outcomes. Lack of CCL2 or CCR2 decreased the hematoma volume early after collagenase-induced ICH, but delayed its recovery. The hematoma size was accompanied by brain edema, neuronal death, and neurological scores. Although microglia activation/migration was attenuated in CCL2−/− or CCR2−/− mice 1 day after injury, more microglia were present at later time points, suggesting that alternative signaling pathways had been activated to recruit them. On the contrary, leukocyte and neutrophil infiltration was decreased in these mice, suggesting a tighter/recovered blood-brain barrier. In addition, we also found that FL- and K104Stop-CCL2 were able to restore the changes found in CCL2−/− mice, but K104A-CCL2 failed to do so. These results suggest that plasmin-mediated truncation of CCL2 may be an indispensable step to fully activate the chemokine in vivo. The data also indicate that CCL2-CCR2 signaling pathway may be a molecular target for the treatment of ICH.
CCL2; CCR2; Intracerebral Hemorrhage
Complement components and their receptors are found within and around Aβ cerebral plaques in Alzheimer’s disease (AD). Microglia defend against pathogens through phagocytosis via complement component C3 and/or engagement of C3 cleavage product iC3b with complement receptor type 3 (CR3, Mac-1). Here we provide direct evidence that C3 and Mac-1 mediate, in part, phagocytosis and clearance of fibrillar amyloid-β (fAβ) by murine microglia in vitro and in vivo. Microglia took up not only synthetic fAβ42 but also amyloid cores from AD patients, transporting them to lysosomes in vitro. Fibrillar Aβ42 uptake was significantly attenuated by the deficiency or knockdown of C3 or Mac-1 and scavenger receptor class A ligands. In addition, C3 or Mac-1 knockdown combined with a scavenger receptor ligand, fucoidan, further attenutated fibrillar Aβ42 uptake by N9 microglia. Fluorescent fibrillar Aβ42 microinjected cortically was significantly higher in C3 and Mac-1 knockout mice compared to wild-type mice 5 days after surgery, indicating reduced clearance in vivo. Together, these results demonstrate that C3 and Mac-1 are involved in phagocytosis and clearance of fAβ by microglia, providing support for a potential beneficial role for microglia and the complement system in AD pathogenesis.
microglia; Aβ; complement component C3; complement receptor type 3; Mac-1; phagocytosis
Chronic inflammation in the central nervous system (CNS) is a central feature of many neurodegenerative and autoimmune diseases. As an immunologically competent cell, the astrocyte plays an important role in CNS inflammation. It is capable of expressing a number of cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) that promote inflammation directly and through the recruitment of immune cells. Checkpoints are therefore in place to keep tight control over cytokine production. Adenylate/uridylate-rich elements (ARE) in the 3′ untranslated region of cytokine mRNAs serve as a major checkpoint by regulating mRNA stability and translational efficiency. Here, we examined the impact of KH-type splicing regulatory protein (KSRP), an RNA binding protein which destabilizes mRNAs via the ARE, on cytokine expression and paracrine phenotypes of primary astrocytes. We identified a network of inflammatory mediators, including TNF-α and IL-1β, whose expression increased 2 to 4-fold at the RNA level in astrocytes isolated from KSRP−/− mice compared to littermate controls. Upon activation, KSRP−/− astrocytes produced TNF-α and IL-1β at levels that exceeded control cells by 15-fold or more. Conditioned media from KSRP−/− astrocytes induced chemotaxis and neuronal cell death in vitro. Surprisingly, we observed a prolongation of half-life in only a subset of mRNA targets and only after selective astrocyte activation. Luciferase reporter studies indicated that KSRP regulates cytokine gene expression at both transcriptional and post-transcriptional levels. Our results outline a critical role for KSRP in regulating pro-inflammatory mediators and have implications for a wide range of CNS inflammatory and autoimmune diseases.
RNA stability; AU-rich element; TNF-α; IL-1β
The recently published comprehensive profiles of genomic alterations in glioma have led to a refinement in our understanding of the molecular events that underlie this cancer. Using state-of-the-art genomic tools, several laboratories have created and characterized accurate genetically engineered mouse models of glioma based on specific genetic alterations observed in human tumors. These in vivo brain tumor models faithfully recapitulate the histopathology, etiology, and biology of gliomas and provide an exceptional experimental system to discover novel therapeutic targets and test therapeutic agents. This review focuses on mouse models of glioma with a special emphasis on genetically engineered models developed around key genetic glioma signature mutations in the PDGFR, EGFR and NF1 genes and pathways. The resulting animal models have provided insight into many fundamental and mechanistic facets of tumor initiation, maintenance and resistance to therapeutic intervention and will continue to do so in the future.
Brain insults, including traumatic and ischemic injuries, are frequently followed by acute seizures and delayed development of epilepsy. Dysfunction of the blood-brain barrier (BBB) is a hallmark of brain insults and is usually surrounding the core lesion. Recent studies from several laboratories confirmed that vascular pathology is involved in the development of epilepsy and demonstrate a key role for astroglia in this process. In this review, we focus on glia-related mechanisms linking vascular pathology, and specifically BBB dysfunction, to seizures and epilepsy. We summarize molecular and physiological experimental data demonstrating that the function of astrocytes is altered due to direct exposure to serum albumin, mediated by transforming growth factor beta signaling. We discuss the reported changes and their potential role in the observed hyperexcitability as well as potential implications of these findings for the future development of new diagnostic modalities and treatments to allow a full implementation of the gained knowledge for the benefit of patients with epilepsy.
buffering; connexins; astroglia; potassium channels; glutamate