Lentiviral vectors for gene delivery to normal and demyelinated white matter

Lentiviral vectors are increasingly used for gene delivery to neurons and in experimental models of neurodegeneration. Their use in gene delivery to white matter and their potential value in preventing or repairing CNS demyelination has received less attention. Here we show using a VSV-G-pseudotyped HIV-derived vector expressing the marker gene LacZ that lentiviral vectors transduce the major macroglial cell types present in normal white matter (astrocytes, oligodendrocytes, and oligodendrocyte progenitors). Injection of lentiviral vectors causes an inflammatory response at the injection site characterized by OX42(+) and ED1(+) macrophages, but only a few CD8(+) and no CD4(+) lymphocytes, and mild demyelination. Injection of lentiviral vectors into areas of toxin-induced demyelination resulted in significant numbers of cells expressing the marker gene and was a more effective means of gene delivery than was a LacZ-expressing murine retroviral vector.     

G protein‐coupled receptor 30 contributes to improved remyelination after cuprizone‐induced demyelination

Estrogen exerts neuroprotective and promyelinating actions. The therapeutic effect has been shown in animal models of multiple sclerosis, in which the myelin sheath is specifically destroyed in the central nervous system. However, it remains unproven whether estrogen is directly involved in remyelination via the myelin producing cells, oligodendrocytes, or which estrogen receptors are involved. In this study, we found that the membrane‐associated estrogen receptor, the G protein‐coupled receptor 30 (GPR30), also known as GPER, was expressed in oligodendrocytes in rat spinal cord and corpus callosum. Moreover, GPR30 was expressed throughout oligodendrocyte differentiation and promyelinating stages in primary oligodendrocyte cultures derived from rat spinal cords and brains. To evaluate the role of signaling via GPR30 in promyelination, a specific agonist for GPR30, G1, was administered to a rat model of demyelination induced by cuprizone treatment. Histological examination of the corpus callosum with oligodendrocyte differentiation stage‐specific markers showed that G1 enhanced oligodendrocyte maturation in corpus callosum of cuprizone‐treated animals. It also enhanced oligodendrocyte ensheathment of dorsal root ganglion (DRG) neurons in co‐culture and myelination in cuprizone‐treated animals. This study is the first evidence that GPR30 signaling promotes remyelination by oligodendrocytes after demyelination. GPR30 ligands may provide a novel therapy for the treatment of multiple sclerosis. © 2012 Wiley Periodicals, Inc.     

Interleukin‐10 is a critical regulator of white matter lesion containment following viral induced demyelination

Neurotropic coronavirus induces an acute encephalomyelitis accompanied by focal areas of demyelination distributed randomly along the spinal column. The initial areas of demyelination increase only slightly after the control of infection. These circumscribed focal lesions are characterized by axonal sparing, myelin ingestion by macrophage/microglia, and glial scars associated with hypertrophic astrocytes, which proliferate at the lesion border. Accelerated virus control in mice lacking the anti‐inflammatory cytokine IL‐10 was associated with limited initial demyelination, but low viral mRNA persistence similar to WT mice and declining antiviral cellular immunity. Nevertheless, lesions exhibited sustained expansion providing a model of dysregulated white matter injury temporally remote from the acute CNS insult. Expanding lesions in the absence of IL‐10 are characterized by sustained microglial activation and partial loss of macrophage/microglia exhibiting an acquired deactivation phenotype. Furthermore, IL‐10 deficiency impaired astrocyte organization into mesh like structures at the lesion borders, but did not prevent astrocyte hypertrophy. The formation of discrete foci of demyelination in IL‐10 sufficient mice correlated with IL‐10 receptor expression exclusively on astrocytes in areas of demyelination suggesting a critical role for IL‐10 signaling to astrocytes in limiting expansion of initial areas of white matter damage. GLIA 2015;63:2106–2120     

Developmental and post‐injury cortical gliogenesis: A Genetic fate‐mapping study with Nestin‐CreER mice

The primary sources of cortical gliogenesis, either during development or after adult brain injury, remain uncertain. We previously generated Nestin‐CreER mice to fate‐map the progeny of radial glial cells (RG), a source of astrocytes and oligodendrocytes in the nervous system. Here, we show that Nestin‐CreER mice label another population of glial progenitors, namely the perinatal subventricular zone (SVZ) glioblasts, if they are crossed with stop‐floxed EGFP mice and receive tamoxifen in late embryogenesis (E16–E18). Quantification showed E18 tamoxifen‐induction labeled more perinatal SVZ glioblasts than RG and transitional RG combined in the newborn brain (54% vs. 22%). Time‐lapse microscopy showed SVZ‐glioblasts underwent complex metamorphosis and often‐reciprocal transformation into transitional RG. Surprisingly, the E10‐dosed RG progenitors produced astrocytes, but no oligodendrocytes, whereas E18‐induction fate‐mapped both astrocytes and NG2+ oligodendrocyte precursors in the postnatal brain. These results suggest that cortical oligodendrocytes mostly derive from perinatal SVZ glioblast progenitors. Further, by combining genetic fate‐mapping and BrdU‐labeling, we showed that cortical astrocytes cease proliferation soon after birth (<P10) and only undergo nonproliferative gliosis (i.e., increased GFAP expression without cell‐division) after stab‐wound injury in adult brains. By contrast, 9.7% of cortical NG2+ progenitors remained mitotic at P29, and the ratio rose to 13.8% after stab‐wound injury. Together, these results suggest NG2+ progenitors, rather than GFAP+ astrocytes, are the primary source of proliferative gliosis after adult brain injury. © 2008 Wiley‐Liss, Inc.     

Expression of distinct splice variants of the stem cell marker prominin‐1 (CD133) in glial cells

Prominin‐1 (CD133) is a cholesterol‐interacting pentaspan membrane glycoprotein specifically associated with plasma membrane protrusions. Prominin‐1 is expressed by various stem and progenitor cells, notably neuroepithelial progenitors found in the developing embryonic brain. Here, we further investigated its expression in the murine brain. Biochemical analyses of brain membranes at early stages of development revealed the expression of two distinct splice variants of prominin‐1, s1 and s3, which have different cytoplasmic C‐terminal domains. The relative abundance of the s3 variant increased toward adulthood, whereas the opposite was observed for the s1 variant. Our combined in situ hybridization and immunohistochemistry revealed the expression of prominin‐1 in a subpopulation of Olig‐2‐positive oligodendroglial cells present within white matter tracts of postnatal and adult brain. Furthermore, immunohistological and biochemical characterization suggested strongly that the s3 variant is a novel component of myelin. Consistent with this, the expression of prominin‐1.s3 was significantly reduced in the brain of myelin‐deficient mice. Finally, oligodendrocytes expressed selectively the s3 variant whereas GFAP‐positive astrocytes expressed the s1 variant in primary glial cell cultures derived from embryonic brains. Collectively, our data demonstrate a complex expression pattern of prominin‐1 molecules in developing adult brain. Given that prominin‐1 is thought to act as an organizer of plasma membrane protrusions, they further suggest that a specific prominin‐1 splice variant might play a role in morphogenesis and/or maintenance of the myelin sheath. © 2008 Wiley‐Liss, Inc.     

Reactive astrocyte COX2‐PGE2 production inhibits oligodendrocyte maturation in neonatal white matter injury

Inflammation is a major risk factor for neonatal white matter injury (NWMI), which is associated with later development of cerebral palsy. Although recent studies have demonstrated maturation arrest of oligodendrocyte progenitor cells (OPCs) in NWMI, the identity of inflammatory mediators with direct effects on OPCs has been unclear. Here, we investigated downstream effects of pro‐inflammatory IL‐1β to induce cyclooxygenase‐2 (COX2) and prostaglandin E2 (PGE2) production in white matter. First, we assessed COX2 expression in human fetal brain and term neonatal brain affected by hypoxic‐ischemic encephalopathy (HIE). In the developing human brain, COX2 was expressed in radial glia, microglia, and endothelial cells. In human term neonatal HIE cases with subcortical WMI, COX2 was strongly induced in reactive astrocytes with “A2” reactivity. Next, we show that OPCs express the EP1 receptor for PGE2, and PGE2 acts directly on OPCs to block maturation in vitro. Pharmacologic blockade with EP1‐specific inhibitors (ONO‐8711, SC‐51089), or genetic deficiency of EP1 attenuated effects of PGE2. In an IL‐1β‐induced model of NWMI, astrocytes also exhibit “A2” reactivity and induce COX2. Furthermore, in vivo inhibition of COX2 with Nimesulide rescues hypomyelination and behavioral impairment. These findings suggest that neonatal white matter astrocytes can develop “A2” reactivity that contributes to OPC maturation arrest in NWMI through induction of COX2‐PGE2 signaling, a pathway that can be targeted for neonatal neuroprotection.     

Short‐ and long‐term functional plasticity of white matter induced by oligodendrocyte depolarization in the hippocampus

Plastic changes in white matter have received considerable attention in relation to normal cognitive function and learning. Oligodendrocytes and myelin, which constitute the white matter in the central nervous system, can respond to neuronal activity with prolonged depolarization of membrane potential and/or an increase in the intracellular Ca²⁺ concentration. Depolarization of oligodendrocytes increases the conduction velocity of an action potential along axons myelinated by the depolarized oligodendrocytes, indicating that white matter shows functional plasticity, as well as structural plasticity. However, the properties and mechanism of oligodendrocyte depolarization‐induced functional plastic changes in white matter are largely unknown. Here, we investigated the functional plasticity of white matter in the hippocampus using mice with oligodendrocytes expressing channelrhodopsin‐2. Using extracellular recordings of compound action potentials at the alveus of the hippocampus, we demonstrated that light‐evoked depolarization of oligodendrocytes induced early‐ and late‐onset facilitation of axonal conduction that was dependent on the magnitude of oligodendrocyte depolarization; the former lasted for approximately 10 min, whereas the latter continued for up to 3 h. Using whole‐cell recordings from CA1 pyramidal cells and recordings of antidromic action potentials, we found that the early‐onset short‐lasting component included the synchronization of action potentials. Moreover, pharmacological analysis demonstrated that the activation of Ba²⁺‐sensitive K⁺ channels was involved in early‐ and late‐onset facilitation, whereas 4‐aminopyridine‐sensitive K⁺ channels were only involved in the early‐onset component. These results demonstrate that oligodendrocyte depolarization induces short‐ and long‐term functional plastic changes in the white matter of the hippocampus and plays active roles in brain functions. GLIA 2014;62:1299–1312     

Substance P-immunoreactive astrocytes related to deep white matter and striatal blood vessels in human brain

Astrocytes immunoreactive for the vasodilator neuropeptide substance P are present adjacent to blood vessels which are contacted by their processes in restricted regions of the deep gray and white matter of the forebrains of human infants. Such astrocytes may play a non-neural role in mediating increases in cerebral blood flow in response to local metabolic changes.     

Role of Rph3A in brain injury induced by experimental cerebral ischemia‐reperfusion model in rats

AimThe aim was to study the role of Rph3A in neuronal injury induced by cerebral ischemia‐reperfusion.MethodsThe protein and mRNA levels of Rph3A in penumbra were detected by Western blot. The localization of Rph3A in different cell types in penumbra was detected by immunofluorescence. Apoptosis in the brain was detected by TUNEL staining. We tested neurobehavioral evaluation using rotarod test, adhesive‐removal test, and Morris Water maze test. We examined the expression and localization of Rph3A in cultured neurons and astrocytes in vitro by Western blot and ELISA, respectively.ResultsThe mRNA and protein levels of Rph3A had significantly increased in brain penumbra of the rat MCAO/R model. Rph3A was mainly distributed in neurons and astrocytes and was significantly increased by MCAO/R. We downregulated Rph3A and found that it further worsened the cerebral infarct, neuronal death and behavioral, cognitive, and memory impairments in rats after MCAO/R. We also found that ischemia‐reperfusion upregulated the in vitro protein level and secretion of Rph3A in astrocytes but led to a decrease in the protein level of Rph3A in neurons.ConclusionThe increase in Rph3A in the brain penumbra may be an endogenous protective mechanism against ischemia‐reperfusion injury, which is mainly dominated by astrocytes.     

Mechanisms of lysophosphatidylcholine‐induced demyelination: A primary lipid disrupting myelinopathy

For decades lysophosphatidylcholine (LPC, lysolecithin) has been used to induce demyelination, without a clear understanding of its mechanisms. LPC is an endogenous lysophospholipid so it may cause demyelination in certain diseases. We investigated whether known receptor systems, inflammation or nonspecific lipid disruption mediates LPC‐demyelination in mice. We found that LPC nonspecifically disrupted myelin lipids. LPC integrated into cellular membranes and rapidly induced cell membrane permeability; in mice, LPC injury was phenocopied by other lipid disrupting agents. Interestingly, following its injection into white matter, LPC was cleared within 24 hr but by five days there was an elevation of endogenous LPC that was not associated with damage. This elevation of LPC in the absence of injury raises the possibility that the brain has mechanisms to buffer LPC. In support, LPC injury in culture was significantly ameliorated by albumin buffering. These results shed light on the mechanisms of LPC injury and homeostasis.     

Brain white matter microstructure is associated with susceptibility to motion‐induced nausea

Nausea is associated with significant morbidity, and there is a wide range in the propensity of individuals to experience nausea. The neural basis of the heterogeneity in nausea susceptibility is poorly understood. Our previous functional magnetic resonance imaging (fMRI) study in healthy adults showed that a visual motion stimulus caused activation in the right MT+/V5 area, and that increased sensation of nausea due to this stimulus was associated with increased activation in the right anterior insula. For the current study, we hypothesized that individual differences in visual motion‐induced nausea are due to microstructural differences in the inferior fronto‐occipital fasciculus (IFOF), the white matter tract connecting the right visual motion processing area (MT+/V5) and right anterior insula. To test this hypothesis, we acquired diffusion tensor imaging data from 30 healthy adults who were subsequently dichotomized into high and low nausea susceptibility groups based on the Motion Sickness Susceptibility Scale. We quantified diffusion along the IFOF for each subject based on axial diffusivity (AD); radial diffusivity (RD), mean diffusivity (MD) and fractional anisotropy (FA), and evaluated between‐group differences in these diffusion metrics. Subjects with high susceptibility to nausea rated significantly (P < 0.001) higher nausea intensity to visual motion stimuli and had significantly (P < 0.05) lower AD and MD along the right IFOF compared to subjects with low susceptibility to nausea. This result suggests that differences in white matter microstructure within tracts connecting visual motion and nausea‐processing brain areas may contribute to nausea susceptibility or may have resulted from an increased history of nausea episodes.     

17β‐estradiol protects against hypoxic/ischemic white matter damage in the neonatal rat brain

Developing oligodendrocytes (pre‐OLs) are highly vulnerable to hypoxic‐ischemic injury and associated excitotoxicity and oxidative stress. 17β‐Estradiol plays an important role in the development and function of the CNS and is neuroprotective. The sudden drop in circulating estrogen after birth may enhance the susceptibility of developing OLs to injury. Estrogen receptor (ER)–α and ER‐β are both expressed in OLs. We examined the effect of 17β‐estradiol on oxygen‐glucose deprivation and oxidative stress–induced cell death in rat pre‐OLs in vitro and on hypoxic‐ischemic brain injury in vivo. Pre‐OLs in culture were subjected to oxygen‐glucose deprivation (OGD) or glutathione depletion in the presence or absence of 17β‐estradiol. LDH release, the Alamar blue assay, and phase‐contrast microscopy were used to assess cell viability. Hypoxic‐ischemic injury was generated in 6‐day‐old rats (P6) by unilateral carotid ligation and hypoxia (6% O₂ for 1 hr). Rat pups received one intraperitoneal injection of 300 or 600 μg/kg 17β‐estradiol or vehicle 12 hr prior to the surgical procedure. Injury was assessed by myelin basic protein (MBP) immunocytochemistry at P10. 17β‐Estradiol produced significant protection against OGD‐induced cell death in primary OLs (EC₅₀ = 1.3 ± 0.46 × 10^?9 M) and against oxidative stress. Moreover, 17β‐estradiol attenuated the loss of MBP labeling in P10 pups ipsilateral to the carotid ligation. These results suggest a potential role for estrogens in attenuation of hypoxic‐ischemic and oxidative injury to developing OLs and in the prevention of periventricular leukomalacia. © 2009 Wiley‐Liss, Inc.     

Gliovascular changes precede white matter damage and long‐term disorders in juvenile mild closed head injury

Traumatic brain injury (TBI) is a leading cause of hospital visits in pediatric patients and often leads to long‐term disorders even in cases of mild severity. White matter (WM) alterations are commonly observed in patients months or years after the injury assessed by magnetic resonance imaging (MRI), but little is known about WM pathophysiology early after mild pediatric TBI. To evaluate the status of the gliovascular unit in this context, mild TBI was induced in postnatal‐day 17 mice using a closed head injury model with two grades of severity (G1, G2). G2 resulted in significant WM edema (increased T2‐signal) and BBB damage (IgG‐extravasation immunostaining) whereas decreased T2 and the increased levels of astrocytic water‐channel AQP4 were observed in G1 mice 1 day post‐injury. Both severities induced astrogliosis (GFAP immunolabeling). No changes in myelin and neurofilament were detected at this acute time point. One month after injury G2 mice exhibited diffusion tensor imaging MRI alterations (decreased fractional anisotropy) accompanied by decreased neurofilament staining in the WM. Both severities induced behavioral impairments at this time point. In conclusion, long‐term deficits and WM changes similar to those found after clinical TBI are preceded by distinct early gliovascular phenotype alterations after juvenile mild TBI, revealing AQP4 as a potential candidate for severity‐based treatments.     

Protective effects of carnosine on white matter damage induced by chronic cerebral hypoperfusion

Carnosine is a dipeptide that scavenges free radicals,inhibits inflammation in the central nervous system,and protects against ischemic and hypoxic brain damage through its anti-oxidative and anti-apoptotic actions.Therefore,we hypothesized that carnosine would also protect against white matter damage caused by subcortical ischemic injury.White matter damage was induced by right unilateral common carotid artery occlusion in mice.The animals were treated with 200,500 or 750 mg/kg carnosine by intraperitoneal injection 30 minutes before injury and every other day after injury.Then,37 days later,Klüver-Barrera staining,toluidine blue staining and immunofluorescence staining were performed.Carnosine(200,500 mg/kg) substantially reduced damage to the white matter in the corpus callosum,internal capsule and optic tract,and it rescued expression of myelin basic protein,and alleviated the loss of oligodendrocytes.However,carnosine at the higher dose of 750 mg/kg did not have the same effects as the 200 and 500 mg/kg doses.These findings show that carnosine,at a particular dose range,protects against white matter damage caused by chronic cerebral ischemia in mice,likely by reducing oligodendroglial cell loss.     

Comparing MRI metrics to quantify white matter microstructural damage in multiple sclerosis

Quantifying white matter damage in vivo is becoming increasingly important for investigating the effects of neuroprotective and repair strategies in multiple sclerosis (MS). While various approaches are available, the relationship between MRI‐based metrics of white matter microstructure in the disease, that is, to what extent the metrics provide complementary versus redundant information, remains largely unexplored. We obtained four microstructural metrics from 123 MS patients: fractional anisotropy (FA), radial diffusivity (RD), myelin water fraction (MWF), and magnetisation transfer ratio (MTR). Coregistration of maps of these four indices allowed quantification of microstructural damage through voxel‐wise damage scores relative to healthy tissue, as assessed in a group of 27 controls. We considered three white matter tissue‐states, which were expected to vary in microstructural damage: normal appearing white matter (NAWM), T2‐weighted hyperintense lesional tissue without T1‐weighted hypointensity (T2L), and T1‐weighted hypointense lesional tissue with corresponding T2‐weighted hyperintensity (T1L). All MRI indices suggested significant damage in all three tissue‐states, the greatest damage being in T1L. The correlations between indices ranged from r = 0.18 to r = 0.87. MWF was most sensitive when differentiating T2L from NAWM, while MTR was most sensitive when differentiating T1L from NAWM and from T2L. Combining the four metrics into one, through a principal component analysis, did not yield a measure more sensitive to damage than any single measure. Our findings suggest that the metrics are (at least partially) correlated with each other, but sensitive to the different aspects of pathology. Leveraging these differences could be beneficial in clinical trials testing the effects of therapeutic interventions.     

Oligodendrogenesis is differentially regulated in gray and white matter of jimpy mice

The factors that regulate oligodendrogenesis have been studied extensively in optic nerve, where oligodendrocyte production and myelination quickly follow colonization of the nerve by progenitor cells. In contrast, oligodendrocyte production in the cerebral cortex begins approximately 1 week after progenitor cell colonization and continues for 3-4 weeks. This and other observations raise the possibility that oligodendrogenesis is regulated by different mechanisms in white and gray matter. The present study examined oligodendrocyte production in the developing cerebral cortex of jimpy (jp) and jimpy(msd) (msd) mice, which exhibit hypomyelination and oligodendrocyte death due to mutations in and toxic accumulations of proteolipid protein, the major structural protein of CNS myelin. Proliferation of oligodendrocyte progenitors and production of myelinating oligodendrocytes was reduced in jp cerebral cortex when compared to wild-type (wt) and msd mice. The incidence of oligodendrocyte cell death was similar in jp and msd cortex, but total dying oligodendrocytes were greater in msd. We confirm previous reports of increased oligodendrocyte production in white matter of both jp and msd mice. The jp mutation, therefore, reduces oligodendrocyte production in cerebral cortex but not in white matter. These data provide additional evidence that oligodendrogenesis is differentially regulated in white matter and gray matter and implicate PLP/DM20 as a modulator of these differences.