Oligodendrocyte-selective deletion of the eIF2α kinase Perk/Eif2ak3 limits functional recovery after spinal cord injury.

After spinal cord injury (SCI), re-establishing cellular homeostasis is critical to optimize functional recovery. Central to that response is PERK signaling, which ultimately initiates a pro-apoptotic response if cellular homeostasis cannot be restored. Oligodendrocyte (OL) loss and white matter damage drive functional consequences and determine recovery potential after thoracic contusive SCI. We examined acute (<48 h post-SCI) and chronic (6 weeks post-SCI) effects of conditionally deleting Perk from OLs prior to SCI. While Perk transcript is expressed in many types of cells in the adult spinal cord, its levels are disproportionately high in OL lineage cells. Deletion of OL-Perk prior to SCI resulted in: (1) enhanced acute phosphorylation of eIF2α, a major PERK substrate and the critical mediator of the integrated stress response (ISR), (2) enhanced acute expression of the downstream ISR genes Atf4, Ddit3/Chop, and Tnfrsf10b/Dr5, (3) reduced acute OL lineage-specific Olig2 mRNA, but not neuronal or astrocytic mRNAs, (4) chronically decreased OL content in the spared white matter at the injury epicenter, (5) impaired hindlimb locomotor recovery, and (6) reduced chronic epicenter white matter sparing. Cultured primary OL precursor cells with reduced PERK expression and activated ER stress response showed: (1) unaffected phosphorylation of eIF2α, (2) enhanced ISR gene induction, and (3) increased cytotoxicity. Therefore, OL-Perk deficiency exacerbates ISR signaling and potentiates white matter damage after SCI. The latter effect is likely mediated by increased loss of Perk-/- OLs.

Oligodendrocyte-specific deletion of Xbp1 exacerbates the endoplasmic reticulum stress response and restricts locomotor recovery after thoracic spinal cord injury.

The endoplasmic reticulum stress response (ERSR) is activated in various neurodegenerative diseases and/or after CNS traumatic injuries. The ERSR is comprised of three major arms, PERK, IRE-1, and activating transcription factor-6, with the latter two contributing to the unfolded protein response (UPR). PERK activity overlaps with the integrated stress response (ISR) kinases, PKR, HRI, and GCN2 which all signal through, eukaryotic initiation factor 2α, ATF4, and CHOP. All initially attempt to restore endoplasmic reticulum (ER) homeostasis, but if ER stress is unresolved, ATF4/CHOP-mediated cell death is initiated. Here, we investigate the contribution of the inositol-requiring protein-1α-X-box binding protein-1 (XBP1)-mediated UPR signaling pathway to the pathogenesis of spinal cord injury (SCI). We demonstrate that deletion of Xbp1 caused an exacerbated ATF4/CHOP signaling in cultured mouse oligodendrocyte (OL) progenitor cells and enhanced their sensitivity to ER stress. Similar effects were also observed with the Xbp1 pathway inhibitor toyocamycin. Furthermore, OL lineage-specific loss of Xbp1 resulted in enhanced ISR in mice that underwent moderate contusive SCI at the T9 level. Consistently, post-injury recovery of hindlimb locomotion and white matter sparing were reduced in OL Xbp1-deficient mice, which correlated with chronically decreased relative density of OPCs and OLs at the injury epicenter at 6 weeks post-SCI. We conclude that the IRE1-XBP1-mediated UPR signaling pathway contributes to restoration of ER homeostasis in OLs and is necessary for enhanced white matter sparing and functional recovery post-SCI.

Blocking Autophagy in Oligodendrocytes Limits Functional Recovery after Spinal Cord Injury.

Autophagy mechanisms are well documented in neurons after spinal cord injury (SCI), but the direct functional role of autophagy in oligodendrocyte (OL) survival in SCI pathogenesis remains unknown. Autophagy is an evolutionary conserved lysosomal-mediated catabolic pathway that ensures degradation of dysfunctional cellular components to maintain homeostasis in response to various forms of stress, including nutrient deprivation, hypoxia, reactive oxygen species, DNA damage, and endoplasmic reticulum (ER) stress. Using pharmacological gain and loss of function and genetic approaches, we investigated the contribution of autophagy in OL survival and its role in the pathogenesis of thoracic contusive SCI in female mice. Although upregulation of Atg5 (an essential autophagy gene) occurs after SCI, autophagy flux is impaired. Purified myelin fractions of contused 8 d post-SCI samples show enriched protein levels of LC3B, ATG5, and BECLIN 1. Data show that, while the nonspecific drugs rapamycin (activates autophagy) and spautin 1 (blocks autophagy) were pharmacologically active on autophagy in vivo, their administration did not alter locomotor recovery after SCI. To directly analyze the role of autophagy, transgenic mice with conditional deletion of Atg5 in OLs were generated. Analysis of hindlimb locomotion demonstrated a significant reduction in locomotor recovery after SCI that correlated with a greater loss in spared white matter. Immunohistochemical analysis demonstrated that deletion of Atg5 from OLs resulted in decreased autophagic flux and was detrimental to OL function after SCI. Thus, our study provides evidence that autophagy is an essential cytoprotective pathway operating in OLs and is required for hindlimb locomotor recovery after thoracic SCI.SIGNIFICANCE STATEMENT This study describes the role of autophagy in oligodendrocyte (OL) survival and pathogenesis after thoracic spinal cord injury (SCI). Modulation of autophagy with available nonselective drugs after thoracic SCI does not affect locomotor recovery despite being pharmacologically active in vivo, indicating significant off-target effects. Using transgenic mice with conditional deletion of Atg5 in OLs, this study definitively identifies autophagy as an essential homeostatic pathway that operates in OLs and exhibits a direct functional role in SCI pathogenesis and recovery. Therefore, this study emphasizes the need to discover novel autophagy-specific drugs that specifically modulate autophagy for further investigation for clinical translation to treat SCI and other CNS pathologies related to OL survival.

Autophagy is essential for oligodendrocyte differentiation, survival, and proper myelination.

Deficient myelination, the spiral wrapping of highly specialized membrane around axons, causes severe neurological disorders. Maturation of oligodendrocyte progenitor cells (OPC) to myelinating oligodendrocytes (OL), the sole providers of central nervous system (CNS) myelin, is tightly regulated and involves extensive morphological changes. Here, we present evidence that autophagy, the targeted isolation of cytoplasm and organelles by the double-membrane autophagosome for lysosomal degradation, is essential for OPC/OL differentiation, survival, and proper myelin development. A marked increase in autophagic activity coincides with OL differentiation, with OL processes having the greatest increase in autophagic flux. Multiple lines of evidence indicate that autophagosomes form in developing myelin sheathes before trafficking from myelin to the OL soma. Mice with conditional OPC/OL-specific deletion of the essential autophagy gene Atg5 beginning on postnatal Day 5 develop a rapid tremor and die around postnatal Day 12. Further analysis revealed apoptotic death of OPCs, reduced differentiation, and reduced myelination. Surviving Atg5-/- OLs failed to produce proper myelin structure. In vitro, pharmacological inhibition of autophagy in OPC/dorsal root ganglion (DRG) co-cultures blocked myelination, producing OLs surrounded by many short processes. Conversely, autophagy stimulation enhanced myelination. These results implicate autophagy as a key regulator of OPC survival, maturation, and proper myelination. Autophagy may provide an attractive target to promote both OL survival and subsequent myelin repair after injury.

Remyelinating Oligodendrocyte Precursor Cell miRNAs from the Sfmbt2 Cluster Promote Cell Cycle Arrest and Differentiation.

Oligodendrocyte (OL) loss contributes to the functional deficits underlying diseases with a demyelinating component. Remyelination by oligodendrocyte progenitor cells (OPCs) can restore these deficits. To understand the role that microRNAs (miRNAs) play in remyelination, 2',3'-cyclic-nucleotide 3'-phosphodiesterase-EGFP(+) mice were treated with cuprizone, and OPCs were sorted from the corpus callosum. Microarray analysis revealed that Sfmbt2 family miRNAs decreased during cuprizone treatment. One particular Sfmbt2 miRNA, miR-297c-5p, increased during mouse OPC differentiation in vitro and during callosal development in vivo. When overexpressed in both mouse embryonic fibroblasts and rat OPCs (rOPCs), cell cycle analysis revealed that miR-297c-5p promoted G1/G0 arrest. Additionally, miR-297c-5p transduction increased the number of O1(+) rOPCs during differentiation. Luciferase reporter assays confirmed that miR-297c-5p targets cyclin T2 (CCNT2), the regulatory subunit of positive transcription elongation factor b, a complex that inhibits OL maturation. Furthermore, CCNT2-specific knockdown promoted rOPC differentiation while not affecting cell cycle status. Together, these data support a dual role for miR-297c-5p as both a negative regulator of OPC proliferation and a positive regulator of OL maturation via its interaction with CCNT2. SIGNIFICANCE STATEMENT: This work describes the role of oligodendrocyte progenitor cell (OPC) microRNAs (miRNAs) during remyelination and development in vivo and differentiation in vitro. This work highlights the importance of miRNAs to OPC biology and describes miR-297c-5p, a novel regulator of OPC function. In addition, we identified CCNT2 as a functional target, thus providing a mechanism by which miR-297c-5p imparts its effects on differentiation. These data are important, given our lack of understanding of OPC miRNA regulatory networks and their potential clinical value. Therefore, efforts to understand the role of miR-297c-5p in pathological conditions and its potential for facilitating repair may provide future therapeutic strategies to treat demyelination.

Reduced Expression of Oligodendrocyte Linage-Enriched Transcripts During the Endoplasmic Reticulum Stress/Integrated Stress Response.

Endoplasmic reticulum (ER) stress in oligodendrocyte (OL) linage cells contributes to several CNS pathologies including traumatic spinal cord injury (SCI) and multiple sclerosis. Therefore, primary rat OL precursor cell (OPC) transcriptomes were analyzed using RNASeq after treatments with two ER stress-inducing drugs, thapsigargin (TG) or tunicamycin (TM). Gene ontology term (GO) enrichment showed that both drugs upregulated mRNAs associated with the general stress response. The GOs related to ER stress were only enriched for TM-upregulated mRNAs, suggesting greater ER stress selectivity of TM. Both TG and TM downregulated cell cycle/cell proliferation-associated transcripts, indicating the anti-proliferative effects of ER stress. Interestingly, many OL lineage-enriched mRNAs were downregulated, including those for transcription factors that drive OL identity such as Olig2. Moreover, ER stress-associated decreases of OL-specific gene expression were found in mature OLs from mouse models of white matter pathologies including contusive SCI, toxin-induced demyelination, and Alzheimer's disease-like neurodegeneration. Taken together, the disrupted transcriptomic fingerprint of OL lineage cells may facilitate myelin degeneration and/or dysfunction when pathological ER stress persists in OL lineage cells.

The ER stress response compromises the transcriptomic identity of the OL lineage. Therefore, persistent, pathological ER stress may have a negative impact on structural and/or functional integrity of the white matter.

Acute Pharmacological Inhibition of Protein Kinase R-Like Endoplasmic Reticulum Kinase Signaling After Spinal Cord Injury Spares Oligodendrocytes and Improves Locomotor Recovery.

Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is a major signal transducer of the endoplasmic reticulum stress response (ERSR) pathway. Outcomes of PERK activation range from abrogating ER stress to induction of cell death, dependent on its level, duration, and cellular context. Current data demonstrate that after mouse spinal cord injury (SCI), acute inhibition of PERK (0-72 h) with the small molecule inhibitor GSK2656157 reduced ERSR while improving white matter sparing and hindlimb locomotion recovery. GSK2656157-treated mice showed increased numbers of oligodendrocytes at the injury epicenter. Moreover, GSK2656157 protected cultured primary mouse oligodendrocyte precursor cells from ER stress-induced cytotoxicity. These findings suggest that in the context of SCI, excessive acute activation of PERK contributes to functionally relevant white matter damage. Pharmacological inhibition of PERK is a potential strategy to protect central nervous system (CNS) white matter following acute injuries, including SCI.

Transplantation of ciliary neurotrophic factor-expressing adult oligodendrocyte precursor cells promotes remyelination and functional recovery after spinal cord injury.

Demyelination contributes to the dysfunction after traumatic spinal cord injury (SCI). We explored whether the combination of neurotrophic factors and transplantation of adult rat spinal cord oligodendrocyte precursor cells (OPCs) could enhance remyelination and functional recovery after SCI. Ciliary neurotrophic factor (CNTF) was the most effective neurotrophic factor to promote oligodendrocyte (OL) differentiation and survival of OPCs in vitro. OPCs were infected with retroviruses expressing enhanced green fluorescent protein (EGFP) or CNTF and transplanted into the contused adult thoracic spinal cord 9 d after injury. Seven weeks after transplantation, the grafted OPCs survived and integrated into the injured spinal cord. The survival of grafted CNTF-OPCs increased fourfold compared with EGFP-OPCs. The grafted OPCs differentiated into adenomatus polyposis coli (APC(+)) OLs, and CNTF significantly increased the percentage of APC(+) OLs from grafted OPCs. Immunofluorescent and immunoelectron microscopic analyses showed that the grafted OPCs formed central myelin sheaths around the axons in the injured spinal cord. The number of OL-remyelinated axons in ventrolateral funiculus (VLF) or lateral funiculus (LF) at the injured epicenter was significantly increased in animals that received CNTF-OPC grafts compared with all other groups. Importantly, 75% of rats receiving CNTF-OPC grafts recovered transcranial magnetic motor-evoked potential and magnetic interenlargement reflex responses, indicating that conduction through the demyelinated axons in VLF or LF, respectively, was partially restored. More importantly, recovery of hindlimb locomotor function was significantly enhanced in animals receiving grafts of CNTF-OPCs. Thus, combined treatment with OPC grafts expressing CNTF can enhance remyelination and facilitate functional recovery after traumatic SCI.

Dual-Viral Transduction Utilizing Highly Efficient Retrograde Lentivirus Improves Labeling of Long Propriospinal Neurons.

The nervous system coordinates pathways and circuits to process sensory information and govern motor behaviors. Mapping these pathways is important to further understand the connectivity throughout the nervous system and is vital for developing treatments for neuronal diseases and disorders. We targeted long ascending propriospinal neurons (LAPNs) in the rat spinal cord utilizing Fluoro-Ruby (FR) [10kD rhodamine dextran amine (RDA)], and two dual-viral systems. Dual-viral tracing utilizing a retrograde adeno-associated virus (retroAAV), which confers robust labeling in the brain, resulted in a small number of LAPNs being labeled, but dual-viral tracing using a highly efficient retrograde (HiRet) lentivirus provided robust labeling similar to FR. Additionally, dual-viral tracing with HiRet lentivirus and tracing with FR may preferentially label different subpopulations of LAPNs. These data demonstrate that dual-viral tracing in the spinal cord employing a HiRet lentivirus provides robust and specific labeling of LAPNs and emphasizes the need to empirically optimize viral systems to target specific neuronal population(s).

Enhanced induction of RPE lineage markers in pluripotent neural stem cells engrafted into the adult rat subretinal space.

PURPOSE: To investigate the differentiation of rat neural stem cells (rNSCs) into cells of retinal pigment epithelial (RPE) lineage both in vitro and in vivo, after subretinal transplantation into normal rats and in a sodium iodate (NaIO(3)) model of RPE loss. METHODS: rNSCs prepared from the cortex of embryonic day (E)14 Fisher F344 rats were cocultured with different concentrations of vasoactive intestinal peptide (VIP), adult rat RPE cells, or neurosensory retina (NSR) for 5 days. Cell morphology and expression of RPE-specific markers (cytokeratin, CD68, microphthalmia-inducing transcription factor [MITF]) were studied. Additional antibodies used to characterize the rNSCs were markers for stem cells (nestin), immature neurons (betaIII-tubulin), astrocytes (glial fibrillary acidic protein [GFAP]), and oligodendrocytes (Rip). In in vivo studies, 10(6) green fluorescent protein [GFP]-labeled rNSCs were injected subretinally in either normal adult Lewis rats or NaIO(3)-treated rats (70 mg/mL NaIO(3) administered intravenously 7 days before transplantation). RESULTS: In vitro VIP-treated rNSCs changed from round cells to glia-like cells with processes that stained for both GFAP and nestin. In addition, small clusters of flattened, polygonal cells with an epithelial-cell-like shape that stained for cytokeratin and CD68 were observed. Coculture of rNSCs with RPE cells, but not with NSR, also led to cells of this phenotype. After transplantation, nestin(+) and GFP(+) rNSCs were visible subretinally as a transplant. In addition, more than 50% of transplanted rNSCs were cytokeratin(+) and CD68(+). CONCLUSIONS: Very few rNSCs differentiate in vitro into epithelial-like cells that express RPE-specific markers. In vivo, this differentiation is remarkably enhanced after subretinal engraftment. Thus, transplantation of NSCs into the subretinal space may be a therapy for retinal diseases involving an RPE abnormality.

Oligodendrocyte precursor cells differentially expressing Nogo-A but not MAG are more permissive to neurite outgrowth than mature oligodendrocytes.

Grafting oligodendrocyte precursor cells (OPCs) has been used as a strategy to repair demyelination of the central nervous system (CNS). Whether OPCs can promote CNS axonal regeneration remains to be tested. If so, they should be permissive to axonal growth and may express less inhibitory molecules on their surface. Here we examined the expression of two oligodendrocyte-associated myelin inhibitors Nogo-A and myelin-associated glycoprotein (MAG) during oligodendrogliogenesis and tested their abilities to promote neurite outgrowth in vitro. Whereas the intracellular domain of Nogo-A was consistently expressed throughout oligodendrocyte differentiation, MAG was expressed only at later stages. Furthermore, the membrane-associated extracellular domain of Nogo-A was not expressed in OPCs but expressed in mature oligodendrocytes. In a dorsal root ganglion (DRG) and OPC/oligodendrocyte co-culture model, significantly greater DRG neurite outgrowth onto OPC monolayer than mature oligodendrocyte was found (1042+/-123 vs. 717+/-342 micrometer; p=0.011). Moreover, DRG neurites elongated as fasciculated fiber tracts and contacted directly on OPCs (133+/-37 cells/fascicle). In contrast, few, if any, direct contacts were found between DRG neurites and mature oligodendrocytes (5+/-3 cells/fascicle, p<0.001). In fact, acellular spaces were found between neurites and surrounding mature oligodendrocytes in contrast to the lack of such spaces in OPC/DRG coculture (51.1+/-16.5 vs. 2.4+/-3.9 micrometer; p<0.001). Thus, OPCs expressing neither extracellular domain of Nogo-A nor MAG are significantly more permissive than mature oligodendrocytes expressing both. Grafting OPCs may thus represent a feasible strategy to foster CNS axonal regeneration.

Gene delivery to the spinal cord: comparison between lentiviral, adenoviral, and retroviral vector delivery systems.

Viral gene delivery for spinal cord injury (SCI) is a promising approach for enhancing axonal regeneration and neuroprotection. An understanding of spatio-temporal transgene expression in the spinal cord is essential for future studies of SCI therapies. Commonly, intracellular marker proteins (e.g., EGFP) were used as indicators of transgene levels after viral delivery, which may not accurately reflect levels of secreted transgene. This study examined transgene expression using ELISA after viral delivery of D15A, a neurotrophin with BDNF and NT-3 activities, at 1, 2, and 4weeks after in vivo and ex vivo delivery using lentiviral, adenoviral, and retroviral vectors. Further, the inflammatory responses and viral infection patterns after in vivo delivery were examined. Lentiviral vectors had the most stable pattern of gene expression, with D15A levels of 536 +/- 38 and 363 +/- 47 pg/mg protein seen at 4 weeks after the in vivo and ex vivo delivery, respectively. Our results show that protein levels downregulate disproportionately to levels of EGFP after adenoviral vectors both in vivo and ex vivo. D15A dropped from initial levels of 422 +/- 87 to 153 +/- 18 pg/mg protein at 4 weeks after in vivo administration. Similarly, ex vivo retrovirus-mediated transgene expression exhibited rapid downregulation by 2 weeks post-grafting. Compared to adenoviral infection, macrophage activation was attenuated after lentiviral infection. These results suggest that lentiviral vectors are most suitable in situations where stable long-term transgene expression is needed. Retroviral ex vivo delivery is optional when transient expression within targeted spinal tissue is desired, with adenoviral vectors in between.

Consequences of noggin expression by neural stem, glial, and neuronal precursor cells engrafted into the injured spinal cord.

Bone morphogenetic proteins (BMPs) are a large class of secreted factors, which serve as modulators of development in multiple organ systems, including the CNS. Studies investigating the potential of stem cell transplantation for restoration of function and cellular replacement following traumatic spinal cord injury (SCI) have demonstrated that the injured adult spinal cord is not conducive to neurogenesis or oligodendrogenesis of engrafted CNS precursors. In light of recent findings that BMP expression is modulated by SCI, we hypothesized that they may play a role in lineage restriction of multipotent grafts. To test this hypothesis, neural stem or precursor cells were engineered to express noggin, an endogenous antagonist of BMP action, prior to transplantation or in vitro challenge with recombinant BMPs. Adult rats were subjected to both contusion and focal ischemic SCI. One week following injury, the animals were transplanted with either EGFP- or noggin-expressing neural stem or precursor cells. Results demonstrate that noggin expression does not antagonize terminal astroglial differentiation in the engrafted stem cells. Furthermore, neutralizing endogenous BMP in the injured spinal cord significantly increased both the lesion volume and the number of infiltrating macrophages in injured spinal cords receiving noggin-expressing stem cell grafts compared with EGFP controls. These data strongly suggest that endogenous factors in the injured spinal microenvironment other than the BMPs restrict the differentiation of engrafted pluripotent neural stem cells as well as suggest other roles for BMPs in tissue protection in the injured CNS.

Differentiation of engrafted neuronal-restricted precursor cells is inhibited in the traumatically injured spinal cord.

Differentiation of pluripotent neural stem cells engrafted into the adult normal and injured spinal cord is restricted to the glial lineage, suggesting that in vitro induction toward a neuronal lineage prior to transplantation and/or modification of the host environment may be necessary to initiate and increase the differentiation of neurons. In the present study, we investigated the differentiation of neuronal-restricted precursors (NRPs) grafted into the normal and contused adult rat spinal cord. NRPs proliferated through multiple passages in the presence of FGF2 and NT3 and differentiated into only neurons in vitro in the presence of retinoic acid and the absence of FGF2. Differentiated NRPs expressed GABA, glycine, glutamate, and ChAT. Two weeks to 2 months after engraftment of undifferentiated NRPs into adult normal spinal cord, large numbers of surviving cells were seen in all of the animals. The majority differentiated into betaIII-tubulin-positive neurons. Some transplanted NRPs expressed GABA and small numbers were glutamate- and ChAT-positive. NRPs were also transplanted into the epicenter of the contused adult rat spinal cord. Two weeks to 2 months after transplantation, some engrafted NRPs remained undifferentiated nestin-positive cells. Small numbers were MAP2- or betaIII-tubulin-positive neurons. However, the expression of GABA, glutamate, or ChAT was not observed. These results show that NRPs can differentiate into different types of neurons in the normal adult rat spinal cord, but that such differentiation is inhibited in the injured spinal cord. Manipulation of the microenvironment in the injured spinal cord will likely be necessary to facilitate neuronal replacement.