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.

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.

Hypoxia-inducible factor prolyl hydroxylase domain (PHD) inhibition after contusive spinal cord injury does not improve locomotor recovery.

Traumatic spinal cord injury (SCI) is a devastating neurological condition that involves both primary and secondary tissue loss. Various cytotoxic events including hypoxia, hemorrhage and blood lysis, bioenergetic failure, oxidative stress, endoplasmic reticulum (ER) stress, and neuroinflammation contribute to secondary injury. The HIF prolyl hydroxylase domain (PHD/EGLN) family of proteins are iron-dependent, oxygen-sensing enzymes that regulate the stability of hypoxia inducible factor-1α (HIF-1α) and also mediate oxidative stress caused by free iron liberated from the lysis of blood. PHD inhibition improves outcome after experimental intracerebral hemorrhage (ICH) by reducing activating transcription factor 4 (ATF4)-driven neuronal death. As the ATF4-CHOP (CCAAT-enhancer-binding protein homologous protein) pathway plays a role in the pathogenesis of contusive SCI, we examined the effects of PHD inhibition in a mouse model of moderate T9 contusive SCI in which white matter damage is the primary driver of locomotor dysfunction. Pharmacological inhibition of PHDs using adaptaquin (AQ) moderately lowers acute induction of Atf4 and Chop mRNAs and prevents the acute decline of oligodendrocyte (OL) lineage mRNAs, but does not improve long-term recovery of hindlimb locomotion or increase chronic white matter sparing. Conditional genetic ablation of all three PHD isoenzymes in OLs did not affect Atf4, Chop or OL mRNAs expression levels, locomotor recovery, and white matter sparing after SCI. Hence, PHDs may not be suitable targets to improve outcomes in traumatic CNS pathologies that involve acute white matter injury.

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.

Acute Neural and Proteostasis Messenger Ribonucleic Acid Levels Predict Chronic Locomotor Recovery after Contusive Spinal Cord Injury.

One of the difficulties in identifying novel therapeutic strategies to manage central nervous system (CNS) trauma is the need for behavioral assays to assess chronic functional recovery. In vitro assays and/or acute behavioral assessments cannot accurately predict long-term functional outcome. Using data from 13 independent T9 moderate contusive spinal cord injury (SCI) studies, we asked whether the ratio of acute (24-72 h post-injury) changes in the levels of neuron-, oligodendrocyte-, astrocyte-specific and/or endoplasmic reticulum stress response (ERSR) messenger ribonucleic acids (mRNAs) could predict the extent of chronic functional recovery. Increased levels of neuron, oligodendrocyte, and astrocyte mRNAs all correlated with enhanced Basso Mouse Scale (BMS) scores. Reduced levels of the ERSR mRNAs Atf4 and Chop correlate with improved chronic locomotor function. Neither neural or ERSR mRNAs were predictive for chronic recovery across all behavioral changes. The ratio of oligodendrocyte/ERSR mRNAs, however, did predict "improved," "no change," or "worse" functional recovery. Neuronal/ERSR mRNA ratios predicted functional improvement, but could not distinguish between worse or no change outcomes. Astrocyte/ERSR mRNA ratios were not predictive. This approach can be used to confirm biological action of injected drugs in vivo and to optimize dose and therapeutic window. It may prove useful in cervical and lumbar SCI and in other traumatic CNS injuries such as traumatic brain injury and stroke, where prevention of neuronal loss is paramount to functional recovery. Although the current analysis was directed toward ERSR whose activity was targeted in all but one study, acute mRNA markers for other pathophysiological cascades may be as predictive of chronic recovery when those cascades are targeted for neuroprotection.

Improved locomotor recovery after contusive spinal cord injury in Bmal1-/- mice is associated with protection of the blood spinal cord barrier.

The transcription factor BMAL1/ARNTL is a non-redundant component of the clock pathway that regulates circadian oscillations of gene expression. Loss of BMAL1 perturbs organismal homeostasis and usually exacerbates pathological responses to many types of insults by enhancing oxidative stress and inflammation. Surprisingly, we observed improved locomotor recovery and spinal cord white matter sparing in Bmal1-/- mice after T9 contusive spinal cord injury (SCI). While acute loss of neurons and oligodendrocytes was unaffected, Bmal1 deficiency reduced the chronic loss of oligodendrocytes at the injury epicenter 6 weeks post SCI. At 3 days post-injury (dpi), decreased expression of genes associated with cell proliferation, neuroinflammation and disruption of the blood spinal cord barrier (BSCB) was also observed. Moreover, intraspinal extravasation of fibrinogen and immunoglobulins was decreased acutely at dpi 1 and subacutely at dpi 7. Subacute decrease of hemoglobin deposition was also observed. Finally, subacutely reduced levels of the leukocyte marker CD45 and even greater reduction of the pro-inflammatory macrophage receptor CD36 suggest not only lower numbers of those cells but also their reduced inflammatory potential. These data indicate that Bmal1 deficiency improves SCI outcome, in part by reducing BSCB disruption and hemorrhage decreasing cytotoxic neuroinflammation and attenuating the chronic loss of oligodendrocytes.

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.

Following spinal cord injury, PDE4B drives an acute, local inflammatory response and a chronic, systemic response exacerbated by gut dysbiosis and endotoxemia.

Emerging evidence links changes in the gut microbiome and intestinal barrier function to alterations in CNS function. We examined the role of endotoxin-responsive, cAMP-specific, Pde4 subfamily b (Pde4b) enzyme in gut dysbiosis induced neuro-inflammation and white matter loss following spinal cord injury (SCI). Using a thoracic contusion model in C57Bl/6 wild type female mice, SCI led to significant shifts in the gut bacterial community including an increase in the phylum Proteobacteria, which consists of endotoxin-harboring, gram-negative bacteria. This was accompanied by increased systemic inflammatory marker, soluble CD14, along with markers of the endoplasmic reticulum stress response (ERSR) and inflammation in the SCI epicenter. Deletion of Pde4b reduced epicenter expression of markers for the ERSR and inflammation, at both acute and chronic time points post-SCI. Correspondingly, expression of oligodendrocyte mRNAs increased. Within the injury penumbra, inflammatory protein markers of activated astrocytes (GFAP), macrophage/microglia (CD11b, Iba1), and the proinflammatory mediator Cox2, were decreased in Pde4b-/- mice. The absence of Pde4b improved white matter sparing and recovery of hindlimb locomotion following injury. Importantly, SCI-induced gut dysbiosis, bacterial overgrowth and endotoxemia were also prevented in Pde4b-/- mice. Taken together, these findings indicate that PDE4B plays an important role in the development of acute and chronic inflammatory response and consequent recovery following 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.

Pharmacological inhibition of spinal cord injury-stimulated ribosomal biogenesis does not affect locomotor outcome.

After unresolved endoplasmic reticulum stress, recovery of protein synthesis including increased expression of ribosomal components and translation factors may induce cell death. Using a mouse model of moderate contusive spinal cord injury (SCI) at the T9 level, upregulation of ribosomal biogenesis was observed in the injury epicenter at 24h after trauma. Such upregulation coincided with endoplasmic reticulum stress response as previously reported in this model. It was also accompanied by changes in expression of many other genes associated with translational regulation. Systemic treatment with a pharmacological inhibitor of RNA-Polymerase-1, BMH-21 reduced rRNA transcription in the spinal cord. Moreover, in the injury epicenter, treatment with BMH-21 increased expression of oligodendrocyte-specific transcripts including Mbp and Cldn11 at 3days post injury. Although such findings may suggest at least transient reduction of oligodendrocyte death, locomotor outcome was mostly unaffected except slightly accelerated recovery of hindlimb function at week 2 post-injury. Therefore, at least in mice, RNA-Polymerase-1 does not appear to be a robust target for therapies to protect spinal cord tissue after contusion. However, these findings raise an interesting possibility that altered rate of ribosomal biogenesis contributes to the apparent translational reprogramming after contusive SCI. Such a reprogramming could be a major regulator of SCI-induced gene expression.

CD36 deletion improves recovery from spinal cord injury.

CD36 is a pleiotropic receptor involved in several pathophysiological conditions, including cerebral ischemia, neurovascular dysfunction and atherosclerosis, and recent reports implicate its involvement in the endoplasmic reticulum stress response (ERSR). We hypothesized that CD36 signaling contributes to the inflammation and microvascular dysfunction following spinal cord injury. Following contusive injury, CD36(-/-) mice demonstrated improved hindlimb functional recovery and greater white matter sparing than CD36(+/+) mice. CD36(-/-) mice exhibited a reduced macrophage, but not neutrophil, infiltration into the injury epicenter. Fewer infiltrating macrophages were either apoptotic or positive for the ERSR marker, phospho-ATF4. CD36(-/-) mice also exhibited significant improvements in injury heterodomain vascularity and function. These microvessels accumulated less of the oxidized lipid product 4-hydroxy-trans-2-nonenal (4HNE) and exhibited a reduced ERSR, as detected by vascular phospho-ATF4, CHOP and CHAC-1 expression. In cultured primary endothelial cells, deletion of CD36 diminished 4HNE-induced phospho-ATF4 and CHOP expression. A reduction in phospho-eIF2α and subsequent increase in KDEL-positive, ER-localized proteins suggest that 4HNE-CD36 signaling facilitates the detection of misfolded proteins upstream of eIF2α phosphorylation, ultimately leading to CHOP-induced apoptosis. We conclude that CD36 deletion modestly, but significantly, improves functional recovery from spinal cord injury by enhancing vascular function and reducing macrophage infiltration. These phenotypes may, in part, stem from reduced ER stress-induced cell death within endothelial and macrophage cells following injury.

Sildenafil improves epicenter vascular perfusion but not hindlimb functional recovery after contusive spinal cord injury in mice.

Nitric oxide (NO) is an important regulator of vasodilation and angiogenesis in the central nervous system (CNS). Signaling initiated by the membrane receptor CD47 antagonizes vasodilation and angiogenesis by inhibiting synthesis of cyclic guanosine monophosphate (cGMP). We recently found that deletion of CD47 led to significant functional locomotor improvements, enhanced angiogenesis, and increased epicenter microvascular perfusion in mice after moderate contusive spinal cord injury (SCI). We tested the hypothesis that improving NO/cGMP signaling within the spinal cord immediately after injury would increase microvascular perfusion, angiogenesis, and functional recovery, with an acute, 7-day administration of the cGMP phosphodiesterase 5 (PDE5) inhibitor sildenafil. PDE5 expression is localized within spinal cord microvascular endothelial cells and smooth muscle cells. While PDE5 antagonism has been shown to increase angiogenesis in a rat embolic stroke model, sildenafil had no significant effect on angiogenesis at 7 days post-injury after murine contusive SCI. Sildenafil treatment increased cGMP concentrations within the spinal cord and improved epicenter microvascular perfusion. Basso Mouse Scale (BMS) and Treadscan analyses revealed that sildenafil treatment had no functional consequence on hindlimb locomotor recovery. These data support the hypothesis that acutely improving microvascular perfusion within the injury epicenter by itself is an insufficient strategy for improving functional deficits following contusive SCI.

CD47 knockout mice exhibit improved recovery from spinal cord injury.

Recent data have implicated thrombospondin-1 (TSP-1) signaling in the acute neuropathological events that occur in microvascular endothelial cells (ECs) following spinal cord injury (SCI) (Benton et al., 2008b). We hypothesized that deletion of TSP-1 or its receptor CD47 would reduce these pathological events following SCI. CD47 is expressed in a variety of tissues, including vascular ECs and neutrophils. CD47 binds to TSP-1 and inhibits angiogenesis. CD47 also binds to the signal regulatory protein (SIRP)α and facilitates neutrophil diapedesis across ECs to sites of injury. After contusive SCI, TSP-1(-/-) mice did not show functional improvement compared to wildtype (WT) mice. CD47(-/-) mice, however, exhibited functional locomotor improvements and greater white matter sparing. Whereas targeted deletion of either CD47 or TSP-1 improved acute epicenter vascularity in contused mice, only CD47 deletion reduced neutrophil diapedesis and increased microvascular perfusion. An ex vivo model of the CNS microvasculature revealed that CD47(-/-)-derived microvessels (MVs) prominently exhibit adherent WT or CD47(-/-) neutrophils on the endothelial lumen, whereas WT-derived MVs do not. This implicates a defect in diapedesis mediated by the loss of CD47 expression on ECs. In vitro transmigration assays confirmed the role of SIRPα in neutrophil diapedesis through EC monolayers. We conclude that CD47 deletion modestly, but significantly, improves functional recovery from SCI via an increase in vascular patency and a reduction of SIRPα-mediated neutrophil diapedesis, rather than the abrogation of TSP-1-mediated anti-angiogenic signaling.