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.

Enhanced oxidative phosphorylation, re-organized intracellular signaling, and epigenetic de-silencing as revealed by oligodendrocyte translatome analysis after contusive spinal cord injury.

Reducing the loss of oligodendrocytes (OLs) is a major goal for neuroprotection after spinal cord injury (SCI). Therefore, the OL translatome was determined in Ribotag:Plp1-CreERT2 mice at 2, 10, and 42 days after moderate contusive T9 SCI. At 2 and 42 days, mitochondrial respiration- or actin cytoskeleton/cell junction/cell adhesion mRNAs were upregulated or downregulated, respectively. The latter effect suggests myelin sheath loss/morphological simplification which is consistent with downregulation of cholesterol biosynthesis transcripts on days 10 and 42. Various regulators of pro-survival-, cell death-, and/or oxidative stress response pathways showed peak expression acutely, on day 2. Many acutely upregulated OL genes are part of the repressive SUZ12/PRC2 operon suggesting that epigenetic de-silencing contributes to SCI effects on OL gene expression. Acute OL upregulation of the iron oxidoreductase Steap3 was confirmed at the protein level and replicated in cultured OLs treated with the mitochondrial uncoupler FCCP. Hence, STEAP3 upregulation may mark mitochondrial dysfunction. Taken together, in SCI-challenged OLs, acute and subchronic enhancement of mitochondrial respiration may be driven by axonal loss and subsequent myelin sheath degeneration. Acutely, the OL switch to oxidative phosphorylation may lead to oxidative stress that is further amplified by upregulation of such enzymes as STEAP3.

Enhanced oxidative phosphorylation, re-organized intracellular signaling, and epigenetic de-silencing as revealed by oligodendrocyte translatome analysis after contusive spinal cord injury.

Reducing the loss of oligodendrocytes (OLs) is a major goal for neuroprotection after spinal cord injury (SCI). Therefore, the OL translatome was determined in Ribotag:Plp1-CreERT2 mice at 2, 10, and 42 days after moderate contusive T9 SCI. At 2 and 42 days, mitochondrial respiration- or actin cytoskeleton/cell junction/cell adhesion mRNAs were upregulated or downregulated, respectively. The latter effect suggests myelin sheath loss/morphological simplification which is consistent with downregulation of cholesterol biosynthesis transcripts on days 10 and 42. Various regulators of pro-survival-, cell death-, and/or oxidative stress response pathways showed peak expression acutely, on day 2. Many acutely upregulated OL genes are part of the repressive SUZ12/PRC2 operon suggesting that epigenetic de-silencing contributes to SCI effects on OL gene expression. Acute OL upregulation of the iron oxidoreductase Steap3 was confirmed at the protein level and replicated in cultured OLs treated with the mitochondrial uncoupler FCCP. Hence, STEAP3 upregulation may mark mitochondrial dysfunction. Taken together, in SCI-challenged OLs, acute and subchronic enhancement of mitochondrial respiration may be driven by axonal loss and subsequent myelin sheath degeneration. Acutely, the OL switch to oxidative phosphorylation may lead to oxidative stress that is further amplified by upregulation of such enzymes as STEAP3.

Opposite modulation of functional recovery following contusive spinal cord injury in mice with oligodendrocyte-selective deletions of Atf4 and Chop/Ddit3.

The integrated stress response (ISR)-activated transcription factors ATF4 and CHOP/DDIT3 may regulate oligodendrocyte (OL) survival, tissue damage and functional impairment/recovery in white matter pathologies, including traumatic spinal cord injury (SCI). Accordingly, in OLs of OL-specific RiboTag mice, Atf4, Chop/Ddit3 and their downstream target gene transcripts were acutely upregulated at 2, but not 10, days post-contusive T9 SCI coinciding with maximal loss of spinal cord tissue. Unexpectedly, another, OL-specific upregulation of Atf4/Chop followed at 42 days post-injury. However, wild type versus OL-specific Atf4-/- or Chop-/- mice showed similar white matter sparing and OL loss at the injury epicenter, as well as unaffected hindlimb function recovery as determined by the Basso mouse scale. In contrast, the horizontal ladder test revealed persistent worsening or improvement of fine locomotor control in OL-Atf4-/- or OL-Chop-/- mice, respectively. Moreover, chronically, OL-Atf-/- mice showed decreased walking speed during plantar stepping despite greater compensatory forelimb usage. Therefore, ATF4 supports, while CHOP antagonizes, fine locomotor control during post-SCI recovery. No correlation between those effects and white matter sparing together with chronic activation of the OL ISR suggest that in OLs, ATF4 and CHOP regulate function of spinal cord circuitries that mediate fine locomotor control during post-SCI recovery.

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.

The Proteostasis Network: A Global Therapeutic Target for Neuroprotection after Spinal Cord Injury.

Proteostasis (protein homeostasis) is critical for cellular as well as organismal survival. It is strictly regulated by multiple conserved pathways including the ubiquitin-proteasome system, autophagy, the heat shock response, the integrated stress response, and the unfolded protein response. These overlapping proteostasis maintenance modules respond to various forms of cellular stress as well as organismal injury. While proteostasis restoration and ultimately organism survival is the main evolutionary driver of such a regulation, unresolved disruption of proteostasis may engage pro-apoptotic mediators of those pathways to eliminate defective cells. In this review, we discuss proteostasis contributions to the pathogenesis of traumatic spinal cord injury (SCI). Most published reports focused on the role of proteostasis networks in acute/sub-acute tissue damage post-SCI. Those reports reveal a complex picture with cell type- and/or proteostasis mediator-specific effects on loss of neurons and/or glia that often translate into the corresponding modulation of functional recovery. Effects of proteostasis networks on such phenomena as neuro-repair, post-injury plasticity, as well as systemic manifestations of SCI including dysregulation of the immune system, metabolism or cardiovascular function are currently understudied. However, as potential interventions that target the proteostasis networks are expected to impact many cell types across multiple organ systems that are compromised after SCI, such therapies could produce beneficial effects across the wide spectrum of highly variable human SCI.

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.