Acute intermittent hypoxia alters disease course and promotes CNS repair including resolution of inflammation and remyelination in the experimental autoimmune encephalomyelitis model of MS.

Remyelination and neurodegeneration prevention mitigate disability in Multiple Sclerosis (MS). We have shown acute intermittent hypoxia (AIH) is a novel, non-invasive and effective therapy for peripheral nerve repair, including remyelination. Thus, we posited AIH would improve repair following CNS demyelination and address the paucity of MS repair treatments. AIH's capacity to enhance intrinsic repair, functional recovery and alter disease course in the experimental autoimmune encephalomyelitis (EAE) model of MS was assessed. EAE was induced by MOG35-55 immunization in C57BL/6 female mice. EAE mice received either AIH (10 cycles-5 min 11% oxygen alternating with 5 min 21% oxygen) or Normoxia (control; 21% oxygen for same duration) once daily for 7d beginning at near peak EAE disease score of 2.5. Mice were followed post-treatment for an additional 7d before assessing histopathology or 14d to examine maintenance of AIH effects. Alterations in histopathological correlates of multiple repair indices were analyzed quantitatively in focally demyelinated ventral lumbar spinal cord areas to assess AIH impacts. AIH begun at near peak disease significantly improved daily clinical scores/functional recovery and associated histopathology relative to Normoxia controls and the former were maintained for at least 14d post-treatment. AIH enhanced correlates of myelination, axon protection and oligodendrocyte precursor cell recruitment to demyelinated areas. AIH also effected a dramatic reduction in inflammation, while polarizing remaining macrophages/microglia toward a pro-repair state. Collectively, this supports a role for AIH as a novel non-invasive therapy to enhance CNS repair and alter disease course following demyelination and holds promise as a neuroregenerative MS strategy.

The Unfolded Protein Response and Cholesterol Biosynthesis Link Luman/CREB3 to Regenerative Axon Growth in Sensory Neurons.

We recently revealed that the axon endoplasmic reticulum resident transcription factor Luman/CREB3 (herein called Luman) serves as a unique retrograde injury signal in regulation of the intrinsic elongating form of sensory axon regeneration. Here, evidence supports that Luman contributes to axonal regeneration through regulation of the unfolded protein response (UPR) and cholesterol biosynthesis in adult rat sensory neurons. One day sciatic nerve crush injury triggered a robust increase in UPR-associated mRNA and protein expression in both neuronal cell bodies and the injured axons. Knockdown of Luman expression in 1 d injury-conditioned neurons by siRNA attenuated axonal outgrowth to 48% of control injured neurons and was concomitant with reduced UPR- and cholesterol biosynthesis-associated gene expression. UPR PCR-array analysis coupled with qRT-PCR identified and confirmed that four transcripts involved in cholesterol regulation were downregulated >2-fold by the Luman siRNA treatment of the injury-conditioned neurons. Further, the Luman siRNA-attenuated outgrowth could be significantly rescued by either cholesterol supplementation or 2 ng/ml of the UPR inducer tunicamycin, an amount determined to elevate the depressed UPR gene expression to a level equivalent of that observed with crush injury. Using these approaches, outgrowth increased significantly to 74% or 69% that of injury-conditioned controls, respectively. The identification of Luman as a regulator of the injury-induced UPR and cholesterol at levels that benefit the intrinsic ability of axotomized adult rat sensory neurons to undergo axonal regeneration reveals new therapeutic targets to bolster nerve repair.

Brief electrical nerve stimulation enhances intrinsic repair capacity of the focally demyelinated central nervous system.

Our lab has shown that brief electrical nerve stimulation (ES) has a dramatic impact on remyelination of lysophosphatidyl choline (LPC)-induced focally demyelinated rat peripheral nerves, while also inducing an axon-protective phenotype and shifting macrophages from a predominantly pro-inflammatory toward a pro-repair phenotype. Whether this same potential exists in the central nervous system is not known. Thus, for proof of principle studies, the peripheral nerve demyelination and ES model was adapted to the central nervous system, whereby a unilateral focal LPC-induced demyelination of the dorsal column at the lumbar enlargement where the sciatic nerve afferents enter was created, so that subsequent ipsilateral sciatic nerve ES results in increased neural activity in the demyelinated axons. Data reveal a robust focal demyelination at 7 days post-LPC injection. Delivery of 1-hour ES at 7 days post-LPC polarizes macrophages/microglia toward a pro-repair phenotype when examined at 14 days post-LPC; results in smaller LPC-associated regions of inflammation compared to non-stimulated controls; results in significantly more cells of the oligodendroglial lineage in the demyelinated region; elevates myelin basic protein levels; and shifts the paranodal protein Caspr along demyelinated axons to a more restricted distribution, consistent with reformation of the paranodes of the nodes of Ranvier. ES also significantly enhanced levels of phosphorylated neurofilaments detected in the zones of demyelination, which has been shown to confer axon protection. Collectively these findings support that strategies that increase neural activity, such as brief electrical stimulation, can be beneficial for promoting intrinsic repair following focal demyelinating insults in demyelinating diseases such as multiple sclerosis. All animal procedures performed were approved by the University of Saskatchewan's Animal Research Ethics Board (protocol# 20090087; last approval date: November 5, 2020).

FOXO3a as a sensor of unilateral nerve injury in sensory neurons ipsilateral, contralateral and remote to injury.

Emerging evidence supports that the stress response to peripheral nerve injury extends beyond the injured neuron, with alterations in associated transcription factors detected both locally and remote to the lesion. Stress-induced nuclear translocation of the transcription factor forkhead class box O3a (FOXO3a) was initially linked to activation of apoptotic genes in many neuronal subtypes. However, a more complex role of FOXO3a has been suggested in the injury response of sensory neurons, with the injured neuron expressing less FOXO3a. To elucidate this response and test whether non-injured sensory neurons also alter FOXO3a expression, the temporal impact of chronic unilateral L4-6 spinal nerve transection on FOXO3a expression and nuclear localization in adult rat dorsal root ganglion neurons ipsilateral, contralateral or remote to injury relative to naïve controls was examined. In naïve neurons, high cytoplasmic and nuclear levels of FOXO3a colocalized with calcitonin gene related peptide, a marker of the nociceptive subpopulation. One hour post-injury, an acute increase in nuclear FOXO3a in small size injured neurons occurred followed by a significant decrease after 1, 2 and 4 days, with levels increasing toward pre-injury levels by 1 week post-injury. A more robust biphasic response to the injury was observed in uninjured neurons contralateral to and those remote to injury. Nuclear levels of FOXO3a peaked at 1 day, decreased by 4 days, then increased by 1 week post-injury, a response mirrored in C4 dorsal root ganglion neurons remote to injury. This altered expression contralateral and remote to injury supports that spinal nerve damage has broader systemic impacts, a response we recently reported for another stress transcription factor, Luman/CREB3. The early decreased expression and nuclear localization of FOXO3a in the injured neuron implicate these changes in the cell body response to injury that may be protective. Finally, the broader systemic changes support the existence of stress/injury-induced humeral factor(s) influencing transcriptional and potentially behavioral changes in uninjured dorsal root ganglion neurons. Approval to conduct this study was obtained from the University of Saskatchewan Animal Research Ethics Board (protocol #19920164).

Axotomy Induces Phasic Alterations in Luman/CREB3 Expression and Nuclear Localization in Injured and Contralateral Uninjured Sensory Neurons: Correlation With Intrinsic Axon Growth Capacity.

Luman/CREB3 is an important early retrograde axotomy signal regulating acute axon outgrowth in sensory neurons through the adaptive unfolded protein response. As the injury response is transcriptionally multiphasic, a spatiotemporal analysis of Luman/CREB3 localization in rat dorsal root ganglion (DRG) with unilateral L4-L6 spinal nerve injury was conducted to determine if Luman/CREB3 expression was similarly regulated. Biphasic alterations in Luman/CREB3 immunofluorescence and nuclear localization occurred in neurons ipsilateral to 1-hour, 1-day, 2-day, 4-day, and 1-week injury, with a largely parallel, but less avid response contralaterally. This biphasic response was not observed at the transcript level. To assess whether changes in neuronal Luman expression corresponded with an altered intrinsic capacity to grow an axon/neurite in vitro, injury-conditioned and contralateral uninjured DRG neurons underwent a 24-hour axon growth assay. Two-day injury-conditioned neurons exhibited maximal outgrowth capacity relative to naïve, declining at later injury-conditioned timepoints. Only neurons contralateral to 1-week injury exhibited significantly higher axon growth capacity than naïve. In conclusion, alterations in neuronal injury-associated Luman/CREB3 expression support that a multiphasic cell body response occurs and reveal a novel contralateral plasticity in axon growth capacity at 1-week post-injury. These adaptive responses have the potential to inform when repair or therapeutic intervention may be most effective.

Neurotrophin-3 suppresses thermal hyperalgesia associated with neuropathic pain and attenuates transient receptor potential vanilloid receptor-1 expression in adult sensory neurons.

Neurotrophin-3 (NT-3) negatively modulates nerve growth factor (NGF) receptor expression and associated nociceptive phenotype in intact neurons, suggesting a beneficial role in treating aspects of neuropathic pain mediated by NGF. We report that NT-3 is effective at suppressing thermal hyperalgesia associated with chronic constriction injury (CCI); however, NT-3 does not alter the mechanical hypersensitivity that also develops with CCI. Thermal hyperalgesia is critically linked to expression and activation of the capsaicin receptor, transient receptor potential vanilloid receptor-1 (TRPV1). Thus, its modulation by NT-3 after CCI was examined. CCI results in elevated TRPV1 expression at both the mRNA and protein levels in predominantly small-to-medium neurons, with the percentage of neurons expressing TRPV1 remaining unchanged at approximately 56%. Attenuation of thermal hyperalgesia mediated by NT-3 correlates with decreased TRPV1 expression such that only approximately 26% of neurons ipsilateral to CCI expressed detectable TRPV1 mRNA. NT-3 effected a decrease in expression of the activated component of the signaling pathway linked to regulation of TRPV1 expression, phospho-p38 MAPK (Ji et al., 2002), in neurons ipsilateral to CCI. Exogenous NT-3 could both prevent the onset of thermal hyperalgesia and reverse established thermal hyperalgesia and elevated TRPV1 expression 1 week after CCI. Continuous infusion is required for suppression of both thermal hyperalgesia and TRPV1 expression, because removal of NT-3 resulted in a prompt reestablishment of the hyperalgesic state and corresponding CCI-associated TRPV1 phenotype. In conclusion, although NGF drives inflammation-associated thermal hyperalgesia via its regulation of TRPV1 expression, NT-3 is now identified as a potent negative modulator of this state.

CGRP peptide and regenerating sensory axons.

CGRP peptide, a widely expressed constituent of sensory neurons, plays important roles in nerve function and repair when axons are severed. CGRP synthesis declines, yet peptide nonetheless accumulates in severed axon endbulbs. In this work we explore an apparent selective and ongoing expression of CGRP peptide in regenerative sensory axon sprouts. Following sural nerve crush in rats out to 14 days, regenerating and branching sensory axons had intense and selective expression of CGRP, not associated with endbulbs. Parent L4 and L5 perikarya and axons in the sural nerve proximal to crush, however, did not exhibit such heightened CGRP presence. Instead, back labeling of regenerating axons with fluorogold or diamidino yellow labeled perikarya with reduced CGRP expression. Similarly, ATF-3, a robust marker of axotomized neurons, was associated with reduced, rather than elevated expression of alphaCGRP mRNA. Unexpectedly, however, we identified an enlarged secondary population of intact uninjured neurons, frequently smaller and projecting to the dorsal horn with new and heightened intense CGRP expression but not ATF-3- or tracer-labeled. Distal regenerating sensory axons selectively express CGRP peptide despite reduced perikaryal content, a phenomenon not explained by simple accumulation. Having an injured neighbor neuron, however, may also paradoxically alter how CGRP is expressed in intact neurons.

p75 neurotrophin receptor is implicated in the ability of neurotrophin-3 to negatively modulate activated ERK1/2 signaling in TrkA-expressing adult sensory neurons.

Neurotrophin-3 (NT-3) can negatively modulate trkA and associated phenotype in intact sensory neurons, while positively regulating trkC and associated phenotype. How NT-3 effects this response is not clear. Whether NT-3 exerts a differential influence on levels of activated ERK1/2 signaling in trkA- versus trkC-mRNA-positive subpopulations of neurons and the role that the common neurotrophin receptor, p75NTR, plays in this response was assessed by examining alterations in the levels of phospho-ERK1/2 immunofluorescence signal over nuclei of sensory neurons expressing trkA alone, trkC alone, or both trkA and trkC mRNA. NT-3 intrathecal infusion differentially modulated nuclear phospho-ERK1/2 levels detected over neurons expressing trkA alone or trkC alone. Levels were significantly decreased over nuclei of neurons expressing trkA alone and significantly increased over the nuclei of neurons expressing trkC alone. Neurons expressing both trkA and trkC or neurons expressing neither trkA nor trkC had no significant alteration in phospho-ERK1/2. Antisense oligonucleotides directed against p75NTR were infused intrathecally with or without NT-3 infusion to examine the impact of suppressing p75NTR expression on the ability of NT-3 to diminish phospho-ERK1/2 signaling in neurons expressing only trkA. NT-3 did not significantly attenuate levels of phospho-ERK1/2 when p75NTR expression was suppressed by antisense infusion, despite being able to do so when NT-3 was infused alone. In conclusion, NT-3's ability to negatively modulate ERK1/2 signaling in a p75-dependent manner in sensory neurons that express trkA to the exclusion of trkC provides a feasible mechanism by which it negatively modulates other aspects of nociceptive phenotype in these neurons.