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.

Spinal control of locomotion before and after spinal cord injury.

Thoracic spinal cord injury affects long propriospinal neurons that interconnect the cervical and lumbar enlargements. These neurons are crucial for coordinating forelimb and hindlimb locomotor movements in a speed-dependent manner. However, recovery from spinal cord injury is usually studied over a very limited range of speeds that may not fully expose circuitry dysfunction. To overcome this limitation, we investigated overground locomotion in rats trained to move over an extended distance with a wide range of speeds both pre-injury and after recovery from thoracic hemisection or contusion injuries. In this experimental context, intact rats expressed a speed-dependent continuum of alternating (walk and trot) and non-alternating (canter, gallop, half-bound gallop, and bound) gaits. After a lateral hemisection injury, rats recovered the ability to locomote over a wide range of speeds but lost the ability to use the highest-speed gaits (half-bound gallop and bound) and predominantly used the limb contralateral to the injury as lead during canter and gallop. A moderate contusion injury caused a greater reduction in maximal speed, loss of all non-alternating gaits, and emergence of novel alternating gaits. These changes resulted from weak fore-hind coupling together with appropriate control of left-right alternation. After hemisection, animals expressed a subset of intact gaits with appropriate interlimb coordination even on the side of the injury, where the long propriospinal connections were severed. These observations highlight how investigating locomotion over the full range of speeds can reveal otherwise hidden aspects of spinal locomotor control and post-injury recovery.

Spinal control of locomotion before and after spinal cord injury.

Thoracic spinal cord injury affects long propriospinal neurons that interconnect the cervical and lumbar enlargements. These neurons are crucial for coordinating forelimb and hindlimb locomotor movements in a speed-dependent manner. However, recovery from spinal cord injury is usually studied over a very limited range of speeds that may not fully expose circuitry dysfunction. To overcome this limitation, we investigated overground locomotion in rats trained to move over an extended distance with a wide range of speeds both pre-injury and after recovery from thoracic hemisection or contusion injuries. In this experimental context, intact rats expressed a speed-dependent continuum of alternating (walk and trot) and non-alternating (canter, gallop, half-bound gallop, and bound) gaits. After a lateral hemisection injury, rats recovered the ability to locomote over a wide range of speeds but lost the ability to use the highest-speed gaits (half-bound gallop and bound) and predominantly used the limb contralateral to the injury as lead during canter and gallop. A moderate contusion injury caused a greater reduction in maximal speed, loss of all non-alternating gaits, and emergence of novel alternating gaits. These changes resulted from weak fore-hind coupling together with appropriate control of left-right alternation. After hemisection, animals expressed a subset of intact gaits with appropriate interlimb coordination even on the side of the injury, where the long propriospinal connections were severed. These observations highlight how investigating locomotion over the full range of speeds can reveal otherwise hidden aspects of spinal locomotor control and post-injury recovery.

Silencing long-descending inter-enlargement propriospinal neurons improves hindlimb stepping after contusive spinal cord injuries.

Spinal locomotor circuitry is comprised of rhythm generating centers, one for each limb, that are interconnected by local and long-distance propriospinal neurons thought to carry temporal information necessary for interlimb coordination and gait control. We showed previously that conditional silencing of the long ascending propriospinal neurons (LAPNs) that project from the lumbar to the cervical rhythmogenic centers (L1/L2 to C6), disrupts right-left alternation of both the forelimbs and hindlimbs without significantly disrupting other fundamental aspects of interlimb and speed-dependent coordination (Pocratsky et al., 2020). Subsequently, we showed that silencing the LAPNs after a moderate thoracic contusive spinal cord injury (SCI) resulted in better recovered locomotor function (Shepard et al., 2021). In this research advance, we focus on the descending equivalent to the LAPNs, the long descending propriospinal neurons (LDPNs) that have cell bodies at C6 and terminals at L2. We found that conditional silencing of the LDPNs in the intact adult rat resulted in a disrupted alternation of each limb pair (forelimbs and hindlimbs) and after a thoracic contusion SCI significantly improved locomotor function. These observations lead us to speculate that the LAPNs and LDPNs have similar roles in the exchange of temporal information between the cervical and lumbar rhythm generating centers, but that the partial disruption of the pathway after SCI limits the independent function of the lumbar circuitry. Silencing the LAPNs or LDPNs effectively permits or frees-up the lumbar circuitry to function independently.

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).

Silencing long ascending propriospinal neurons after spinal cord injury improves hindlimb stepping in the adult rat.

Long ascending propriospinal neurons (LAPNs) are a subpopulation of spinal cord interneurons that directly connect the lumbar and cervical enlargements. Previously we showed, in uninjured animals, that conditionally silencing LAPNs disrupted left-right coordination of the hindlimbs and forelimbs in a context-dependent manner, demonstrating that LAPNs secure alternation of the fore- and hindlimb pairs during overground stepping. Given the ventrolateral location of LAPN axons in the spinal cord white matter, many likely remain intact following incomplete, contusive, thoracic spinal cord injury (SCI), suggesting a potential role in the recovery of stepping. Thus, we hypothesized that silencing LAPNs after SCI would disrupt recovered locomotion. Instead, we found that silencing spared LAPNs post-SCI improved locomotor function, including paw placement order and timing, and a decrease in the number of dorsal steps. Silencing also restored left-right hindlimb coordination and normalized spatiotemporal features of gait such as stance and swing time. However, hindlimb-forelimb coordination was not restored. These data indicate that the temporal information carried between the spinal enlargements by the spared LAPNs post-SCI is detrimental to recovered hindlimb locomotor function. These findings are an illustration of a post-SCI neuroanatomical-functional paradox and have implications for the development of neuronal- and axonal-protective therapeutic strategies and the clinical study/implementation of neuromodulation strategies.

Long ascending propriospinal neurons provide flexible, context-specific control of interlimb coordination.

Within the cervical and lumbar spinal enlargements, central pattern generator (CPG) circuitry produces the rhythmic output necessary for limb coordination during locomotion. Long propriospinal neurons that inter-connect these CPGs are thought to secure hindlimb-forelimb coordination, ensuring that diagonal limb pairs move synchronously while the ipsilateral limb pairs move out-of-phase during stepping. Here, we show that silencing long ascending propriospinal neurons (LAPNs) that inter-connect the lumbar and cervical CPGs disrupts left-right limb coupling of each limb pair in the adult rat during overground locomotion on a high-friction surface. These perturbations occurred independent of the locomotor rhythm, intralimb coordination, and speed-dependent (or any other) principal features of locomotion. Strikingly, the functional consequences of silencing LAPNs are highly context-dependent; the phenotype was not expressed during swimming, treadmill stepping, exploratory locomotion, or walking on an uncoated, slick surface. These data reveal surprising flexibility and context-dependence in the control of interlimb coordination during locomotion.