Combined chondroitinase and KLF7 expression reduce net retraction of sensory and CST axons from sites of spinal injury

Wang, Zimei; Winsor, Kristen; Nienhaus, Christopher; Hess, Evan; Blackmore, Murray G.

doi:10.1016/j.nbd.2016.12.010

Cited by 33 publications

(38 citation statements)

References 73 publications

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“…2C,D). These data indicate that when confronted with a deeper and more severe injury, KLF6stimulated axons are largely unable to regenerate, a finding reminiscent of prior findings with closely related KLF7 (21,23). Thus, although KLF-based manipulations elevate intrinsic regenerative ability in CST axons (22), extrinsic barriers continue to constrain growth.…”

Section: Resultssupporting

confidence: 63%

“…A transcription factor called KLF6 has emerged as a particularly potent promoter of CST axon growth by activating a set of complementary gene networks involved in axon extension (22). Yet these neuron-intrinsic interventions only partially restore growth ability, and stimulated axons generally circumvent partial injuries, but fail to traverse complete injuries, suggesting persistent environmental inhibition (23). To address extrinsic inhibition, an important advance has been the demonstration that grafts of neural progenitor cells (NPCs) placed into sites of spinal injury can attract regenerative ingrowth from CST axons (10).…”

Section: Introductionmentioning

confidence: 99%

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Restoration of Direct Corticospinal Communication Across Sites of Spinal Injury

Jayaprakash

Nowak

Eastwood

et al. 2019

Preprint

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Injury to the spinal cord often disrupts long-distance axon tracts that link the brain and spinal cord, causing permanent disability. Axon regeneration is then prevented by a combination of inhibitory signals that emerge at the injury site and by a low capacity for regeneration within injured neurons. The corticospinal tract (CST) is essential for fine motor control but has proven refractory to many attempted pro-regenerative treatments. Although strategies are emerging to create relay or detour circuits that reroute cortical motor commands through spared circuits, these have only partially met the challenge of restoring motor control. Here, using a murine model of spinal injury, we elevated the intrinsic regenerative ability of CST neurons by supplying a pro-regenerative transcription factor, KLF6, while simultaneously supplying injured CST axons with a growth-permissive graft of neural progenitor cells (NPCs) transplanted into a site of spinal injury. The combined treatment produced robust CST regeneration directly through the grafts and into distal spinal cord. Moreover, selective optogenetic stimulation of regenerated CST axons and single-unit electrophysiology revealed extensive synaptic integration by CST axons with spinal neurons beyond the injury site. Finally, when KLF6 was delivered to injured neurons with a highly effective retrograde vector, combined KLF6/NPC treatment yielded significant improvements in forelimb function. These findings highlight the utility of retrograde gene therapy as a strategy to treat CNS injury and establish conditions that restore functional CST communication across a site of spinal injury. Significance StatementDamage to the spinal cord results in incurable paralysis because axons that carry descending motor commands are unable to regenerate. Here we deployed a two-pronged strategy in a rodent model of spinal injury to promote regeneration by the corticospinal tract, a critical mediator of fine motor control. Delivering pro-regenerative KLF6 to injured neurons while simultaneously transplanting neural progenitor cells to injury sites resulted in robust regeneration directly through sites of spinal injury, accompanied by extensive synapse formation with spinal neurons. In addition, when KLF6 was delivered with improved retrograde gene therapy vectors, the combined treatment significantly improved forelimb function in injured animals. This work represents important progress toward restoring regeneration and motor function after spinal injury.

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Section: Resultssupporting

confidence: 63%

Section: Introductionmentioning

confidence: 99%

Restoration of Direct Corticospinal Communication Across Sites of Spinal Injury

Jayaprakash

Nowak

Eastwood

et al. 2019

Preprint

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“…For example, knockout of growth-inhibitory KLF4 promotes axon regeneration by retinal ganglion cells in the injured optic nerve (Moore et al, 2009; Qin et al, 2013). In contrast, forced expression of pro-regenerative KLF7 constructs improves axon regeneration by injured corticospinal tract (CST) neurons, and reduces dieback of axotomized sensory neurons (Blackmore et al, 2012; Wang et al, 2017). Importantly, exogenous KLF7 is poorly expressed in neurons, likely due to rapid protein degradation from an N-terminal targeting domain (Blackmore et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

KLF6 and STAT3 co-occupy regulatory DNA and functionally synergize to promote axon growth in CNS neurons

Wang¹,

Mehra²,

Simpson³

et al. 2018

Preprint

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Members of the KLF family of transcription factors can exert both positive and negative effects on axon regeneration in the central nervous system, but the underlying mechanisms are unclear. KLF6 and −7 share nearly identical DNA binding domains and stand out as the only known growth-promoting family members. Here we confirm that similar to KLF7, expression of KLF6 declines during postnatal cortical development and that forced re-expression of KLF6 in corticospinal tract neurons of adult female mice enhances axon regeneration after cervical spinal injury. Unlike KLF7, however, these effects were achieved with wildtype KLF6, as opposed constitutively active mutants, thus simplifying the interpretation of mechanistic studies. To clarify the molecular basis of growth promotion, RNA sequencing identified 454 genes whose expression changed upon forced KLF6 expression in cortical neurons. Network analysis of these genes revealed sub-networks of downregulated genes that were highly enriched for synaptic functions, and sub-networks of upregulated genes with functions relevant to axon extension including cytoskeleton remodeling, lipid synthesis and transport, and bioenergetics. The promoter regions of KLF6-sensitive genes showed enrichment for the binding sequence of STAT3, a previously identified regeneration-associated gene. Notably, co-expression of constitutively active STAT3 along with KLF6 in cortical neurons produced synergistic increases in neurite length. Finally, genome-wide ATAC-seq footprinting detected frequent co-binding by the two factors in pro-growth gene networks, indicating co-occupancy as an underlying mechanism for the observed synergy. These findings advance understanding of KLF-stimulated axon growth and indicate functional synergy of KLF6 transcriptional effects with those of STAT3.SIGNIFICANCE STATEMENTThe failure of axon regeneration in the CNS limits recovery from damage and disease. These findings show the transcription factor KLF6 to be a potent promoter of axon growth after spinal injury, and more importantly clarify the underlying transcriptional changes. In addition, bioinformatics analysis predicted a functional interaction between KLF6 and a second transcription factor, STAT3, and genome-wide footprinting confirmed frequent co-occupancy. Co-expression of the two factors yielded synergistic elevation of neurite growth in primary neurons. These data point the way toward novel transcriptional interventions to promote CNS regeneration.

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“…Neuroprotective agents (e.g., methylprednisolone, gacyclidine, thyrotropin releasing hormone, nimodipine, etc.) against secondary SCI, together with neuroregenerative agents (e.g., gangliosides, Rho antagonist, anti-Nogo antibodies, acidic fibroblast growth factor) have been used in SCI (Table I) with mixed outcomes (28–39). Recent studies also evaluated the potential beneficial effects of estrogen, which could be a neuroprotective agent in the treatment of SCI and TBI (40–43).…”

Section: Introductionmentioning

confidence: 99%

Targeting Enolase in Reducing Secondary Damage in Acute Spinal Cord Injury in Rats

et al. 2017

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Spinal cord injury (SCI) is a complex debilitating condition leading to permanent life-long neurological deficits. The complexity of SCI suggests that a concerted multi-targeted therapeutic approach is warranted to optimally improve function. Damage to spinal cord is complicated by an increased detrimental response from secondary injury factors mediated by activated glial cells and infiltrating macrophages. While elevation of enolase especially Neuron Specific Enolase (NSE) in glial and neuronal cells is believed to trigger inflammatory cascades in acute SCI, alteration of NSE and its subsequent effects in acute SCI remains unknown. This study measured NSE expression levels and key inflammatory mediators after acute SCI and investigated the role of ENOblock, a novel small molecule inhibitor of enolase, in a male Sprague-Dawley (SD) rat SCI model. Serum NSE levels as well as cytokines/chemokines and metabolic factors were evaluated in injured animals following treatment with vehicle alone or ENOblock using Discovery assay. Spinal cord samples were also analyzed for NSE and MMPs 2 and 9 as well as glial markers by Western blotting. The results indicated a significant decrease in serum inflammatory cytokines/chemokines and NSE, alterations of metabolic factors and expression of MMPs in spinal cord tissues after treatment with ENOblock (100μg/kg, twice). These results support the hypothesis that activation of glial cells and inflammation status can be modulated by regulation of NSE expression and activity. Analysis of SCI tissue samples by immunohistochemistry confirmed that ENOblock decreased gliosis which may have occurred through reduction of elevated NSE in rats. Overall, elevation of NSE is deleterious as it promotes extracellular degradation and production of inflammatory cytokines/chemokines and metabolic factors which activates glia and damages neurons. Thus, reduction of NSE by ENOblock may have potential therapeutic implications in acute SCI.

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Combined chondroitinase and KLF7 expression reduce net retraction of sensory and CST axons from sites of spinal injury

Cited by 33 publications

References 73 publications

Restoration of Direct Corticospinal Communication Across Sites of Spinal Injury

Restoration of Direct Corticospinal Communication Across Sites of Spinal Injury

KLF6 and STAT3 co-occupy regulatory DNA and functionally synergize to promote axon growth in CNS neurons

Targeting Enolase in Reducing Secondary Damage in Acute Spinal Cord Injury in Rats

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