The role of RhoA in retrograde neuronal death and axon regeneration after spinal cord injury

Abstract

Paralysis following spinal cord injury (SCI) is due to interruption of axons and their failure to regenerate. It has been suggested that the small GTPase RhoA may be an intracellular signaling convergence point for several types of growth-inhibiting extracellular molecules. Even if this is true in vitro, it is not clear from studies in mammalian SCI, whether the effects of RhoA manipulations on axon growth in vivo are due to a RhoA-mediated inhibition of true regeneration or only of collateral sprouting from spared axons, since work on SCI generally is performed with partial injury models. RhoA also has been implicated in local neuronal apoptosis after SCI, but whether this reflects an effect on axotomy-induced cell death or an effect on other pathological mechanisms is not known. In order to resolve these ambiguities, we studied the effects of RhoA knockdown in the sea lamprey central nervous system (CNS), where after complete spinal cord transection (TX), robust but incomplete regeneration of large axons belonging to individually identified reticulospinal (RS) neurons occurs, and where some RS neurons show unambiguous delayed retrograde apoptosis after axotomy. RhoA protein was detected in neurons and axons of the lamprey brain and spinal cord, and its expression was increased post-TX. Knockdown of RhoA in vivo by retrogradely-delivered morpholino antisense oligonucleotides (MOs) to the RS neurons significantly reduced retrograde apoptosis signaling in identified RS neurons post-SCI, as indicated by Fluorochrome Labeled Inhibitor of Caspases (FLICA) in brain wholemounts. In individual RS neurons, the reduction of caspase activation by RhoA knockdown began at 2 weeks post-TX and was still seen at 8 weeks. RhoA knockdown slowed axon retraction and possibly increased early axon regeneration in the proximal stump. The number of axons regenerating beyond the lesion more than 5 mm at 10 weeks post-TX also was increased. Thus RhoA knockdown both enhanced true axon regeneration and inhibited retrograde apoptosis signaling after SCI.

PTPσ Knockdown in Lampreys Impairs Reticulospinal Axon Regeneration and Neuronal Survival After Spinal Cord Injury

“…Thus, PTPσ must be continually synthesized to maintain expression levels. Previous studies demonstrated that MOs applied to the cut ends of lamprey RS axons immediately after TX reach their perikarya in the brain within 1 week of injury and are effective in reducing protein expression of their target RNAs Hu et al, 2017). After an initial 10-day period of retraction, axons of the identified RS neurons begin to regenerate, reaching the edge of the CSPG-rich scar by 3 weeks post-TX (Yin and Selzer, 1983).…”

“…Antisense MOs, which bind complementary target RNA via Watson-Crick base paring, have proven to be reliable vectors for inducing protein knockdown in lampreys Hu et al, 2017;Sobrido-Cameán et al, 2019). Importantly, MOs applied to the spinal cord stumps after TX are readily taken up by severed axons and retrogradely transported to their cell bodies in the brain .…”

“…Additionally, the long delay between confirmed MO biological activity in this study (i.e., within 2 weeks post-TX) and neuron loss (10-20 weeks post-TX) seems uncharacteristic of acute toxicity. Finally, it is important to note, that MOs have been used repeatedly in lamprey studies, and, to date, no off-target effects have been reported Hu et al, 2017;Sobrido-Cameán et al, 2019).…”

GABA promotes survival and axonal regeneration in identifiable descending neurons after spinal cord injury in larval lampreys

“…1), and a 5-base pair mismatch gabab1 negative control morpholino (5'-ACcTCTcCAACcGAGAcTCATcAGA-3'). During recovery, the morpholinos are retrogradely transported to the cell soma of descending neurons where they can knockdown the expression of the target mRNA (Zhang et al, 2015;Fogerson et al, 2016;Chen et al, 2017;Hu et al, 2017). Animals were allowed to recover for 10 wpl to analyse the effect of gabab1 knockdown (KD) in axonal regeneration of identifiable descending neurons.…”

RhoA-GTPase Modulates Neurite Outgrowth by Regulating the Expression of Spastin and p60-Katanin

“…In the micro-environment of growing neurites, especially in the injured nerve system, there are many kinds of extracellular inhibitory molecules, such as chondroitin sulfate proteoglycans (CSPGs), myelin-associated glycoprotein (MAG), Lingo-1, Nogo, oligodendrocyte-myelin glycoprotein (OMgp), and so on [11][12][13]. Existing evidence has suggested that the small GTPase RhoA subfamily (RhoA) is an intracellular convergence point for most extracellular inhibitory signals [14][15][16].…”