Knocking out non-muscle myosin II in retinal ganglion cells promotes long-distance optic nerve regeneration

Abstract

23In addition to altered gene expression, pathological cytoskeletal dynamics in the axon are another 24 key intrinsic barrier for axon regeneration in the central nervous system (CNS). Here we showed that 25 knocking out myosin IIA/B in retinal ganglion cells alone either before or after optic nerve crush induced 26 marked and sustained optic nerve regeneration. Combined Lin28 overexpression and myosin IIA/B 27 knockout led to synergistic promoting effect and long-distance axon regeneration. Immunostaining, RNA-28 seq and western blot analyses revealed that myosin II deletion did not affect known axon regeneration 29 signaling pathways or the expression of regeneration associated genes. Instead, it abolished the retraction 30 bulb formation and significantly enhanced the axon extension efficiency. The study provided clear 31 evidence that directly targeting neuronal cytoskeleton was sufficient to induce strong CNS axon 32 regeneration, and combining gene expression in the soma and modified cytoskeletal dynamics in the axon 33 was a promising approach for long-distance CNS axon regeneration. 34 35 Keywords 36Axon regeneration, optic nerve regeneration, non-muscle myosin II, Lin28, growth cone, cytoskeleton 37 38 42 hostile environment created by inhibitors in the scar tissues and degenerating myelin, and the other is the 43 diminished intrinsic neural regeneration ability of mature CNS neurons (1, 2). Therefore, the widely 44 accepted view is that combination strategies that target both intrinsic growth ability and inhibitory 45 environment are likely the best option for successful CNS axon regeneration and function recovery. Early 46 studies (3-6) using peripheral nerve graft transplants have shown that some mature CNS neurons, such as 47 spinal cord neurons and retinal ganglion cells (RGCs), could regenerate their axons into the permissive 48 nerve grafts, indicating clearly that these neurons still retain limited intrinsic regeneration ability. However, 49 to date, many studies targeting selected inhibitory molecules resulted in no or very modest CNS 50 regeneration (7, 8). A likely reason is that there are multiple classes of inhibitory molecules, potentially 51 including unidentified ones, which inhibit axon regeneration via distinct cellular and molecular 52 mechanisms. Thus, targeting a few inhibitory signals while leaving the others intact may not result in a 53 permissive environment similar to that in the peripheral nerve grafts. 54In contrast, studies targeting the intrinsic axon growth ability have produced very promising results. 55In the optic nerve regeneration model, for example, Pten, Socs3, Klf4 loss of function, and Lin28 gain of 56 function all achieved strong optic nerve regeneration (9-13). However, tissue clearing and 3D imaging 57 studies have revealed that many of the regenerating RGC axons make U-turns in the optic nerve, at the 58 optic chiasm, or make wrong guidance decisions after the chiasm (14, 15). In the corticospinal tract (CST) 59 regeneration model, although modulation of the in...

Neural Cadherin Plays Distinct Roles for Neuronal Survival and Axon Growth under Different Regenerative Conditions

“…To further examine the mechanisms by which NCAD deletion reduces axon regeneration, we assessed the level of mTORC1 activity in the RGCs of shPTEN-treated animals. To this end, we performed immunohistochemistry on sectioned retinas using an antibody against the phosphorylated ribosomal protein S6 (pS6), a known indicator of mTORC1 activity (Park et al, 2008;Yang et al, 2014;Wang et al, 2020). The numbers of pS6+ RGCs after injury were similar between the WT and NCAD-deleted animals (Figure 7).…”

“…For the determination of mTORC1 activity in RGCs, several retina sections were immunostained with an antibody against the phosphorylated ribosomal protein S6 (pS6), a widely used marker for mTORC1 activity (Park et al, 2008;Yang et al, 2014;Wang et al, 2020). The number of pS6+ RGCs in the ganglion cell layer of the injured retina was expressed as a percentage of the total number obtained in the contralateral intact retina.…”

“…Several studies have demonstrated that mTORC1 acts downstream of PTEN, and mediates axon regeneration (Park et al, 2008;Yang et al, 2014;Wang et al, 2020). To further examine the mechanisms by which NCAD deletion reduces axon regeneration, we assessed the level of mTORC1 activity in the RGCs of shPTEN-treated animals.…”

Advances in the Signaling Pathways Downstream of Glial-Scar Axon Growth Inhibitors

“…Moreover, myosin II, an ATP-dependent motor protein, probably mediates CSPG inhibition on axonal growth (Hur et al, 2011 ; Yu et al, 2012 ). CSPGs increase phosphorylation of nonmuscle myosin II regulatory light chains, while pharmacological or genetic inhibition of myosin II promotes axon growth in vitro on inhibitory substrates including CSPGs and axon regeneration after optic nerve injury (Wang et al, 2020 ).…”

Structural engraftment and topographic spacing of transplanted human stem cell-derived retinal ganglion cells

“…Studies of endogenous RGC axon regeneration following injury identify numerous molecular pathways that can be targeted to drive optic nerve regeneration and efferent visual signal propagation by exogenous transplanted RGCs. [12][13][14][15][16][17] However, factors limiting transplanted RGC somal migration, spatial patterning, or dendrite integration within the recipient mammalian retina, all of which are necessary for achieving afferent input, are equally important to functional vision restoration and remain comparatively understudied.…”