Functional Genome-wide Screen Identifies Pathways Restricting Central Nervous System Axonal Regeneration

Sekine, Yoshifumi; Lin-Moore, Alexander T.; Chenette, Devon M.; Wang, Xingxing; Jiang, Zhaoxin; Cafferty, William B. J.; Hammarlund, Marc; Strittmatter, Stephen M.

doi:10.1016/j.celrep.2018.03.058

Cited by 51 publications

(62 citation statements)

References 61 publications

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“…These results indicated that myosin IIA/B dKO per se did not significantly change the transcriptome in sensory neurons. In addition, in wild type and myosin IIA/B dKO neurons, we closely examined and compared the FPKMs of many classic regeneration-associated genes (RAGs) and genes well-known to control axon regeneration, such as Atf3 , Sox11 , Lin28a , Gap43 , Pten , Klf9 , and Rab27 , etc ( 12, 28–32 ). The results showed that the mRNA levels of these genes were up- or downregulated by SNI as expected, but were not largely affected by myosin IIA/B dKO (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Wang

Yang

Zhang

et al. 2019

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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...

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Section: Resultsmentioning

confidence: 99%

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

Wang

Yang

Zhang

et al. 2019

Preprint

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“…The Ngr1 Ϫ/Ϫ mouse line has been described previously (32,33) and was backcrossed for more than 10 generations to C57BL/6 WT (Ngr1 ϩ/ϩ ) mice. Primary neuron cultures were obtained from these mice.…”

Section: Cortical Axon Regeneration Assaymentioning

confidence: 99%

A proteolytic C-terminal fragment of Nogo-A (reticulon-4A) is released in exosomes and potently inhibits axon regeneration

Sekine¹,

Lindborg

Strittmatter

2020

Journal of Biological Chemistry

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Glial signals are known to inhibit axonal regeneration and functional recovery after mammalian central nervous system trauma, including spinal cord injury. Such signals include membrane-associated proteins of the oligodendrocyte plasma membrane and astrocyte-derived, matrix-associated proteins. Here, using cell lines and primary cortical neuron cultures, recombinant protein expression, immunoprecipitation and immunoblot assays, transmission EM of exosomes, and axon regeneration assays, we explored the secretion and activity of the myelin-associated neurite outgrowth inhibitor Nogo-A and observed exosomal release of a 24-kDa C-terminal Nogo-A fragment from cultured cells. We found that the cleavage site in this 1192-amino-acid-long fragment is located between amino acids 961–971. We also detected a Nogo-66 receptor (NgR1)–interacting Nogo-66 domain on the exosome surface. Enzyme inhibitor treatment and siRNA knockdown revealed that β-secretase 1 (BACE1) is the protease responsible for Nogo-A cleavage. Functionally, exosomes with the Nogo-66 domain on their surface potently inhibited axonal regeneration of mechanically injured cerebral cortex neurons from mice. Production of this fragment was observed in the exosomal fraction from neuronal tissue lysates after spinal cord crush injury of mice. We also noted that, relative to the exosomal marker Alix, a Nogo-immunoreactive, 24-kDa protein is enriched in exosomes 2-fold after injury. We conclude that membrane-associated Nogo-A produced in oligodendrocytes is processed proteolytically by BACE1, is released via exosomes, and is a potent diffusible inhibitor of regenerative growth in NgR1-expressing axons.

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“…As PlexinA2 has a functional role in Nogo-A-mediated NgR1 signaling transduction in non-neuronal Cos7 cell, we conducted a neuronal in vitro axon regeneration assessment with cultured cortical neurons (Huebner et al, 2011;Sekine et al, 2018b). E17 mouse derived cortical neurons were cultured for 8 d and scraped with a metal pin tool for axotomy, and then incubated with Nogo22 for 3 d to assess axon regeneration.…”

Section: Ngr1/plexina2 Signaling Mediates Nogo-a-mediated Axon Regenementioning

confidence: 99%

Plexina2 and CRMP2 Signaling Complex Is Activated by Nogo-A-Liganded Ngr1 to Restrict Corticospinal Axon Sprouting after Trauma

et al. 2019

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After brain or spinal cord trauma, interaction of Nogo-A with neuronal NgR1 limits regenerative axonal sprouting and functional recovery. Cellular signaling by lipid-anchored NgR1 requires a coreceptor but the relevant partner in vivo is not clear. Here, we examined proteins enriched in NgR1 immunoprecipitates by Nogo-A exposure, identifying CRMP2, a cytosolic protein implicated in axon growth inhibition by Semaphorin/Plexin complexes. The Nogo-A-induced association of NgR1 with CRMP2 requires PlexinA2 as a coreceptor. Non-neuronal cells expressing both NgR1 and PlexinA2, but not either protein alone, contract upon Nogo-A exposure. Inhibition of cortical axon regeneration by Nogo-A depends on a NgR1/PlexinA2 genetic interaction because double-heterozygous NgR1 ϩ/Ϫ , PlexinA2 ϩ/Ϫ neurons, but not single-heterozygote neurons, are rescued from Nogo-A inhibition. NgR1 and PlexinA2 also interact genetically in vivo to restrict corticospinal sprouting in mouse cervical spinal cord after unilateral pyramidotomy. Greater post-injury sprouting in NgR1 ϩ/Ϫ , PlexinA2 ϩ/Ϫ mice supports enhanced neurological recovery of a mixed female and male double-heterozygous cohort. Thus, a NgR1/PlexinA2/CRMP2 ternary complex limits neural repair after adult mammalian CNS trauma. Significance StatementSeveral decades of molecular research have suggested that developmental regulation of axon growth is distinct in most regards from titration of axonal regenerative growth after adult CNS trauma. Among adult CNS pathways, the oligodendrocyte Nogo-A inhibition of growth through NgR1 is thought to have little molecular relationship to axonal guidance mechanisms active embryonically. Here, biochemical analysis of NgR1 function uncovered a physical complex with CRMP cytoplasmic mediators, and this led to appreciation of a role for PlexinA2 in concert with NgR1 after adult trauma. The data extend molecular understanding of neural repair after CNS trauma and link it to developmental processes.

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Functional Genome-wide Screen Identifies Pathways Restricting Central Nervous System Axonal Regeneration

Cited by 51 publications

References 61 publications

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

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

A proteolytic C-terminal fragment of Nogo-A (reticulon-4A) is released in exosomes and potently inhibits axon regeneration

Plexina2 and CRMP2 Signaling Complex Is Activated by Nogo-A-Liganded Ngr1 to Restrict Corticospinal Axon Sprouting after Trauma

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