2b. Neuronal Molecular Signaling of Nerve Injury
There are two distinct phases of the neuronal signalling of the nerve injury: (i) a rapid phase dictated by a retrograde calcium wave to the neuronal soma and (ii) a later slow signaling phase characterized by the retrograde transport of signaling molecules by motor proteins [
88,
89,
90].
In the
first rapid phase, the disruption of the axonal membrane at the site of the nerve crush or transection, exposes the axonal cytoplasm to external ionic concentrations. Within seconds of the injury, calcium ions enter the proximal nerve stump from the external fluid and, as membrane depolarization activates voltage-gated calcium channels, the intracellular calcium concentrations rise and trigger rapid sealing of the axon membrane at the injury site [
91,
92,
93,
94,
95]. Local protein translation proceeds, long-range retrograde signaling is activated, and the local cytoskeleton contributes to the formation of the growth cone [
96,
97,
98,
99]. The growth cone is composed primarily of a microtubule cytoskeleton and F-actin (filamentous actin) with the central domain containing microtubules, organelles, and vesicles, and the P domain, composed of dynamic microtubules and F-actin [
100,
101]. The F-actin bundles comprise the finger-like projections of the filopodia.
The axonal calcium activates the nucleotide cAMP (cyclic adenosine monophosphate), via the calcium-dependent adenylyl cyclase enzyme which, in turn, activates the pro-regenerative kinase DLK (dual leucine zipper-bearing kinase) via PKA (protein kinase A), and the transcription factor, CREB1 (cAMP-responsive element-binding protein 1), amongst others [
102,
103]. DLK is a key sensor of local injury that informs the soma about the injury [
102,
103]. JNK (JUN N-terminal kinase) downstream of DLK signaling, is also transported to the soma where it activates STAT3 (signal transducer and activator of transcription factor 3) and c-Jun {an immediate early gene of the AP-1 (activator protein-1 transcription factor) transcription complex of Jun, c-Fos, and the ATF/CREB families [
105]}, to promote nerve regeneration [
106,
107]. A wavefront of the calcium ions propagates anterograde to the cell body to reach millimolar concentrations in the soma. Calcium is also raised throughout the stump by additional calcium entry that follows the reversal of the sodium-calcium exchange pump by the calcium load [
92]. The accumulating calcium ions activate local intracellular calpain (a serine-threonine protein) and oxidative species, resulting in axon swelling, rapid granular disintegration of the axonal cytoskeleton [
108,
109,
110,
111,
112,
113], and dieback to the first node of Ranvier [
114,
115,
116]. The local calpain at the sealed end of the stump cleaves the submembranous spectrin complex, restructures the cytoskeleton by microtubule and actin depolymerization, and, in turn, allows the elaboration of the growth cone [
95,
117,
118,
119].
The local calcium activates many other signaling pathways. These include CaMK (Ca
2+/calmodulin-dependent protein kinase) that phosphorylates CREB in the nucleus. The phosphorylated CREB later influences gene expression directly. It does so by mediating cAMP-induced transcription [
120,
121] via PKA and MEK/Erk (extracellular signal-regulated kinase) pathways [
122], and the translation of the transcription factor, HIF-1a (hypoxia-inducible factor), that, in turn, activates HIF-1a responsive genes for axonal regeneration [
123].
There is an initial and local translational burst of mTOR (mammalian target of rapamycin) that controls ~250 localized mRNAs. These in turn, transcribe several proteins that are transported to the cell body via retrograde axonal transport. The transport is facilitated by the local increase in tyrosinated α-tubulin in the microtubular cytoskeleton [
124]. The transported proteins include importin-β1, the adaptor protein that transports cytoplasmic proteins via dynein, the retrograde motor on the microtubules [
125], vimentin (the intermediate filament protein), STAT3, ZBP-1 (Z-DNA-binding protein 1), RanBP1 (Ran binding protein 1), Ran being the Ras-related nuclear protein ligand-activated nuclear receptor, and PPARƔ, the peroxisome proliferator-activated receptor ([
126,
127,
128], reviewed by [
129]). Luman/ATF3, an endoplasmic reticulum (ER) transmembrane basic leucine zipper transcription factor, is also transported in an importin-dependent manner to the cell body where it is critical regulator of sensory axon regeneration, linking the unfolded protein response and the ensuing endoplasmic stress response to axon repair [
130,
131]. There is an interesting coordination of the temporal phases of the luman protein levels. They are co-ordinated with the three phases of the growth responsive response of sensory neurons, (1) the stress response phase immediately after injury, (2) the pre-regenerative phase, 9 to 24 hours thereafter when transcription factor activity regulates DNA replication and transcription, and (3) the regenerative phase at 4 days [
132].
The transported proteins activate pro-regenerative pathways in the
second slow signaling phase of nerve injury. These pathways include the translation and activation of transcription factors, specific epigenetic modifiers, and additional signaling molecules [
129].
Transcription factors There are more than 1500 transcription factors in the genome [
133] of which c-Jun is markedly increased in axotomized motor and sensory neurons [
134]. The genes that coordinate the transcription of many other genes, often the transcription factors, are referred to as Hub genes [
102,
135,
136]. The transcription factors bind to selective DNA promotor regions [
137,
138,
139,
140] to increase or repress the transcription of specific target genes, thereby coordinating the expression of multiple RAGs [
76,
129]. The transcription factor c-JUN was the first to be identified in a RAG network [134,141). CREB serves to coordinate the transcription of many RAGs because the CREB protein not only regulates transcription of BDNF and arginase 1, amongst other genes, but also drives the transcription of both the AP1 and the ATF3 hub genes, being a highly connected node [
101]. One study distilled the RAG network to ~40 transcription factors downstream of multiple parallel signaling pathways [
142], of which the calcium-dependent cAMP activates only a fraction of injury-induced genes, at least in sensory neurons [
143].
Transcription factors that were identified independently, include ATF3 (cAMP-dependent transcription factor 3) [
144,
145,
146,
147,
148], STAT3 [
149,
150], SOX11 {(SRY-Box Transcription Factor 11) [
151,
152,
153,
154]}, SMAD1 (Suppressor of Mothers against Decapentaplegic family member 1) [
155,
156,
157,
158,
159], C/EBPβ (CCAAT/enhancer binding protein β) [
160,
161], p53 (tumor protein p53) [
162,
162], GSK3β (glycogen synthase kinase 3β [
163]), c-Myc [
151,
152,
153,
154], and KLF4 (Krüppel-like factor 4) [
163,
164]. ATF-3, a cAMP-dependent transcription factor, is upregulated rapidly in injured motor and sensory neurons following JNK signalling [
87,
146,
147,
148]. The factor is used frequently and reliably as a biomarker of axotomy [27;87,145]. Both Jun and ATF3 mediate peripheral nerve regeneration in vitro [
151] and in vivo [
152]. Phosphorylated STAT3 stimulates growth initiation but does not perpetuate axonal growth [
150]. SOX11 is also elevated in injured nerves and promotes their regeneration [
151,
153]. It does so via the activation of the transcription factors, ATF3 and c-Jun, and the RAGs, Arpc3 and Sprr1a [
151,
153], and by increasing the responsiveness of neurotrophic factors [
153]. Elevated SMADs are phosphorylated and accumulate in the nuclei of injured DRGs and motoneurons, SMAD1 in the sensory neurons [
155,
156,
157,
158] and SMADs 1,2, and 4 in the motoneurons [
159]. They act as modulators of activated GSK3β (glycogen synthase kinase 3) downstream of P13K (phophoinositide-3-kinase)/Akt pathway, by interacting with the transcriptional coregulator p300 HAT (histone acetyltransferase p300), to promote expression of several pro-regenerative target genes and in turn, nerve regeneration [
155,
156,
158]. The transcription factor C/EBP (CCAAT/enhancer binding protein) that is induced in injured neurons, is also an essential transcription factor for nerve regeneration. It binds to the promoters of Tα1-tubulin of the microtubules and the growth cone protein, GAP-43 [
161]. It was reported that p53 stimulates regeneration of transected sciatic nerve fibres with reduced reinnervation of target muscle fibres after facial nerve injury in p53 knockout mice [
162,
163]. The c-Myc transcription factor stimulates peripheral nerve regeneration by elevating expression of the RAGs arpc3 and Sprr1a and the transcription factors c-Jun and Atf3, in the nucleus of axotomized DRG neurons, and by increasing the responsiveness of the neurons to neurotrophic factors [
151,
152,
153,
154]. In contrast to the other factors, KLF4 is downregulated after injury, thereby negating its inhibitory effect on nerve regeneration [
164,
165].
Epigenetic modifications, or “tags,” regulate patterns of gene expression by altering DNA accessibility and chromatin structure without altering the DNA sequence [
166]. They include the miRNA (microRNA), and the non-coding RNAs, namely the long chain and circular RNAs. They can affect nerve regeneration by altering the access of transcription factors to DNA by DNA methylation, post-translational modification of the histone protein that wraps around nuclear DNA, or by controlling ncRNAs (noncoding RNA) that silence genes [
101,
130,
167].
DNA methylation generally, is associated with repression of transcription [
129]. There are >200 identified histone modifications [
168]. An important example is the p300/CBP-associated factor (PCAF)-dependent acetylation of H3K9ac (histone 3 lysine 9) that is paralleled by the reduction in methylation of H3K9 (H3K9me2). This reduced methylation relaxes the chromatin environment surrounding the promoters of several pro-regenerative genes. In DRG sensory neurons, this modification results in the expression of the growth-associated genes
GAP-43, galanin, and
BDNF [
169]. The acetylation requires retrograde ERK signaling. Also, PCAF overexpression promotes axonal regeneration of the central axons of the DRG neurons across injured spinal cord.
MicroRNAs (miRNA), of which lncRNA (long chain non-coding RNAs) account for 60 to 80% of the mammalian genome transcriptome [
170], are differentially expressed after peripheral nerve injury [
101,
171]. They target specific mRNAs with resulting repression or degradation of their translation. For example, miR-21 is upregulated after sciatic nerve injury and by targeting Sprouty2 (a specific inhibitor of the Ras/Raf/Erk pathway), promotes axonal growth from adult DRG neurons [
172,
173]. A second example is the nerve regeneration that results from miR-26a that specifically targets GSK3β to rescue axon regeneration, the miR26a-GSK3β pathway regulating axon regeneration at the neuronal soma by controlling the expression of the regeneration-associated transcription factor, SMAD1 [
160].
2c. Wallerian Degeneration
The peripheral nerve fibres distal to the site of crush or transection, are isolated from their neuronal cell bodies and, in turn are deprived, for all intents and purposes, of their source of synthesis of proteins, lipids, glycoproteins, and carbohydrates [
15,
72,
109]. They undergo the self-destructive process of Wallerian degeneration of nerve fibres and their myelin, including the axons that die back to the first node of Ranvier in the proximal nerve stump that prevents the scarring that occurs in CNS injury [
114,
115,
116,
174]. Ramon Y Cajal [
1], using his silver staining technique, elucidated the degeneration of the axons distal to the nerve injury, myelin breakdown, and the proliferation of the remaining SCs. The calcium ions that enter the nerve via calcium channels, activate calpain proximal and distal to the injury site, that mediates proteolysis with degeneration of axon segments several hundred micrometers from the injury site [
175,
176]. The expression of SARM1 (sterile alpha TIR motif-containing protein 1), the essential protein for axon degeneration in the distal nerve stump, is elevated due to the loss of the anterograde transport by the calcium-dependent proteolysis of the cytoskeletal structures in the stump, followed by a rapid depletion of NMNAT2 (NAD-synthesizing enzyme, nicotinamide mononucleotide acetyltransferase 2; reviewed by [
18]). The downstream steps to axon degeneration remain to be determined. Meanwhile, the remaining fast axonal transport allows for continued propagation of action potentials in the distal stump for hours and even days [
108,
177,
178,
179,
180].
Ras/raf/ERK signaling in SCs is evident immediately after injury with ERK levels returning to lower levels just prior to SC proliferation [
181]. Activation of Raf-kinase drives SC dedifferentiation as well as inducing much of the inflammatory response important for nerve repair, including breakdown of the blood nerve barrier and the delayed recruitment of macrophages into the denervated nerve stump [
182]. SCs break their myelin sheaths down through autophagy [
183,
184,
185] with ~50% of the total myelin thought to be broken down by the SCs [
186]. The SC expression of proinflammatory cytokines and chemokines within 3 to 5 hours of nerve injury, contribute to myelin and axon breakdown and the phagocytosis of their debris within the first 3 days of injury [
187]. The cytokines, including IL-1α (interleukin 1α), and LIF (leukemia inhibitory factor), their receptors IL-6R and gp130 respectively, and tumor necrosis factor α (TNF-α), stimulate the expression of the two cystolic forms of PLA
2 (phospholipase enzyme-A
2) [
188]. These remain high for two weeks [
187]. TNF-α hydrolyses the phosphatidylcholines in the myelin membranes, thereby releasing the potent myelinolytic agent, lysophosphotidylcholine. Lysophosphotidylcholine also feeds back to sustain cytokine expression and the expression of the chemokines, including MIP-1 (macrophages inflammatory protein-1), MMP-1α or CCL2 (monocyte chemoattractant protein-1α) and IL-lβ [
17,
189,
190,
191,
192,
193,
194,
195,
196]. Within a day of the nerve injury, IL-6 and TNF-α induce the expression of MMP-9 (matrix metallopeptidase 9) that contributes to myelinolysis [
197,
198].
The recruited macrophages also express these cytokines and chemokines and are responsible for the bulk of the myelin and axonal phagocytosis over the following ~3 weeks [
116,
199,
200,
201,
202]. They play the major role in removing inhibitory molecules, including MAGs (myelin associated glycoproteins), from the degenerating axons [
202]. Their release of nitric oxide has been implicated in the myelin breakdown [
203] and their phagocytosis of the myelin debris includes the myelin that is opsonized by complement components binding to the complement receptor type 3 on the macrophages [
204]. Antibodies to non-opsonized myelin are phagocytosed via the macrophage Fc receptor [
205]. The third macrophage receptor used is the scavenger receptor-AI,II [
206]. The spectrum of macrophages has been separated into two “polarizing” phenotypes, M1 and M2 with the M1 macrophages associated with pro-inflammatory and neurodegenerative functions and the M2 macrophages broadly viewed as anti-inflammatory and promoting cellular repair [
207]. Although an early increase in M1 macrophages 1-2 days after injury with M2 macrophages replacing these from days 3 to 7, the majority of the accumulating M2 macrophages may be of a mixed phenotype because these mixed type macrophages were not included in their analysis [
207,
208]. In addition to their essential role in phagocytosis of degenerating axons and their myelin, the macrophages sense hypoxic conditions and stimulate angiogenesis for a polarized vasculature that guides SCs and elongating axons [
209,
210].
2d. Schwann Cell Response to Nerve Injury
Once the SCs lose their myelin, they re-enter the cell cycle, undergo mitosis, and differentiate toward a state supportive of nerve regeneration [
5,
15,
75,
108,
139,
211]. The transient expression of Cdc2 (cyclin-dependent kinase, cell cycle division 2) by the denervated SCs, possibly induced by c-Jun [
212], is involved in their proliferation and migration [
213]. The SC genes that transcribe myelin proteins, including MAP (myelin associated protein), MBP (myelin basic protein), P
0 (myelin protein zero), and PLP (proteolipid protein), are downregulated in the denervated distal stump with the RAGs associated with nerve regeneration, upregulated as the SCs dedifferentiate and acquire the ability to survive without axonal interactions (
Figure 1A; [
214]). This change in SC transcription occurs with their transition from their myelinating state to the growth supportive state [
75]. The transcription factor, c-Jun, programs the SCs to generate the repair cells that are essential for nerve regeneration, c-Jun accelerating the downregulation of myelin genes, promoting myelin breakdown, and amplifying the upregulation of a broad spectrum of repair-supportive features, including the expression of trophic factors ([
137,
215]; reviewed by Jessen and Arthur-Farraj [
216]). As early as 1991, the upregulation of hundreds of growth-supportive RAGs was reported in denervated SCs [
217] with more genes differentially expressed in the SCs than in sensory neurons [
218].
IL-6, synthesized in the SCs within 24 hours [
190,
192,
219,
220], signals the expression of RAGs via its receptor [
191]. The SCs express NRG-1 (neuregulin-1), a member of the family of glial growth factors, and its ErbB2/3 receptor [
221,
222,
223,
224]. The NRG-1 levels remain elevated for at least 30 days [
225,
226,
227]. NRG-1 strongly inhibits the expression of genes involved in myelination and in glial cell differentiation, suggesting that it might be involved in the dedifferentiation process of SCs from the myelinating to the repair phenotype [
228]. In addition, NRG-1 likely mediates, at least in part, the second phase of SC proliferation that is stimulated when regenerating axons contact the SCs in the Bands of Büngner [
229,
230,
231,
232,
233,
234,
235]. The scaffolding oncoprotein Gab2 (Grb2-associated binder-2) is required for SC proliferation after nerve injury, its activation leading to the migration of the SCs [
236], possibly through actin modulation [
237]. In the model based on their findings, autocrine/paracrine activation by NRG of its erbB2 receptor on the SCs, promotes the SC migration by leading to transcriptional Gab2 expression via the Rac-JNK-cJun pathway, and the phosphorylation of Gab2 via the paracrine HGF (hepatocyte growth factor) from fibroblasts [
236].
The non-coding RNAs, namely the long chain and circular RNAs, play important roles in SC proliferation and migration [
101]. Examples of the long chain RNAs include NEAT1 (nuclear enriched abundant transcript 1) that promotes SC proliferation and migration [
101], MALAT1 (metastasis associated lung adenocarcinoma transcript 1) that elevates BDNF [
238], and Loc680254 that promotes nerve regeneration by inducing SC proliferation [
239]. BC088259 which showed the most significant upregulation after sciatic nerve injury, interacts with vimentin to regulate SC migration [
240,
241]. Downregulation of some long chain RNAs also enhances SC proliferation and migration. An example is MEG-3 (maternally expressed gene 3) that increases SC proliferation and migration and facilitates nerve regeneration through the PTEN/P13K/ADT pathway [
242]. Some circular RNAs are also upregulated in the denervated nerve stumps and are associated with SC proliferation. For example, cirRNA-Spidr targets P13K-Akt to promote nerve regeneration after rat sciatic nerve crush injury [
243].
Within the first week of nerve injury, the denervated SC’s express several neurotrophic factors and their receptors, their levels peaking within a month. The factors include the neurotrophins, NGF (nerve growth factor), BDNF, and their p75 receptor, NT-3 and NT-4/5 (neurotrophin-3 and 4/5) and the TrkC receptor, the GDNF (glial derived neurotrophic factor) family and their receptors, GDFRα1 and ret, and other factors including IGF-1 and IGF-II (insulin-like neurotrophic factor I and II), VEGF (vascular endothelial growth factor), HGF (hepatocyte growth factor), platelet-rederived growth factor-BB, FGF (fibroblast growth factor), TFG-β (transforming growth factor β), and their receptors, and pleiotrophin [
16,
244,
245,
246,
247,
248,
249] and Xu et al 2023 unpublished data. The expression of GDNF and pleiotrophin is specific for the denervated SCs in the motor pathways of the quadriceps nerve branch of the femoral nerve, whereas the remaining neurotrophic factors, HGF, BDNF, NGF, and IGF I and II, are more specific for the denervated SCs located in the sensory pathways of the saphenous nerve branch [
245,
246,
247]. The time course of expression varies for different trophic factors. NGF rises rapidly, then declines prior to a 5-fold upregulation possibly in response to macrophage release of IL-1β and persisting for at least 3 weeks [
250,
251]. The upregulation of BDNF is much slower, detectable at 7 days and increasing up to 28 days after nerve injury to levels much higher than NGF [
16,
251]. On the other hand, GDNF and its GDFRα1, but not its coreceptor Ret, are upregulated and reach a peak within 7 days [
16,
252]. The expression is not sustained, declining with time when the denervation period of the distal nerve stump is prolonged for more than a month [
16,
244,
245,
247,
252]. NT-3 upregulates c-Jun [
215] and regulates the levels of the upregulated p75 receptor in the SCs [
182,
254,
255,
256]. The transcription factor, Notch, is also upregulated in the SCs [
137,
138,
139,
140].
2e. Axonal Regeneration
Regenerating axonal sprouts emanate from the first node of Ranvier proximal to the injury site [
1,
257,
258]. The formation of the growth cones occurs without direct support from the cell body and depending on material that is locally available in the axons in the proximal nerve stump that includes the preexisting cytoskeletal elements of actin and tubulin [
258,
259]. The major source of materials for subsequent axonal elongation comes from the cell body via anterograde axonal transport [
79]. As described above in
Section 2d. Schwann cell response to nerve injury, the growth cones link to ECM glycoproteins that become organized at the injury site, navigate across the site, and regenerate axons along the bands of Bungner in the denervated distal stumps. A single regenerating axon can give rise to as many of 50-100 branches [
260] but, it is an average of 5 daughter axons that regenerate with more regenerating into the distal stump after crush than after transection injuries [
261,
262,
263].
Following the nerve injury, the SCs migrate to form the repair SC layer of the Bands of Bungner on the endothelial laminal sheath [
214]. They do so by their sorting through intercellular N-cadherin linkage mediated by EphB-Sox signaling [
264]. The growing axons contact the SC basement membrane glycoproteins that are secreted by both SCs and fibroblasts. These glycoproteins include collagen, fibronectin, tenascin C, and laminin ([
265,
266,
268,
269]; reviewed by [
5,
214,
267,
270,
272]). Via adaptor molecules on the SC membranes, including N-cadherin and integrins, the interaction of the axons with the glycoproteins mediates the progression of the growth cones as the axons regenerate [
271,
273,
274,
275,
276,
277,
278,
279,
280].
Contact between the axolemma and SCs initiates remyelination of regenerating axons with the SCs forming myelin layers (lamellae) in proportion to the size of the regenerating axons [
281]. Both NRG-III from axons and NRG1-I from SCs play a pivotal role in remyelination [
226,
282,
283] with tyrosine phosphorylation of GAB1 in the denervated SCs, principally regulated by NRG-1, being essential for remyelination [
236]. The internodal distances between the formed myelin sheaths are shorter than normal as a result of the 3-fold increase in SC numbers after denervation [
284] but they lengthen with time [
281]. The regenerating axons increase their diameters in proportion to the size of their parent nerve [
285], recovering their normal size if and when they make functional connections [
286].