Neuritin accelerates Schwann cell dedifferentiation via PI3K/Akt/mTOR signalling pathway during Wallerian degeneration.

Neuritin, also known as candidate plasticity gene 15 (CPG15), was first identified as one of the activity-dependent gene products in the brain. Previous studies have been reported that Neuritin induces neuritogenesis, neurite arborization, neurite outgrowth and synapse formation, which are involved in the development and functions of the central nervous system. However, the role of Neuritin in peripheral nerve injury is still unknown. Given the importance and necessity of Schwann cell dedifferentiation response to peripheral nerve injury, we aim to investigate the molecular mechanism of Neuritin steering Schwann cell dedifferentiation during Wallerian degeneration (WD) in injured peripheral nerve. Herein, using the explants of sciatic nerve, an ex vivo model of nerve degeneration, we provided evidences indicating that Neuritin vividly accelerates Schwann cell dedifferentiation. Moreover, we found that Neuritin promotes Schwann cell demyelination as well as axonal degeneration, phagocytosis, secretion capacity. In summary, we first described Neuritin acts as a positive regulator for Schwann cell dedifferentiation and WD after peripheral nerve injury.

Tempol, a Superoxide Dismutase Mimetic, Inhibits Wallerian Degeneration Following Spinal Cord Injury by Preventing Glutathione Depletion and Aldose Reductase Activation.

Spinal cord contusion injury results in Wallerian degeneration of spinal cord axonal tracts, which are necessary for locomotor function. Axonal swelling and loss of axonal density at the contusion site, characteristic of Wallerian degeneration, commence within hours of injury. Tempol, a superoxide dismutase mimetic, was previously shown to reduce the loss of spinal cord white matter and improve locomotor function in an experimental model of spinal cord contusion, suggesting that tempol treatment might inhibit Wallerian degeneration of spinal cord axons. Here, we report that tempol partially inhibits Wallerian degeneration, resulting in improved locomotor recovery. We previously reported that Wallerian degeneration is reduced by inhibitors of aldose reductase (AR), which converts glucose to sorbitol in the polyol pathway. We observed that tempol inhibited sorbitol production in the injured spinal cord to the same extent as the AR inhibitor, sorbinil. Tempol also prevented post-contusion upregulation of AR (AKR1B10) protein expression within degenerating axons, as previously observed for AR inhibitors. Additionally, we hypothesized that tempol inhibits axonal degeneration by preventing loss of the glutathione pool due to polyol pathway activity. Consistent with our hypothesis, tempol treatment resulted in greater glutathione content in the injured spinal cord, which was correlated with increased expression and activity of gamma glutamyl cysteine ligase (γGCL; EC 6.3.2.2), the rate-limiting enzyme for glutathione synthesis. Administration of the γGCL inhibitor buthionine sulfoximine abolished all observed effects of tempol administration. Together, these results support a pathological role for polyol pathway activation in glutathione depletion, resulting in Wallerian degeneration after spinal cord injury (SCI). Interestingly, methylprednisolone, oxandrolone, and clenbuterol, which are known to spare axonal tracts after SCI, were equally effective in inhibiting polyol pathway activation. These results suggest that prevention of AR activation is a common target of many disparate post-SCI interventions.

The SARM1 TIR domain produces glycocyclic ADPR molecules as minor products.

Sterile alpha and TIR motif-containing 1 (SARM1) is a protein involved in programmed death of injured axons. Following axon injury or a drug-induced insult, the TIR domain of SARM1 degrades the essential molecule nicotinamide adenine dinucleotide (NAD+), leading to a form of axonal death called Wallerian degeneration. Degradation of NAD+ by SARM1 is essential for the Wallerian degeneration process, but accumulating evidence suggest that other activities of SARM1, beyond the mere degradation of NAD+, may be necessary for programmed axonal death. In this study we show that the TIR domains of both human and fruit fly SARM1 produce 1''-2' and 1''-3' glycocyclic ADP-ribose (gcADPR) molecules as minor products. As previously reported, we observed that SARM1 TIR domains mostly convert NAD+ to ADPR (for human SARM1) or cADPR (in the case of SARM1 from Drosophila melanogaster). However, we now show that human and Drosophila SARM1 additionally convert ~0.1-0.5% of NAD+ into gcADPR molecules. We find that SARM1 TIR domains produce gcADPR molecules both when purified in vitro and when expressed in bacterial cells. Given that gcADPR is a second messenger involved in programmed cell death in bacteria and likely in plants, we propose that gcADPR may play a role in SARM1-induced programmed axonal death in animals.

Elevated intracellular Ca2+ functions downstream of mitodysfunction to induce Wallerian-like degeneration and necroptosis in organophosphorus-induced delayed neuropathy.

Neurotoxic organophosphorus compounds can induce a type of delayed neuropathy in humans and sensitive animals, known as organophosphorus-induced delayed neuropathy (OPIDN). OPIDN is characterized by axonal degeneration akin to Wallerian-like degeneration, which is thought to be caused by increased intra-axonal Ca2+ concentrations. This study was designed to investigate that deregulated cytosolic Ca2+ may function downstream of mitodysfunction in activating Wallerian-like degeneration and necroptosis in OPIDN. Adult hens were administrated a single dosage of 750 mg/kg tri-ortho-cresyl phosphate (TOCP), and then sacrificed at 1 day, 5 day, 10 day and 21 day post-exposure, respectively. Sciatic nerves and spinal cords were examined for pathological changes and proteins expression related to Wallerian-like degeneration and necroptosis. In vitro experiments using differentiated neuro-2a (N2a) cells were conducted to investigate the relationship among mitochondrial dysfunction, Ca2+ influx, axonal degeneration, and necroptosis. The cells were co-administered with the Ca2+-chelator BAPTA-AM, the TRPA1 channel inhibitor HC030031, the RIPK1 inhibitor Necrostatin-1, and the mitochondrial-targeted antioxidant MitoQ along with TOCP. Results demonstrated an increase in cytosolic calcium concentration and key proteins associated with Wallerian degeneration and necroptosis in both in vivo and in vitro models after TOCP exposure. Moreover, co-administration with BATPA-AM or HC030031 significantly attenuated the loss of NMNAT2 and STMN2 in N2a cells, as well as the upregulation of SARM1, RIPK1 and p-MLKL. In contrast, Necrostatin-1 treatment only inhibited the TOCP-induced elevation of p-MLKL. Notably, pharmacological protection of mitochondrial function with MitoQ effectively alleviated the increase in intracellular Ca2+ following TOCP and mitigated axonal degeneration and necroptosis in N2a cells, supporting mitochondrial dysfunction as an upstream event of the intracellular Ca2+ imbalance and neuronal damage in OPIDN. These findings suggest that mitochondrial dysfunction post-TOCP intoxication leads to an elevated intracellular Ca2+ concentration, which plays a pivotal role in the initiation and development of OPIDN through inducing SARM1-mediated axonal degeneration and activating the necroptotic signaling pathway.

Iron role paradox in nerve degeneration and regeneration.

Iron accumulates in the neural tissue during peripheral nerve degeneration. Some studies have already been suggested that iron facilitates Wallerian degeneration (WD) events such as Schwann cell de-differentiation. On the other hand, intracellular iron levels remain elevated during nerve regeneration and gradually decrease. Iron enhances Schwann cell differentiation and axonal outgrowth. Therefore, there seems to be a paradox in the role of iron during nerve degeneration and regeneration. We explain this contradiction by suggesting that the increase in intracellular iron concentration during peripheral nerve degeneration is likely to prepare neural cells for the initiation of regeneration. Changes in iron levels are the result of changes in the expression of iron homeostasis proteins. In this review, we will first discuss the changes in the iron/iron homeostasis protein levels during peripheral nerve degeneration and regeneration and then explain how iron is related to nerve regeneration. This data may help better understand the mechanisms of peripheral nerve repair and find a solution to prevent or slow the progression of peripheral neuropathies.

Next-generation RNA sequencing elucidates transcriptomic signatures of pathophysiologic nerve regeneration.

The cellular and molecular underpinnings of Wallerian degeneration have been robustly explored in laboratory models of successful nerve regeneration. In contrast, there is limited interrogation of failed regeneration, which is the challenge facing clinical practice. Specifically, we lack insight on the pathophysiologic mechanisms that lead to the formation of neuromas-in-continuity (NIC). To address this knowledge gap, we have developed and validated a novel basic science model of rapid-stretch nerve injury, which provides a biofidelic injury with NIC development and incomplete neurologic recovery. In this study, we applied next-generation RNA sequencing to elucidate the temporal transcriptional landscape of pathophysiologic nerve regeneration. To corroborate genetic analysis, nerves were subject to immunofluorescent staining for transcripts representative of the prominent biological pathways identified. Pathophysiologic nerve regeneration produces substantially altered genetic profiles both temporally and in the mature neuroma microenvironment, in contrast to the coordinated genetic signatures of Wallerian degeneration and successful regeneration. To our knowledge, this study presents as the first transcriptional study of NIC pathophysiology and has identified cellular death, fibrosis, neurodegeneration, metabolism, and unresolved inflammatory signatures that diverge from pathways elaborated by traditional models of successful nerve regeneration.

Glycolytic System in Axons Supplement Decreased ATP Levels after Axotomy of the Peripheral Nerve.

Wallerian degeneration (WD) occurs in the early stages of numerous neurologic disorders, and clarifying WD pathology is crucial for the advancement of neurologic therapies. ATP is acknowledged as one of the key pathologic substances in WD. The ATP-related pathologic pathways that regulate WD have been defined. The elevation of ATP levels in axon contributes to delay WD and protects axons. However, ATP is necessary for the active processes to proceed WD, given that WD is stringently managed by auto-destruction programs. But little is known about the bioenergetics during WD. In this study, we made sciatic nerve transection models for GO-ATeam2 knock-in rats and mice. We presented the spatiotemporal ATP distribution in the injured axons with in vivo ATP imaging systems, and investigated the metabolic source of ATP in the distal nerve stump. A gradual decrease in ATP levels was observed before the progression of WD. In addition, the glycolytic system and monocarboxylate transporters (MCTs) were activated in Schwann cells following axotomy. Interestingly, in axons, we found the activation of glycolytic system and the inactivation of the tricarboxylic acid (TCA) cycle. Glycolytic inhibitors, 2-deoxyglucose (2-DG) and MCT inhibitors, a-cyano-4-hydroxycinnamic acid (4-CIN) decreased ATP and enhanced WD progression, whereas mitochondrial pyruvate carrier (MPC) inhibitors (MSDC-0160) did not change. Finally, ethyl pyruvate (EP) increased ATP levels and delayed WD. Together, our findings suggest that glycolytic system, both in Schwann cells and axons, is the main source of maintaining ATP levels in the distal nerve stump.

NMNAT2: An important metabolic enzyme affecting the disease progression.

Nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) is an evolutionarily conserved nicotinamide adenine dinucleotide (NAD+) synthase located in the cytoplasm and Golgi apparatus. NMNAT2 has an important role in neurodegenerative diseases, malignant tumors, and other diseases that seriously endanger human health. NMNAT2 exerts a neuroprotective function through its NAD synthase activity and chaperone function. Among them, the NMNAT2-NAD+-Sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1) axis is closely related to Wallerian degeneration. Physical injury or pathological stimulation will cause a decrease in NMNAT2, which activates SARM1, leading to axonal degeneration and the occurrence of amyotrophic lateral sclerosis (ALS), Alzheimer's disease, peripheral neuropathy, and other neurodegenerative diseases. In addition, NMNAT2 exerts a cancer-promoting role in solid tumors, including colorectal cancer, lung cancer, ovarian cancer, and glioma, and is closely related to tumor occurrence and development. This paper reviews the chromosomal and subcellular localization of NMNAT2 and its basic biological functions. We also summarize the NMNAT2-related signal transduction pathway and the role of NMNAT2 in diseases. We aimed to provide a new perspective to comprehensively understand the relationship between NMNAT2 and its associated diseases.

Tri-ortho-cresyl phosphate induces axonal degeneration in chicken DRG neurons by the NAD+ pathway.

Wallerian degeneration (WD) is a well-known process by which degenerating axons and myelin are cleared after nerve injury. Although organophosphate-induced delayed neuropathy (OPIDN) is characterized by Wallerian-like degeneration of long axons in human and sensitive animals, the precise pathological mechanism remains unclear. In this study, we cultured embryonic chicken dorsal root ganglia (DRG) neurons, the model of OPIDN in vitro, to investigate the underlying mechanism of axon degeneration induced by tri-ortho-cresyl phosphate (TOCP), an OPIDN inducer. The results showed that TOCP exposure time- and concentration-dependently induced a serious degeneration and fragmentation of the axons from the DRG neurons. A collapse of mitochondrial membrane potential and a dramatic depletion of ATP levels were found in the DRG neurons after TOCP treatment. In addition, nicotinamide nucleotide adenylyl transferase 2 (NMNAT2) expression and nicotinamide adenine dinucleotide (NAD+) level was also found to be decreased in the DRG neurons exposed to TOCP. However, the TOCP-induced Wallerian degeneration in the DRG neurons could be inhibited by ATP supplementation. And exogenous NAD+ or NAD+ processor nicotinamide riboside can rescue TOCP-induced ATP deficiency and prevent TOCP-induced axon degeneration of the DRG neurons. These findings may shed light on the pathophysiological mechanism of TOCP-induced axonal damages, and implicate the potential application of NAD+ to treat OPIDN.

SIRT6 Negatively Regulates Schwann Cells Dedifferentiation via Targeting c-Jun During Wallerian Degeneration After Peripheral Nerve Injury.

Silent information regulator 6 (SIRT6) is a mammalian homolog of the nicotinamide adenine dinucleotide (NAD)-dependent deacetylase sirtuin family. Previous studies have been reported a pro-regenerative role of SIRT6 in central nervous system injury. However, the role of SIRT6 in peripheral nerve injury is still unknown. Given the importance and necessity of Schwann cell dedifferentiation response to peripheral nerve injury, we aim to investigate the molecular mechanism of SIRT6 steering Schwann cell dedifferentiation during Wallerian degeneration in injured peripheral nerve. Herein, we first examined the expression pattern of SIRT6 after peripheral nerve injury. Using the explants of sciatic nerve, an ex vivo model of nerve degeneration, we provided evidences indicating that SIRT6 inhibitor accelerates Schwann cell dedifferentiation as well as axonal and myelin degeneration, while SIRT6 activator attenuates this process. Moreover, in an in vitro Schwann cell dedifferentiation model, we found SIRT6 inhibitor promotes Schwann cell dedifferentiation through upregulating the expression of c-Jun. In addition, downregulation of c-Jun reverse the effects of SIRT6 inhibition on the Schwann cells dedifferentiation and axonal and myelin degeneration. In summary, we first described SIRT6 acts as a negative regulator for Schwann cells dedifferentiation during Wallerian degeneration and c-Jun worked as a direct downstream partner of SIRT6 in injured peripheral nerve.

Macrophage-specific RhoA knockout delays Wallerian degeneration after peripheral nerve injury in mice.

BACKGROUND: Plenty of macrophages are recruited to the injured nerve to play key roles in the immunoreaction and engulf the debris of degenerated axons and myelin during Wallerian degeneration, thus creating a conducive microenvironment for nerve regeneration. Recently, drugs targeting the RhoA pathway have been widely used to promote peripheral axonal regeneration. However, the role of RhoA in macrophage during Wallerian degeneration and nerve regeneration after peripheral nerve injury is still unknown. Herein, we come up with the hypothesis that RhoA might influence Wallerian degeneration and nerve regeneration by affecting the migration and phagocytosis of macrophages after peripheral nerve injury. METHODS: Immunohistochemistry, Western blotting, H&E staining, and electrophysiology were performed to access the Wallerian degeneration and axonal regeneration after sciatic nerve transection and crush injury in the LyzCre+/-; RhoAflox/flox (cKO) mice or Lyz2Cre+/- (Cre) mice, regardless of sex. Macrophages' migration and phagocytosis were detected in the injured nerves and the cultured macrophages. Moreover, the expression and potential roles of ROCK and MLCK were also evaluated in the cultured macrophages. RESULTS: 1. RhoA was specifically knocked out in macrophages of the cKO mice; 2. The segmentation of axons and myelin, the axonal regeneration, and nerve conduction in the injured nerve were significantly impeded while the myoatrophy was more severe in the cKO mice compared with those in Cre mice; 3. RhoA knockout attenuated the migration and phagocytosis of macrophages in vivo and in vitro; 4. ROCK and MLCK were downregulated in the cKO macrophages while inhibition of ROCK and MLCK could weaken the migration and phagocytosis of macrophages. CONCLUSIONS: Our findings suggest that RhoA depletion in macrophages exerts a detrimental effect on Wallerian degeneration and nerve regeneration, which is most likely due to the impaired migration and phagocytosis of macrophages resulted from disrupted RhoA/ROCK/MLCK pathway. Since previous research has proved RhoA inhibition in neurons was favoring for axonal regeneration, the present study reminds us of that the cellular specificity of RhoA-targeted drugs is needed to be considered in the future application for treating peripheral nerve injury.

p53 is required for nuclear but not mitochondrial DNA damage-induced degeneration.

While the consequences of nuclear DNA damage have been well studied, the exact consequences of acute and selective mitochondrial DNA (mtDNA) damage are less understood. DNA damaging chemotherapeutic drugs are known to activate p53-dependent apoptosis in response to sustained nuclear DNA damage. While it is recognized that whole-cell exposure to these drugs also damages mtDNA, the specific contribution of mtDNA damage to cellular degeneration is less clear. To examine this, we induced selective mtDNA damage in neuronal axons using microfluidic chambers that allow for the spatial and fluidic isolation of neuronal cell bodies (containing nucleus and mitochondria) from the axons (containing mitochondria). Exposure of the DNA damaging drug cisplatin selectively to only the axons induced mtDNA damage in axonal mitochondria, without nuclear damage. We found that this resulted in the selective degeneration of only the targeted axons that were exposed to DNA damage, where ROS was induced but mitochondria were not permeabilized. mtDNA damage-induced axon degeneration was not mediated by any of the three known axon degeneration pathways: apoptosis, axon pruning, and Wallerian degeneration, as Bax-deficiency, or Casp3-deficiency, or Sarm1-deficiency failed to protect the degenerating axons. Strikingly, p53, which is essential for degeneration after nuclear DNA damage, was also not required for degeneration induced with mtDNA damage. This was most evident when the p53-deficient neurons were globally exposed to cisplatin. While the cell bodies of p53-deficient neurons were protected from degeneration in this context, the axons farthest from the cell bodies still underwent degeneration. These results highlight how whole cell exposure to DNA damage activates two pathways of degeneration; a faster, p53-dependent apoptotic degeneration that is triggered in the cell bodies with nuclear DNA damage, and a slower, p53-independent degeneration that is induced with mtDNA damage.

Cisplatin induced neurotoxicity is mediated by Sarm1 and calpain activation.

Cisplatin is a commonly used chemotherapy agent with significant dose-limiting neurotoxicity resulting in peripheral neuropathy. Although it is postulated that formation of DNA-platinum adducts is responsible for both its cytotoxicity in cancer cells and side effects in neurons, downstream mechanisms that lead to distal axonal degeneration are unknown. Here we show that activation of calpains is required for both neurotoxicity and formation of DNA-platinum adduct formation in neurons but not in cancer cells. Furthermore, we show that neurotoxicity of cisplatin requires activation of Sarm1, a key regulator of Wallerian degeneration, as mice lacking the Sarm1 gene do not develop peripheral neuropathy as evaluated by both behavioral or pathological measures. These findings indicate that Sarm1 and/or specific calpain inhibitors could be developed to prevent cisplatin induced peripheral neuropathy.

Measuring Bioenergetic Signatures of Peripheral Nerve Segments by Extracellular Flux Analysis.

Changes of energy metabolism in axons and their adjacent glia as well as alterations in metabolic axon-glia cross talk are emerging as central mechanistic components underlying axon degeneration. The analysis of extracellular flux with commercial metabolic analyzers greatly facilitates the measurement of key parameters of glycolytic and mitochondrial energy metabolism in cells and tissues. In this chapter, I describe a straightforward method to capture bioenergetic profiles of acutely isolated peripheral nerve segments using the Agilent Seahorse XFe24 platform.

Deletion of SIRPα (signal regulatory protein-α) promotes phagocytic clearance of myelin debris in Wallerian degeneration, axon regeneration, and recovery from nerve injury.

BACKGROUND: Recovery of function from traumatic nerve injury depends on the ability of severed axons to grow/regenerate back to their target tissues. This is achieved by successfully crossing the lesion site where physical impact severed axons, determined by the type of trauma, followed by successfully growing throughout the Wallerian degenerating nerve segment located distal to and beyond the lesion site, determined by the nature of Wallerian degeneration. The protracted removal of myelin debris in Wallerian degeneration, which leads residual myelin debris to slow down axon growth, impedes recovery of function. We focused in this study on mechanism(s) that delay the removal of myelin debris in Wallerian degeneration and so impede recovery. Previously, we showed that myelin debris inhibited its own phagocytosis in primary cultured macrophages and microglia as CD47 on myelin ligated SIRPα (signal regulatory protein-α) on phagocytes, and sequentially, SIRPα generated "don't eat me" signaling. We also demonstrated that serum inhibited phagocytosis in a SIRPα-dependent manner. Herein, we aimed to determine whether SIRPα-dependent inhibition of phagocytosis in macrophages impedes the in vivo removal of myelin debris in Wallerian degeneration, further leading to impaired healing. METHODS: Using SIRPα null (SIRPα-/-) and littermate wild-type (SIRPα+/+) mice, we studied the recovery of sensory and motor functions from nerve injury and, further, axon regeneration, SIRPα expression, myelin debris removal, and the phagocytic capacity and presence of macrophages in Wallerian degeneration. RESULTS: Myelin debris removal, axon regeneration, and the recovery of functions were all faster in SIRPα-/- mice than in wild-type mice. Between the two cell types that mostly scavenge myelin debris, macrophages but not Schwann cells expressed SIRPα in wild-type mice, and furthermore, SIRPα-/- macrophages phagocytosed significantly more than wild-type macrophages. CONCLUSIONS: Our findings suggest an intrinsic normally occurring SIRPα-dependent mechanism that impedes the in vivo removal of myelin debris in Wallerian degeneration by inhibiting the phagocytosis of myelin debris in macrophages, hence preventing fast growing axons from fully implementing their regenerative potential. Thus, accelerating the removal of myelin debris by eliminating SIRPα-dependent inhibition of phagocytosis will most likely advance recovery of functions from nerve injury.

FGF9 alters the Wallerian degeneration process by inhibiting Schwann cell transformation and accelerating macrophage infiltration.

In vitro experiments have proved that Fibroblast Growth Factor 9 (FGF9) was decreased in Schwann cells (SCs) in which Wallerian degeneration (WD) occurred after nerve injury. We hypothesize that FGF9 downregulation in WD has some biological influence on Schwann cells (SCs) and macrophages - the two most important cell components involved in WD. In this study, we employed strategies to regulate FGF9 in sciatic nerve crush by generating a mouse model, wherein Fgf9 was specifically knocked-out in SCs, and an intraneural injection of human FGF9 protein administered to overexpress FGF9 independently. Furthermore, an inhibitor of extracellular-regulated kinases 1/2 (ERK1/2), PD0325901, was used to clarify the underlying downstream mechanism of ERK1/2 activated by FGF9. Analysis of WD revealed the novel features of FGF9: (i) FGF9 was widely expressed in axons and SCs, and was decreased during WD process. (ii) Fgf9 knockout in SCs impaired the debris clearance and eventually impeded the regeneration of nerve fibers after damage. (iii) Fgf9 knockout in SCs promoted the dedifferentiation of SCs and delayed the infiltration of macrophages by decreasing Mcp1, Tnfα, Il1ß levels and leaky blood-nerve-barrier (BNB) in WD. (iv) FGF9 injection preserved the nerve fibers, inhibited SCs dedifferentiation and accelerated macrophages infiltration. (v) ERK1/2 phosphorylation was increased by exogenous FGF9 injection. P75, Cyclin D1, Mcp1, Tnfα, Il1ß, c-Jun changes by FGF9 intraneural injection were partially reversed by the ERK1/2 inhibitor. Conclusion was that FGF9 inhibited the dedifferentiation of SCs and accelerated the accumulation of macrophages in WD, and exogenous FGF9 took effects partially by ERK1/2.

Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration.

Apoptotic cells expose Phosphatidylserine (PS), that serves as an "eat me" signal for engulfing cells. Previous studies have shown that PS also marks degenerating axonsduring developmental pruning or in response to insults (Wallerian degeneration), but the pathways that control PS exposure on degenerating axons are largely unknown. Here, we used a series of in vitro assays to systematically explore the regulation of PS exposure during axonal degeneration. Our results show that PS exposure is regulated by the upstream activators of axonal pruning and Wallerian degeneration. However, our investigation of signaling further downstream revealed divergence between axon degeneration and PS exposure. Importantly, elevation of the axonal energetic status hindered PS exposure, while inhibition of mitochondrial activity caused PS exposure, without degeneration. Overall, our results suggest that the levels of PS on the outer axonal membrane can be dissociated from the degeneration process and that the axonal energetic status plays a key role in the regulation of PS exposure.

Granulocyte-macrophage colony-stimulating factor improves mouse peripheral nerve regeneration following sciatic nerve crush.

Peripheral nerve injuries severely impair patients' quality of life as full recovery is seldom achieved. Upon axonal disruption, the distal nerve stump undergoes fragmentation, and myelin breaks down; the subsequent regeneration progression is dependent on cell debris removal. In addition to tissue clearance, macrophages release angiogenic and neurotrophic factors that contribute to axon growth. Based on the importance of macrophages for nerve regeneration, especially during the initial response to injury, we treated mice with granulocyte-macrophage colony-stimulating factor (GM-CSF) at various intervals after sciatic nerve crushing. Sciatic nerves were histologically analyzed at different time intervals after injury for the presence of macrophages and indicators of regeneration. Functional recovery was followed by an automated walking track test. We found that GM-CSF potentiated early axon growth, as indicated by the enhanced expression of growth-associated protein at 7 days postinjury. Inducible nitric oxide synthase expression increased at the beginning and at the end of the regenerative process, suggesting that nitric oxide is involved in axon growth and pruning. As expected, GM-CSF treatment stimulated macrophage infiltration, which increased at 7 and 14 days; however, it did not improve myelin clearance. Instead, GM-CSF stimulated early brain-derived neurotrophic factor (BDNF) production, which peaked at 7 days. Locomotor recovery pattern was not improved by GM-CSF treatment. The present results suggest that GM-CSF may have beneficial effects on early axonal regeneration.

Effect of Spp1 on nerve degeneration and regeneration after rat sciatic nerve injury.

BACKGROUND: Wallerian degeneration (WD) in injured peripheral nerves is associated with a large number of up- or down-regulated genes, but the effects of these changes are poorly understood. In our previous studies, we reported some key factors that are differentially expressed to activate nerve degeneration and regeneration during WD. Here, we determined the effects of secreted phosphoprotein 1 (Spp1) on WD after rat sciatic nerve injury. RESULTS: Spp1 was upregulated from 6 h to 14 days after sciatic nerve injury. Altered expression of Spp1 in Schwann cells (SC) resulted in altered mRNA and protein expression levels for cytokines, c-Fos, PKCα and phospho-ERK/ERK and affected SC apoptosis in vitro. Silencing of Spp1 expression in SCs using siRNA technology reduced proliferation and promoted migration of SCs in vitro. By contrast, overexpression of Spp1 promoted proliferation and reduced migration in SCs in vitro. Differential expression of Spp1 after sciatic nerve injury in vivo altered the expression of cytokines, c-Fos, PKCα, and the p-ERK/ERK pathway. CONCLUSIONS: Spp1 is a key regulatory factor that affects nerve degeneration and regeneration through c-Fos, PKCα and p-ERK/ERK pathways after rat sciatic nerve injury. These results shed new light on the role of Spp1 in nerve degeneration and regeneration during WD.

Oxidative stress-dependent phosphorylation activates ZNRF1 to induce neuronal/axonal degeneration.

Oxidative stress is a well-known inducer of neuronal apoptosis and axonal degeneration. We previously showed that the E3 ubiquitin ligase ZNRF1 promotes Wallerian degeneration by degrading AKT to induce GSK3B activation. We now demonstrate that oxidative stress serves as an activator of the ubiquitin ligase activity of ZNRF1 by inducing epidermal growth factor receptor (EGFR)-mediated phosphorylation at the 103rd tyrosine residue and that the up-regulation of ZNRF1 activity by oxidative stress leads to neuronal apoptosis and Wallerian degeneration. We also show that nicotinamide adenine dinucleotide phosphate-reduced oxidase activity is required for the EGFR-dependent phosphorylation-induced activation of ZNRF1 and resultant AKT degradation via the ubiquitin proteasome system to induce Wallerian degeneration. These results indicate the pathophysiological significance of the EGFR-ZNRF1 pathway induced by oxidative stress in the regulation of neuronal apoptosis and Wallerian degeneration. A deeper understanding of the regulatory mechanism for ZNRF1 catalytic activity via phosphorylation will provide a potential therapeutic avenue for neurodegeneration.