Hallmarks of peripheral nerve injury and regeneration.

Peripheral nerves are functional networks in the body. Disruption of these networks induces varied functional consequences depending on the types of nerves and organs affected. Despite the advances in microsurgical repair and understanding of nerve regeneration biology, restoring full functions after severe traumatic nerve injuries is still far from achieved. While a blunted growth response from axons and errors in axon guidance due to physical barriers may surface as the major hurdles in repairing nerves, critical additional cellular and molecular aspects challenge the orderly healing of injured nerves. Understanding the systematic reprogramming of injured nerves at the cellular and molecular levels, referred to here as "hallmarks of nerve injury regeneration," will offer better ideas. This chapter discusses the hallmarks of nerve injury and regeneration and critical points of failures in the natural healing process. Potential pharmacological and nonpharmacological intervention points for repairing nerves are also discussed.

Neuron-specific RNA-sequencing reveals different responses in peripheral neurons after nerve injury.

Peripheral neurons are heterogeneous and functionally diverse, but all share the capability to switch to a pro-regenerative state after nerve injury. Despite the assumption that the injury response is similar among neuronal subtypes, functional recovery may differ. Understanding the distinct intrinsic regenerative properties between neurons may help to improve the quality of regeneration, prioritizing the growth of axon subpopulations to their targets. Here, we present a comparative analysis of regeneration across four key peripheral neuron populations: motoneurons, proprioceptors, cutaneous mechanoreceptors, and nociceptors. Using Cre/Ai9 mice that allow fluorescent labeling of neuronal subtypes, we found that nociceptors showed the greater regeneration after a sciatic crush, followed by motoneurons, mechanoreceptors, and, finally, proprioceptors. By breeding these Cre mice with Ribotag mice, we isolated specific translatomes and defined the regenerative response of these neuronal subtypes after axotomy. Only 20% of the regulated genes were common, revealing a diverse response to injury among neurons, which was also supported by the differential influence of neurotrophins among neuron subtypes. Among differentially regulated genes, we proposed MED12 as a specific regulator of the regeneration of proprioceptors. Altogether, we demonstrate that the intrinsic regenerative capacity differs between peripheral neuron subtypes, opening the door to selectively modulate these responses.

Manipulation of the Myc Interactome to Enhance Nerve Regeneration in a Murine Model.

OBJECTIVE: This study was undertaken to explore manipulation of the Myc protein interactome, members of an oncogene group, in enhancing the intrinsic growth of injured peripheral adult postmitotic neurons and the nerves they supply. New approaches to enhance adult neuron growth properties are a key strategy in improving nerve regeneration. METHODS: Expression and impact of Myc interactome members c-Myc, N-Myc, Mad1, and Max were evaluated within naive and "preconditioned" adult sensory neurons and Schwann cells (SCs), using siRNA and transfection of CRISPR/Cas9 or luciferase reporter in vitro. Morphological, behavioral, and electrophysiological indices of nerve regeneration were analyzed in vivo. RESULTS: c-Myc, N-Myc, Max, and Mad were expressed in adult sensory neurons and in partnering SCs. In vitro knockdown (KD) of either Mad1 or Max, competitive inhibitors of Myc, unleashed heightened neurite outgrowth in both naive uninjured or preconditioned adult neurons. In contrast, KD or inhibition of both isoforms of Myc was required to suppress growth. In SCs, Mad1 KD not only enhanced migratory behavior but also conditioned increased outgrowth in separately cultured adult sensory neurons. In vivo, local Mad1 KD improved electrophysiological, behavioral, and structural indices of nerve regeneration out to 60 days of follow-up. INTERPRETATION: Members of the Myc interactome, specifically Mad1, are novel targets for improving nerve regeneration. Unleashing of Myc growth signaling through Mad1 KD enhances the regrowth of both peripheral neurons and SCs to facilitate better regrowth of nerves. ANN NEUROL 2024.

Axillary and musculocutaneous neuropathies.

This chapter covers axillary and musculocutaneous neuropathies, with a focus on clinically relevant anatomy, electrodiagnostic approaches, etiologic considerations, and management principles. Disorders of the lateral antebrachial cutaneous nerve, a derivative of the musculocutaneous nerve, are also reviewed. We emphasize the importance of objective findings, including the physical examination and electrodiagnostic evaluation in confirming the isolated involvement of each nerve which, along with the clinical history, informs etiologic considerations. Axillary and musculocutaneous neuropathies are both rare in isolation and most frequently occur in the setting of trauma. Less commonly encountered etiologies include external compression or entrapment, neoplastic involvement, or immune-mediated disorders including neuralgic amyotrophy, postsurgical inflammatory neuropathy, multifocal motor neuropathy, vasculitic neuropathy, and multifocal chronic inflammatory demyelinating polyradiculoneuropathy.

The Role of Protocadherin γ in Adult Sensory Neurons and Skin Reinnervation.

The clustered protocadherins (cPcdhs) play a critical role in the patterning of several CNS axon and dendritic arbors, through regulation of homophilic self and neighboring interactions. While not explored, primary peripheral sensory afferents that innervate the epidermis may require similar constraints to convey spatial signals with appropriate fidelity. Here, we show that members of the γ-Pcdh (Pcdhγ) family are expressed in both adult sensory neuron axons and in neighboring keratinocytes that have close interactions during skin reinnervation. Adult mice of both sexes were studied. Pcdhγ knock-down either through small interfering RNA (siRNA) transduction or AAV-Cre recombinase transfection of adult mouse primary sensory neurons from floxed Pcdhγ mice was associated with a remarkable rise in neurite outgrowth and branching. Rises in outgrowth were abrogated by Rac1 inhibition. Moreover, AAV-Cre knock-down in Pcdhγ floxed neurons generated a rise in neurite self-intersections, and a robust rise in neighbor intersections or tiling, suggesting a role in sensory axon repulsion. Interestingly, preconditioned (3-d axotomy) neurons with enhanced growth had temporary declines in Pcdhγ and lessened outgrowth from Pcdhγ siRNA. In vivo, mice with local hindpaw skin Pcdhγ knock-down by siRNA had accelerated reinnervation by new epidermal axons with greater terminal branching and reduced intra-axonal spacing. Pcdhγ knock-down also had reciprocal impacts on keratinocyte density and nuclear size. Taken together, this work provides evidence for a role of Pcdhγ in attenuating outgrowth of sensory axons and their interactions, with implications in how new reinnervating axons following injury fare amid skin keratinocytes that also express Pcdhγ.SIGNIFICANCE STATEMENT The molecular mechanisms and potential constraints that govern skin reinnervation and patterning by sensory axons are largely unexplored. Here, we show that γ-protocadherins (Pcdhγ) may help to dictate interaction not only among axons but also between axons and keratinocytes as the former re-enter the skin during reinnervation. Pcdhγ neuronal knock-down enhances outgrowth in peripheral sensory neurons, involving the growth cone protein Rac1 whereas skin Pcdhγ knock-down generates rises in terminal epidermal axon growth and branching during re-innervation. Manipulation of sensory axon regrowth within the epidermis offers an opportunity to influence regenerative outcomes following nerve injury.

Growth factors and molecular-driven plasticity in neurological systems.

It has been almost 70 years since the discovery of nerve growth factor (NGF), a period of a dramatic evolution in our understanding of dynamic growth, regeneration, and rewiring of the nervous system. In 1953, the extraordinary finding that a protein found in mouse submandibular glands generated a halo of outgrowing axons has now redefined our concept of the nervous system connectome. Central and peripheral neurons and their axons or dendrites are no longer considered fixed or static "wiring." Exploiting this molecular-driven plasticity as a therapeutic approach has arrived in the clinic with a slate of new trials and ideas. Neural growth factors (GFs), soluble proteins that alter the behavior of neurons, have expanded in numbers and our understanding of the complexity of their signaling and interactions with other proteins has intensified. However, beyond these "extrinsic" determinants of neuron growth and function are the downstream pathways that impact neurons, ripe for translational development and potentially more important than individual growth factors that may trigger them. Persistent and ongoing nuances in clinical trial design in some of the most intractable and irreversible neurological conditions give hope for connecting new biological ideas with clinical benefits. This review is a targeted update on neural GFs, their signals, and new therapeutic ideas, selected from an expansive literature.

Combined PTEN Knockdown and Local Insulin in Chronic Experimental Diabetic Neuropathy.

Diabetic polyneuropathy (DPN) renders progressive sensory neurodegeneration linked to hyperglycemia and its associated metabolopathy. We hypothesized that there may be additive impacts of direct insulin signaling, independent of glycemia and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) knockdown on neuropathy. Our targets for combined interventions were neurons and Schwann cells (SCs) in vitro and chronic type 1 DPN in mice. Insulin receptor expression was not altered by high-glucose conditions in neurons or SCs, and insulin promoted survival of neurons and proliferation of SCs in vitro. There were additive impacts between insulin signaling and PTEN knockdown in sensory neuron outgrowth and in axon myelination by SCs. In a chronic mouse model of experimental DPN, unilateral intra-hind paw injections of a PTEN siRNA and local insulin had additive impacts on correcting key features of chronic experimental DPN independent of glycemia, including motor axon conduction and thermal and mechanical sensory loss. Moreover, combined interventions improved sural and tibial nerve myelin thickness, hind paw epidermal innervation, and pAkt expression in dorsal root ganglion sensory neurons. We conclude that local PTEN inhibition or knockdown and insulin provide additive trophic support for sensory neurons and SCs while reversing key abnormalities of experimental DPN but without requiring metabolic correction. ARTICLE HIGHLIGHTS: Impaired growth and plasticity of neurons may contribute to chronic diabetic polyneuropathy. Both direct insulin signaling of neurons and neuron knockdown of the protein phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a roadblock to neuronal regenerative growth, offer direct support of neurons. Direct insulin and PTEN knockdown using siRNA had additive impacts on neuron survival, Schwann cell proliferation, neuron outgrowth, and myelination in vitro. Combined local insulin and PTEN siRNA hind paw injections improved abnormalities in chronic experimental diabetic polyneuropathy, including sensory axon loss, independently of glycemia.

Diabetic Polyneuropathy: New Strategies to Target Sensory Neurons in Dorsal Root Ganglia.

Diabetic polyneuropathy (DPN) is the most common type of diabetic neuropathy, rendering a slowly progressive, symmetrical, and length-dependent dying-back axonopathy with preferential sensory involvement. Although the pathogenesis of DPN is complex, this review emphasizes the concept that hyperglycemia and metabolic stressors directly target sensory neurons in the dorsal root ganglia (DRG), leading to distal axonal degeneration. In this context, we discuss the role for DRG-targeting gene delivery, specifically oligonucleotide therapeutics for DPN. Molecules including insulin, GLP-1, PTEN, HSP27, RAGE, CWC22, and DUSP1 that impact neurotrophic signal transduction (for example, phosphatidylinositol-3 kinase/phosphorylated protein kinase B [PI3/pAkt] signaling) and other cellular networks may promote regeneration. Regenerative strategies may be essential in maintaining axon integrity during ongoing degeneration in diabetes mellitus (DM). We discuss specific new findings that relate to sensory neuron function in DM associated with abnormal dynamics of nuclear bodies such as Cajal bodies and nuclear speckles in which mRNA transcription and post-transcriptional processing occur. Manipulating noncoding RNAs such as microRNA and long-noncoding RNA (specifically MALAT1) that regulate gene expression through post-transcriptional modification are interesting avenues to consider in supporting neurons during DM. Finally, we present therapeutic possibilities around the use of a novel DNA/RNA heteroduplex oligonucleotide that provides more efficient gene knockdown in DRG than the single-stranded antisense oligonucleotide.

Survival of compromised adult sensory neurons involves macrovesicular formation.

Adult neurons are recognized as post-mitotically arrested cells with limited regenerative potential. Given these restraints, it is perplexing how neurons sustain routine physiological and occasional reparative stress without compromising their density and integrity. We observed that specific insults or physiological alterations drive adult sensory neurons to attempt cell cycle entry. In this context, we demonstrate that at least a small population of sensory neurons modify their cytoskeleton as a survival mechanism in settings of growth arrest and associated stress. Most notably, among their apparent survival modifications is included a unique, and uncharacterized form of macrovesicle shedding and a subsequent neuron size adjustment. Using time-lapse imaging, we demonstrate macrovesicle shedding in some neurons subjected to growth restraint, but not associated with apoptosis. In axotomized neurons in vivo, cell cycle entry was rare to absent and macrovesicles were not observed, but we nonetheless identified changes in mRNA associated with autophagy. In vivo, neighbouring macrophages may have a role in modifying the neuron cytoskeleton after axotomy. Overall, the findings identify previously unrecognized structural adaptations in adult sensory neurons that may provide resilience to diverse insults.

Unleashing Intrinsic Growth Pathways in Regenerating Peripheral Neurons.

Common mechanisms of peripheral axon regeneration are recruited following diverse forms of damage to peripheral nerve axons. Whether the injury is traumatic or disease related neuropathy, reconnection of axons to their targets is required to restore function. Supporting peripheral axon regrowth, while not yet available in clinics, might be accomplished from several directions focusing on one or more of the complex stages of regrowth. Direct axon support, with follow on participation of supporting Schwann cells is one approach, emphasized in this review. However alternative approaches might include direct support of Schwann cells that instruct axons to regrow, manipulation of the inflammatory milieu to prevent ongoing bystander axon damage, or use of inflammatory cytokines as growth factors. Axons may be supported by a growing list of growth factors, extending well beyond the classical neurotrophin family. The understanding of growth factor roles continues to expand but their impact experimentally and in humans has faced serious limitations. The downstream signaling pathways that impact neuron growth have been exploited less frequently in regeneration models and rarely in human work, despite their promise and potency. Here we review the major regenerative signaling cascades that are known to influence adult peripheral axon regeneration. Within these pathways there are major checkpoints or roadblocks that normally check unwanted growth, but are an impediment to robust growth after injury. Several molecular roadblocks, overlapping with tumour suppressor systems in oncology, operate at the level of the perikarya. They have impacts on overall neuron plasticity and growth. A second approach targets proteins that largely operate at growth cones. Addressing both sites might offer synergistic benefits to regrowing neurons. This review emphasizes intrinsic aspects of adult peripheral axon regeneration, emphasizing several molecular barriers to regrowth that have been studied in our laboratory.

Corneal nerve and nerve conduction abnormalities in children with type 1 diabetes.

OBJECTIVE: In vivo corneal confocal microscopy (CCM) is a novel, rapid, and non-invasive technique that identifies early small fiber damage and can predict the progression and development of clinical neuropathy in adults with type 1 diabetes. However, its usefulness in children is not well established. This study compared corneal confocal microscopy with neuropathic symptoms, signs, and objective measures of neuropathy for the diagnosis of diabetic neuropathy in children with type 1 diabetes. RESEARCH DESIGN AND METHODS: A total of 83 children with type 1 diabetes and 83 healthy participants of similar age underwent assessment of neuropathy symptoms, signs, nerve conduction studies, quantitative sensory and autonomic function testing, and in vivo CCM. RESULTS: Only of 3/83 (4%) children with type 1 diabetes had subclinical neuropathy. However, corneal nerve fiber density (p = 0.001), branch density (p = 0.006), fiber length (p = 0.002), tibial motor nerve amplitude and conduction velocity, and sural sensory nerve amplitude and conduction velocity (all p < 0.004) were lower in participants with type 1 diabetes than in the controls. Vibration, cooling, and warm perception thresholds and deep breathing heart rate variability were not found to be different (all p > 0.05) between children with type 1 diabetes and healthy controls. Multivariate regression analysis identified a possible association between body mass index and decreased corneal nerves. CONCLUSIONS: Decreased corneal nerves and abnormal nerve conduction were found in children with type 1 diabetes. CCM may allow rapid objective detection of subclinical diabetic neuropathy in children and adolescents with type 1 diabetes.

Repurposed major urinary protein pheromones and adult sensory neurons: roles in neuron plasticity and experimental diabetes.

Major urinary proteins (MUPs), members of the broader lipocalin protein family, are classified as pheromones that are excreted in male rodent urine to define conspecific territoriality. In screening for differentially regulated mRNA transcripts in a mouse model of type 1 experimental diabetes mellitus (DM), we identified an unexpected upregulation of several closely related MUP transcripts within diabetic sensory dorsal root ganglia (DRG). Both sexes expressed overall MUP protein content as identified by an antibody widely targeting these upregulated family members, and immunohistochemistry identified expression within neurons, satellite glial cells, and Schwann cells. In dissociated adult sensory neurons, knockdown by an siRNA targeting upregulated MUP mRNAs, enhanced neurite outgrowth, indicating a growth-suppressive role, an impact that was synergistic with subnanomolar insulin neuronal signaling. While MUP knockdown did not generate rises in insulin signaling transcripts, the protein did bind to several mitochondrial and glial targets in DRG lysates. Analysis of a protein closely related to MUPs but that is expressed in humans, lipocalin-2, also suppressed growth, but its impact was unrelated to insulin. In a model of chronic type 1 DM, MUP siRNA knockdown improved electrophysiological and behavioral abnormalities of experimental neuropathy. MUPs have actions beyond pheromone signaling in rodents that involve suppression of growth plasticity of sensory neurons. Its hitherto unanticipated actions overlap with those of lipocalin-2 and may identify a common and widely mediated impact on neuron growth properties by members of the lipocalin family. Knockdown of MUP supports the trophic actions of insulin as a strategy that may improve features of type 1 experimental diabetic neuropathy.NEW & NOTEWORTHY New molecular mechanisms are important to unravel and understand diabetic polyneuropathy, a disorder prevalent in over half of persons with diabetes mellitus (DM). MUPs, members of the lipocalin family of molecules, have an unexpected impact on the plasticity of sensory neurons that are targeted in type 1 experimental diabetic neuropathy. This work explores this potential target in neuropathy in the context of the lipocalin family of molecules.

The Role of Long Noncoding RNA MALAT1 in Diabetic Polyneuropathy and the Impact of Its Silencing in the Dorsal Root Ganglion by a DNA/RNA Heteroduplex Oligonucleotide.

Diabetic polyneuropathy (DPN) is the most common complication of diabetes, yet its pathophysiology has not been established. Accumulating evidence suggests that long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays pivotal roles in the regulation of cell growth and survival during diabetic complications. This study aimed to investigate the impact of MALAT1 silencing in dorsal root ganglion (DRG) sensory neurons, using an α-tocopherol-conjugated DNA/RNA heteroduplex oligonucleotide (Toc-HDO), on the peripheral nervous system of diabetic mice. We identified MALAT1 upregulation in the DRG of chronic diabetic mice that suggested either a pathological change or one that might be protective, and systemic intravenous injection of Toc-HDO effectively inhibited its gene expression. However, we unexpectedly noted that this intervention paradoxically exacerbated disease with increased thermal and mechanical nociceptive thresholds, indicating further sensory loss, greater sciatic-tibial nerve conduction slowing, and additional declines of intraepidermal nerve fiber density in the hind paw footpads. Serine/arginine-rich splicing factors, which are involved in pre-mRNA splicing by interacting with MALAT1, reside in nuclear speckles in wild-type and diabetic DRG neurons; MALAT1 silencing was associated with their disruption. The findings provide evidence for an important role that MALAT1 plays in DPN, suggesting neuroprotection and regulation of pre-mRNA splicing in nuclear speckles. This is also the first example in which a systemically delivered nucleotide therapy had a direct impact on DRG diabetic neurons and their axons.

Neurobiological Opportunities in Diabetic Polyneuropathy.

This review highlights a selection of potential translational directions for the treatment of diabetic polyneuropathy (DPN) currently irreversible and without approved interventions beyond pain management. The list does not include all diabetic targets that have been generated over several decades of research but focuses on newer work. The emphasis is firstly on approaches that support the viability and growth of peripheral neurons and their ability to withstand a barrage of diabetic alterations. We include a section describing Schwann cell targets and finally how mitochondrial damage has been a common element in discussing neuropathic damage. Most of the molecules and pathways described here have not yet reached clinical trials, but many trials have been negative to date. Nonetheless, these failures clear the pathway for new thoughts over reversing DPN.

Major urinary protein excreted in rodent hindpaw sweat.

Alternative roles for sweat production beyond thermoregulation, considered less frequently, include chemical signaling. We identified the presence of a well-established rodent urinary pheromone, major urinary protein (MUP) in sweat ductules of the footpad dermal skin of mice. A hindpaw sweat proteomic analysis in hindpaw sweat samples collected in rats and generated by unmyelinated axon activation, identified seven lipocalin family members including MUP and 19 additional unique proteins. Behavioural responses to sniffing male mouse foot protein lysates suggested avoidance in a subset of male mice, but were not definitive. Rodent hindpaw sweat glands secrete a repertoire of proteins that include MUPs known to have roles in olfactory communication.

Dual Specificity Phosphatases Support Axon Plasticity and Viability.

In peripheral neuropathies, axonal degeneration (AxD) impairs the prognosis for recovery. Here, we describe a role for dual specificity phosphatases (DUSPs; MAP kinase phosphatases, MKPs), in supporting autonomous axon plasticity and viability. Both DUSPs 1 and 4 were identified within intact or axotomized sensory neurons. Knockdown of DUSP 1 or 4 independently or combined impaired neurite outgrowth in adult dissociated sensory neurons. Furthermore, adult sensory neurons with DUSP knockdown were rendered sensitive to axonopathy in vitro following exposure to low, subtoxic TrpV1 (transient receptor potential cation channel subfamily V member 1) activation by capsaicin, an intervention normally supportive of growth. This was not prevented by concurrent DLK (dual leucine zipper kinase) knockdown. Ex vivo neurofilament dissolution was heightened by DUSP inhibition within explanted nerves. In vivo DUSP knockdown or inhibition was associated with more rapid loss of motor axon excitability. The addition of SARM1 (sterile alpha and TIR motif containing 1) siRNA abrogated DUSP1 and 4 mediated loss of excitability. DUSP knockdown accelerated neurofilament breakdown and there was earlier morphological evidence of myelinated axon degeneration distal to axotomy. Taken together, the findings identify a key role for DUSPs in supporting axon plasticity and survival.

In vitro priming response in dorsal root ganglia partially mimics injury-driven pre-conditioning response and reprograms neurons for enhanced outgrowth.

Peripheral nerve injuries have the potential to bring about long-term disabilities in individuals. The major issue in repairing nerve injuries is the poor growth rate of axons. Although several molecules have been identified as potential candidates for improving axon growth, their potential translation into clinical practice is preliminary and largely unexplored. This necessitates identifying additional molecular candidates with superior potential to improve axon growth. Lack of a simple non-surgical screening model also poses a hurdle in rapidly screening potential candidate molecules. In this work, we developed a novel, rapid screening model for nerve regeneration therapeutics that retains a focus on adult neurons. The model involves simple incubation of sensory ganglia over a period of 24 h prior to dissociation. Surprisingly, this model features unique events that reprogram both sensory neurons and supporting glia favoring axon growth. Moreover, several associated cellular and molecular changes involved in this model partially mimic classic axotomy-induced changes in sensory ganglia. Overall, this model presents with a platform that not only allows rapid screening of drug candidates but offers opportunities in studying novel intrinsic molecular changes in both neurons and glial cells directed towards improving the pace of axon growth.