In adults, peripheral nerves comprise bundles of disseminated motor, sensory and autonomic axons that are considered stable neuroanatomical units. The only exceptions to this established wiring are very distal terminal branches in target organs, such as skin. Here we provide a remarkable deviation from this state of affairs in the peripheral nerves of mice with a conditional knockout of sensory neuron PTEN (phosphatase and tensin homolog deleted on chromosome ten). PTEN is normally expressed in adult sensory neurons, particularly small IB4 nonpeptidergic subtypes and its knockdown after injury or during experimental diabetes improves axon regrowth. We studied Advillin Cre;PTEN null mice lacking PTEN in their sensory neurons. Harvested and cultured DRG neurons displayed enhanced neurite outgrowth in vitro. In vivo, these mice were healthy and had a normal sensory behavioural phenotype. However, the nerves of mice lacking sensory neuron PTEN were highly abnormal, with augmented clusters of small myelinated and unmyelinated axons populating endoneurial fascicles of their peripheral nerve trunks. The axon clusters did not disrupt normal fascicular anatomy but invested the epidermis with greater axon numbers. Within endoneurial fascicles, supernumerary axons formed regenerative units and expressed selected growth-related markers, unlike normal adult axons. This was not accompanied by rises in dorsal root ganglia (DRG) neuron numbers, indicating enhanced distal sprouting from parent neurons. Additionally, sprouting axons were electrophysiologically intact, generating rises in the amplitudes of sensory nerve action potentials. Despite this extensive activity resembling active regeneration of intact nerves, regeneration indices after superimposed injury were only modestly enhanced or unchanged. This unusual behaviour of adult sensory axons lacking a single growth-suppressive molecule may identify insights into what molecular constraints the nervous system normally utilizes to suppress inappropriate plasticity.

The role of local of growth cone (GC) manipulation in adult regenerative systems is largely unexplored despite substantial translational importance. Here we investigated collaboration among Rac1 GTPase, its partnering ERM proteins and PTEN in adult sensory neurons and adult nerve regeneration. We confirmed expression of both Rac1 and ERM in adults and noted substantial impacts on neurite outgrowth in naïve and pre-injured adult sensory neurons. PTEN inhibition added to this outgrowth. Rac1 activation acted directly on adult GCs facilitating both attractive turning and advancement. In vivo regeneration indices including electrophysiological recovery, return of sensation, walking, repopulation of myelinated axons and reinnervation of the target epidermis indicated benefits of local Rac1 activation. These indices suggested maximal GC activation whereas local PTEN inhibition offered only limited added improvement. Our findings provide support for the concept of manipulating adult GCs, by emphasizing local Rac1 activation in directing therapy for nerve repair.

ObjectiveThis 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.MethodsExpression 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.Resultsc‐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.InterpretationMembers 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;96:216–230

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.

Diabetic polyneuropathy (DPN) renders progressive sensory neurodegeneration linked to hyperglycemia and its associated metabolopathy. Here we hypothesized that there may be additive impacts of direct insulin signaling, independent of glycemia and PTEN (phosphatase and tensin homolog deleted on chromosome 10) knockdown on neuropathy. Our targets for combined interventions were neurons and Schwann cells in vitro and chronic Type 1 DPN in mice. IR (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 long term mouse model of experimental DPN unilateral intra-hindpaw 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, thermal and mechanical sensory loss. Moreover, combined interventions improved sural and tibial nerve myelin thickness, hindpaw epidermal innervation and pAkt expression in dorsal root ganglia sensory neurons. We conclude that local PTEN inhibition or knockdown and insulin provide additive trophic support for sensory neurons and SCs whilst reversing key abnormalities of experimental DPN but without requiring metabolic correction.

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.

Neuropathic pain is a maladaptive pain phenotype that results from injury or damage to the somatosensory nervous system and is proposed to be linked to a cascade of events including excitotoxicity, oxidative stress, mitochondrial dysfunction, neuroinflammation and apoptosis. Oxidative/nitrosative stress is a critical link between neuroinflammation and neurodegeneration through poly (ADP) ribose polymerase (PARP) overactivation. Hence, the present study investigated the antioxidant and anti-inflammatory effects of peroxynitrite decomposition catalyst; FeTMPyP in chronic constriction injury (CCI) of sciatic nerve-induced neuropathy in rats. CCI of the sciatic nerve manifested significant deficits in behavioral, biochemical, functional parameters and was markedly reversed by administration of FeTMPyP. After 14 days of CCI induction, oxidative/nitrosative stress and inflammatory markers such as iNOS, NF-kB, TNF-alpha and IL-6 were elevated in sciatic nerves of CCI rats along with depleted levels of ATP and elevated levels of poly (ADP) ribose (PAR) in both sciatic nerves in ipsilateral (L4-L5) dorsal root ganglions (DRG's), suggesting over activation of PARP. Additionally, CCI resulted in aberrations in mitochondrial function as evident by decreased Mn-SOD levels and respiratory complex activities with increased mitochondrial fission protein DRP-1. These changes were reversed by treatment with FeTMPyP (1 & 3 mg/kg, p.o.). Findings of this study suggest that FeTMPyP, by virtue of its antioxidant properties, reduced both PARP over-activation and subsequent neuroinflammation resulted in protection against CCI-induced functional, behavioral and biochemical deficits.

While several new translational strategies to enhance regrowth of peripheral axons have been identified, combined approaches with different targets are rare. Moreover, few have been studied after a significant delay when growth programs are already well established and regeneration-related protein expression has waned. Here we study two approaches, Rb1 (Retinoblastoma 1) knockdown that targets overall neuron plasticity, and near nerve insulin acting as a growth factor. Both are validated to boost regrowth only at the outset of regeneration. We show that local delivery of Rb1 siRNA alone, with electroporation to an area of prior sciatic nerve injury generated knockdown of Rb1 mRNA in ipsilateral lumbar dorsal root ganglia. While mice treated with Rb1-targeted siRNA, compared with scrambled control siRNA, starting 2 weeks after the onset of regeneration, had only limited behavioural or electrophysiological benefits, they had enhanced reinnervation of epidermal axons. We next confirmed that intrinsic Rb1 knockdown combined with exogenous insulin had dramatic synergistic impacts on the growth patterns of adult sensory neurons studied in vitro, prompting analysis of a combined approach in vivo. Using an identical delayed post-injury protocol, we noted that added insulin not only augmented epidermal reinnervation rendered by Rb1 knockdown alone but also improved indices of mechanical sensation and motor axon recovery. The findings illustrate that peripheral neurons that are well into attempted regrowth retain their responsiveness to both intrinsic and exogenous approaches that improve their recovery. We also identify a novel local approach to manipulate gene expression and outcome in regrowing axons.

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.

Dendrimers therapy is one of the emerging strategies for several diseases either alone or in combinations. It has been proved its efficacy with intriguing results against different diseases in different conditions with various combinations. Portfolios of dendrimers were developed based on differential procedures with a wide variety of sequences. Their end terminal functionality makes them work uniquely. In this chapter, we restricted our discussion to explain the use of dendrimers in immunotherapy and hormone therapy.

The evaluation of human epidermal innervation and its impact by disease has largely focused on rigorous immunohistochemical counts of PGP 9.5 labelled axons. In this brief and preliminary report, we expand the repertoire of epidermal axon markers to include those with an influence on their regenerative plasticity. We studied human lower limb punch skin samples with tandem analyses of their mRNA content using qRT-PCR. Normal human subjects (n = 11) and two patients with newly diagnosed CIDP were sampled with the latter undergoing serial tandem biopsies before and after 3 months of immunotherapy. Controls expressed regeneration proteins within dermal and epidermal axons: GAP43 (growth associated protein 43), Shh (sonic hedgehog) and SCG (superior cervical ganglion-10; stathmin 2). Moreover, this expression accompanied intraepidermal nerve fiber density (IENF) within normal established values. CIDP patients had lower IENF but also expressed GAP43, Shh, and SCG. Tandem qRT-PCR identified confirmed the presence not only of these plasticity markers but of additional regeneration related mRNAs. CIDP patients had marked elevation of several mRNAs, with improvement following treatment. The findings support the concept of dynamic skin axon plasticity in humans is relevant toward consideration of newer therapeutic approaches.

Current strategies to enhance regeneration of peripheral neurons involve broad activation of sensory, autonomic, and motor axons. Peripheral neuron regeneration is limited in persons with damage or disease of peripheral axons. Here, we provide evidence that subtoxic activation of TRPV1 channels in sensory neurons is associated with activation of growth and subtle changes in skin reinnervation. We identify a bidirectional, dose-related impact of capsaicin, a TRPV1 agonist, on sensory neurons and their axons with rises in their outgrowth plasticity at low doses and toxic neurodegeneration at high doses. Moreover, its impact on growth added to that of preconditioning. Neither outcome was observed in TRPV1 null neurons. We confirmed that low dose activation was associated with rises in neuronal calcium, as well as rises in TRPV1 mRNA transcripts. In mice with a sciatic nerve crush followed by a single application of capsaicin directly to the injury site, there was no impact on motor or myelinated axon recovery but there was evidence of better recovery of thermal sensation toward baseline with hyperalgesia. Moreover, skin reinnervation by epidermal axons approached contralateral levels. TRPV1 null mice displayed loss of thermal sensation during later recovery. In sensory axons innervating the pinna of the ear, local capsaicin rendered early axon loss followed by later hyperinnervation. Taken together, TRPV1 activation alters the regenerative behavior of adult neurons and their axons both in vitro and during epidermal reinnervation in vivo. The findings identify a selective manipulation that augments cutaneous innervation by thermosensitive axons.

Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting complication which develops as a consequence of treatment with chemotherapeutic agents like oxaliplatin and is a mainstay of therapy for colorectal cancer. Ever since CIPN was identified, understanding its exact pathomechanisms remains a clinical challenge. The role of mitochondrial dysfunction and glial cell activation has surfaced in the etiology of CIPN. Rosmarinic acid (RA), a known mitoprotectant exerts neuroprotection against the oxidative stress and neuroinflammation in various disease conditions. Hence, in the present study, we investigated the effect using rosmarinic acid (25 and 50 mg/kg, po) in the experimental model of oxaliplatin-induced peripheral neuropathy (OIPN) in rats. Results showed that RA significantly (p < 0.001) prevented the functional deficits, reversed oxaliplatin-induced mechanical allodynia and cold hyperalgesia in rats. It reduced the oxidative stress, improved the mitochondrial function, and prevented the oxaliplatin-induced loss of ATP levels. RA significantly (p < 0.01) inhibited the spinal glial cell activation and suppressed the expression of inflammatory markers. RA treatment also resulted in the activation of adenosine monophosphate-activated protein kinase (AMPK) in the peripheral nerves and dorsal root ganglion (DRG) which also might have contributed to its neuroprotective actions. In vitro screening also revealed that RA did not compromise the anti-cancer activity of oxaliplatin in colon cancer cells (HT-29). Taken together, the above results demonstrate the therapeutic activity of RA against the oxaliplatin-induced mitochondrial dysfunction and neuroinflammation and thus, suggest its potential for the management of OIPN.

Oxaliplatin, an organoplatinum compound, is used in the treatment of colorectal cancer, but its clinical use can be limited due to the development of peripheral neuropathy. Whilst mitochondrial dysfunction has been implicated as a major pathomechanism for oxaliplatin‐induced neurotoxicity, the prevention of autophagy may also aggravate neuronal cell death. Melatonin, a well‐known mitoprotectant and autophagy inducer, was used to examine its neuroprotective role in oxaliplatin‐induced peripheral neuropathy (OIPN). Melatonin prevented the loss of mitochondrial membrane potential (Ψm) and promoted neuritogenesis in oxaliplatin‐challenged neuro‐2a cells. It did not interfere with the cytotoxic activity of oxaliplatin in human colon cancer cell line, HT‐29. Melatonin treatment significantly alleviated oxaliplatin‐induced pain behavior and neuropathic deficits in rats. It also ameliorated nitro‐oxidative stress mediated by oxaliplatin, thus prevented nitrosylation of proteins and loss of antioxidant enzymes, and therefore, it improved mitochondrial electron transport chain function and maintained cellular bioenergetics by improving the ATP levels. The protective effects of melatonin were attributed to preventing oxaliplatin‐induced neuronal apoptosis by increasing the autophagy pathway (via LC3A/3B) in peripheral nerves and dorsal root ganglion (DRG). Hence, it preserved the epidermal nerve fiber density in oxaliplatin‐induced neuropathic rats. Taken together, we provide detailed molecular mechanisms for the neuroprotective effect of melatonin and suggest it has translational potential for oxaliplatin‐induced neuropathy.

Oxaliplatin use as chemotherapeutic agent is frequently limited by cumulative neurotoxicity which may compromise quality of life. Reports relate this neurotoxic effect to oxidative stress and mitochondrial dysfunction in peripheral nerves and dorsal root ganglion (DRG). Carvedilol is an antihypertensive drug, has also been appreciated for its antioxidant and mitoprotective properties. Carvedilol co-treatment did not reduce the anti-tumor effects of oxaliplatin in human colon cancer cells (HT-29), but exhibited free radical scavenging activity against oxaliplatin-induced oxidative stress in neuronal cells (Neuro-2a). Hence, the present study was designed to investigate the effect of carvedilol in the experimental model of oxaliplatin-induced peripheral neuropathy (OIPN) in Sprague-Dawley rats. Oxaliplatin reduced the sensory nerve conduction velocity and produced the thermal and mechanical nociception. Carvedilol significantly (P<0.001) attenuated these functional and sensorimotor deficits. It also counteracted oxidative/nitrosative stress by reducing the levels of nitrotyrosine and improving the mitochondrial superoxide dismutase expression in both sciatic nerve and DRG tissues. It improved the mitochondrial function and prevented the oxaliplatin-induced alteration in mitochondrial membrane potential in sciatic nerve thus prevented loss of intra epidermal nerve fiber density in the foot pads. Together the results prompt the use of carvedilol along with chemotherapy with oxaliplatin to prevent the peripheral neuropathy.

Neuropathic pain is triggered by the lesions to peripheral nerves which alter their structure and function. Neuroprotective approaches that limit the pathological changes and improve the behavioral outcome have been well explained in different experimental models of neuropathy but translation of such strategies to clinics has been disappointing. Experimental evidences revealed the role of free radicals, especially peroxynitrite after the nerve injury. They provoke oxidative DNA damage and consequent over-activation of the poly(ADP-ribose) polymerase (PARP) upregulates pro-inflammatory pathways, causing bioenergetic crisis and neuronal death. Along with these changes, it causes mitochondrial dysfunction leading to neuronal apoptosis. In related preclinical studies agents that neutralize the free radicals and pharmacological inhibitors of PARP have shown benefits in treating experimental neuropathy. This article reviews the involvement of PARP over-activation in trauma induced neuropathy and therapeutic significance of PARP inhibitors in the experimental neuropathy and neuropathic pain.

BACKGROUND:Peripheral neuropathies are a group of diseases characterized by malfunctioning of peripheral nervous system. Neuropathic pain, one of the core manifestations of peripheral neuropathy remains as the most severe disabling condition affecting the social and daily routine life of patients suffering from peripheral neuropathy.METHOD:The current review is aimed at unfolding the possible role of mitochondrial dysfunction in peripheral nerve damage and to discuss on the probable therapeutic strategies against neuronal mitotoxicity. The article also highlights the therapeutic significance of maintaining a healthy mitochondrial environment in neuronal cells via pharmacological management in context of peripheral neuropathies.RESULTS:Aberrant cellular signaling coupled with changes in neurotransmission, peripheral and central sensitization are found to be responsible for the pathogenesis of variant toxic neuropathies. Current research reports have indicated the possible involvement of mitochondria mediated redox imbalance as one of the principal causes of neuropathy aetiologies. In addition to imbalance in redox homeostasis, mitochondrial dysfunction is also responsible for alterations in physiological bioenergetic metabolism, apoptosis and autophagy pathways.CONCLUSIONS:In spite of various etiological factors, mitochondrial dysfunction has been found to be a major pathomechanism underlying the neuronal dysfunction associated with peripheral neuropathies. Pharmacological modulation of mitochondria either directly or indirectly is expected to yield therapeutic relief from various primary and secondary mitochondrial diseases.