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;96:216-230.

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.

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.

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.

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.