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Diabetes represents a spectrum of disease in which metabolic dysfunction damages multiple organ systems including liver, kidneys and peripheral nerves1,2. Although the onset and progression of these co-morbidities are linked with insulin resistance, hyperglycaemia and dyslipidaemia3–7, aberrant non-essential amino acid (NEAA) metabolism also contributes to the pathogenesis of diabetes8–10. Serine and glycine are closely related NEAAs whose levels are consistently reduced in patients with metabolic syndrome10–14, but the mechanistic drivers and downstream consequences of this metabotype remain unclear. Low systemic serine and glycine are also emerging as a hallmark of macular and peripheral nerve disorders, correlating with impaired visual acuity and peripheral neuropathy15,16. Here we demonstrate that aberrant serine homeostasis drives serine and glycine deficiencies in diabetic mice, which can be diagnosed with a serine tolerance test that quantifies serine uptake and disposal. Mimicking these metabolic alterations in young mice by dietary serine or glycine restriction together with high fat intake markedly accelerates the onset of small fibre neuropathy while reducing adiposity. Normalization of serine by dietary supplementation and mitigation of dyslipidaemia with myriocin both alleviate neuropathy in diabetic mice, linking serine-associated peripheral neuropathy to sphingolipid metabolism. These findings identify systemic serine deficiency and dyslipidaemia as novel risk factors for peripheral neuropathy that may be exploited therapeutically.

We investigated the application of rate-dependent depression (RDD) of the Hoffmann (H) wave as a predictor of treatment efficacy in patients with painful diabetic peripheral neuropathy (DPN). General medical information, scales, and nerve conduction data were collected from 73 healthy subjects, 50 subjects with type 2 diabetes and painless DPN, and 71 subjects with type 2 diabetes and painful DPN. The left tibial nerve was stimulated, and RDD was calculated by the decline in amplitude of the third H wave relative to the first one. Gabapentin treatment was initiated after baseline evaluation, and the RDD and visual analog scale (VAS) score were both evaluated regularly during the 2-week study period. At baseline, the painful DPN group exhibited significant RDD impairment across all stimulation frequencies. Gabapentin treatment significantly reduced the VAS score and restored RDD during the 2-week observation period. RDD was found to be an independent factor of minimal VAS score improvement, such that the benefit increased by 1.27 times per 1% decrease in the RDD value. In conclusion, this study demonstrates that diabetes-induced loss of RDD can be modified by gabapentin and suggests that RDD may be valuable for predicting the initial efficacy of gabapentin therapy in patients with painful DPN.

The dominant sensory phenotype in patients with diabetic polyneuropathy and neuropathic pain is a loss of function. This raises questions as to which mechanisms underlie pain generation in the face of potentially reduced afferent input. One potential mechanism is spinal disinhibition, whereby a loss of spinal inhibition leads to increased ascending nociceptive drive due to amplification of, or a failure to suppress, incoming signals from the periphery. We aimed to explore whether a putative biomarker of spinal disinhibition, impaired rate-dependent depression of the Hoffmann reflex, is associated with a mechanistically appropriate and distinct pain phenotype in patients with painful diabetic neuropathy. In this cross-sectional study, 93 patients with diabetic neuropathy underwent testing of Hoffmann reflex rate-dependent depression and detailed clinical and sensory phenotyping, including quantitative sensory testing. Compared to neuropathic patients without pain, patients with painful diabetic neuropathy had impaired Hoffmann reflex rate-dependent depression at 1, 2 and 3 Hz (P ≤ 0.001). Patients with painful diabetic neuropathy exhibited an overall loss of function profile on quantitative sensory testing. However, within the painful diabetic neuropathy group, cluster analysis showed evidence of greater spinal disinhibition associated with greater mechanical pain sensitivity, relative heat hyperalgesia and higher ratings of spontaneous burning pain. These findings support spinal disinhibition as an important centrally mediated pain amplification mechanism in painful diabetic neuropathy. Furthermore, our analysis indicates an association between spinal disinhibition and a distinct phenotype, arguably akin to hyperpathia, with combined loss and relative gain of function leading to increasing nociceptive drive.

Rapamycin treatment has positive and negative effects on progression of type 2 diabetes (T2D) in a recombinant inbred polygenic mouse model, male NONcNZO10/LtJ (NcZ10). Here, we show that combination treatment with metformin ameliorates negative effects of rapamycin while maintaining its benefits. From 12 to 30 weeks of age, NcZ10 males were fed a control diet or diets supplemented with rapamycin, metformin, or a combination of both. Rapamycin alone reduced weight gain, adiposity, HOMA-IR, and inflammation, and prevented hyperinsulinemia and pre-steatotic hepatic lipidosis, but exacerbated hyperglycemia, hypertriglyceridemia, and pancreatic islet degranulation. Metformin alone reduced hyperinsulinemia and circulating c-reactive protein, but exacerbated nephropathy. Combination treatment retained the benefits of both while preventing many of the deleterious effects. Importantly, the combination treatment reversed effects of rapamycin on markers of hepatic insulin resistance and normalized systemic insulin sensitivity in this inherently insulin-resistant model. In adipose tissue, rapamycin attenuated the expression of genes associated with adipose tissue expansion (Mest, Gpam), inflammation (Itgam, Itgax, Hmox1, Lbp), and cell senescence (Serpine1). In liver, the addition of metformin counteracted rapamycin-induced alterations of G6pc, Ppara, and Ldlr expressions that promote hyperglycemia and hypertriglyceridemia. Both rapamycin and metformin treatment reduced hepatic Fasn expression, potentially preventing lipidosis. These results delineate a state of "insulin signaling restriction" that withdraws endocrine support for further adipogenesis, progression of the metabolic syndrome, and the development of its comorbidities. Our results are relevant for the treatment of T2D, the optimization of current rapamycin-based treatments for posttransplant rejection and various cancers, and for the development of treatments for healthy aging.

Introduction: Metformin is currently first line therapy for type 2 diabetes (T2D). The mechanism of action of metformin involves activation of AMP-activated protein kinase (AMPK) to enhance mitochondrial function (for example, biogenesis, refurbishment and dynamics) and autophagy. Many neurodegenerative diseases of the central and peripheral nervous systems arise from metabolic failure and toxic protein aggregation where activated AMPK could prove protective. Areas covered: The authors review literature on metformin treatment in Parkinson's disease, Huntington's disease and other neurological diseases of the CNS along with neuroprotective effects of AMPK activation and suppression of the mammalian target of rapamycin (mTOR) pathway on peripheral neuropathy and neuropathic pain. The authors compare the efficacy of metformin with the actions of resveratrol. Expert opinion: Metformin, through activation of AMPK and autophagy, can enhance neuronal bioenergetics, promote nerve repair and reduce toxic protein aggregates in neurological diseases. A long history of safe use in humans should encourage development of metformin and other AMPK activators in preclinical and clinical research. Future studies in animal models of neurological disease should strive to further dissect in a mechanistic manner the pathways downstream from metformin-dependent AMPK activation, and to further investigate mTOR dependent and independent signaling pathways driving neuroprotection.

Corneal confocal microscopy (CCM) is emerging as a tool for identifying small fiber neuropathy in both peripheral neuropathies and neurodegenerative disease of the central nervous system (CNS). The value of corneal nerves as biomarkers for efficacy of clinical interventions against small fiber neuropathy and neurodegenerative disease is less clear but may be supported by preclinical studies of investigational agents. We, therefore, used diverse investigational agents to assess concordance of efficacy against corneal nerve loss and peripheral neuropathy in a mouse model of diabetes. Ocular delivery of the peptides ciliary neurotrophic factor (CNTF) or the glucagon-like peptide (GLP) analog exendin-4, both of which prevent diabetic neuropathy when given systemically, restored corneal nerve density within 2 weeks. Similarly, ocular delivery of the muscarinic receptor antagonist cyclopentolate protected corneal nerve density while concurrently reversing indices of systemic peripheral neuropathy. Conversely, systemic delivery of the muscarinic antagonist glycopyrrolate, but not gallamine, prevented multiple indices of systemic peripheral neuropathy and concurrently protected against corneal nerve loss. These data highlight the potential for use of corneal nerve quantification by confocal microscopy as a bridging assay between in vitro and whole animal assays in drug development programs for neuroprotectants and support its use as a biomarker of efficacy against peripheral neuropathy.

Aberrant insulin-like growth factor 1 (IGF-1) signaling has been proposed as a contributing factor to the development of neurodegenerative disorders including diabetic neuropathy, and delivery of exogenous IGF-1 has been explored as a treatment for Alzheimer’s disease and amyotrophic lateral sclerosis. However, the role of autocrine/paracrine IGF-1 in neuroprotection has not been well established. We therefore used in vitro cell culture systems and animal models of diabetic neuropathy to characterize endogenous IGF-1 in sensory neurons and determine the factors regulating IGF-1 expression and/or affecting neuronal health. Single-cell RNA sequencing (scRNA-Seq) and in situ hybridization analyses revealed high expression of endogenous IGF-1 in non-peptidergic neurons and satellite glial cells (SGCs) of dorsal root ganglia (DRG). Brain cortex and DRG had higher IGF-1 gene expression than sciatic nerve. Bidirectional transport of IGF-1 along sensory nerves was observed. Despite no difference in IGF-1 receptor levels, IGF-1 gene expression was significantly (P db/db type 2 diabetic mice. Hyperglycemia suppressed IGF-1 gene expression in cultured DRG neurons and this was reversed by exogenous IGF-1 or the aldose reductase inhibitor sorbinil. Transcription factors, such as NFAT1 and CEBPβ, were also less enriched at the IGF-1 promoter in DRG from diabetic rats vs control rats. CEBPβ overexpression promoted neurite outgrowth and mitochondrial respiration, both of which were blunted by knocking down or blocking IGF-1. Suppression of endogenous IGF-1 in diabetes may contribute to neuropathy and its upregulation at the transcriptional level by CEBPβ can be a promising therapeutic approach.

Mitochondrial dysfunction is implicated in a variety of neurodegenerative diseases of the nervous system. Peroxisome proliferator–activated receptor-γ coactivator-1α (PGC-1α) is a regulator of mitochondrial function in multiple cell types. In sensory neurons, AMP-activated protein kinase (AMPK) augments PGC-1α activity and this pathway is depressed in diabetes leading to mitochondrial dysfunction and neurodegeneration. Antimuscarinic drugs targeting the muscarinic acetylcholine type 1 receptor (M1R) prevent/reverse neurodegeneration by inducing nerve regeneration in rodent models of diabetes and chemotherapy-induced peripheral neuropathy (CIPN). Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) is an upstream regulator of AMPK activity. We hypothesized that antimuscarinic drugs modulate CaMKKβ to enhance activity of AMPK, and PGC-1α, increase mitochondrial function and thus protect from neurodegeneration. We used the specific M1R antagonist muscarinic toxin 7 (MT7) to manipulate muscarinic signaling in the dorsal root ganglia (DRG) neurons of normal rats or rats with streptozotocin-induced diabetes. DRG neurons treated with MT7 (100 nM) or a selective muscarinic antagonist, pirenzepine (1 μM), for 24 h showed increased neurite outgrowth that was blocked by the CaMKK inhibitor STO-609 (1 μM) or short hairpin RNA to CaMKKβ. MT7 enhanced AMPK phosphorylation which was blocked by STO-609 (1 μM). PGC-1α reporter activity was augmented up to 2-fold (p p p 1R-modulated, CaMKKβ-dependent pathway in neurons that represents a therapeutic target to enhance nerve repair in two of the most common forms of peripheral neuropathy.

Background Peripheral neuropathy is a common and progressive disorder in the elderly that interferes with daily activities. It is of importance to find efficient treatments to treat or delay this age-related neurodegeneration. Silencing macrophages by reducing foamy macrophages showed significant improvement of age-related degenerative changes in peripheral nerves of aged mice. We previously demonstrated that activation of the cholesterol sensor Liver X receptor (LXR) with the potent agonist, GW3965, alleviates pain in a diet-induced obesity model. We sought to test whether LXR activation may improve neuropathy in aged mice. Methods 21-month-old mice were treated with GW3965 (25 mg/Kg body weight) for 3 months while testing for mechanical allodynia and thermal hyperalgesia. At termination, flow cytometry was used to profile dorsal root ganglia and sciatic nerve cells. Immune cells were sorted and analyzed for cholesterol and gene expression. Nerve fibers of the skin from the paws were analyzed. Some human sural nerves were also evaluated. Comparisons were made using either t test or one-way ANOVA. Results Treatment with GW3965 prevented the development of mechanical hypersensitivity and thermal hyperalgesia over time in aged mice. We also observed change in polarization and cholesterol content of sciatic nerve macrophages accompanied by a significant increase in nerve fibers of the skin. Conclusions These results suggest that activation of the LXR may delay the PNS aging by modifying nerve-immune cell lipid content. Our study provides new potential targets to treat or delay neuropathy during aging.

Peripheral neuropathy is a common and progressive disorder in the elderly that interferes with daily activities and increases the risk of injury. It is of importance to find efficient treatments to treat or delay this age-related neurodegeneration. We previously demonstrated that activation of the cholesterol sensor Liver X receptor (LXR) with the potent agonist GW3965, alleviates pain in a diet-induced obesity model. Because cholesterol had also been linked to neuropathy during aging, we sought to test whether LXR activation may improve neuropathy and pain in aged mice by treating 21-month-old mice for 3 months with GW3965. Treatment resulted in a significant increase in nerve fibers of the sub-basal plexus, accompanied by a change in polarization, metabolism, and cholesterol content of macrophages in the sciatic nerve. These results suggest that activation of the LXR may block the progression of neuropathy associated with aging by modifying nerve-immune cell cholesterol, thereby providing new pathways to target in efforts to delay neuropathy during aging.

We investigated rate-dependent depression (RDD) of the Hoffman reflex (H-reflex) in patients with amyotrophic lateral sclerosis (ALS), a degenerative disease with ventral horn involvement.In this case-control study, we enrolled 27 patients with ALS and 30 matched healthy control subjects. Clinical and electrophysiological assessments, as well as RDD in response to various stimulation frequencies (0.5 Hz, 1 Hz, 3 Hz and 5 Hz), were compared between groups. Multiple clinical and electrophysiological factors were also explored to determine any underlying associations with RDD.The ALS group showed a significant loss of RDD across all frequencies compared to the control group, most notably following 1 Hz stimulation (19.1 ± 20.3 vs. 34.0 ± 13.7%, p = 0.003). Among factors that might influence RDD, the enlargement of the motor unit potential (MUP) showed a significant relationship with RDD following multifactor analysis of variance (p = 0.007) and Pearson correlation analysis (ρ = - 0.70, p 0.001), while various upper motor neuron manifestations were not correlated with RDD values (p 0.05).We report a loss of RDD in patients with ALS. The strong correlation detected between the RDD deficit and increased MUP suggests that RDD is a sensitive indicator of underlying spinal disinhibition in ALS.ChiCTR2000038848, 10/7/2020 (retrospectively registered), http://www.chictr.org.cn/ .

Impaired rate-dependent depression (RDD) of the spinal H-reflex occurs in diabetic rodents and a sub-set of patients with painful diabetic neuropathy. RDD is unaffected in animal models of painful neuropathy associated with peripheral pain mechanisms and diabetic patients with painless neuropathy, suggesting RDD could serve as a biomarker for individuals in whom spinal disinhibition contributes to painful neuropathy and help identify therapies that target impaired spinal inhibitory function. The spinal pharmacology of RDD was investigated in normal rats and rats after 4 and 8 weeks of streptozotocin-induced diabetes. In normal rats, dependence of RDD on spinal GABAergic inhibitory function encompassed both GABAA and GABAB receptor sub-types. The time-dependent emergence of impaired RDD in diabetic rats was preceded by depletion of potassium-chloride co-transporter 2 (KCC2) protein in the dorsal, but not ventral, spinal cord and by dysfunction of GABAA receptor-mediated inhibition. GABAB receptor-mediated spinal inhibition remained functional and initially compensated for loss of GABAA receptor-mediated inhibition. Administration of the GABAB receptor agonist baclofen restored RDD and alleviated indices of neuropathic pain in diabetic rats, as did spinal delivery of the carbonic anhydrase inhibitor acetazolamide. Pharmacological manipulation of RDD can be used to identify potential therapies that act against neuropathic pain arising from spinal disinhibition.

The worldwide diabetes epidemic is estimated to currently afflict almost 500 million persons. Long-term diabetes damages multi ple organ systems with the blood vessels, eyes, kidneys and nervous systems being particularly vulnerable. These complications of diabetes reduce lifespan, impede quality of life and impose a huge social and economic burden on both the individual and society. Peripheral neuropathy is a debilitating complication that will impact over half of all persons with diabetes. There is no treatment for diabetic neuropathy and a disturbingly long history of therapeutic approaches showing promise in preclinical studies but fail ing to translate to the clinic. These failures have prompted re-examination of both the animal models and clinical trial design. This review focuses on the functional and structural parameters used as indices of peripheral neuropathy in preclinical and clinical studies and the extent to which they share a common pathogenesis and presentation. Nerve conduction studies in large myelinat ed fibers have long been the mainstay of preclinical efficacy screening programs and clinical trials, supplemented by quantitative sensory tests. However, a more refined approach is emerging that incorporates measures of small fiber density in the skin and cornea alongside these traditional assays at both preclinical and clinical phases.

Purpose Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes. Small and large peripheral nerve fibers can be involved in DPN. Large nerve fiber damage causes paresthesia, sensory loss, and muscle weakness, and small nerve fiber damage is associated with pain, anesthesia, foot ulcer, and autonomic symptoms. Treatments for DPN and painful DPN (pDPN) pose considerable challenges due to the lack of effective therapies. To meet these challenges, there is a major need to develop biomarkers that can reliably diagnose and monitor progression of nerve damage and, for pDPN, facilitate personalized treatment based on underlying pain mechanisms. Methods This study involved a comprehensive literature review, incorporating article searches in electronic databases (Google Scholar, PubMed, and OVID) and reference lists of relevant articles with the authors’ substantial expertise in DPN. This review considered seminal and novel research and summarizes emerging biomarkers of DPN and pDPN that are based on neurophysiological methods. Findings From the evidence gathered from 145 papers, this submission describes emerging clinical neurophysiological methods with potential to act as biomarkers for the diagnosis and monitoring of DPN as well as putative future roles as predictors of response to antineuropathic pain medication in pDPN. Nerve conduction studies only detect large fiber damage and do not capture pathology or dysfunction of small fibers. Because small nerve fiber damage is prominent in DPN, additional biomarkers of small nerve fiber function are needed. Activation of peripheral nociceptor fibers using laser, heat, or targeted electrical stimuli can generate pain-related evoked potentials, which are an objective neurophysiological measure of damage along the small fiber pathways. Assessment of nerve excitability, which provides a surrogate of axonal properties, may detect alterations in function before abnormalities are detected by nerve conduction studies. Microneurography and rate-dependent depression of the Hoffmann-reflex can be used to dissect underlying pain-generating mechanisms arising from the periphery and spinal cord, respectively. Their role in informing mechanistic-based treatment of pDPN as well as facilitating clinical trials design is discussed. Implications The neurophysiological methods discussed, although currently not practical for use in busy outpatient settings, detect small fiber and early large fiber damage in DPN as well as disclosing dominant pain mechanisms in pDPN. They are suited as diagnostic and predictive biomarkers as well as end points in mechanistic clinical trials of DPN and pDPN.

Obesity affects over 2 billion people worldwide and is accompanied by peripheral neuropathy (PN) and an associated poorer quality of life. Despite high prevalence, the molecular mechanisms underlying the painful manifestations of PN are poorly understood, and therapies are restricted to use of painkillers or other drugs that do not address the underlying disease. Studies have demonstrated that the gut microbiome is linked to metabolic health and its alteration is associated with many diseases, including obesity. Pathologic changes to the gut microbiome have recently been linked to somatosensory pain, but any relationships between gut microbiome and PN in obesity have yet to be explored. Our data show that mice fed a Western diet developed indices of PN that were attenuated by concurrent fecal microbiome transplantation (FMT). In addition, we observed changes in expression of genes involved in lipid metabolism and calcium handling in cells of the peripheral nerve system (PNS). FMT also induced changes in the immune cell populations of the PNS. There was a correlation between an increase in the circulating short-chain fatty acid butyrate and pain improvement following FMT. Additionally, butyrate modulated gene expression and immune cells in the PNS. Circulating butyrate was also negatively correlated with distal pain in 29 participants with varied body mass index. Our data suggest that the metabolite butyrate, secreted by the gut microbiome, underlies some of the effects of FMT. Targeting the gut microbiome, butyrate, and its consequences may represent novel viable approaches to prevent or relieve obesity-associated neuropathies.

Neuropathy is a common complication of long-term diabetes that impairs quality of life by producing pain, sensory loss and limb amputation. The presence of neuropathy in both insulin-deficient (type 1) and insulin resistant (type 2) diabetes along with the slowing of progression of neuropathy by improved glycemic control in type 1 diabetes has caused the majority of preclinical and clinical investigations to focus on hyperglycemia as the initiating pathogenic lesion. Studies in animal models of diabetes have identified multiple plausible mechanisms of glucotoxicity to the nervous system including post-translational modification of proteins by glucose and increased glucose metabolism by aldose reductase, glycolysis and other catabolic pathways. However, it is becoming increasingly apparent that factors not necessarily downstream of hyperglycemia can also contribute to the incidence, progression and severity of neuropathy and neuropathic pain. For example, peripheral nerve contains insulin receptors that transduce the neurotrophic and neurosupportive properties of insulin, independent of systemic glucose regulation, while the detection of neuropathy and neuropathic pain in patients with metabolic syndrome and failure of improved glycemic control to protect against neuropathy in cohorts of type 2 diabetic patients has placed a focus on the pathogenic role of dyslipidemia. This review provides an overview of current understanding of potential initiating lesions for diabetic neuropathy and the multiple downstream mechanisms identified in cell and animal models of diabetes that may contribute to the pathogenesis of diabetic neuropathy and neuropathic pain.

Muscarinic antagonists promote sensory neurite outgrowth in vitro and prevent and/or reverse multiple indices of peripheral neuropathy in rodent models of diabetes, chemotherapy-induced peripheral neuropathy, and HIV protein-induced neuropathy when delivered systemically. We measured plasma concentrations of the M(1) receptor–selective muscarinic antagonist pirenzepine when delivered by subcutaneous injection, oral gavage, or topical application to the skin and investigated efficacy of topically delivered pirenzepine against indices of peripheral neuropathy in diabetic mice. Topical application of 2% pirenzepine to the paw resulted in plasma concentrations 6 hours postdelivery that approximated those previously shown to promote neurite outgrowth in vitro. Topical delivery of pirenzepine to the paw of mice with streptozotocin-induced diabetes dose-dependently (0.1%–10.0%) prevented tactile allodynia, thermal hypoalgesia, and loss of epidermal nerve fibers in the treated paw and attenuated large fiber motor nerve conduction slowing in the ipsilateral limb. Efficacy against some indices of neuropathy was also noted in the contralateral limb, indicating systemic effects following local treatment. Topical pirenzepine also reversed established paw heat hypoalgesia, whereas withdrawal of treatment resulted in a gradual decline in efficacy over 2–4 weeks. Efficacy of topical pirenzepine was muted when treatment was reduced from 5 to 3 or 1 day/wk. Similar local effects were noted with the nonselective muscarinic receptor antagonist atropine when applied either to the paw or to the eye. Topical delivery of muscarinic antagonists may serve as a practical therapeutic approach to treating diabetic and other peripheral neuropathies. SIGNIFICANCE STATEMENT: Muscarinic antagonist pirenzepine alleviates diabetic peripheral neuropathy when applied topically in mice.

The distance between nodes of Ranvier, referred to as internode length, positively correlates with axon diameter, and is optimized during development to ensure maximal neuronal conduction velocity. Following myelin loss, internode length is reestablished through remyelination. However, remyelination results in short internode lengths and reduced conduction rates. We analyzed the potential role of neurofilament phosphorylation in regulating internode length during remyelination and myelination. Following ethidium bromide induced demyelination, levels of neurofilament medium (NF-M) and heavy (NF-H) phosphorylation were unaffected. Preventing NF-M lysine-serine-proline (KSP) repeat phosphorylation increased internode length by 30% after remyelination. To further analyze the role of NF-M phosphorylation in regulating internode length, gene replacement was used to produce mice in which all KSP serine residues were replaced with glutamate to mimic constitutive phosphorylation. Mimicking constitutive KSP phosphorylation reduced internode length by 16% during myelination and motor nerve conduction velocity by ~27% without altering sensory nerve structure or function. Our results suggest that NF-M KSP phosphorylation is part of a cooperative mechanism between axons and Schwann cells that together determine internode length, and suggest motor and sensory axons utilize different mechanisms to establish internode length.

Impaired rate-dependent depression of the Hoffman reflex (HRDD) is a potential biomarker of impaired spinal inhibition in patients with painful diabetic neuropathy. However, the optimum stimulus-response parameters that identify patients with spinal disinhibition are currently unknown. We systematically compared HRDD, performed using trains of 10 stimuli at five stimulation frequencies (0.3, 0.5, 1, 2 and 3 Hz), in 42 subjects with painful and 62 subjects with painless diabetic neuropathy with comparable neuropathy severity, and 34 healthy controls. HRDD was calculated using individual and mean responses compared to the initial response. At stimulation frequencies of 1, 2 and 3 Hz, HRDD was significantly impaired in patients with painful diabetic neuropathy compared to patients with painless diabetic neuropathy for all parameters and for most parameters when compared to healthy controls. HRDD was significantly enhanced in patients with painless diabetic neuropathy compared to controls for responses towards the end of the 1 Hz stimulation train. Receiver operating characteristic curve analysis in patients with and without pain showed that the area under the curve was greatest for response averages of stimuli 2-4 and 2-5 at 1 Hz, AUC = 0.84 (95%CI 0.76-0.92). Trains of 5 stimuli delivered at 1 Hz can segregate patients with painful diabetic neuropathy and spinal disinhibition, whereas longer stimulus trains are required to segregate patients with painless diabetic neuropathy and enhanced spinal inhibition. ispartof: DIAGNOSTICS vol:11 issue:7 ispartof: location:Switzerland status: published

Introduction: Deletion of myostatin in mice (MSTN-/-) alters structural properties of peripheral axons. However, properties like axon diameter and myelin thickness were analyzed in mixed nerves, so it is unclear if loss of myostatin affects motor, sensory or both types of axons. Methods: Using the MSTN-/- mouse model, we analyzed the effects of increasing the number of muscle fibers on axon diameter, myelin thickness, and internode length in motor and sensory axons. Results: Axon diameter and myelin thickness were increased in motor axons of MSTN-/- mice without affecting internode length or axon number. The number of sensory axons was increased without affecting their structural properties. Discussion: These results suggest that motor and sensory axons establish structural properties by independent mechanisms. Moreover, in motor axons, instructive cues from the neuromuscular junction may play a role in co-regulating axon diameter and myelin thickness, while internode length is established independently. This article is protected by copyright. All rights reserved.

Impaired rate-dependent depression (RDD) of the Hoffman reflex is associated with reduced dorsal spinal cord potassium chloride cotransporter expression and impaired spinal γ-aminobutyric acid type A receptor function, indicative of spinal inhibitory dysfunction. We have investigated the pathogenesis of impaired RDD in diabetic rodents exhibiting features of painful neuropathy and the translational potential of this marker of spinal inhibitory dysfunction in human painful diabetic neuropathy. Impaired RDD and allodynia were present in type 1 and type 2 diabetic rats but not in rats with type 1 diabetes receiving insulin supplementation that did not restore normoglycemia. Impaired RDD in diabetic rats was rapidly normalized by spinal delivery of duloxetine acting via 5-hydroxytryptamine type 2A receptors and temporally coincident with the alleviation of allodynia. Deficits in RDD and corneal nerve density were demonstrated in patients with painful diabetic neuropathy compared with healthy control subjects and patients with painless diabetic neuropathy. Spinal inhibitory dysfunction and peripheral small fiber pathology may contribute to the clinical phenotype in painful diabetic neuropathy. Deficits in RDD may help identify patients with spinally mediated painful diabetic neuropathy who may respond optimally to therapies such as duloxetine.

The prevalence of diabetes worldwide is at pandemic levels, with the number of patients increasing by 5% annually. The most common complication of diabetes is peripheral neuropathy, which has a prevalence as high as 50% and is characterized by damage to neurons, Schwann cells and blood vessels within the nerve. The pathogenic mechanisms of diabetic neuropathy remain poorly understood, impeding the development of targeted therapies to treat nerve degeneration and its most disruptive consequences of sensory loss and neuropathic pain. Involvement of Schwann cells has long been proposed, and new research techniques are beginning to unravel a complex interplay between these cells, axons and microvessels that is compromised during the development of diabetic neuropathy. In this Review, we discuss the evolving concept of Schwannopathy as an integral factor in the pathogenesis of diabetic neuropathy, and how disruption of the interactions between Schwann cells, axons and microvessels contribute to the disease.

Mounting evidence suggests that antiretroviral therapy (ART) drugs may contribute to the prevalence of HIV-associated neurological dysfunction. The HIV envelope glycoprotein (gp120) is neurotoxic and has been linked to alterations in mitochondrial function and increased inflammatory gene expression, which are common neuropathological findings in HIV+ cases on ART with neurological disorders. Tenofovir disproxil fumarate (TDF) has been shown to affect neurogenesis in brains of mice and mitochondria in neurons. In this study, we hypothesized that TDF contributes to neurotoxicity by modulating mitochondrial biogenesis and inflammatory pathways. TDF administered to wild-type (wt) and GFAP-gp120 transgenic (tg) mice caused peripheral neuropathy, as indicated by nerve conduction slowing and thermal hyperalgesia. Conversely TDF protected gp120-tg mice from cognitive dysfunction. In the brains of wt and gp120-tg mice, TDF decreased expression of mitochondrial transcription factor A (TFAM). However, double immunolabelling revealed that TFAM was reduced in neurons and increased in astroglia in the hippocampi of TDF-treated wt and gp120-tg mice. TDF also increased expression of GFAP and decreased expression of IBA1 in the wt and gp120-tg mice. TDF increased tumor necrosis factor (TNF) α in wt mice. However, TDF reduced interleukin (IL) 1β and TNFα mRNA in gp120-tg mouse brains. Primary human astroglia were exposed to increasing doses of TDF for 24 hours and then analyzed for mitochondrial alterations and inflammatory gene expression. In astroglia, TDF caused a dose-dependent increase in oxygen consumption rate, extracellular acidification rate and spare respiratory capacity, changes consistent with increased metabolism. TDF also reduced IL-1β-mediated increases in IL-1β and TNFα mRNA. These data demonstrate that TDF causes peripheral neuropathy in mice and alterations in inflammatory signaling and mitochondrial activity in the brain.

While peripheral neuropathy is the most common complication of long-term diabetes, cognitive deficits associated with encephalopathy and myelopathy also occur. Diabetes is a risk factor for Alzheimer disease (AD) and increases the risk of progression from mild cognitive impairment to AD. The only current recommendation for preventing or slowing the progression of peripheral neuropathy is to maintain close glycemic control, while there is no recommendation for central nervous system disorders. NSI-189 is a new chemical entity that when orally administered promotes neurogenesis in the adult hippocampus, increases hippocampal volume, enhances synaptic plasticity, and reduces cognitive dysfunction. To establish the potential for impact on peripheral neuropathy, we first showed that NSI-189 enhances neurite outgrowth and mitochondrial functions in cultured adult rat primary sensory neurons. Oral delivery of NSI-189 to murine models of type 1 (female) and type 2 (male) diabetes prevented multiple functional and structural indices of small and large fiber peripheral neuropathy, increased hippocampal neurogenesis, synaptic markers and volume, and protected long-term memory. NSI-189 also halted progression of established peripheral and central neuropathy. NSI-189, which is currently in clinical trials for treatment of major depressive disorder, offers the opportunity for the development of a single therapeutic agent against multiple indices of central and peripheral neuropathy.

Dichloroacetate (DCA) has been the focus of research by both environmental toxicologists and biomedical scientists for over 50 years. As a product of water chlorination and a metabolite of certain industrial chemicals, DCA is ubiquitous in our biosphere at low μg/kg body weight daily exposure levels without obvious adverse effects in humans. As an investigational drug for numerous congenital and acquired diseases, DCA is administered orally or parenterally, usually at doses of 10-50mg/kg per day. As a therapeutic, its principal mechanism of action is to inhibit pyruvate dehydrogenase kinase (PDK). In turn, PDK inhibits the key mitochondrial energy homeostat, pyruvate dehydrogenase complex (PDC), by reversible phosphorylation. By blocking PDK, DCA activates PDC and, consequently, the mitochondrial respiratory chain and ATP synthesis. A reversible sensory/motor peripheral neuropathy is the clinically limiting adverse effect of chronic DCA exposure and experimental data implicate the Schwann cell as a toxicological target. It has been postulated that stimulation of PDC and respiratory chain activity by DCA in normally glycolytic Schwann cells causes uncompensated oxidative stress from increased reactive oxygen species production. Additionally, the metabolism of DCA interferes with the catabolism of the amino acids phenylalanine and tyrosine and with heme synthesis, resulting in accumulation of reactive molecules capable of forming adducts with DNA and proteins and also resulting in oxidative stress. Preliminary evidence in rodent models of peripheral neuropathy suggest that DCA-induced neurotoxicity may be mitigated by naturally occurring antioxidants and by a specific class of muscarinic receptor antagonists. These findings generate a number of testable hypotheses regarding the etiology and treatment of DCA peripheral neuropathy.

Objective Diabetic sensorimotor polyneuropathy (DSPN) affects approximately half of diabetic patients leading to significant morbidity. There is impaired neurotrophic growth factor signaling, AMP-activated protein kinase (AMPK) activity and mitochondrial function in dorsal root ganglia (DRG) of animal models of type 1 and type 2 diabetes. We hypothesized that sub-optimal insulin-like growth factor 1 (IGF-1) signaling in diabetes drives loss of AMPK activity and mitochondrial function, both contributing to development of DSPN. Methods Age-matched control Sprague-Dawley rats and streptozotocin (STZ)-induced type 1 diabetic rats with/without IGF-1 therapy were used for in vivo studies. For in vitro studies, DRG neurons from control and STZ-diabetic rats were cultured and treated with/without IGF-1 in the presence or absence of inhibitors or siRNAs. Results Dysregulation of mRNAs for IGF-1, AMPKα2, ATP5a1 (subunit of ATPase), and PGC-1β occurred in DRG of diabetic vs. control rats. IGF-1 up-regulated mRNA levels of these genes in cultured DRGs from control or diabetic rats. IGF-1 treatment of DRG cultures significantly (P, Highlights • IGF-1 activates AMPK to drive mitochondrial phenotype in sensory neurons. • AMPK isoforms differentially regulate mitochondrial gene expression and respiratory activity. • IGF-1 treatment of type 1 diabetic rodents protected from neuropathy. • DRG of diabetic rats treated with IGF-1 exhibited raised activity and gene expression of AMPK and respiratory complexes.

Dichloroacetate (DCA) has been the focus of research by both environmental toxicologists and biomedical scientists for over 50 years. As a product of water chlorination and a metabolite of certain industrial chemicals, DCA is ubiquitous in our biosphere at low μg/kg body weight daily exposure levels without obvious adverse effects in humans. As an investigational drug for numerous congenital and acquired diseases, DCA is administered orally or parenterally, usually at doses of 10-50mg/kg per day. As a therapeutic, its principal mechanism of action is to inhibit pyruvate dehydrogenase kinase (PDK). In turn, PDK inhibits the key mitochondrial energy homeostat, pyruvate dehydrogenase complex (PDC), by reversible phosphorylation. By blocking PDK, DCA activates PDC and, consequently, the mitochondrial respiratory chain and ATP synthesis. A reversible sensory/motor peripheral neuropathy is the clinically limiting adverse effect of chronic DCA exposure and experimental data implicate the Schwann cell as a toxicological target. It has been postulated that stimulation of PDC and respiratory chain activity by DCA in normally glycolytic Schwann cells causes uncompensated oxidative stress from increased reactive oxygen species production. Additionally, the metabolism of DCA interferes with the catabolism of the amino acids phenylalanine and tyrosine and with heme synthesis, resulting in accumulation of reactive molecules capable of forming adducts with DNA and proteins and also resulting in oxidative stress. Preliminary evidence in rodent models of peripheral neuropathy suggest that DCA-induced neurotoxicity may be mitigated by naturally occurring antioxidants and by a specific class of muscarinic receptor antagonists. These findings generate a number of testable hypotheses regarding the etiology and treatment of DCA peripheral neuropathy.

Peripheral neuropathy is a frequent complication of chronic diabetes that most commonly presents as a distal degenerative polyneuropathy with sensory loss. Around 20% to 30% of such patients may also experience neuropathic pain. The underlying pathogenic mechanisms are uncertain, and therapeutic options are limited. Rodent models of diabetes have been used for more than 40 years to study neuropathy and evaluate potential therapies. For much of this period, streptozotocin-diabetic rats were the model of choice. The emergence of new technologies that allow relatively cheap and routine manipulations of the mouse genome has prompted increased use of mouse models of diabetes to study neuropathy. In this article, we describe the commonly used mouse models of type 1 and type 2 diabetes, and provide protocols to phenotype the structural, functional, and behavioral indices of peripheral neuropathy, with a particular emphasis on assays pertinent to the human condition. © 2016 by John Wiley & Sons, Inc.

It has been recently proposed that α5-subunit containing GABAA receptors (α5-GABAA receptors) that mediate tonic inhibition might be involved in pain. The purpose of this study was to investigate the contribution of α5-GABAA receptors in the loss of GABAergic inhibition and in formalin-induced, complete Freund's adjuvant (CFA)-induced and L5 and L6 spinal nerve ligation-induced long-lasting hypersensitivity. Formalin or CFA injection and L5 and L6 spinal nerve ligation produced long-lasting allodynia and hyperalgesia. Moreover, formalin injection impaired the rate-dependent depression of the Hofmann reflex. Peripheral and intrathecal pretreatment or post-treatment with the α5-GABAA receptor antagonist, L-655,708 (0.15-15 nmol), prevented and reversed, respectively, these long-lasting behaviors. Formalin injection increased α5-GABAA receptor mRNA expression in the spinal cord and dorsal root ganglia (DRG) mainly at 3 days. The α5-GABAA receptors were localized in the dorsal spinal cord and DRG colabeling with NeuN, CGRP, and IB4 which suggests their presence in peptidergic and nonpeptidergic neurons. These receptors were found mainly in small and medium sized neurons. Formalin injection enhanced α5-GABAA receptor fluorescence intensity in spinal cord and DRG at 3 and 6 days. Intrathecal administration of L-655,708 (15 nmol) prevented and reversed formalin-induced impairment of rate-dependent depression. These results suggest that α5-GABAA receptors play a role in the loss of GABAergic inhibition and contribute to long-lasting secondary allodynia and hyperalgesia.

A missense point mutation in nerve growth factor (NGFR100W) is associated with hereditary sensory autonomic neuropathy V (HSAN V), originally discovered in a Swedish family. These patients develop severe loss of perception to deep pain but with apparently normal cognitive functions. To better understand the disease mechanism, we have generated the first NGFR100Wknockin mouse model of HSAN V. Mice homozygous for the NGFR100Wmutation (NGFfln/fln) showed significant structural deficits in intra-epidermal nerve fibers (IENFs) at birth. These mice had a total loss of pain perception at ∼2 months of age and they often failed to survive to full adulthood. Heterozygous mice (NGF+/fln) developed a progressive degeneration of small sensory fibers both behaviorally and functionally: they showed a progressive loss of IENFs starting at the age of 9 months accompanied with progressive loss of perception to painful stimuli such as noxious temperature. Quantitative analysis of lumbar 4/5 dorsal root ganglia (DRG) revealed a significant reduction in small size neurons positive for calcitonin gene-related peptide, while analysis of sciatic nerve fibers revealed the mutant NGF+/flnmice had no reduction in myelinated nerve fibers. Significantly, the amount of NGF secreted from fibroblasts were reduced in heterozygous and homozygous mice compared to their wild-type littermates. Interestingly, NGF+/flnshowed no apparent structural alteration in the brain: neither the anterior cingulate cortex nor the medial septum including NGF-dependent basal forebrain cholinergic neurons. Accordingly, these animals did not develop appreciable deficits in tests for central nervous system function. Our study provides novel insights into the selective impact of NGFR100Wmutation on the development and function of the peripheral sensory system.

Degeneration of nerve fibers due to diabetic peripheral neuropathy (DPN) has been linked to mitochondrial dysfunction. Manipulation of mitochondrial dysfunction through antagonism of muscarinic receptors (MR) promotes neurite outgrowth in adult sensory neurons in vitro and provides neuroprotection in rodent models of DPN. The aim of the study was to assess the efficacy of MR antagonist topical 3% oxybutynin in structural and functional measures of nerve fiber function in subjects with type 2 diabetes (T2DM) and DPN. Pilot, randomized, placebo-controlled, double-blinded study in 40 subjects assessed at baseline and after 20 weeks of treatment with oxybutynin or placebo with the following: intraepidermal nerve fiber density (IENFD) on proximal and distal leg; neuropathy scores and quality of life (Norfolk QoL DN) questionnaire. Baseline demographic characteristics were similar between the treatment groups. IENFD improved significantly after 20 weeks for the treatment group. Neuropathy scores and Norfolk QoL DN also improved significantly in the treatment group (Table 1). No improvements were seen in the placebo group. In this study, oxybutynin proves to be efficacious in improving structural and functional measures of small fiber function, and quality of life in T2DM subjects. These results offer a promising novel therapeutic approach for DPN that needs to be explored further. Disclosure A.I. Vinik: None. N.A. Calcutt: Stock/Shareholder; Self; WinSanTor, Inc.. J.F. Edwards: None. J.R. Weaver: None. M.D. Bailey: None. P. Fernyhough: Stock/Shareholder; Self; WinSanTor, Inc.. L.B. Cundra: None. K.E. Frizzi: None. H. Parson: None. C.M. Casellini: None.

PURPOSE OF REVIEW: Neuropathic pain may arise from multiple mechanisms and locations. Efficacy of current treatments for painful diabetic neuropathy is limited to an unpredictable sub-set of patients, possibly reflecting diversity of pain generator mechanisms, and there is a lack of targeted treatments for individual patients. This review summarizes preclinical evidence supporting a role for spinal disinhibition in painful diabetic neuropathy, the physiology and pharmacology of rate dependent depression (RDD) of the spinal H-reflex and the translational potential of using RDD as a biomarker of spinally-mediated pain. RECENT FINDINGS: Impaired RDD occurs in animal models of diabetes and was also detected in diabetic patients with painful versus painless neuropathy. SUMMARY: RDD status can be determined using standard neurophysiological equipment. Loss of RDD may provide a clinical biomarker of spinal disinhibition, thereby enabling a personalized medicine approach to selection of current treatment options and enrichment of future clinical trial populations.

Diabetic neuropathy affects approximately 50% of diabetic patients. Down-regulation of mitochondrial gene expression and function has been reported in both human tissues and in dorsal root ganglia (DRG) from animal models of type 1 and type 2 diabetes. We hypothesized that loss of direct insulin signaling in diabetes contributes to loss of mitochondrial function in DRG neurons and to development of neuropathy. Sensory neurons obtained from age-matched adult control or streptozotocin (STZ)-induced type 1 diabetic rats were cultured with or without insulin before determining mitochondrial respiration and expression of mitochondrial respiratory chain and insulin signaling-linked proteins. For in vivo studies age-matched control rats and diabetic rats with or without trace insulin supplementation were maintained for 5months before DRG were analyzed for respiratory chain gene expression and cytochrome c oxidase activity. Insulin (10nM) significantly (P

Neuropathy is a common complication of long-term diabetes. Proposed mechanisms of neuronal damage caused by diabetes that are downstream of hyperglycemia and/or loss of insulin signaling include ischemic hypoxia, inflammation and loss of neurotrophic support. The curcumin derivative J147 is a potent neurogenic and neuroprotective drug candidate initially developed for the treatment of neurodegenerative conditions associated with aging that impacts many pathways implicated in the pathogenesis of diabetic neuropathy. Here, we demonstrate efficacy of J147 in ameliorating multiple indices of neuropathy in the streptozotocin-induced mouse model of type 1 diabetes. Diabetes was determined by blood glucose, HbA1c, and insulin levels and efficacy of J147 by behavioral, physiologic, biochemical, proteomic, and transcriptomic assays. Biological efficacy of systemic J147 treatment was confirmed by its capacity to decrease TNFα pathway activation and several other markers of neuroinflammation in the CNS. Chronic oral treatment with J147 protected the sciatic nerve from progressive diabetes-induced slowing of large myelinated fiber conduction velocity while single doses of J147 rapidly and transiently reversed established touch-evoked allodynia. Conduction slowing and allodynia are clinically relevant markers of early diabetic neuropathy and neuropathic pain, respectively. RNA expression profiling suggests that one of the pathways by which J147 imparts its protection against diabetic induced neuropathy may be through activation of the AMP kinase pathway. The diverse biological and therapeutic effects of J147 suggest it as an alternative to the polypharmaceutical approaches required to treat the multiple pathogenic mechanisms that contribute to diabetic neuropathy.

Sensory neurons have the capacity to produce, release, and respond to acetylcholine (ACh), but the functional role of cholinergic systems in adult mammalian peripheral sensory nerves has not been established. Here, we have reported that neurite outgrowth from adult sensory neurons that were maintained under subsaturating neurotrophic factor conditions operates under cholinergic constraint that is mediated by muscarinic receptor-dependent regulation of mitochondrial function via AMPK. Sensory neurons from mice lacking the muscarinic ACh type 1 receptor (M1R) exhibited enhanced neurite outgrowth, confirming the role of M1R in tonic suppression of axonal plasticity. M1R-deficient mice made diabetic with streptozotocin were protected from physiological and structural indices of sensory neuropathy. Pharmacological blockade of M1R using specific or selective antagonists, pirenzepine, VU0255035, or muscarinic toxin 7 (MT7) activated AMPK and overcame diabetes-induced mitochondrial dysfunction in vitro and in vivo. These antimuscarinic drugs prevented or reversed indices of peripheral neuropathy, such as depletion of sensory nerve terminals, thermal hypoalgesia, and nerve conduction slowing in diverse rodent models of diabetes. Pirenzepine and MT7 also prevented peripheral neuropathy induced by the chemotherapeutic agents dichloroacetate and paclitaxel or HIV envelope protein gp120. As a variety of antimuscarinic drugs are approved for clinical use against other conditions, prompt translation of this therapeutic approach to clinical trials is feasible.