Nerve Regeneration Using Compartmentalized System in CIPN Model.

The analysis of nerve regeneration in the chemotherapy-induced peripheral neuropathy (CIPN) model can be achieved using the compartmentalized culture system. This system enables us to isolate the cell body from the axon physically and fluidically, therefore allowing for the independent manipulation of the cell body and axons. Compartmentalized chambers mimic the human body conditions, and can be used to study axonal degeneration, disease modeling, and drug screening. This culture system is applied to the CIPN model to study and analyze axonal behavior in response to paclitaxel (PTX) with and without fluocinolone acetonide (FA) and to better understand the site-specific target of PTX. Therefore, this compartmentalized system allows for the independent treatment of chemotherapy drugs to the cell body or axonal side which enables monitoring their reaction as a result of the treatment.

Identification of novel neuroprotectants against vincristine-induced neurotoxicity in iPSC-derived neurons.

Chemotherapy-induced peripheral neuropathy (CIPN) is a disabling side effect of cancer chemotherapy that can often limit treatment options for cancer patients or have life-long neurodegenerative consequences that reduce the patient's quality of life. CIPN is caused by the detrimental actions of various chemotherapeutic agents on peripheral axons. Currently, there are no approved preventative measures or treatment options for CIPN, highlighting the need for the discovery of novel therapeutics and improving our understanding of disease mechanisms. In this study, we utilized human-induced pluripotent stem cell (hiPSC)-derived motor neurons as a platform to mimic axonal damage after treatment with vincristine, a chemotherapeutic used for the treatment of breast cancers, osteosarcomas, and leukemia. We screened a total of 1902 small molecules for neuroprotective properties in rescuing vincristine-induced axon growth deficits. From our primary screen, we identified 38 hit compounds that were subjected to secondary dose response screens. Six compounds showed favorable pharmacological profiles - AZD7762, A-674563, Blebbistatin, Glesatinib, KW-2449, and Pelitinib, all novel neuroprotectants against vincristine toxicity to neurons. In addition, four of these six compounds also showed efficacy against vincristine-induced growth arrest in human iPSC-derived sensory neurons. In this study, we utilized high-throughput screening of a large library of compounds in a therapeutically relevant assay. We identified several novel compounds that are efficacious in protecting different neuronal subtypes from the toxicity induced by a common chemotherapeutic agent, vincristine which could have therapeutic potential in the clinic.

Advances and challenges in modeling inherited peripheral neuropathies using iPSCs.

Inherited peripheral neuropathies (IPNs) are a group of diseases associated with mutations in various genes with fundamental roles in the development and function of peripheral nerves. Over the past 10 years, significant advances in identifying molecular disease mechanisms underlying axonal and myelin degeneration, acquired from cellular biology studies and transgenic fly and rodent models, have facilitated the development of promising treatment strategies. However, no clinical treatment has emerged to date. This lack of treatment highlights the urgent need for more biologically and clinically relevant models recapitulating IPNs. For both neurodevelopmental and neurodegenerative diseases, patient-specific induced pluripotent stem cells (iPSCs) are a particularly powerful platform for disease modeling and preclinical studies. In this review, we provide an update on different in vitro human cellular IPN models, including traditional two-dimensional monoculture iPSC derivatives, and recent advances in more complex human iPSC-based systems using microfluidic chips, organoids, and assembloids.

Augustus Waller's foresight realized: SARM1 in peripheral neuropathies.

Peripheral neuropathy is a common neurodegenerative condition characterized by numbness, tingling, pain, and weakness that frequently starts in the distal limbs. Arising from multiple etiologies, many peripheral neuropathies exhibit a slowly progressive course due to axon degeneration for which no effective treatments exist. During the past decade, numerous crucial insights into mechanisms of axon degeneration in peripheral neuropathies emerged from experiments involving nerve-cutting procedures, revealing the central role of the SARM1 axon degeneration pathway in both. Here I review commonalities and differences in the role of SARM1 after nerve cut and in several acquired and inherited peripheral neuropathies. This new knowledge now paves the way for the development of therapeutics that directly address root causes of various kinds of neuropathies.

CORRECTION OF PATHOLOGICAL CHANGES IN SALIVARY GLANDS OF ANIMALS WITH PACLITAXEL-INDUCED NEUROPATHY.

BACKGROUND: Paclitaxel is a highly effective chemotherapeutic agent used to treat breast, ovarian, and other cancers. At the same time, paclitaxel causes peripheral neuropathy as a side effect in 45%-70% of patients. AIM: The aim of the study was to investigate the effect of paclitaxel-induced peripheral neuropathy on the development of pathological changes in the salivary glands of animals and to explore the possibility of correction of the identified changes with vitamin B/ATP complex. MATERIALS AND METHODS: To simulate toxic neuropathy, animals were injected i/p with paclitaxel 2 mg/kg for 4 days. In order to correct the identified changes, rats were injected i/m with vitamin B/ATP complex (1 mg/ kg) for 9 days. In the homogenate of the submandibular salivary glands, α-amylase activity, total proteolytic activity, total antitryptic activity, the content of medium mass molecules, thiobarbituric acid reactive substances (TBARS), oxidatively modified proteins, and catalase activity were determined. RESULTS: A significant increase in the content of oxidatively modified proteins, medium mass molecules, and the content of TBARS and significant decrease in the activity of catalase and amylase were determined in the salivary glands of animals with toxic neuropathy compared to these parameters in intact animals. Administration of vitamin B/ATP complex for 9 days against the background of paclitaxel-induced neuropathy led to normalization of antitryptic activity and amylase activity, a significant decrease in the content of oxidatively modified proteins, medium mass molecules, and TBARS along with a significant increase in catalase activity in the salivary glands of animals compared to the untreated rats with neuropathy. CONCLUSION: Paclitaxel-induced neuropathy caused the development of pathological changes in the salivary glands of rats, which was evidenced by a carbonyl- oxidative stress and impaired protein synthetic function. The correction with vitamin B/ATP complex restored the protein-synthetic function and the proteinase-inhibitor balance, suppressed the oxidative stress and normalized free radical processes in the salivary glands of rats.

[Utilization of Peripheral Neuropathology Tests].

Neuropathological findings rarely lead to a definitive diagnosis of autoimmune and inflammatory peripheral nerve diseases, and indications for invasive nerve biopsy with subsequent disability should be carefully determined. In addition to disease-specific pathological findings, identifying findings that facilitate differential diagnosis in clinical practice is necessary. This article reviews the neuropathological findings that are valuable in the differential diagnosis of autoimmune and inflammatory peripheral nerve diseases.

CLUH maintains functional mitochondria and translation in motoneuronal axons and prevents peripheral neuropathy.

Transporting and translating mRNAs in axons is crucial for neuronal viability. Local synthesis of nuclear-encoded mitochondrial proteins protects long-lived axonal mitochondria from damage; however, the regulatory factors involved are largely unknown. We show that CLUH, which binds mRNAs encoding mitochondrial proteins, prevents peripheral neuropathy and motor deficits in the mouse. CLUH is enriched in the growth cone of developing spinal motoneurons and is required for their growth. The lack of CLUH affects the abundance of target mRNAs and the corresponding mitochondrial proteins more prominently in axons, leading to ATP deficits in the growth cone. CLUH interacts with ribosomal subunits, translation initiation, and ribosome recycling components and preserves axonal translation. Overexpression of the ribosome recycling factor ABCE1 rescues the mRNA and translation defects, as well as the growth cone size, in CLUH-deficient motoneurons. Thus, we demonstrate a role for CLUH in mitochondrial quality control and translational regulation in axons, which is essential for their development and long-term integrity and function.

Neurological features of Hansen disease: a retrospective, multicenter cohort study.

To elucidate the neurological features of Hansen disease. The medical records of patients with confirmed Hansen disease transferred from the neurology department were reviewed, and all medical and neurological manifestations of Hansen disease were assessed. Eleven patients with confirmed Hansen disease, 10 with newly detected Hansen disease and 1 with relapsed Hansen disease, who visited neurology departments were enrolled. The newly detected patients with Hansen disease were classified as having lepromatous leprosy (LL, n = 1), borderline lepromatous leprosy (BL, n = 2), borderline leprosy (BB, n = 2), borderline tuberculoid leprosy (BT, n = 1), tuberculoid leprosy (TT, n = 2), or pure neural leprosy (PNL, n = 2). All of the patients with confirmed Hansen were diagnosed with peripheral neuropathy (100.00%, 11/11). The symptoms and signs presented were mainly limb numbness (100.00%, 11/11), sensory and motor dysfunction (100.00%, 11/11), decreased muscle strength (90.90%, 10/11), and skin lesions (81.81%, 9/11). Nerve morphological features in nerve ultrasonography (US) included peripheral nerve asymmetry and segmental thickening (100.00%, 9/9). For neuro-electrophysiology feature, the frequency of no response of sensory nerves was significantly higher than those of motor nerves [(51.21% 42/82) vs (24.70%, 21/85)(P = 0.0183*)] by electrodiagnostic (EDX) studies. Nerve histological features in nerve biopsy analysis included demyelination (100.00%, 5/5) and axonal damage (60.00%, 3/5). In addition to confirmed diagnoses by acid-fast bacteria (AFB) staining (54.54%, 6/11) and skin pathology analysis (100.00%, 8/8), serology and molecular technology were positive in 36.36% (4/11) and 100.00% (11/11) of confirmed patients of Hansen disease, respectively. It is not uncommon for patients of Hansen disease to visit neurology departments due to peripheral neuropathy. The main pathological features of affected nerves are demyelination and axonal damage. The combination of nerve US, EDX studies, nerve biopsy, and serological and molecular tests can improve the diagnosis of Hansen disease.

No Evidence of Sensory Neuropathy in a Traditional Mouse Model of Idiopathic Parkinson's Disease.

Parkinson's disease (PD) is the second-most common neurodegenerative disorder worldwide and is diagnosed based on motor impairments. Non-motor symptoms are also well-recognised in this disorder, and peripheral neuropathy is a frequent but poorly appreciated non-motor sign. Studying how central and peripheral sensory systems are affected can contribute to the development of targeted therapies and deepen our understanding of the pathophysiology of PD. Although the cause of sporadic PD is unknown, chronic exposure to the pesticide rotenone in humans increases the risk of developing the disease. Here, we aimed to investigate whether peripheral neuropathy is present in a traditional model of PD. Mice receiving intrastriatal rotenone showed greatly reduced dopamine terminals in the striatum and a reduction in tyrosine hydroxylase-positive neurons in the Substantia nigra pars compacta and developed progressive motor impairments in hindlimb stepping and rotarod but no change in spontaneous activity. Interestingly, repeated testing using gold-standard protocols showed no change in gut motility, a well-known non-motor symptom of PD. Importantly, we did not observe any change in heat, cold, or touch sensitivity, again based upon repeated testing with well-validated protocols that were statistically well powered. Therefore, this traditional model fails to replicate PD, and our data again reiterate the importance of the periphery to the disorder.

Conduction slowing, conduction block and temporal dispersion in demyelinating, dysmyelinating and axonal neuropathies: Electrophysiology meets pathology.

Nerve conduction studies are usually the first diagnostic step in peripheral nerve disorders and their results are the basis for planning further investigations. However, there are some commonplaces in the interpretation of electrodiagnostic findings in peripheral neuropathies that, although useful in the everyday practice, may be misleading: (1) conduction block and abnormal temporal dispersion are distinctive features of acquired demyelinating disorders; (2) hereditary neuropathies are characterized by uniform slowing of conduction velocity; (3) axonal neuropathies are simply diagnosed by reduced amplitude of motor and sensory nerve action potentials with normal or slightly slow conduction velocity. In this review, we reappraise the occurrence of uniform and non-uniform conduction velocity slowing, conduction block and temporal dispersion in demyelinating, dysmyelinating and axonal neuropathies attempting, with a translational approach, a correlation between electrophysiological and pathological features as derived from sensory nerve biopsy in patients and animal models. Additionally, we provide some hints to navigate in this complex field.

A missense mutation in human INSC causes peripheral neuropathy.

PAR3/INSC/LGN form an evolutionarily conserved complex required for asymmetric cell division in the developing brain, but its post-developmental function and disease relevance in the peripheral nervous system (PNS) remains unknown. We mapped a new locus for axonal Charcot-Marie-Tooth disease (CMT2) and identified a missense mutation c.209 T > G (p.Met70Arg) in the INSC gene. Modeling the INSCM70R variant in Drosophila, we showed that it caused proprioceptive defects in adult flies, leading to gait defects resembling those in CMT2 patients. Cellularly, PAR3/INSC/LGN dysfunction caused tubulin aggregation and necrotic neurodegeneration, with microtubule-stabilizing agents rescuing both morphological and functional defects of the INSCM70R mutation in the PNS. Our findings underscore the critical role of the PAR3/INSC/LGN machinery in the adult PNS and highlight a potential therapeutic target for INSC-associated CMT2.

Recessive GNE Mutations in Korean Nonaka Distal Myopathy Patients with or without Peripheral Neuropathy.

Autosomal recessive Nonaka distal myopathy is a rare autosomal recessive genetic disease characterized by progressive degeneration of the distal muscles, causing muscle weakness and decreased grip strength. It is primarily associated with mutations in the GNE gene, which encodes a key enzyme of sialic acid biosynthesis (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase). This study was performed to find GNE mutations in six independent distal myopathy patients with or without peripheral neuropathy using whole-exome sequencing (WES). In silico pathogenic prediction and simulation of 3D structural changes were performed for the mutant GNE proteins. As a result, we identified five pathogenic or likely pathogenic missense variants: c.86T>C (p.Met29Thr), c.527A>T (p.Asp176Val), c.782T>C (p.Met261Thr), c.1714G>C (p.Val572Leu), and c.1771G>A (p.Ala591Thr). Five affected individuals showed compound heterozygous mutations, while only one patient revealed a homozygous mutation. Two patients revealed unreported combinations of combined heterozygous mutations. We observed some specific clinical features, such as complex phenotypes of distal myopathy with distal hereditary peripheral neuropathy, an earlier onset of weakness in legs than that of hands, and clinical heterogeneity between two patients with the same set of compound heterozygous mutations. Our findings on these genetic causes expand the clinical spectrum associated with the GNE mutations and can help prepare therapeutic strategies.

Elevated liver enzymes and fasting glucose levels correlate with neuropathy in patients diagnosed with alcohol use disorder independently of the blood thiamine levels.

AIMS: Chronic alcohol consumption is well known to cause peripheral neuropathy, affecting both small and large nerve fibers. The aim of this study was to correlate biochemical and neurophysiological findings and investigate possible biomarkers and risk factors for pathogenetic mechanisms of neuropathy in patients diagnosed with alcohol use disorder (AUD). METHODS: Ninety patients diagnosed with AUD were enrolled in this prospective study over a period of 3 years. Serum biochemical parameters, as well as thiamine blood levels, were determined upon admission. Every subject was assessed by clinical neurological examination, followed by Nerve Conduction Studies, Quantitative Sensory Testing, and Sympathetic Skin Response. Fifty age and gender-matched patients without a diagnosis of AUD were used as the control group. RESULTS: Peripheral neuropathy was diagnosed in 54 patients (60%). Among them, pure large fiber neuropathy was found in 18 patients, pure small fiber neuropathy in 12 patients, and both large and small fiber neuropathy was diagnosed in 24 patients. Elevated liver enzymes and fasting glucose levels upon admission were significantly correlated with neuropathy. Lower blood thiamine levels (than reference) were found in seven patients and were not correlated with neuropathy. CONCLUSIONS: Our study suggests that alcohol-related liver dysfunction and hyperglycemia may contribute as risk factors of peripheral neuropathy in patients diagnosed with AUD, while blood thiamine levels do not correlate with neuropathy. Moreover, we suggest that liver enzymes and the De Ritis ratio could be potentially used as biomarkers for the incidence and severity of alcohol-related neuropathy.

Transcriptomic profiling of sciatic nerves and dorsal root ganglia reveals site-specific effects of prediabetic neuropathy.

Peripheral neuropathy (PN) is a severe and frequent complication of obesity, prediabetes, and type 2 diabetes characterized by progressive distal-to-proximal peripheral nerve degeneration. However, a comprehensive understanding of the mechanisms underlying PN, and whether these mechanisms change during PN progression, is currently lacking. Here, gene expression data were obtained from distal (sciatic nerve; SCN) and proximal (dorsal root ganglia; DRG) injury sites of a high-fat diet (HFD)-induced mouse model of obesity/prediabetes at early and late disease stages. Self-organizing map and differentially expressed gene analyses followed by pathway enrichment analysis identified genes and pathways altered across disease stage and injury site. Pathways related to immune response, inflammation, and glucose and lipid metabolism were consistently dysregulated with HFD-induced PN, irrespective of injury site. However, regulation of oxidative stress was unique to the SCN while dysregulated Hippo and Notch signaling were only observed in the DRG. The role of the immune system and inflammation in disease progression was supported by an increase in the percentage of immune cells in the SCN with PN progression. Finally, when comparing these data to transcriptomic signatures from human patients with PN, we observed conserved pathways related to metabolic dysregulation across species, highlighting the translational relevance of our mouse data. Our findings demonstrate that PN is associated with distinct site-specific molecular re-programming in the peripheral nervous system, identifying novel, clinically relevant therapeutic targets.

SORD-deficient rats develop a motor-predominant peripheral neuropathy unveiling novel pathophysiological insights.

Biallelic SORD mutations cause one of the most frequent forms of recessive hereditary neuropathy, estimated to affect ∼10 000 patients in North America and Europe alone. Pathogenic SORD loss-of-function changes in the encoded enzyme sorbitol dehydrogenase result in abnormally high sorbitol levels in cells and serum. How sorbitol accumulation leads to peripheral neuropathy remains to be elucidated. A reproducible animal model for SORD neuropathy is essential to illuminate the pathogenesis of SORD deficiency and for preclinical studies of potential therapies. Therefore, we have generated a Sord knockout (KO), Sord-/-, Sprague Dawley rat, to model the human disease and to investigate the pathophysiology underlying SORD deficiency. We have characterized the phenotype in these rats with a battery of behavioural tests as well as biochemical, physiological and comprehensive histological examinations. Sord-/- rats had remarkably increased levels of sorbitol in serum, CSF and peripheral nerve. Moreover, serum from Sord-/- rats contained significantly increased levels of neurofilament light chain, an established biomarker for axonal degeneration. Motor performance significantly declined in Sord-/- animals starting at ∼7 months of age. Gait analysis evaluated with video motion-tracking confirmed abnormal gait patterns in the hindlimbs. Motor nerve conduction velocities of the tibial nerves were slowed. Light and electron microscopy of the peripheral nervous system revealed degenerating myelinated axons, de- and remyelinated axons, and a likely pathognomonic finding-enlarged 'ballooned' myelin sheaths. These findings mainly affected myelinated motor axons; myelinated sensory axons were largely spared. In summary, Sord-/- rats develop a motor-predominant neuropathy that closely resembles the human phenotype. Our studies revealed novel significant aspects of SORD deficiency, and this model will lead to an improved understanding of the pathophysiology and the therapeutic options for SORD neuropathy.

Label-Free Visualization and Morphological Profiling of Neuronal Differentiation and Axonal Degeneration through Quantitative Phase Imaging.

Understanding the intricate processes of neuronal growth, degeneration, and neurotoxicity is paramount for unraveling nervous system function and holds significant promise in improving patient outcomes, especially in the context of chemotherapy-induced peripheral neuropathy (CIPN). These processes are influenced by a broad range of entwined events facilitated by chemical, electrical, and mechanical signals. The progress of each process is inherently linked to phenotypic changes in cells. Currently, the primary means of demonstrating morphological changes rely on measurements of neurite outgrowth and axon length. However, conventional techniques for monitoring these processes often require extensive preparation to enable manual or semi-automated measurements. Here, a label-free and non-invasive approach is employed for monitoring neuronal differentiation and degeneration using quantitative phase imaging (QPI). Operating on unlabeled specimens and offering little to no phototoxicity and photobleaching, QPI delivers quantitative maps of optical path length delays that provide an objective measure of cellular morphology and dynamics. This approach enables the visualization and quantification of axon length and other physical properties of dorsal root ganglion (DRG) neuronal cells, allowing greater understanding of neuronal responses to stimuli simulating CIPN conditions. This research paves new avenues for the development of more effective strategies in the clinical management of neurotoxicity.

Exploration for Blood Biomarkers of Human Long Non-coding RNAs Predicting Oxaliplatin-Induced Chronic Neuropathy Through iPS Cell-Derived Sensory Neuron Analysis.

Oxaliplatin, a platinum-based chemotherapeutic agent, frequently causes acute and chronic peripheral sensory neuropathy, for which no effective treatment has been established. In particular, chronic neuropathy can persist for years even after treatment completion, thus worsening patients' quality of life. To avoid the development of intractable adverse effects, a predictive biomarker early in treatment is awaited. In this study, we explored extracellular long non-coding RNAs (lncRNAs) released from primary sensory neurons as biomarker candidates for oxaliplatin-induced peripheral neuropathy. Because many human-specific lncRNA genes exist, we induced peripheral sensory neurons from human induced pluripotent stem cells. Oxaliplatin treatment changed the levels of many lncRNAs in extracellular vesicles (EVs) released from cultured primary sensory neurons. Among them, the levels of release of lncRNAs that were considered to be selectively expressed in dorsal root ganglia were correlated with those of lncRNAs in plasma EV obtained from healthy individuals. Several lncRNAs in plasma EVs early after the initiation of treatment showed greater changes in patients who did not develop chronic neuropathy that persisted for more than 1 year than in those who did. Therefore, these extracellular lncRNAs in plasma EVs may represent predictive biomarkers for the development of chronic peripheral neuropathy induced by oxaliplatin.

Docking protein 6 (DOK6) selectively docks the neurotrophic signaling transduction to restrain peripheral neuropathy.

The appropriate and specific response of nerve cells to various external cues is essential for the establishment and maintenance of neural circuits, and this process requires the proper recruitment of adaptor molecules to selectively activate downstream pathways. Here, we identified that DOK6, a member of the Dok (downstream of tyrosine kinases) family, is required for the maintenance of peripheral axons, and that loss of Dok6 can cause typical peripheral neuropathy symptoms in mice, manifested as impaired sensory, abnormal posture, paw deformities, blocked nerve conduction, and dysmyelination. Furthermore, Dok6 is highly expressed in peripheral neurons but not in Schwann cells, and genetic deletion of Dok6 in peripheral neurons led to typical peripheral myelin outfolding, axon destruction, and hindered retrograde axonal transport. Specifically, DOK6 acts as an adaptor protein for selectivity-mediated neurotrophic signal transduction and retrograde transport for TrkC and Ret but not for TrkA and TrkB. DOK6 interacts with certain proteins in the trafficking machinery and controls their phosphorylation, including MAP1B, Tau and Dynein for axonal transport, and specifically activates the downstream ERK1/2 kinase pathway to maintain axonal survival and homeostasis. This finding provides new clues to potential insights into the pathogenesis and treatment of hereditary peripheral neuropathies and other degenerative diseases.

Absence of functional deficits in rats following systemic administration of an AAV9 vector despite moderate peripheral nerve and dorsal root ganglia findings: A clinically silent peripheral neuropathy.

Adeno-associated virus (AAV)-based vectors are commonly used for delivering transgenes in gene therapy studies, but they are also known to cause dorsal root ganglia (DRG) and peripheral nerve toxicities in animals. However, the functional implications of these pathologic findings and their time course remain unclear. At 2, 4, 6, and 8 weeks following a single dose of an AAV9 vector carrying human frataxin transgene in rats, non-standard functional assessments, including von Frey filament, electrophysiology, and Rotarod tests, were conducted longitudinally to measure allodynia, nerve conduction velocity, and coordination, respectively. Additionally, DRGs, peripheral nerves, brain and spinal cord were evaluated histologically and circulating neurofilament light chain (NfL) was quantified at 1, 2, 4, and 8 weeks, respectively. At 2 and 4 weeks after dosing, minimal-to-moderate nerve fiber degeneration and neuronal degeneration were observed in the DRGs in some of the AAV9 vector-dosed animals. At 8 weeks, nerve fiber degeneration was observed in DRGs, with or without neuronal degeneration, and in sciatic nerves of all AAV9 vector-dosed animals. NfL values were higher in AAV9 vector-treated animals at weeks 4 and 8 compared with controls. However, there were no significant differences in the three functional endpoints evaluated between the AAV9 vector- and vehicle-dosed animals, or in a longitudinal comparison between baseline (predose), 4, and 8 week values in the AAV9 vector-dose animals. These findings demonstrate that there is no detectable functional consequence to the minimal-to-moderate neurodegeneration observed with our AAV9 vector treatment in rats, suggesting a functional tolerance or reserve for loss of DRG neurons after systemic administration of AAV9 vector.