Improvement of Charcot-Marie-Tooth Phenotype with a Nanocomplex Treatment in Two Transgenic Models of CMT1A.

Curcumin has been shown to exert beneficial effects in peripheral neuropathies. Despite its known biological activities, curcumin has unfavorable pharmacokinetics. Its instability has been linked to its failure in clinical trials of curcumin for the treatment of human pathologies. For this reason, we developed curcumin-loaded cyclodextrin/cellulose nanocrystals (NanoCur) to improve its pharmacokinetics. The present study aims to assess the potency of a low dose of NanoCur in 2 Charcot-Marie-Tooth disease type 1A (CMT1A) rodent models at different stages of the disease. The efficiency of NanoCur is also compared to that of Theracurmin (Thera), a commercially available curcumin formulation. The toxicity of a short-term and chronic exposure to the treatment is investigated both in vitro and in vivo, respectively. Furthermore, the entry route, the mechanism of action and the effect on the nerve phenotype are dissected in this study. Overall, the data support an improvement in sensorimotor functions, associated with amelioration in peripheral myelination in NanoCur-treated animals; an effect that was not evident in the Thera-treated group. That was combined with a high margin of safety both in vivo and in vitro. Furthermore, NanoCur appears to inhibit inflammatory pathways that normally include macrophage recruitment to the diseased nerve. This study shows that NanoCur shows therapeutic benefits with minimal systemic toxicity, suggesting that it is a potential therapeutic candidate for CMT1A and, possibly, for other neuropathies.

Readthrough Activators and Nonsense-Mediated mRNA Decay Inhibitor Molecules: Real Potential in Many Genetic Diseases Harboring Premature Termination Codons.

Nonsense mutations that generate a premature termination codon (PTC) can induce both the accelerated degradation of mutated mRNA compared with the wild type version of the mRNA or the production of a truncated protein. One of the considered therapeutic strategies to bypass PTCs is their "readthrough" based on small-molecule drugs. These molecules promote the incorporation of a near-cognate tRNA at the PTC position through the native polypeptide chain. In this review, we detailed the various existing strategies organized according to pharmacological molecule types through their different mechanisms. The positive results that followed readthrough molecule testing in multiple neuromuscular disorder models indicate the potential of this approach in peripheral neuropathies.

Amlexanox: Readthrough Induction and Nonsense-Mediated mRNA Decay Inhibition in a Charcot-Marie-Tooth Model of hiPSCs-Derived Neuronal Cells Harboring a Nonsense Mutation in GDAP1 Gene.

Nonsense mutations are involved in multiple peripheral neuropathies. These mutations induce the presence of a premature termination codon (PTC) at the mRNA level. As a result, a dysfunctional or truncated protein is synthesized, or even absent linked to nonsense-mediated mRNA degradation (NMD) system activation. Readthrough molecules or NMD inhibitors could be innovative therapies in these hereditary neuropathies, particularly molecules harboring the dual activity as amlexanox. Charcot-Marie-Tooth (CMT) is the most common inherited pathology of the peripheral nervous system, affecting 1 in 2500 people worldwide. Nonsense mutations in the GDAP1 gene have been associated with a severe form of CMT, prompting us to investigate the effect of readthrough and NMD inhibitor molecules. Although not clearly defined, GDAP1 could be involved in mitochondrial functions, such as mitophagy. We focused on the homozygous c.581C>G (p.Ser194*) mutation inducing CMT2H using patient human induced pluripotent stem cell (hiPSC)-derived neuronal cells. Treatment during 20 h with 100 µM of amlexanox on this cell model stabilized GDAP1 mRNAs carrying UGA-PTC and induced a restoration of the mitochondrial morphology. These results highlight the potential of readthrough molecules associated to NMD inhibitors for the treatment of genetic alterations in CMT, opening the way for future investigations and a potential therapy.

GDAP1 Involvement in Mitochondrial Function and Oxidative Stress, Investigated in a Charcot-Marie-Tooth Model of hiPSCs-Derived Motor Neurons.

Mutations in the ganglioside-induced differentiation associated protein 1 (GDAP1) gene have been associated with demyelinating and axonal forms of Charcot-Marie-Tooth (CMT) disease, the most frequent hereditary peripheral neuropathy in humans. Previous studies reported the prevalent GDAP1 expression in neural tissues and cells, from animal models. Here, we described the first GDAP1 functional study on human induced-pluripotent stem cells (hiPSCs)-derived motor neurons, obtained from normal subjects and from a CMT2H patient, carrying the GDAP1 homozygous c.581C>G (p.Ser194*) mutation. At mRNA level, we observed that, in normal subjects, GDAP1 is mainly expressed in motor neurons, while it is drastically reduced in the patient's cells containing a premature termination codon (PTC), probably degraded by the nonsense-mediated mRNA decay (NMD) system. Morphological and functional investigations revealed in the CMT patient's motor neurons a decrease of cell viability associated to lipid dysfunction and oxidative stress development. Mitochondrion is a key organelle in oxidative stress generation, but it is also mainly involved in energetic metabolism. Thus, in the CMT patient's motor neurons, mitochondrial cristae defects were observed, even if no deficit in ATP production emerged. This cellular model of hiPSCs-derived motor neurons underlines the role of mitochondrion and oxidative stress in CMT disease and paves the way for new treatment evaluation.

Deciphering the links between psychological stress, depression, and neurocognitive decline in patients with Down syndrome.

The relationships between psychological stress and cognitive functions are still to be defined despite some recent progress. Clinically, we noticed that patients with Down syndrome (DS) may develop rapid neurocognitive decline and Alzheimer's disease (AD) earlier than expected, often shortly after a traumatic life event (bereavement over the leave of a primary caregiver, an assault, modification of lifestyle, or the loss of parents). Of course, individuals with DS are naturally prone to develop AD, given the triplication of chromosome 21. However, the relatively weak intensity of the stressful event and the rapid pace of cognitive decline after stress in these patients have to be noticed. It seems DS patients react to stress in a similar manner normal persons react to a very intense stress, and thereafter develop a state very much alike post-traumatic stress disorders. Unfortunately, only a few studies have studied stress-induced regression in patients with DS. Thus, we reviewed the biochemical events involved in psychological stress and found some possible links with cognitive impairment and AD. Interestingly, these links could probably be also applied to non-DS persons submitted to an intense stress. We believe these links should be further explored as a better understanding of the relationships between stress and cognition could help in many situations including individuals of the general population.

Focus on cell therapy to treat corneal endothelial diseases.

The cornea is a multi-layered structure which allows fine refraction and provides both resistance to external insults and adequate transparency. The corneal endothelium ensures stromal hydration, failure of which, such as in Fuchs endothelial corneal dystrophy, after trauma or in aging, may lead to loss of corneal transparency and induce blindness. Currently, no efficient therapeutic alternatives exist except for corneal grafting. Thus corneal tissue engineering represents a valuable alternative approach, which may overcome cornea donor shortage. Several studies describe protocols to isolate, differentiate, and cultivate corneal endothelial cells (CEnCs) in vitro. Two main in vitro strategies can be described: expansion of eye-native cell populations, such as CEnCs, or the production and expansion of CEnCs from non-eye native cell populations, such as induced Pluripotent Stem Cells (iPSCs). The challenge with these cells is to obtain a monolayer of CEnCs on a biocompatible carrier, with a specific morphology (flat hexagonal cells), and with specific functions such as programmed cell cycle arrest. Another issue for this cell culture methodology is to define the adapted protocol (media, trophic factors, timeframe) that can mimic physiological development. Additionally, contamination by other cell types still represents a huge problem. Thus, purification methods, such as Fluorescence Activated Cell Sorting (FACS), Magnetic Ativated Cell Sorting (MACS) or Sedimentation Field Flow Fractionation (SdFFF) are useful. Animal models are also crucial to provide a translational approach for these therapies, integrating macro- and microenvironment influences, systemic hormonal or immune responses, and exogenous interactions. Non-eye native cell graft protocols are constantly improving both in efficacy and safety, with the aim of being the most suitable candidate for corneal therapies in future routine practice. The aim of this work is to review these different aspects with a special focus on issues facing CEnC culture in vitro, and to highlight animal graft models adapted to screen the efficacy of these different protocols.

One Multilocus Genomic Variation Is Responsible for a Severe Charcot-Marie-Tooth Axonal Form.

Charcot-Marie-Tooth (CMT) disease is a heterogeneous group of inherited disorders affecting the peripheral nervous system, with a prevalence of 1/2500. So far, mutations in more than 80 genes have been identified causing either demyelinating forms (CMT1) or axonal forms (CMT2). Consequentially, the genotype-phenotype correlation is not always easy to assess. Diagnosis could require multiple analysis before the correct causative mutation is detected. Moreover, it seems that approximately 5% of overall diagnoses for genetic diseases involves multiple genomic loci, although they are often underestimated or underreported. In particular, the combination of multiple variants is rarely described in CMT pathology and often neglected during the diagnostic process. Here, we present the complex genetic analysis of a family including two CMT cases with various severities. Interestingly, next generation sequencing (NGS) associated with Cov'Cop analysis, allowing structural variants (SV) detection, highlighted variations in MORC2 (microrchidia family CW-type zinc-finger 2) and AARS1 (alanyl-tRNA-synthetase) genes for one patient and an additional mutation in MFN2 (Mitofusin 2) in the more affected patient.

Pharmacoresistant Epilepsy in Childhood: Think of the Cerebral Folate Deficiency, a Treatable Disease.

Cerebral folate deficiency (CFD) is a neurological disorder characterized by low levels of 5-methyltetrahydrofolate (5-MTHF) in the cerebrospinal fluid (CSF). The prevalence of this autosomal recessive disorder is estimated to be <1/1,000,000. Fifteen different pathogenic variants in the folate receptor 1 gene (FOLR1) encoding the receptor of folate α (FRα) have already been described. We present a new pathogenic variation in the FOLR1 in a childhood-stage patient. We aim to establish the core structure of the FRα protein mandatory for its activity. A three-year-old child was admitted at hospital for a first febrile convulsions episode. Recurrent seizures without fever also occurred a few months later, associated with motor and cognitive impairment. Various antiepileptic drugs failed to control seizures. Magnetic resonance imaging (MRI) showed central hypomyelination and biological analysis revealed markedly low levels of 5-MTHF in CSF. Next generation sequencing (NGS) confirmed a CFD with a FOLR1 homozygous variation (c.197 G > A, p.Cys66Tyr). This variation induces an altered folate receptor α protein and underlines the role of a disulfide bond: Cys66-Cys109, essential to transport 5-MTHF into the central nervous system. Fortunately, this severe form of CFD had remarkably responded to high doses of oral folinic acid combined with intravenous administrations.

Curcumin-cyclodextrin/cellulose nanocrystals improve the phenotype of Charcot-Marie-Tooth-1A transgenic rats through the reduction of oxidative stress.

The most prevalent form of Charcot-Marie-Tooth disease (CMT type 1A) is characterized by duplication of the PMP22 gene, peripheral dysmyelination and decreased nerve conduction velocities leading to muscle weakness. Recently, oxidative stress was reported as a feature in CMT1A patients. Curcumin exhibits antioxidant activities and has shown beneficial properties on peripheral nerves. However, curcumin presents unfavorable pharmacokinetics. We developed curcumin-cyclodextrin/cellulose nanocrystals (Nano-Cur) to bypass this limitation. The present study investigated the therapeutic potential of Nano-Cur in vitro in Schwann cells (SCs) and in vivo in the transgenic CMT1A rat model. In vitro, Nano-Cur treatment (0.01 µM for 8 h) reduced reactive oxygen species and improved mitochondrial membrane potential in CMT1A SCs. Moreover, Nano-Cur treatment (0.01 µM for 1 week) increased the expression of myelin basic protein in SC/neuron co-cultures. Preliminary in vivo experiments carried out in WT rats showed that intraperitoneal (i.p.) injection of Nano-Cur treatment containing 0.2 mg/kg of curcumin strongly enhanced the bioavailability of curcumin. Afterwards, in 1-month-old male CMT1A rats, Nano-Cur treatment (0.2 mg/kg/day, i.p. for 8 weeks) significantly improved sensori-motor functions (grip strength, balance performance, and mechanical and thermal sensitivities). Importantly, sensory and motor nerve conduction velocities were improved. Further histological and biochemical analyses indicated that myelin sheath thickness and myelin protein expression (myelin protein zero and PMP22) were increased. In addition, oxidative stress markers were decreased in the sciatic nerve and gastrocnemius muscle. Finally, Nrf2 expression and some major antioxidant enzymes were increased in sciatic nerve. Therefore, Nano-Cur significantly improved cellular, electrophysiological, and functional features of CMT1A rats.

A mutation can hide another one: Think Structural Variants!

Next Generation Sequencing (NGS) using capture or amplicons strategies allows the detection of a large number of mutations increasing the rate of positive diagnosis for the patients. However, most of the detected mutations are Single Nucleotide Variants (SNVs) or small indels. Structural Variants (SVs) are often underdiagnosed in inherited genetic diseases, probably because few user-friendly tools are available for biologists or geneticists to identify them easily. We present here the diagnosis of two brothers presenting a demyelinating motor-sensitive neuropathy: a presumed homozygous c.5744_5745delAT in exon 10 of SACS gene was initially detected, while actually these patients were heterozygous for this mutation and harbored a large deletion of SACS exon 10 in the other allele. This hidden mutation has been detected thanks to the user-friendly CovCopCan software. We recommend to systematically use such a software to screen NGS data in order to detect SVs, such as Copy Number Variations, to improve diagnosis of the patients.

Optimized Protocol to Generate Spinal Motor Neuron Cells from Induced Pluripotent Stem Cells from Charcot Marie Tooth Patients.

Modelling rare neurogenetic diseases to develop new therapeutic strategies is highly challenging. The use of human-induced pluripotent stem cells (hiPSCs) is a powerful approach to obtain specialized cells from patients. For hereditary peripheral neuropathies, such as Charcot-Marie-Tooth disease (CMT) Type II, spinal motor neurons (MNs) are impaired but are very difficult to study. Although several protocols are available to differentiate hiPSCs into neurons, their efficiency is still poor for CMT patients. Thus, our goal was to develop a robust, easy, and reproducible protocol to obtain MNs from CMT patient hiPSCs. The presented protocol generates MNs within 20 days, with a success rate of 80%, using specifically chosen molecules, such as Sonic Hedgehog or retinoic acid. The timing and concentrations of the factors used to induce differentiation are crucial and are given hereby. We then assessed the MNs by optic microscopy, immunocytochemistry (Islet1/2, HB9, Tuj1, and PGP9.5), and electrophysiological recordings. This method of generating MNs from CMT patients in vitro shows promise for the further development of assays to understand the pathological mechanisms of CMT and for drug screening.

Tannic Acid Improves Renal Function Recovery after Renal Warm Ischemia-Reperfusion in a Rat Model.

BACKGROUND AND PURPOSE: Ischemia-reperfusion injury is encountered in numerous processes such as cardiovascular diseases or kidney transplantation; however, the latter involves cold ischemia, different from the warm ischemia found in vascular surgery by arterial clamping. The nature and the intensity of the processes induced by ischemia types are different, hence the therapeutic strategy should be adapted. Herein, we investigated the protective role of tannic acid, a natural polyphenol in a rat model reproducing both renal warm ischemia and kidney allotransplantation. The follow-up was done after 1 week. EXPERIMENTAL APPROACH: To characterize the effect of tannic acid, an in vitro model of endothelial cells subjected to hypoxia-reoxygenation was used. KEY RESULTS: Tannic acid statistically improved recovery after warm ischemia but not after cold ischemia. In kidneys biopsies, 3h after warm ischemia-reperfusion, oxidative stress development was limited by tannic acid and the production of reactive oxygen species was inhibited, potentially through Nuclear Factor erythroid-2-Related factor 2 (NRF2) activation. In vitro, tannic acid and its derivatives limited cytotoxicity and the generation of reactive oxygen species. Molecular dynamics simulations showed that tannic acid efficiently interacts with biological membranes, allowing efficient lipid oxidation inhibition. Tannic acid also promoted endothelial cell migration and proliferation during hypoxia. CONCLUSIONS: Tannic acid was able to improve renal recovery after renal warm ischemia with an antioxidant effect putatively extended by the production of its derivatives in the body and promoted cell regeneration during hypoxia. This suggests that the mechanisms induced by warm and cold ischemia are different and require specific therapeutic strategies.

A case report of a mild form of multiple acyl-CoA dehydrogenase deficiency due to compound heterozygous mutations in the ETFA gene.

BACKGROUND: Multiple acyl-CoA dehydrogenase deficiency (MADD), previously called glutaric aciduria type II, is a rare congenital metabolic disorder of fatty acids and amino acids oxidation, with recessive autosomal transmission. The prevalence in the general population is estimated to be 9/1,000,000 and the prevalence at birth approximately 1/200,000. The clinical features of this disease are divided into three groups of symptoms linked to a defect in electron transfer flavoprotein (ETF) metabolism. In this case report, we present new pathogenic variations in one of the two ETF protein subunits, called electron transfer flavoprotein alpha (ETFA), in a childhood-stage patient with no antecedent. CASE PRESENTATION: A five-year-old child was admitted to the paediatric emergency unit for seizures without fever. He was unconscious due to hypoglycaemia confirmed by laboratory analyses. At birth, he was a eutrophic full-term new-born with a normal APGAR index (score for appearance, pulse, grimace, activity, and respiration). He had one older brother and no parental consanguinity was reported. A slight speech acquisition delay was observed a few months before his admission, but he had no schooling problems. MADD was suspected based on urinary organic acids and plasma acylcarnitine analyses and later confirmed by genetic analysis, which showed previously unreported ETFA gene variations, both heterozygous (c.354C > A (p.Asn118Lys) and c.652G > A (p.Val218Met) variations). Treatment was based on avoiding fasting and a slow carbohydrate-rich evening meal associated with L-carnitine supplementation (approximately 100 mg/kg/day) for several weeks. This treatment was maintained and associated with riboflavin supplementation (approximately 150 mg/day). During follow up, the patient exhibited normal development and normal scholastic performance, with no decompensation. CONCLUSION: This case report describes new pathogenic variations of the ETFA gene. These compound heterozygous mutations induce the production of altered proteins, leading to a mild form of MADD.

Focus on 1,25-Dihydroxyvitamin D3 in the Peripheral Nervous System.

In this review, we draw attention to the roles of calcitriol (1,25-dihydroxyvitamin D3) in the trophicity of the peripheral nervous system. Calcitriol has long been known to be crucial in phosphocalcium homeostasis. However, recent discoveries concerning its involvement in the immune system, anti-cancer defenses, and central nervous system development suggest a more pleiotropic role than previously thought. Several studies have highlighted the impact of calcitriol deficiency as a promoting factor of various central neurological diseases, such as multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease. Based on these findings and recent publications, a greater role for calcitriol may be envisioned in the peripheral nervous system. Indeed, calcitriol is involved in myelination, axonal homogeneity of peripheral nerves, and neuronal-cell differentiation. This may have useful clinical consequences, as calcitriol supplementation may be a simple means to avoid the onset and/or development of peripheral nervous-system disorders.

Local low dose curcumin treatment improves functional recovery and remyelination in a rat model of sciatic nerve crush through inhibition of oxidative stress.

Traumatic injuries to peripheral nerves are frequent, however, specific pharmacological treatments are currently lacking. Curcumin has antioxidant, anti-inflammatory and neuroprotective properties but high oral doses are required for therapeutic use, particularly due to its low bioavailability. The aim of the present study was to investigate the effects of local and continuous treatment using low curcumin doses on functional recovery and nerve regeneration after rat sciatic nerve crush (SNC). Curcumin was administered by osmotic pumps with a catheter delivering the drug at the injury site (0.2 mg/day for 4 weeks). Functionally, early improvements in mechanical sensitivity, finger spacing of the injured paw, skilful walking and grip strength were observed in curcumin-treated animals. The curcumin treatment increased expression of compact myelin proteins (MPZ and PMP22), myelin sheath thickness and, correspondingly, increased motor and sensitive nerve conduction velocity. Microscopic analysis of gastrocnemius muscle indicated a curcumin-induced decrease in neurogenic lesions. Curcumin treatment reduced the production of reactive oxygen species (ROS) (which were notably produced by macrophages), lipid peroxidation and increased expression of transcription factor Nrf2. In silico analyses indicated that curcumin combines all the characteristics required to be an efficient lipid peroxidation inhibitor at the heart of biological membranes, hence protecting their degradation due to ROS. This antioxidant capacity is likely to contribute to the beneficial effects of curcumin after SNC injury. These results demonstrate that, when administrated locally, low doses of curcumin represent a promising therapy for peripheral nerve regeneration.

Engulfment of ceramic particles by fibroblasts does not alter cell behavior.

Despite many studies, the impact of ceramic particles on cell behavior remains unclear. The aim of the present study was to investigate the effects of nano-sized ceramic particles on fibroblastic cells. Fibroblasts (dermal fibroblasts freshly isolated from skin samples and WI26 fibroblastic cells) were cultured in a monolayer in the presence of alumina or cerium-zirconia particles (≈50 nm diameter) at two concentrations (100 or 500 µg ml-1). Fluorescent alumina particles were also used. The following properties were analyzed: cell morphology, cytoplasmic ceramic incorporation (using confocal and transmission electron microscopy) and migration (using a silicon insert). Sedimentation field-flow fractionation (SdFFF) was also used to evaluate the rate of incorporation of ceramic particles into the cells. Finally, after treatment with various concentrations of ceramic particles, fibroblasts were also included in a collagen type I lattice constituting a dermal equivalent (DE), and the collagen lattice retraction and cell proliferation were evaluated. In monolayer conditions, the presence of both alumina and cerium-zirconia ceramic particles did not cause any deleterious effects on cultured cells (dermal fibroblast and WI26 cells) and cell fate was not affected in any way by the presence of ceramic particles in the cytoplasm. Confocal (using fluorescent alumina particles) and electron microscopy (using both alumina and cerium-zirconia particles) showed that ceramic particles were internalized in the WI26 cells. Using fluorescent membrane labeling and fluorescent alumina particles, a membrane was observed around the particle-containing vesicles present in the cytoplasm. Electron microscopy on WI26 cells showed the presence of a classical bilayer membrane around the ceramic particles. Interestingly, SdFFF confirmed that some dermal fibroblasts contained many alumina ceramic particles while others contained very few; in WI26 cells, the uptake of alumina ceramic was more homogeneous. In DE, collagen lattice retraction and cell proliferation were unchanged when WI26 fibroblastic cells contained alumina or cerium-zirconia ceramic particles. Our data suggest that ceramic particles are internalized in the cells by endocytosis. The presence of ceramic particles in the cytoplasm has no affect on cell behavior, confirming the excellent biocompatibility of this material and anticipating a minimal harmful effect of potential wear debris.

New Method for Sorting Endothelial and Neural Progenitors from Human Induced Pluripotent Stem Cells by Sedimentation Field Flow Fractionation.

Human induced pluripotent stem cells (hiPSc) are a very useful solution to create and observe the behavior of specific and usually inaccessible cells, such as human motor neurons. Obtained from a patient biopsy by reprograming dermal fibroblasts (DF), hiPSc present the same properties as embryonic stem cells and can generate any cell type after several weeks of differentiation. Today, there are numerus protocols which aim to control hiPSC differentiation. The principal challenge is to obtain a sufficiently enriched specific cell population to study disease pathophysiology and to provide a good model for further investigation and drug screening. The differentiation process is very costly and time-consuming, because many specific factors and different culture media must be used. In this study, we used Sedimentation Field Flow Fractionation (SdFFF) to prepare enriched populations derived from hiPSc after only 10 days of culture in a classical medium. Based on phenotypic and proteomic characterization, "hyperlayer" elution resulted in a fraction expressing markers of endothelial progenitors while another fraction expressed markers of neural progenitors. The isolation of subpopulations representing various differentiation lineages is of major interest for the production of specialized, cell-enriched fractions and in the preparation of increasingly complex models for the development of new therapeutic tools.