Alleviating Severe Cytoskeletal Destruction of Spinal Motor Neurons: Another Effect of Docosahexaenoic Acid in Spinal Cord Injury.

Spinal cord injury (SCI) treatment remains a major challenge. Spinal motor neurons (MNs) are seriously injured in the early stage after SCI, but this has not received sufficient attention. Oxidative stress is known to play a crucial role in SCI pathology. Our studies demonstrated that oxidative stress can cause severe damage to the cytoskeleton of spinal MNs. Docosahexaenoic acid (DHA) has been shown to have beneficial effects on SCI, but the mechanism remains unclear, and no study has investigated the effect of DHA on oxidative stress-induced spinal MN injury. Here, we investigated the effect of DHA on spinal MN injury through in vivo and in vitro experiments, focusing on the cytoskeleton. We found that DHA not only promoted spinal MN survival but, more importantly, alleviated the severe cytoskeletal destruction of these neurons induced by oxidative stress in vitro and in mice with SCI in vivo. In addition, the mechanisms involved were investigated and elucidated. These results not only suggested a beneficial role of DHA in spinal MN cytoskeletal destruction caused by oxidative stress and SCI but also indicated the important role of the spinal MN cytoskeleton in the recovery of motor function after SCI. Our study provides new insights for the formulation of SCI treatment.

Human Motor Neurons Elicit Pathological Hallmarks of ALS and Reveal Potential Biomarkers of the Disease in Response to Prolonged IFNγ Exposure.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder marked by progressive motor neuron degeneration and muscle denervation. A recent transcriptomic study integrating a wide range of human ALS samples revealed that the upregulation of p53, a downstream target of inflammatory stress, is commonly detected in familial and sporadic ALS cases by a mechanism linked to a transactive response DNA-binding protein 43 (TDP-43) dysfunction. In this study, we show that prolonged interferon-gamma (IFNγ) treatment of human induced pluripotent stem cell-derived spinal motor neurons results in a severe cytoplasmic aggregation of TDP-43. TDP-43 dysfunction resulting from either IFNγ exposure or an ALS-associated TDP-43 mutation was associated with the activation of the p53 pathway. This was accompanied by the hyperactivation of neuronal firing, followed by the complete loss of their electrophysiological function. Through a comparative single-cell transcriptome analysis, we have identified significant alterations in ALS-associated genes in motor neurons exposed to IFNγ, implicating their direct involvement in ALS pathology. Interestingly, IFNγ was found to induce significant levels of programmed death-ligand 1 (PD-L1) expression in motor neurons without affecting the levels of any other immune checkpoint proteins. This finding suggests a potential role of excessive PD-L1 expression in ALS development, given that PD-L1 was recently reported to impair neuronal firing ability in mice. Our findings suggest that exposing motor neurons to IFNγ could directly derive ALS pathogenesis, even without the presence of the inherent genetic mutation or functional glia component. Furthermore, this study provides a comprehensive list of potential candidate genes for future immunotherapeutic targets with which to treat sporadic forms of ALS, which account for 90% of all reported cases.

Enhanced axon outgrowth of spinal motor neurons in co-culturing with dorsal root ganglions antagonizes the growth inhibitory environment.

Introduction: Forming a bridge made of functional axons to span the lesion is essential to reconstruct the motor circuitry following spinal cord injury (SCI). Dorsal root ganglion (DRG) axons are robust in axon growth and have been proved to facilitate the growth of cortical neurons in a process of axon-facilitated axon regeneration. However, whether DRG transplantation affects the axon outgrowth of spinal motor neurons (SMNs) that play crucial roles in motor circuitry remains unclear. Methods: We investigated the axonal growth patterns of co-cultured DRGs and SMN aggregates (SMNAs) taking advantage of a well-designed 3D-printed in vitro system. Chondroitin sulphate proteoglycans (CSPG) induced inhibitory matrix was introduced to imitate the inhibitory environment following SCI. Axonal lengths of DRG, SMNA or DRG & SMNA cultured on the permissive or CSPG induced inhibitory matrix were measured and compared. Results: Our results indicated that under the guidance of full axonal connection generated from two opposing populations of DRGs, SMNA axons were growth-enhanced and elongated along the DRG axon bridge to distances that they could not otherwise reach. Quantitatively, the co-culture increased the SMNA axonal length by 32.1 %. Moreover, the CSPG matrix reduced the axonal length of DRGs and SMNAs by 46.2 % and 17.7 %, respectively. This inhibitory effect was antagonized by the co-culture of DRGs and SMNAs. Especially for SMNAs, they extended the axons across the CSPG-coating matrix, reached the lengths close to those of SMNAs cultured on the permissive matrix alone. Conclusions: This study deepens our understanding of axon-facilitated reconstruction of the motor circuitry. Moreover, the results support SCI treatment utilizing the enhanced outgrowth of axons to restore functional connectivity in SCI patients.

A novel protocol to derive cervical motor neurons from induced pluripotent stem cells for amyotrophic lateral sclerosis.

Sporadic amyotrophic lateral sclerosis (sALS) is the majority of ALS, and the lack of appropriate disease models has hindered its research. Induced pluripotent stem cell (iPSC) technology now permits derivation of iPSCs from somatic cells of sALS patients to investigate disease phenotypes and mechanisms. Most existing differentiation protocols are time-consuming or low efficient in generating motor neurons (MNs). Here we report a rapid and simple protocol to differentiate MNs in monolayer culture using small molecules, which led to nearly pure neural stem cells in 6 days, robust OLIG2+ pMNs (73%-91%) in 12 days, enriched CHAT+ cervical spinal MNs (sMNs) (88%-97%) in 18 days, and functionally mature sMNs in 28 days. This simple and reproducible protocol permitted the identification of hyperexcitability phenotypes in our sALS iPSC-derived sMNs, and its application in neurodegenerative diseases should facilitate in vitro disease modeling, drug screening, and the development of cell therapy.

Dysregulated TDP-43 proteostasis perturbs excitability of spinal motor neurons during brainstem-mediated fictive locomotion in zebrafish.

Spinal motor neurons (SMNs) are the primary target of degeneration in amyotrophic lateral sclerosis (ALS). Degenerating motor neurons accumulate cytoplasmic TAR DNA-binding protein 43 (TDP-43) aggregates in most ALS cases. This SMN pathology can occur without mutation in the coding sequence of the TDP-43-encoding gene, TARDBP. Whether and how wild-type TDP-43 drives pathological changes in SMNs in vivo remains largely unexplored. In this study, we develop a two-photon calcium imaging setup in which tactile-evoked neural responses of motor neurons in the brainstem and spinal cord can be monitored using the calcium indicator GCaMP. We devise a piezo-assisted tactile stimulator that reproducibly evokes a brainstem descending neuron upon tactile stimulation of the head. A direct comparison between caudal primary motor neurons (CaPs) with or without TDP-43 overexpression in contiguous spinal segments demonstrates that CaPs overexpressing TDP-43 display attenuated Ca2+ transients during fictive escape locomotion evoked by the tactile stimulation. These results show that excessive amounts of TDP-43 protein reduce the neuronal excitability of SMNs and potentially contribute to asymptomatic pathological lesions of SMNs and movement disorders in patients with ALS.

Fetuses and infants with Amyoplasia congenita in congenital Zika syndrome: The evidence of a viral cause. A narrative review of 144 cases.

OBJECTIVES: Amyoplasia congenita is the most frequent type of arthrogryposis causing fetal hypokinesia, leading to congenital contractures at birth. The pathogenesis is thought to be impaired blood circulation to the fetus early in pregnancy, with hypotension and hypoxia damaging the anterior horn cells. In animal studies however a prenatal infection with a poliomyelitis-like viral agent was demonstrated. Congenital Zika virus syndrome (CZVS) has recently been described in infants with severe microcephaly, and in 10-25% of cases arthrogryposis. METHODS: A search in PubMed for CZVS yielded 124 studies. After a selection for arthrogryposis, 35 papers were included, describing 144 cases. The studies were divided into two categories. 1) Those (87 cases) focussing on imaging or histological data of congenital brain defects, contained insufficient information to link arthrogryposis specifically to lesions of the brain or spinal motor neuron. 2) In the other 57 cases detailed clinical data could be linked to neurophysiological, imaging or histological data. RESULTS: In category 1 the most frequent brain abnormalities in imaging studies were ventriculomegaly, calcifications (subcortical, basal ganglia, cerebellum), hypoplasia of the brainstem and cerebellum, atrophy of the cerebral cortex, migration disorders and corpus callosum anomalies. In category 2, in 38 of 57 cases clinical data were indicative of Amyoplasia congenita. This diagnosis was confirmed by electromyographic findings (13 cases), by MRI (37 cases) or histology (12 cases) of the spinal cord. The latter showed small or absent lateral corticospinal tracts, and cell loss and degeneration of motor neuron cells. Zika virus-proteins and flavivirus-like particles were detected in cytoplasm of spinal neurons. CONCLUSION: The phenotype of arthrogryposis in CZVS is consistent with Amyoplasia congenita. These findings warrant search for an intrauterine infection with any neurotropic viral agent with affinity to spinal motor neurons in neonates with Amyoplasia.

Spastic muscle stiffness evaluated using ultrasound elastography and evoked electromyogram in patients following severe traumatic brain injury: an observational study.

OBJECTIVE: To determine the relationship between muscle stiffness assessed using ultrasound shear wave elastography, spinal motor neuron excitability assessed using the F wave, and clinical findings of spasticity in patients with spastic muscle overactivity following severe traumatic brain injury. METHODS: This study enrolled 17 inpatients with severe traumatic brain injury and 20 healthy volunteers. Biceps brachii muscle stiffness was then evaluated using ultrasound shear wave speed. Spinal motor neuron excitability was evaluated using the F/M ratio recorded from abductor pollicis brevis muscle. Clinical parameters, such as the modified Ashworth scale and modified Tardieu scale, were assessed in the patient with traumatic brain injury. RESULTS: The patients with traumatic brain injury group had a significantly higher shear wave speed and F/M ratio compared with the healthy group. A higher shear wave speed was correlated with higher clinical spastic severity in patients with traumatic brain injury. The F/M ratio was not significantly correlated with clinical spastic severity. CONCLUSION: Ultrasound shear wave elastography might be helpful for assessing muscle stiffness in patients with spastic muscle overactivity following severe traumatic brain injury. Further studies comprising larger cohorts are warranted.

Human stem cell-derived neurons and neural circuitry therapeutics: Next frontier in spinal cord injury repair.

Spinal cord injury (SCI) remains a life-altering event that devastates those injured and the families that support them. Numerous laboratories are engaged in preclinical and clinical trials to repair the injured spinal cord with stem cell-derived therapeutics. A new developmental paradigm reveals early bifurcation of brain or trunk neurons in mammals via neuromesodermal progenitors (NMPs) relevant to therapies requiring homotypic spinal cord neural populations. Human-induced pluripotent stem cell (hiPSC) NMP-derived spinal motor neurons generated ex vivo following this natural developmental route demonstrate robust survival in vivo when delivered as suspension grafts or as in vitro preformed encapsulated neuronal circuitry when transplanted into a rat C4-C5 hemicontusion injury site. Use of in vitro matured neurons avoids in vivo differentiation challenges of using pluripotent hiPSC or multipotent neural stem cell (NSC) or mesenchymal stem cell therapeutics. In this review, we provide an injury to therapeutics overview focusing on how stem cell and developmental fields are merging to generate exquisitely matched spinal motor neurons for SCI therapeutic studies. The complexity of the SCI microenvironment generated by trauma to neurons and vasculature, along with infiltrating inflammatory cells and scarring, underlies the challenging cytokine microenvironment that therapeutic cells encounter. An overview of evolving but limited stem cell-based SCI therapies that have progressed from preclinical to clinical trials illustrates the challenges and need for additional stem cell-based therapeutic approaches. The focus here on neurons describes how NMP-based neurotechnologies are advancing parallel strategies such as transplantation of preformed neuronal circuitry as well as human in vitro gastruloid multicellular models of trunk central and peripheral nervous system integration with organs. NMP-derived neurons are expected to be powerful drivers of the next generation of SCI therapeutics and integrate well with combination therapies that may utilize alternate biomimetic scaffolds for bridging injuries or flexible biodegradable electronics for electrostimulation.

A Therapeutic Strategy for Lower Motor Neuron Disease and Injury Integrating Neural Stem Cell Transplantation and Functional Electrical Stimulation in a Rat Model.

Promising treatments for upper motor neuron disease are emerging in which motor function is restored by brain-computer interfaces and functional electrical stimulation. At present, such technologies and procedures are not applicable to lower motor neuron disease. We propose a novel therapeutic strategy for lower motor neuron disease and injury integrating neural stem cell transplantation with our new functional electrical stimulation control system. In a rat sciatic nerve transection model, we transplanted embryonic spinal neural stem cells into the distal stump of the peripheral nerve to reinnervate denervated muscle, and subsequently demonstrated that highly responsive limb movement similar to that of a healthy limb could be attained with a wirelessly powered two-channel neurostimulator that we developed. This unique technology, which can reinnervate and precisely move previously denervated muscles that were unresponsive to electrical stimulation, contributes to improving the condition of patients suffering from intractable diseases of paralysis and traumatic injury.

Prolyl Isomerase Pin1 Expression in the Spinal Motor Neurons of Patients With Sporadic Amyotrophic Lateral Sclerosis.

BACKGROUND AND PURPOSE: Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease. Selective deficiency of edited adenosine deaminase acting on RNA 2 (ADAR2), a key molecule in the acquisition of Ca2+ resistance in motor neurons, has been reported in sporadic ALS (sALS) spinal motor neurons. Since ADAR2 activity is positively regulated by prolyl isomerase Protein never in mitosis gene A interacting-1 (Pin1), a known phosphorylation-dependent peptidyl-prolyl cis/trans isomerase, we investigated Pin1 expression in spinal motor neurons in sALS. METHODS: Specimens of the spinal cord were obtained from the lumbar region in eight sALS patients and age-matched five controls after postmortem examinations. The specimens were double stained with anti-Pin1 and anti-TAR DNA-binding protein of 43 kDa (TDP-43) antibodies, and examined under a fluorescence microscope. RESULTS: This study analyzed 254 and 422 spinal motor neurons from 8 sALS patients and 5 control subjects, respectively. The frequency of motor neurons with high cytoplasmic Pin1 expression from the spinal cord did not differ significantly between sALS specimens without cytoplasmic TDP-43 inclusions and control specimens. However, in sALS specimens, neurons for which the Pin1 immunoluminescence intensity in the cytoplasm was at least twice that in the background were more common in specimens with cytoplasmic TDP-43 inclusions (p<0.05 in χ² test). CONCLUSIONS: In sALS, neurons with higher expression levels of Pin1 levels had more TDP-43 inclusions. Despite the feedback mechanism between Pin1 and ADAR2 being unclear, since Pin1 positively regulates ADAR2, our results suggest that higher Pin1 expression levels in motor neurons with TDP-43 pathology from sALS patients represent a compensatory mechanism.

Thenar Muscle Motor Imagery Increases Spinal Motor Neuron Excitability of the Abductor Digiti Minimi Muscle.

When a person attempts intended finger movements, unintended finger movement also occur, a phenomenon called "enslaving". Given that motor imagery (MI) and motor execution (ME) share a common neural foundation, we hypothesized that the enslaving effect on the spinal motor neuron excitability occurs during MI. To investigate this hypothesis, electromyography (EMG) and F-wave analysis were conducted in 11 healthy male volunteers. Initially, the EMG activity of the left abductor digiti minimi (ADM) muscle during isometric opposition pinch movement by the left thumb and index finger at 50% maximal effort was compared with EMG activity during the Rest condition. Next, the F-wave and background EMG recordings were performed under the Rest condition, followed by the MI condition. Specifically, in the Rest condition, subjects maintained relaxation. In the MI condition, they imagined isometric left thenar muscle activity at 50% maximal voluntary contraction (MVC). During ME, ADM muscle activity was confirmed. During the MI condition, both F-wave persistence and the F-wave/M-wave amplitude ratio obtained from the ADM muscle were significantly increased compared with that obtained during the Rest condition. No difference was observed in the background EMG between the Rest and MI conditions. These results suggest that MI of isometric intended finger muscle activity at 50% MVC facilitates spinal motor neuron excitability corresponding to unintended finger muscle. Furthermore, MI may induce similar modulation of spinal motor neuron excitability as actual movement.

Therapeutic Effects of Risperidone against Spinal Cord Injury in a Rat Model of Asphyxial Cardiac Arrest: A Focus on Body Temperature, Paraplegia, Motor Neuron Damage, and Neuroinflammation.

Cardiac arrest (CA) causes severe spinal cord injury and evokes spinal cord disorders including paraplegia. It has been reported that risperidone, an antipsychotic drug, effectively protects neuronal cell death from transient ischemia injury in gerbil brains. However, until now, studies on the effects of risperidone on spinal cord injury after asphyxial CA (ACA) and cardiopulmonary resuscitation (CPR) are not sufficient. Therefore, this study investigated the effect of risperidone on hind limb motor deficits and neuronal damage/death in the lumbar part of the spinal cord following ACA in rats. Mortality, severe motor deficits in the hind limbs, and the damage/death (loss) of motor neurons located in the anterior horn were observed two days after ACA/CPR. These symptoms were significantly alleviated by risperidone (an atypical antipsychotic) treatment after ACA. In vehicle-treated rats, the immunoreactivities of tumor necrosis factor-alpha (TNF-α) and interleukin 1-beta (IL-1ß), as pro-inflammatory cytokines, were increased, and the immunoreactivities of IL-4 and IL-13, as anti-inflammatory cytokines, were reduced with time after ACA/CPR. In contrast, in risperidone-treated rats, the immunoreactivity of the pro-inflammatory cytokines was significantly decreased, and the anti-inflammatory cytokines were enhanced compared to vehicle-treated rats. In brief, risperidone treatment after ACA/CPR in rats significantly improved the survival rate and attenuated paralysis, the damage/death (loss) of motor neurons, and inflammation in the lumbar anterior horn. Thus, risperidone might be a therapeutic agent for paraplegia by attenuation of the damage/death (loss) of spinal motor neurons and neuroinflammation after ACA/CPR.

MFN2 Deficiency Impairs Mitochondrial Transport and Downregulates Motor Protein Expression in Human Spinal Motor Neurons.

Charcot-Marie-Tooth (CMT) disease is one of the most common genetically inherited neurological disorders and CMT type 2A (CMT 2A) is caused by dominant mutations in the mitofusin-2 (MFN2) gene. MFN2 is located in the outer mitochondrial membrane and is a mediator of mitochondrial fusion, with an essential role in maintaining normal neuronal functions. Although loss of MFN2 induces axonal neuropathy, the detailed mechanism by which MFN2 deficiency results in axonal degeneration of human spinal motor neurons remains largely unknown. In this study, we generated MFN2-knockdown human embryonic stem cell (hESC) lines using lentivirus expressing MFN2 short hairpin RNA (shRNA). Using these hESC lines, we found that MFN2 loss did not affect spinal motor neuron differentiation from hESCs but resulted in mitochondrial fragmentation and dysfunction as determined by live-cell imaging. Notably, MFN2-knockodwn spinal motor neurons exhibited CMT2A disease-related phenotypes, including extensive perikaryal inclusions of phosphorylated neurofilament heavy chain (pNfH), frequent axonal swellings, and increased pNfH levels in long-term cultures. Importantly, MFN2 deficit impaired anterograde and retrograde mitochondrial transport within axons, and reduced the mRNA and protein levels of kinesin and dynein, indicating the interfered motor protein expression induced by MFN2 deficiency. Our results reveal that MFN2 knockdown induced axonal degeneration of spinal motor neurons and defects in mitochondrial morphology and function. The impaired mitochondrial transport in MFN2-knockdown spinal motor neurons is mediated, at least partially, by the altered motor proteins, providing potential therapeutic targets for rescuing axonal degeneration of spinal motor neurons in CMT2A disease.

Fully Characterized Mature Human iPS- and NMP-Derived Motor Neurons Thrive Without Neuroprotection in the Spinal Contusion Cavity.

Neural cell interventions in spinal cord injury (SCI) have focused predominantly on transplanted multipotent neural stem/progenitor cells (NSPCs) for animal research and clinical use due to limited information on survival of spinal neurons. However, transplanted NSPC fate is unpredictable and largely governed by injury-derived matrix and cytokine factors that are often gliogenic and inflammatory. Here, using a rat cervical hemicontusion model, we evaluate the survival and integration of hiPSC-derived spinal motor neurons (SMNs) and oligodendrocyte progenitor cells (OPCs). SMNs and OPCs were differentiated in vitro through a neuromesodermal progenitor stage to mimic the natural origin of the spinal cord. We demonstrate robust survival and engraftment without additional injury site modifiers or neuroprotective biomaterials. Ex vivo differentiated neurons achieve cervical spinal cord matched transcriptomic and proteomic profiles, meeting functional electrophysiology parameters prior to transplantation. These data establish an approach for ex vivo developmentally accurate neuronal fate specification and subsequent transplantation for a more streamlined and predictable outcome in neural cell-based therapies of SCI.

Differential Changed Excitability of Spinal Motor Neurons Innervating Tibialis Anterior and Peroneus Muscles Cause Foot Inversion After Stroke.

Objective: To study differential post-stroke changes of excitability of spinal motor neurons innervating a group of antagonist muscles of ankle and their effects on foot inversion. Methods: F waves in tibialis anterior (TA) and peroneus muscles (PN) were recorded. The condition of spasticity and foot inversion in stroke patients were also evaluated. The differences of F wave parameters between patients and healthy controls (HC), as well as TA and PN, were investigated. Results: There were natural differences in the persistence of the F waves (Fp) and F/M amplitude ratio (F/M) between TA and PN in HC. Stroke patients showed significantly higher F/M in TA and PN, while there was no difference in Fp comparing to HC. The natural differences in F wave parameters between TA and PN were differentially retained after stroke. The natural difference of the two muscles in Fp remained unchanged and the F/M difference disappeared in those without spasticity or foot inversion, while the Fp difference disappeared and the F/M difference remained in those with spasticity or foot inversion. Conclusion: Based on the natural difference of the number and size of spinal motor neurons innervating TA and PN, their excitability may change differently according to the severity of the stroke, which may be the reason of foot inversion.

Early motor deficits in the phalangeal fine movements induced by chronic AMPA infusion in the rat spinal cord assessed by a novel method: Phalangeal tension recording test.

Motor behavior alterations are a shared hallmark of neurodegenerative diseases affecting motor circuits, such as amyotrophic lateral sclerosis (ALS), Parkinson's, and Huntington's diseases. In patients and transgenic animal models of amyotrophic lateral sclerosis fine movements controlled by distal muscles are the first to be affected, but its study and knowledge remain poorly understood, mainly because most of the tests used for describing the motor alterations are focused on the function of proximal muscles and gross movements. In this study we demonstrate that alterations of phalangeal fine movements can be quantitatively evaluated using a novel procedure designed by us, phalangeal tension recording test, which showed high sensitivity to detect such alterations. The evaluation was carried out during the motor neuron (MN) degenerative process induced by the acute and chronic overactivation of AMPA receptors in the lumbar rat spinal cord, using previously described models. The new method allowed the quantification of significant alterations of the fine movements of the hindpaws phalanges when AMPA was infused in the lumbar segment controlling the distal muscles, but not when a more rostral spinal segment was infused, and these alterations were not detected by the rotarod or the stride tests. These changes occurred before the paralysis of the hindlimbs. Studying the early distal motor alterations before the total paralysis at late stages is essential for understanding the initial consequences of MN degeneration and therefore for designing new strategies for the control, treatment and prevention of MN diseases.

Identification of Novel Microsatellite Markers Flanking the SMN1 and SMN2 Duplicated Region and Inclusion Into a Single-Tube Tridecaplex Panel for Haplotype-Based Preimplantation Genetic Testing of Spinal Muscular Atrophy.

Preimplantation genetic testing for the monogenic disorder (PGT-M) spinal muscular atrophy (SMA) is significantly improved by supplementation of SMN1 deletion detection with marker-based linkage analysis. To expand the availability of informative markers for PGT-M of SMA, we identified novel non-duplicated and highly polymorphic microsatellite markers closely flanking the SMN1 and SMN2 duplicated region. Six of the novel markers within 0.5 Mb of the 1.7 Mb duplicated region containing SMN1 and SMN2 (SMA6863, SMA6873, SMA6877, SMA7093, SMA7115, and SMA7120) and seven established markers (D5S1417, D5S1413, D5S1370, D5S1408, D5S610, D5S1999, and D5S637), all with predicted high heterozygosity values, were selected and optimized in a tridecaplex PCR panel, and their polymorphism indices were determined in two populations. Observed marker heterozygosities in the Chinese and Caucasian populations ranged from 0.54 to 0.86, and 98.4% of genotyped individuals (185 of 188) were heterozygous for ≥2 markers on either side of SMN1. The marker panel was evaluated for disease haplotype phasing using single cells from two parent-child trios after whole-genome amplification, and applied to a clinical IVF (in vitro fertilization) PGT-M cycle in an at-risk couple, in parallel with SMN1 deletion detection. Both direct and indirect test methods determined that none of five tested embryos were at risk for SMA, with haplotype analysis further identifying one embryo as unaffected and four as carriers. Fresh transfer of the unaffected embryo did not lead to implantation, but subsequent frozen-thaw transfer of a carrier embryo produced a pregnancy, with fetal genotype confirmed by amniocentesis, and a live birth at term.

Survival Motor Neuron (SMN) Protein Insufficiency Exacerbates Renal Ischemia/Reperfusion Injury.

The survival of motor neuron (SMN) protein is ubiquitously involved in spliceosome assembly and ribonucleoprotein biogenesis. SMN protein is expressed in kidney and can affect cell death processes. However, the role of SMN in acute kidney injury (AKI) is largely unknown. In the current study, we found that the expression of SMN in the kidney was significantly reduced in both clinical ischemic AKI and a mouse model of renal ischemia-reperfusion injury (IRI). We then used SMN heterozygous knockout (SMN+/-) mice and found that the declines in renal function, tubular injury, and tubular cell apoptosis after experimental IRI were significantly more severe in SMN+/- mice than those in their wild-type littermates. Concomitantly, the canonical transcription factor nuclear factor-κb (NFκb) signaling was enhanced in ischemic SMN+/- mice. In vitro, cobalt dichloride (CoCl2) treatment reduced SMN expression in proximal tubular epithelial cells. In addition, CoCl2-induced apoptosis and activation of NFκb signaling pathway were enhanced by transient transfection of a small-interfering RNA (siRNA) against SMN while attenuated by transient transfection of a full-length SMN plasmid. Taken together, this study for the first time supported the protective role of SMN in ischemic AKI.

Bi-allelic mutations in PRUNE lead to neurodegeneration with spinal motor neuron involvement and hyperCKaemia.

Okur D, Daimagüler HS, Ersen Danyeli A, Tekgül H, Wang H, Wunderlich G, Çirak S, Yis U. Bi-allelic mutations in PRUNE lead to neurodegeneration with spinal motor neuron involvement and hyperCKaemia. Turk J Pediatr 2019; 61: 931-936. We aimed to systematically investigate the neuromuscular involvement of individuals with PRUNE mutations who may have a major spinal motor neuron involvement as part of the PRUNE-associated neurodegenerative phenotype. The complex neurological phenotypes associated with Prune mutations include microcephaly with brain abnormalities, spasticity, seizures, severe developmental delay and developmental regression. We used whole exome sequencing to identify the mutation and electrophysiological and muscle biopsy studies to evaluate the signs of spinal motor neuron involvement. The affected individuals carry homozygous PRUNE mutation (NM_021222.1, c.316G > A, p.D106N), showing the signs of spinal motor neuron involvement supported by electrophysiological and muscle biopsy findings and also persistent high creatine kinase levels. We confirm that individuals with PRUNE mutations may have a major spinal motor neuron involvement as part of the PRUNE-associated neurodegenerative phenotype. The PRUNE gene should be considered in all the individuals with non-5q spinal muscular atrophy. High creatine kinase values may be a part of PRUNE disease spectrum.

Estimation of voluntary elicited motor neuron discharge using a peripheral nerve collision technique at different contraction strengths.

OBJECTIVE: To estimate non-invasively the amount, recruitment pattern and discharge frequency of spinal motor neurons (MN) at contraction strength >20% of maximal voluntary contraction (MVC) of small hand muscles. METHODS: A peripheral collision technique was used and consisted of supramaximal electrical stimuli at Erb's point and at the wrist, synchronizing descending volleys of action potential during voluntary isometric contractions of the abductor digiti minimi muscle at 20-80% of MVC strength and 1-8 s of contraction duration. Responses of 13 healthy volunteers were quantified and analysed using a recently described model of MN behaviour. RESULTS: A linear relationship between MN discharge and force generation was noticed with R2 = 0.996, and was confirmed using the simulation results (R2 = 0.997) for contraction durations up to 8 s. For each investigated force level, discharge frequency and recruitment pattern were calculated for individual MN. CONCLUSIONS: Using this method, MN discharge properties during voluntary activity can be estimated non-invasively. SIGNIFICANCE: This method provides new opportunities for the non-invasive study of MN behaviour, and could be expanded to patients with conduction failure and during fatigue.