Degeneration Of Spinal Motor Neurons Research Articles

Axonal transcriptome reveals upregulation of PLK1 as a protective mechanism in response to increased DNA damage in FUS P525L spinal motor neurons.

Mutations in the gene FUSED IN SARCOMA ( FUS ) are among the most frequently occurring genetic forms of amyotrophic lateral sclerosis (ALS). Early pathogenesis of FUS -ALS involves impaired DNA damage response and axonal degeneration. However, it is still poorly understood how these gene mutations lead to selective spinal motor neuron (MN) degeneration and how nuclear and axonal phenotypes are linked. To specifically address this, we applied a compartment specific RNA-sequencing approach using microfluidic chambers to generate axonal as well as somatodendritic compartment-specific profiles from isogenic induced pluripotent stem cells (iPSCs)-derived MNs. We demonstrate high purity of axonal and soma fractions and show that the axonal transcriptome is unique and distinct from that of somas including significantly fewer number of transcripts. Functional enrichment analysis revealed that differentially expressed genes (DEGs) in axons were mainly enriched in key pathways like RNA metabolism and DNA damage, complementing our knowledge of early phenotypes in ALS pathogenesis and known functions of FUS. In addition, we demonstrate a strong enrichment for cell cycle associated genes including significant upregulation of polo-like kinase 1 (PLK1) in FUS P525L mutant MNs. PLK1 was increased upon DNA damage induction and PLK1 inhibition further increased the number of DNA damage foci in etoposide-treated cells, an effect that was diminished in case of FUS mutant MNs. In contrast, inhibition of PLK1 increased late apoptotic or necrosis-induced neuronal cell death in mutant neurons. Taken together, our findings provide insights into compartment-specific transcriptomics in human FUS -ALS MNs and we propose that specific upregulation of PLK1 might represent an early event in the pathogenesis of ALS, possibly modulating DNA damage response and other associated pathways.

Ubiquitination Insight from Spinal Muscular Atrophy-From Pathogenesis to Therapy: A Muscle Perspective.

Spinal muscular atrophy (SMA) is one of the most frequent causes of death in childhood. The disease's molecular basis is deletion or mutations in the SMN1 gene, which produces reduced survival motor neuron protein (SMN) levels. As a result, there is spinal motor neuron degeneration and a large increase in muscle atrophy, in which the ubiquitin-proteasome system (UPS) plays a significant role. In humans, a paralogue of SMN1, SMN2 encodes the truncated protein SMNΔ7. Structural differences between SMN and SMNΔ7 affect the interaction of the proteins with UPS and decrease the stability of the truncated protein. SMN loss affects the general ubiquitination process by lowering the levels of UBA1, one of the main enzymes in the ubiquitination process. We discuss how SMN loss affects both SMN stability and the general ubiquitination process, and how the proteins involved in ubiquitination could be used as future targets for SMA treatment.

Morphometric analysis of spinal motor neuron degeneration in sporadic amyotrophic lateral sclerosis

ObjectivesThis study aimed to clarify the relationship between 43-kDa TAR DNA-binding protein (TDP-43) pathology and spinal cord anterior horn motor neuron (AHMN) atrophy in sporadic amyotrophic lateral sclerosis (SALS). MethodsEight patients with SALS and 12 controls were included in this study. Formalin-fixed specimens of lumbar spinal cord samples were paraffin-embedded and sectioned at the level of the fourth lumbar spinal cord with a 4 μm thickness. Using a microscope, the long diameters of the neurons with nucleoli were measured in spinal AHMNs stained with an anti-SMI-32 antibody. AHMNs were divided into medial and lateral nuclei for statistical analysis. We also used previously reported data to measure the long diameter of AHMNs with initial TDP-43 pathology, in which TDP-43 was present both in the nucleus and cytoplasm. ResultsThe long diameter of the lumbar spinal AHMNs in patients with SALS was smaller in the medial nucleus (42.54 ± 9.33 μm, n = 24) and the lateral nucleus (49.41 ± 13.86 μm, n = 129) than in controls (medial nucleus: 55.84 ± 13.49 μm, n = 85, p < 0.001; lateral nucleus: 62.39 ± 13.29 μm, n = 756, p < 0.001, Mann–Whitney U test). All 21 motor neurons with initial TDP-43 pathology were in the lateral nucleus, and their long diameter (67.60 ± 18.3 μm, p = 0.352) was not significantly different from that of controls. ConclusionMotor neuron atrophy in SALS does not occur during the initial stages of TDP-43 pathology, and TDP-43 pathology is already advanced in the atrophied motor neurons.

Integrated Approaches and Practical Recommendations in Patient Care Identified with 5q Spinal Muscular Atrophy through Newborn Screening.

In recent years, significant progress has been made in 5q Spinal Muscular Atrophy therapeutics, emphasizing the importance of early diagnosis and intervention for better clinical outcomes. Characterized by spinal cord motor neuron degeneration, 5q-SMA leads to muscle weakness, swallowing difficulties, respiratory insufficiency, and skeletal deformities. Recognizing the pre-symptomatic phases supported by screening and confirmatory genetic tests is crucial for early diagnosis. This work addresses key considerations in implementing 5q-SMA screening within the Brazilian National Newborn Screening Program and explores Brazil's unique challenges and opportunities, including genetic tests, time-to-patient referral to specialized centers, program follow-up, and treatment algorithms. We aim to guide healthcare professionals and policymakers, facilitating global discussions, including Latin American countries, and knowledge-sharing on this critical subject to improve the care for newborns identified with 5q SMA.

Non-Invasive Spinal Cord Stimulation for Motor Rehabilitation of Patients with Spinal Muscular Atrophy Treated with Orphan Drugs.

Spinal muscular atrophy (SMA) is an orphan disease characterized by the progressive degeneration of spinal alpha motor neurons. In recent years, nusinersen and several other drugs have been approved for the treatment of this disease. Transcutaneous spinal cord stimulation (tSCS) modulates spinal neuronal networks, resulting in changes in locomotion and posture in patients with severe spinal cord injury and stroke. We hypothesize that tSCS can activate motor neurons that are intact and restored by medication, slow the decline in motor activity, and contribute to the development of motor skills in SMA patients. Thirty-seven children and adults with SMA types 2 and 3 participated in this study. The median duration of drug treatment was over 20 months. The application of tSCS was performed during physical therapy for 20-40 min per day for ~12 days. Outcome measures were specific SMA motor scales, goniometry of contractured joints, and forced vital capacity. Significant increases in motor function, improved respiratory function, and decreased contracture were observed in both type 2 and 3 SMA participants. The magnitude of functional changes was not associated with participant age. Further studies are needed to elucidate the reasons for the beneficial effects of spinal cord electrical stimulation on SMA.

Analysis of Free Circulating Messenger Ribonucleic Acids in Serum Samples from Late-Onset Spinal Muscular Atrophy Patients Using nCounter NanoString Technology.

5q-related Spinal muscular atrophy (SMA) is a hereditary multi-systemic disorder leading to progressive muscle atrophy and weakness caused by the degeneration of spinal motor neurons (MNs) in the ventral horn of the spinal cord. Three SMN-enhancing drugs for SMA treatment are available. However, even if these drugs are highly effective when administrated early, several patients do not benefit sufficiently or remain non-responders, e.g., adults suffering from late-onset SMA and starting their therapy at advanced disease stages characterized by long-standing irreversible loss of MNs. Therefore, it is important to identify additional molecular targets to expand therapeutic strategies for SMA treatment and establish prognostic biomarkers related to the treatment response. Using high-throughput nCounter NanoString technology, we analyzed serum samples of late-onset SMA type 2 and type 3 patients before and six months under nusinersen treatment. Four genes (AMIGO1, CA2, CCL5, TLR2) were significantly altered in their transcript counts in the serum of patients, where differential expression patterns were dependent on SMA subtype and treatment response, assessed with outcome scales. No changes in gene expression were observed six months after nusinersen treatment, compared to healthy controls. These alterations in the transcription of four genes in SMA patients qualified those genes as potential SMN-independent therapeutic targets to complement current SMN-enhancing therapies.

Onasemnogene Abeparvovec: Post-infusion Efficacy and Safety in Patients With Spinal Muscular Atrophy (SMA)-A Fondazione Policlinico Gemelli IRCCS Experience.

Objective: The term Spinal Muscular Atrophy (SMA) identifies a group of genetic disorders affecting spinal motor neurons. It is caused by the loss of the SMN1 gene, resulting in degeneration of spinal alpha motor neurons and muscle atrophy. This study is focused on innovative gene therapies with onasemnogene abeparvovec approved in Italy in March 2021 with full reimbursement by the National Health Service. The objective pursued is verify, by means of the CHOP-INTEND scores obtained, whether therapy with onasemnogene abeparvovec led to an improvement in the clinical picture of the treated subjects and any adverse reactions that occurred. Methods: this study was conducted by evaluating the scores in the different re-evaluations of individual patients treated in our hospital (Fondazione Policlinico Universitario Agostino Gemelli IRCCS - Rome) and comparing them with the results of the CL-303 study described in SPC (Summary of Product Characteristics). The data were extracted from the patients' clinical records on the AIFA (Agenzia Italiana del Farmaco - Italian Medicines Agency) registries, also collecting information on any post-infusion ADRs. Everything was then represented graphically to have a clear comparison with the data from the study registered for drug approval. Results: from the data obtained, 7 out of 8 patients improved their health status post infusion with, in some cases, a significant increase in score. Conclusions: this result allows us to understand how crucial it is to start treatment as soon as possible after the diagnosis of the condition as the greatest improvements were seen in subjects who received treatment within 2 months of birth.

Agonist of growth hormone-releasing hormone improves the disease features of spinal muscular atrophy mice

Spinal muscular atrophy (SMA) is a severe autosomal recessive neuromuscular disease affecting children and young adults, caused by mutations of the survival motor neuron 1 gene (SMN1). SMA is characterized by the degeneration of spinal alpha motor neurons (αMNs), associated with muscle paralysis and atrophy, as well as other peripheral alterations. Both growth hormone-releasing hormone (GHRH) and its potent agonistic analog, MR-409, exert protective effects on muscle atrophy, cardiomyopathies, ischemic stroke, and inflammation. In this study, we aimed to assess the protective role of MR-409 in SMNΔ7 mice, a widely used model of SMA. Daily subcutaneous treatment with MR-409 (1 or 2 mg/kg), from postnatal day 2 (P2) to euthanization (P12), increased body weight and improved motor behavior in SMA mice, particularly at the highest dose tested. In addition, MR-409 reduced atrophy and ameliorated trophism in quadriceps and gastrocnemius muscles, as determined by an increase in fiber size, as well as upregulation of myogenic genes and inhibition of proteolytic pathways. MR-409 also promoted the maturation of neuromuscular junctions, by reducing multi-innervated endplates and increasing those mono-innervated. Finally, treatment with MR-409 delayed αMN death and blunted neuroinflammation in the spinal cord of SMA mice. In conclusion, the present study demonstrates that MR-409 has protective effects in SMNΔ7 mice, suggesting that GHRH agonists are promising agents for the treatment of SMA, possibly in combination with SMN-dependent strategies.

Astrocyte heterogeneity within white matter tracts and a unique subpopulation of optic nerve head astrocytes.

Much of what we know about astrocyte form and function is derived from the study of gray matter protoplasmic astrocytes, whereas white matter fibrous astrocytes remain relatively unexplored. Here, we used the ribotag approach to isolate ribosome-associated mRNA and investigated the transcriptome of uninjured fibrous astrocytes from three regions: unmyelinated optic nerve head, myelinated optic nerve proper, and corpus callosum. Astrocytes from each region were transcriptionally distinct and we identified region-specific astrocyte genes and pathways. Energy metabolism, particularly oxidative phosphorylation and mitochondrial protein translation emerged as key differentiators of astrocyte populations. Optic nerve astrocytes expressed higher levels of neuroinflammatory pathways than corpus callosum astrocytes and we further identified CARTPT as a new marker of optic nerve head astrocytes. These previously uncharacterized transcriptional profiles of white matter astrocyte types reveal their functional diversity and a greater heterogeneity than previously appreciated.

Microvasculopathy in spinal muscular atrophy is driven by a reversible autonomous endothelial cell defect.

Spinal muscular atrophy (SMA) is a neuromuscular disorder due to degeneration of spinal cord motor neurons caused by deficiency of the ubiquitously expressed SMN protein. Here, we present a retinal vascular defect in patients, recapitulated in SMA transgenic mice, driven by failure of angiogenesis and maturation of blood vessels. Importantly, the retinal vascular phenotype was rescued by early, systemic SMN restoration therapy in SMA mice. We also demonstrate in patients an unfavorable imbalance between endothelial injury and repair, as indicated by increased circulating endothelial cell counts and decreased endothelial progenitor cell counts in blood circulation. The cellular markers of endothelial injury were associated with disease severity and improved following SMN restoration treatment in cultured endothelial cells from patients. Finally, we demonstrated autonomous defects in angiogenesis and blood vessel formation, secondary to SMN deficiency in cultured human and mouse endothelial cells, as the underlying cellular mechanism of microvascular pathology. Our cellular and vascular biomarker findings indicate microvasculopathy as a fundamental feature of SMA. Our findings provide mechanistic insights into previously described SMA microvascular complications, and highlight the functional role of SMN in the periphery, including the vascular system, where deficiency of SMN can be addressed by systemic SMN-restoring treatment.

Generation of FOUR iPSC lines (CRICKi004-A; CRICKi005-A; CRICKi006-A, CRICKi007-A) from Spinal muscle atrophy patients with lower extremity dominant (SMALED) phenotype

Spinal muscular atrophy with lower extremity dominant (SMALED) is a hereditary neuromuscular disorder characterized by degeneration of spinal cord motor neurons resulting in lower limbs muscle weakness and paralysis. Mutations in DYNC1H1, which encodes BICD2, a multifunctional adaptor for microtubule motor proteins, cause the disorder. Here, we generated four induced pluripotent stem cell (iPSC) lines from patients with SMALED. Dermal fibroblasts were obtained from the MRC neuromuscular disease biobank and reprogrammed using non-integrating mRNA-based protocol. Characterization of the four iPSC lines included karyotyping and Sanger sequencing, while the expression of associated markers confirmed pluripotency and differentiation potential.

P.52 Determination of DLC1 isoform 1 (DLC1-i1) as a gene therapy for the treatment of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a motor neuron (MN) disease caused by loss of the ubiquitously expressed survival motor neuron (SMN) spliceosome protein, resulting in selective degeneration of spinal motor neurons (MNs) but the mechanism underlying the specific loss of MNs remains unknown. A previous report showed that Deleted in Liver Cancer 1 (DLC1) is the most down-regulated gene in MNs derived from a SMA patient but its roles in MN development and SMA pathogenesis remain to be elucidated. Here, we found a specific increase in the expression level of DLC1 isoform 1 (DLC1-i1) in human induced pluripotent stem cells (hiPSCs)-derived MNs compared to other isoforms. Knockdown of DLC1-i1 in MN progenitors resulted in reduced MN formation, axonal outgrowth, synapse number and cell survival, whereas overexpression of DLC1-i1 promoted MN differentiation with extensive neurite projections. Consistent with previous findings, we detected downregulated expression of DLC1-i1 in MNs derived from SMA patients' urine-derived iPSCs compared with healthy controls, likely due to intron retention of DLC1-i1 pre-mRNA. In addition, SMA patients-derived neuromuscular organoids (NMOs) showed reduced MNs and skeletal muscles generation, resulting in defective formation of neuromuscular junctions. Overexpression of DLC1-i1 partly restored both neural and muscle fibre formation in SMA NMOs. Similarly, DLC1-i1 expression was markedly reduced in the lumber spinal cord of SMA mice compared with that of wild-type, implying conservation of dysregulated DLC1-i1 expression in both the human SMA MNs and the spinal neurons of SMA mice. Injection of AAV9-SMN into the brain of SMA mice not only restored their weight and locomotor function but also DLC1-i1 expression level in the lumber spinal cord. Importantly, SMA mice treated with AAV9-DLC1-i1 together with AAV9-SMN resulted in synergistic improvement of their motor activity and lifespan compared with single treatment. Altogether, these findings demonstrate that MN-specific DLC1-i1 functions downstream of SMN to regulate MN maturation, survival, and neuromuscular junctions formation, revealing a new therapeutic target for the treatment of SMA.

VP.54 Amifampridine safety and efficacy in spinal muscular atrophy ambulatory patients: a randomized placebo-controlled, crossover, phase 2 trial

Spinal muscular atrophy (SMA) is an autosomal recessive disease where a deficient amount of SMN protein leads to progressive degeneration of bulbar and spinal motor neurons. Therapies for the restoration of SMN production are now available. Yet, fatigue and signs of impaired neuromuscular junction (NMJ) transmission have been documented as possible contributors to SMA phenotype. Amifampridine (3,4-diaminopyridine), a voltage-dependent K+ channel blocker, prolongs depolarization of the presynaptic NMJ terminal, enhancing neuromuscular transmission. Here, we evaluated the safety and efficacy of amifampridine in ambulatory patients with SMA Type 3. SMA-001 was a phase 2, 1:1 randomized, double-blind, placebo-controlled, 2-period, 2-treatment, crossover study. Genetically confirmed SMA Type 3, able to walk unaided for 30 m, not treated with nusinersen in the previous 6 months, entered the run-in phase for at least 21 days during which amifampridine was titrated up to 80 mg/day to reach an optimized stable dose. Then, patients with at least 3-points improvement in Hammersmith Functional Motor Score Extended (HFMSE) were randomized to receive either amifampridine or placebo for 2 weeks, alternatively, for a total of 28 days of double-blind treatment. Safety was evaluated from the first day of Run-In to the end of the study (primary safety outcome). Efficacy was evaluated by changes from randomization of HFMSE (primary efficacy outcome), quality of life, 6-minute walk test, and timed test (secondary outcomes). Descriptive analyses and a mixed effects linear model were used for statistic. Thirteen adult patients were included in the study, no serious adverse events (SAEs) attributed to Amifampridine were reported. Transient paresthesias (33,3%) were the only amifampridine-related AEs reported. Six patients for each sequence of treatment were randomized. Amifampridine treatment led to a statistically significant improvement in HFMSE (LS Mean Difference 0.792; 95% CI from 0.22 to 1.37; p=0.0083), compared to placebo, but not in the secondary endpoints. SMA-001 study provided Class I evidence that AP was safe and effective in treating ambulatory patients affected by SMA type 3.

R-loop Mediated DNA Damage and Impaired DNA Repair in Spinal Muscular Atrophy.

Defects in DNA repair pathways are a major cause of DNA damage accumulation leading to genomic instability and neurodegeneration. Efficient DNA damage repair is critical to maintain genomicstability and support cell function and viability. DNA damage results in the activation of cell death pathways, causing neuronal death in an expanding spectrum of neurological disorders, such as amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), Alzheimer’s disease (AD), and spinal muscular atrophy (SMA). SMA is a neurodegenerative disorder caused by mutations in the Survival Motor Neuron 1 (SMN1) gene. SMA is characterized by the degeneration of spinal cord motor neurons due to low levels of the SMN protein. The molecular mechanism of selective motor neuron degeneration in SMA was unclear for about 20 years. However, several studies have identified biochemical and molecular mechanisms that may contribute to the predominant degeneration of motor neurons in SMA, including the RhoA/ROCK, the c-Jun NH2-terminal kinase (JNK), and p53-mediated pathways, which are involved in mediating DNA damage-dependent cell death. Recent studies provided insight into selective degeneration of motor neurons, which might be caused by accumulation of R-loop-mediated DNA damage and impaired non-homologous end joining (NHEJ) DNA repair pathway leading to genomic instability. Here, we review the latest findings involving R-loop-mediated DNA damage and defects in neuron-specific DNA repair mechanisms in SMA and discuss these findings in the context of other neurodegenerative disorders linked to DNA damage.

Amyotrophic lateral sclerosis mimics.

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disorder characterized by progressive degeneration of cortical, bulbar, and spinal motor neurons. When a patient presents with a progressive upper and/or lower motor syndrome, clinicians must pay particular attention to any atypical features in the history and/or clinical examination suggesting an alternate diagnosis, as up to 10% percent of patients initially diagnosed with ALS have a mimic of ALS. ALS is a clinical diagnosis and requires the exclusion of other disorders that may have similar presentations but a more favorable prognosis or an effective therapy. Because there is currently no specific diagnostic biomarker that is sensitive or specific for ALS, understanding the spectrum of clinical presentations of ALS and its mimics is paramount. While true mimics of ALS are rare, the clinician must correctly identify these disorders to avoid the misdiagnosis of ALS and to initiate effective treatment where available.

The mechanism of the WNT5A and FZD4 receptor mediated WNT/β–catenin pathway in the degeneration of ALS spinal cord motor neurons

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with unknown etiology, characterized by motor neuron degeneration, and there is no highly effective treatment. The canonical WNT/β-catenin signaling pathway has a critical role in the physiological and pathophysiological processes of the central nervous system. In this study, we investigated the regulatory mechanism of the WNT/β-catenin signaling pathway from the perspective of ligand-receptor binding and its relationship with the degeneration of ALS motor neurons. We used hSOD1-G93A mutant ALS transgenic mice and hSOD1-G93A mutant NSC34 cells combined with morphological and molecular biology techniques to determine the role of the WNT/β–catenin pathway in ALS. Our findings demonstrated that WNT5A regulates the WNT/β-catenin signaling pathway by binding to the FZD4 receptor in the pathogenesis of ALS and affects the proliferation and apoptosis of ALS motor neurons. Therefore, these findings may lead to the development of novel therapies to support the survival of ALS motor neurons.

Cortical Hyperexcitability in the Driver’s Seat in ALS

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by the degeneration of cortical and spinal motor neurons. With no effective treatment available to date, patients face progressive paralysis and eventually succumb to the disease due to respiratory failure within only a few years. Recent research has revealed the multifaceted nature of the mechanisms and cell types involved in motor neuron degeneration, thereby opening up new therapeutic avenues. Intriguingly, two key features present in both ALS patients and rodent models of the disease are cortical hyperexcitability and hyperconnectivity, the mechanisms of which are still not fully understood. We here recapitulate current findings arguing for cell autonomous and non-cell autonomous mechanisms causing cortical excitation and inhibition imbalance, which is involved in the degeneration of motor neurons in ALS. Moreover, we will highlight recent evidence that strongly indicates a cardinal role for the motor cortex as a main driver and source of the disease, thus arguing for a corticofugal trajectory of the pathology.

Upper motor neurons are a target for gene therapy and UCHL1 is necessary and sufficient to improve cellular integrity of diseased upper motor neurons.

There are no effective cures for upper motor neuron (UMN) diseases, such as amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, and hereditary spastic paraplegia. Here, we show UMN loss occurs independent of spinal motor neuron degeneration and that UMNs are indeed effective cellular targets for gene therapy, which offers a potential solution especially for UMN disease patients.UCHL1 (ubiquitin C-terminal hydrolase-L1) is a deubiquitinating enzyme crucial for maintaining free ubiquitin levels. Corticospinal motor neurons (CSMN, a.k.a UMNs in mice) show early, selective, and profound degeneration in Uchl1nm3419 (UCHL1−/−) mice, which lack all UCHL1 function. When UCHL1 activity is ablated only from spinal motor neurons, CSMN remained intact. However, restoring UCHL1 specifically in CSMN of UCHL1−/− mice via directed gene delivery, was sufficient to improve CSMN integrity to the healthy control levels. In addition, when UCHL1 gene was delivered selectively to CSMN that are diseased due to misfolded SOD1 toxicity and TDP-43 pathology via AAV-mediated retrograde transduction, the disease causing misfolded SOD1 and mutant human TDP-43 were reduced in hSOD1G93A and prpTDP-43A315T models, respectively. Diseased CSMN retained their neuronal integrity and cytoarchitectural stability in two different mouse models that represent two distinct causes of neurodegeneration in ALS.

Novel mutation of EXOSC3 presenting as hereditary spastic paraplegia plus syndrome

RNA processing protein 40 (RRP40) is an exosome complex involved in processing RNA, of which EXOSC3 forms a non-catalytic subunit. Homozygous mutation in EXOSC3 causes Pontocerebellar hypoplasia-1 (PCH-1), characterised by postnatal psychomotor retardation with spinal motor neuron degeneration. Imaging in PCH-1 shows severe cerebellar and variable pontine atrophy. Here we report an adolescent male with EXOSC3 mutation who presented with spastic paraparesis and nystagmus since childhood with severe vermian atrophy and normal pons suggestive phenotypically Hereditary spastic paraplegia plus syndrome (HSP-Plus).

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.