Curing SMA: Are we there yet?

Loss or deletion of survival motor neuron 1 gene (SMN1) is causative for a severe and devastating neuromuscular disease, Spinal Muscular Atrophy (SMA). SMN1 produces SMN, a ubiquitously expressed protein, that is essential for the development and survival of motor neurons. Major advances and developments in SMA therapeutics are shifting the natural history of the disease. With three relatively new available therapies, nusinersen (Spinraza), onasemnogene abeparvovec (Zolgensma), and risdiplam (Evrysdi), patients survive longer and have improved outcomes. However, patients and families continue to face many challenges associated with use of these therapies, including poor treatment response and a variability in the benefits to those that do respond, suggesting that the quest for the SMA cure is not over. In this review, we discuss the current therapies, their limitations, and highlight necessary gaps that need to be addressed to guarantee the best outcomes for SMA patients.

Survival motor neuron protein deficiency alters microglia reactivity.

Survival motor neuron (SMN) protein deficiency results in loss of alpha motor neurons and subsequent muscle atrophy in patients with spinal muscular atrophy (SMA). Reactive microglia have been reported in SMA mice and depleting microglia rescues the number of proprioceptive synapses, suggesting a role in SMA pathology. Here, we explore the contribution of lymphocytes on microglia reactivity in SMA mice and investigate how SMN deficiency alters the reactive profile of human induced pluripotent stem cell (iPSC)-derived microglia. We show that microglia adopt a reactive morphology in spinal cords of SMA mice. Ablating lymphocytes did not alter the reactive morphology of SMA microglia and did not improve the survival or motor function of SMA mice, indicating limited impact of peripheral immune cells on the SMA phenotype. We found iPSC-derived SMA microglia adopted an amoeboid morphology and displayed a reactive transcriptome profile, increased cell migration, and enhanced phagocytic activity. Importantly, cell morphology and electrophysiological properties of motor neurons were altered when they were incubated with conditioned media from SMA microglia. Together, these data reveal that SMN-deficient microglia adopt a reactive profile and exhibit an exaggerated inflammatory response with potential impact on SMA neuropathology.

Metabolic Dysfunction in Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder leading to paralysis, muscle atrophy, and death. Significant advances in antisense oligonucleotide treatment and gene therapy have made it possible for SMA patients to benefit from improvements in many aspects of the once devastating natural history of the disease. How the depletion of survival motor neuron (SMN) protein, the product of the gene implicated in the disease, leads to the consequent pathogenic changes remains unresolved. Over the past few years, evidence toward a potential contribution of gastrointestinal, metabolic, and endocrine defects to disease phenotype has surfaced. These findings ranged from disrupted body composition, gastrointestinal tract, fatty acid, glucose, amino acid, and hormonal regulation. Together, these changes could have a meaningful clinical impact on disease traits. However, it is currently unclear whether these findings are secondary to widespread denervation or unique to the SMA phenotype. This review provides an in-depth account of metabolism-related research available to date, with a discussion of unique features compared to other motor neuron and related disorders.

Performance report for a 10-year-old MD/PhD Program: A survey of trainees at the University of Ottawa.

PURPOSE: Integrated MD/PhD programs are relatively new in Canada and represent a platform to train the next generation of clinician-scientists. However, MD/PhD programs vary substantially by structure, funding and mentorship opportunities, and there exists a paucity of data on the overall students' successes and challenges. The purpose of this study is to assess objective and subjective metrics of the MD/PhD Program at the University of Ottawa. METHODS: Students in all years of the program were invited to complete a 58- question survey, and the resulting data were analyzed by descriptive statistics. RESULTS: Our survey had an 88.5% (23/26) participation rate. The program has been gaining interest and the number of applications increased by 178% between 2013 and 2018. Tuition support was considered an essential element in accepting the admission offer, as 47.8% of students would have declined admission without full tuition coverage. The MD/PhD students were heavily engaged in scholarly activities, with an average of 8.3 presentations/ publications per respondent. Respondents indicated low satisfaction with formal career planning advice (28.6% satisfied/very satisfied) and program transition guidance (22.2%). When delivered informally by peers, both career planning advice and program transition guidance were experienced as more satisfying (65.2% and 63.6%, respectively). Only 34.8% of survey respondents identified as female, highlighting the challenge of achieving diversity in clinician-scientist training programs. CONCLUSION: Our report contributes to the body of knowledge on concrete obstacles experienced by students within MD/PhD programs and key areas that can be improved upon-locally, provincially and nationally-to further advance student success.

Acute exposure to environmentally relevant levels of DDT alters muscle mitochondrial function in vivo in rats but not in vitro in L6 myotubes: A pilot study.

Under insulin-stimulated conditions, skeletal muscle is the largest glucose consumer in the body. Mitochondrial dysfunction and damage to this tissue from oxidative stress are linked to the pathogenesis of type 2 diabetes. Environmental exposure to dichlorodiphenyltrichloroethane (DDT) and its metabolite, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), has been associated with the incidence of type 2 diabetes as well as altered oxidative stress and mitochondrial dysfunction in non-muscle tissues. We hypothesized that energy metabolism and insulin sensitivity in skeletal muscle will be altered with exposure to DDT and DDE. In this pilot study, mitochondrial function was measured in permeabilized muscle fibers from Sprague-Dawley rats after one week of exposure to a single injection of DDT (40 µg/kg), a dose comparable to DDT levels in the diets of the Inuit of Northern Canada. The levels of oxidative phosphorylation chain complexes and ROS detoxification enzymes were measured in muscle tissue from these specimens. This acute in vivo exposure to DDT decreased muscle mitochondrial function by 45% without affecting the levels of mitochondrial oxidative phosphorylation chain complexes nor levels of ROS detoxification enzymes. To isolate the effects of DDT and DDE exposure on muscle, L6 myotubes were exposed to DDT or DDE (0, 10, 100, 1000, 10 000 nM) for 24 h. Only very high concentrations of DDT and DDE (1 000 - 10 000 nM) altered maximal respiration with only DDT altering basal glucose uptake in L6 myotubes. This did not alter levels of ROS detoxification enzymes or malondialdehyde (MDA) in L6 myotubes. Altogether, acute exposure to environmentally relevant doses of DDT resulted in muscle mitochondrial dysfunction in vivo in rats, but not when muscle cells were directly exposed to the pollutant or its metabolite.

Suppression of the necroptotic cell death pathways improves survival in Smn 2B/- mice.

Spinal muscular atrophy (SMA) is a monogenic neuromuscular disease caused by low levels of the Survival Motor Neuron (SMN) protein. Motor neuron degeneration is the central hallmark of the disease. However, the SMN protein is ubiquitously expressed and depletion of the protein in peripheral tissues results in intrinsic disease manifestations, including muscle defects, independent of neurodegeneration. The approved SMN-restoring therapies have led to remarkable clinical improvements in SMA patients. Yet, the presence of a significant number of non-responders stresses the need for complementary therapeutic strategies targeting processes which do not rely solely on restoring SMN. Dysregulated cell death pathways are candidates for SMN-independent pathomechanisms in SMA. Receptor-interacting protein kinase 1 (RIPK1) and RIPK3 have been widely recognized as critical therapeutic targets of necroptosis, an important form of programmed cell death. In addition, Caspase-1 plays a fundamental role in inflammation and cell death. In this study, we evaluate the role of necroptosis, particularly RIPK3 and Caspase-1, in the Smn 2B/- mouse model of SMA. We have generated a triple mutant (TKO), the Smn 2B/-; Ripk3 -/-; Casp1 -/- mouse. TKO mice displayed a robust increase in survival and improved motor function compared to Smn 2B/- mice. While there was no protection against motor neuron loss or neuromuscular junction pathology, larger muscle fibers were observed in TKO mice compared to Smn 2B/- mice. Our study shows that necroptosis modulates survival, motor behavior and muscle fiber size independent of SMN levels and independent of neurodegeneration. Thus, small-molecule inhibitors of necroptosis as a combinatorial approach together with SMN-restoring drugs could be a future strategy for the treatment of SMA.

SMN Depleted Mice Offer a Robust and Rapid Onset Model of Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is considered a health epidemic with potential devastating effects on the patients and the healthcare systems. Current preclinical models of NAFLD are invariably imperfect and generally take a long time to develop. A mouse model of survival motor neuron (SMN) depletion (Smn2B/- mice) was recently shown to develop significant hepatic steatosis in less than 2 weeks from birth. The rapid onset of fatty liver in Smn2B/- mice provides an opportunity to identify molecular markers of NAFLD. Here, we investigated whether Smn2B/- mice display typical features of NAFLD/nonalcoholic steatohepatitis (NASH). METHODS: Biochemical, histologic, electron microscopy, proteomic, and high-resolution respirometry were used. RESULTS: The Smn2B/- mice develop microvesicular steatohepatitis within 2 weeks, a feature prevented by AAV9-SMN gene therapy. Although fibrosis is not overtly apparent in histologic sections of the liver, there is molecular evidence of fibrogenesis and presence of stellate cell activation. The consequent liver damage arises from mitochondrial reactive oxygen species production and results in hepatic dysfunction in protein output, complement, coagulation, iron homeostasis, and insulin-like growth factor-1 metabolism. The NAFLD phenotype is likely due to non-esterified fatty acid overload from peripheral lipolysis subsequent to hyperglucagonemia compounded by reduced muscle use and insulin resistance. Despite the low hepatic mitochondrial content, isolated mitochondria show enhanced ß-oxidation, likely as a compensatory response, resulting in the production of reactive oxygen species. In contrast to typical NAFLD/NASH, the Smn2B/- mice lose weight because of their associated neurological condition (spinal muscular atrophy) and develop hypoglycemia. CONCLUSIONS: The Smn2B/- mice represent a good model of microvesicular steatohepatitis. Like other models, it is not representative of the complete NAFLD/NASH spectrum. Nevertheless, it offers a reliable, low-cost, early-onset model that is not dependent on diet to identify molecular players in NAFLD pathogenesis and can serve as one of the very few models of microvesicular steatohepatitis for both adult and pediatric populations.

The effects of acute BPA exposure on skeletal muscle mitochondrial function and glucose metabolism.

Bisphenol A (BPA) is an environmental pollutant that has been associated with adverse health effects including skeletal muscle insulin resistance, a major contributor to the pathogenesis of type 2 diabetes (T2D). Early mitochondrial dysfunction and oxidative stress are linked to impaired glucose metabolism in skeletal muscle. In this study, we investigated the effects of BPA on skeletal muscle mitochondrial function and insulin sensitivity. L6 myotubes were treated with BPA (1 nM-105 nM) during the last 24 h of differentiation. Following exposure to 105 nM of BPA, resting and maximal oxygen consumption rates were decreased, whereas mitochondrial proton leak was increased. Overall metabolic activity, measured by redox ability, was decreased in L6 myotubes exposed to 105 nM of BPA. At this concentration, insulin-stimulated glucose uptake was increased, which corresponded to an increased phosphorylation of the insulin signaling protein Akt, and increased glycolysis measured by extracellular acidification rate (ECAR). Acute BPA exposure did not alter levels of oxidative stress markers in muscle cells, but significantly increased mitochondrial proton leak, which is known to be involved in decreased ROS production. The effects of BPA on glucose uptake, but not mitochondrial function, were reversed by the use of an estrogen receptor antagonist. These results suggest that acute exposure of L6 myotubes at only high concentrations of BPA alters glucose metabolism, which is likely a compensatory response to reduced mitochondrial energy production capacity.

Motor transmission defects with sex differences in a new mouse model of mild spinal muscular atrophy.

BACKGROUND: Mouse models of mild spinal muscular atrophy (SMA) have been extremely challenging to generate. This paucity of model systems has limited our understanding of pathophysiological events in milder forms of the disease and of the effect of SMN depletion during aging. METHODS: A mild mouse model of SMA, termed Smn2B/-;SMN2+/-, was generated by crossing Smn-/-;SMN2 and Smn2B/2B mice. This new model was characterized using behavioral testing, histology, western blot, muscle-nerve electrophysiology as well as ultrasonography to study classical SMA features and extra-neuronal involvement. FINDINGS: Smn2B/-;SMN2+/- mice have normal survival, mild but sustained motor weakness, denervation and neuronal/neuromuscular junction (NMJ) transmission defects, and neurogenic muscle atrophy that are more prominent in male mice. Increased centrally located nuclei, intrinsic contractile and relaxation muscle defects were also identified in both female and male mice, with some male predominance. There was an absence of extra-neuronal pathology. INTERPRETATION: The Smn2B/-;SMN2+/- mouse provides a model of mild SMA, displaying some hallmark features including reduced weight, sustained motor weakness, electrophysiological transmission deficit, NMJ defects, and muscle atrophy. Early and prominent increase central nucleation and intrinsic electrophysiological deficits demonstrate the potential role played by muscle in SMA disease. The use of this model will allow for the understanding of the most susceptible pathogenic molecular changes in motor neurons and muscles, investigation of the effects of SMN depletion in aging, sex differences and most importantly will provide guidance for the currently aging SMA patients treated with the recently approved genetic therapies. FUNDING: This work was supported by Cure SMA/Families of SMA Canada (grant numbers KOT-1819 and KOT-2021); Muscular Dystrophy Association (USA) (grant number 575466); and Canadian Institutes of Health Research (CIHR) (grant number PJT-156379).

Low fat diets increase survival of a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disorder leading to paralysis and death. Recent evidence shows increased susceptibility to dyslipidemia and liver steatosis in patients. Here, we provide evidence that low fat diets nearly double survival in Smn2B/- mice, a model for SMA, independent of changes in SMN levels, liver steatosis, or enhanced hepatic functions. Liver damage and ketone levels were reduced, implying a lower reliance on fatty acid oxidation. This preclinical proof of concept study provides grounds for controlled clinical investigation of dietary needs and offers evidence to inform nutritional guidelines specific to SMA.

ERK8 is a novel HuR kinase that regulates tumour suppressor PDCD4 through a miR-21 dependent mechanism.

Programmed cell death 4 (PDCD4) is a tumour suppressor implicated in cancer development and progression and was recently identified as a repressor of cap-independent translation of specific genes involved in the regulation of apoptosis. We show that the RNA-binding protein HuR binds to the PDCD4 3'UTR to protect it from miR-21-induced silencing. However, following H2O2 treatment, PDCD4 mRNA is degraded via miR-21 binding. Importantly, we identify HuR as a novel substrate of the ERK8 kinase pathway in response to H2O2 treatment. We show that phosphorylation of HuR by ERK8 prevents it from binding to PDCD4 mRNA and allows miR-21-mediated degradation of PDCD4.