Publications by authors named "Rashmi Kothary"

120 Publications

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

Cell Mol Gastroenterol Hepatol 2021 Feb 2. Epub 2021 Feb 2.

Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada; Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada; Centre for Neuromuscular Disease, University of Ottawa, Ottawa, Ontario, Canada; Department of Medicine, University of Ottawa, Ottawa, Ontario, Canada. Electronic address:

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 (Smn mice) was recently shown to develop significant hepatic steatosis in less than 2 weeks from birth. The rapid onset of fatty liver in Smn mice provides an opportunity to identify molecular markers of NAFLD. Here, we investigated whether Smn mice display typical features of NAFLD/nonalcoholic steatohepatitis (NASH).

Methods: Biochemical, histologic, electron microscopy, proteomic, and high-resolution respirometry were used.

Results: The Smn 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 nonesterified 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 β-oxidation, likely as a compensatory response, resulting in the production of reactive oxygen species. In contrast to typical NAFLD/NASH, the Smn mice lose weight because of their associated neurological condition (spinal muscular atrophy) and develop hypoglycemia.

Conclusions: The Smn 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jcmgh.2021.01.019DOI Listing
February 2021

Report on the 5th Ottawa International Conference on Neuromuscular Disease & Biology -October 17-19, 2019, Ottawa, Canada.

J Neuromuscul Dis 2021 Jan 20. Epub 2021 Jan 20.

Department of Medicine, The Ottawa Hospital and University of Ottawa, Canada.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3233/JND-219001DOI Listing
January 2021

Dystonin loss-of-function leads to impaired autophagy-endolysosomal pathway dynamics.

Biochem Cell Biol 2020 Dec 21. Epub 2020 Dec 21.

Ottawa Hospital Research Institute, 10055, Ottawa, Canada.

The neuronal dystonin protein (DST-a) is a large cytoskeletal linker important for integrating the various components of the cytoskeleton. Recessive Dst mutations lead to a sensory neuropathy in mice known as dystonia musculorum (Dstdt). The disease is characterized by ataxia, autonomic disturbances, and ultimately death, which are associated with massive dorsal root ganglion (DRG) sensory neuron degeneration. Recent investigation of Dstdt sensory neurons revealed an accumulation in autophagosomes and a disruption in autophagic flux, which was believed to be due to insufficient motor protein availability. Motor protein levels and the endolysosomal pathway were assessed in pre-symptomatic (postnatal day 5; P5) and symptomatic (P15) stage wild type and Dstdt DRGs. Levels of mRNA encoding molecular motors are reduced, although no significant reduction protein level is detected. An increase in lysosomal marker LAMP1 in medium-large size Dstdt-27J sensory neurons is observed, along with an accumulation of electron-light single-membraned vesicles in Dstdt-27J DRG tissue at late stages of disease. These vesicles are likely to be autolysosomes, and their presence in only late stage Dstdt-27J sensory neurons is suggestive of a pathological defect in autophagy. Further investigation is necessary to confirm vesicle identity, and to determine the role of Dst-a in normal autophagic flux.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1139/bcb-2020-0557DOI Listing
December 2020

Spinal muscular atrophy type III complicated by spinal superficial siderosis: a case report with molecular and neuropathological findings.

Acta Neuropathol Commun 2020 11 9;8(1):188. Epub 2020 Nov 9.

Department of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Canada.

Spinal muscular atrophy (SMA) is largely linked to deletion or mutation of the Survival motor neuron 1 (SMN1) gene located on chromosome 5q13. Type III (Kugelberg-Welander disease) is the mildest childhood form and patients may become ambulatory and have a normal life expectancy. We report the clinical history and morphological findings of a 55-year-old woman who began to experience motor problems at the age of two. She was never fully ambulatory, and her severe scoliosis required the insertion of surgical rod at age 19. Unexpectedly, around 35 years of age, she began to experience sensory symptoms best characterized as a myelo-radiculo-neuropathy with pain as the dominant symptom. Investigations never clarified the etiology of these symptoms. Molecular confirmation of SMA type III was done post-mortem. Neuropathological examination showed classic changes of lower motor neuron neurodegeneration, in line with those reported in the single molecularly confirmed case published so far, and with findings in rare cases reported prior to the discovery of the gene defect. A key autopsy finding was the presence of a severe superficial siderosis of the lower half of the spinal cord. In recent years, the concept of duropathy was put forward, associating superficial siderosis of the spinal cord with various spinal abnormalities, some of which were present in our patient. The presence of significant hemosiderin deposits in the spinal cord and sensory nerve roots with associated tissue and axonal damage provide a plausible explanation for the unexpected sensory symptomatology in this mild lower motor neurodegeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40478-020-01063-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7653749PMC
November 2020

Metformin promotes CNS remyelination and improves social interaction following focal demyelination through CBP Ser436 phosphorylation.

Exp Neurol 2020 12 30;334:113454. Epub 2020 Aug 30.

Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada; University of Ottawa Brain and Mind Research Institute, Ottawa, ON K1H 8M5, Canada; Canadian Partnership for Stroke Recovery, Ottawa, ON K1G 5Z3, Canada. Electronic address:

Individuals with demyelinating diseases often experience difficulties during social interactions that are not well studied in preclinical models. Here, we describe a novel juvenile focal corpus callosum demyelination murine model exhibiting a social interaction deficit. Using this preclinical murine demyelination model, we discover that application of metformin, an FDA-approved drug, in this model promotes oligodendrocyte regeneration and remyelination and improves the social interaction. This beneficial effect of metformin acts through stimulating Ser436 phosphorylation in CBP, a histone acetyltransferase. In addition, we found that metformin acts through two distinct molecular pathways to enhance oligodendrocyte precursor (OPC) proliferation and differentiation, respectively. Metformin enhances OPC proliferation through early-stage autophagy inhibition, while metformin promotes OPC differentiation into mature oligodendrocytes through activating CBP Ser436 phosphorylation. In summary, we identify that metformin is a promising remyelinating agent to improve juvenile demyelination-associated social interaction deficits by promoting oligodendrocyte regeneration and remyelination.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.expneurol.2020.113454DOI Listing
December 2020

XIAP Protects Retinal Ganglion Cells in the Mutant ND4 Mouse Model of Leber Hereditary Optic Neuropathy.

Invest Ophthalmol Vis Sci 2020 07;61(8):49

,.

Purpose: Leber hereditary optic neuropathy (LHON) is a genetic form of vision loss that occurs primarily owing to mutations in the nicotinamide adenine dinucleotide dehydrogenase (ND) subunits that make up complex I of the electron transport chain. LHON mutations result in the apoptotic death of retinal ganglion cells. We tested the hypothesis that gene therapy with the X-linked inhibitor of apoptosis (XIAP) would prevent retinal ganglion cell apoptosis and reduce disease progression in a vector-induced mouse model of LHON that carries the ND4 mutation.

Methods: Adeno-associated virus (AAV) encoding full length hemagglutinin-tagged XIAP (AAV2.HA-XIAP) or green fluorescent protein (AAV2.GFP) was injected into the vitreous of DBA/1J mice. Two weeks later, the LHON phenotype was induced by AAV delivery of mutant ND4 (AAV2.mND4FLAG) to the vitreous. Retinal function was assessed by pattern electroretinography. Optic nerves were harvested at 4 months, and the effects of XIAP therapy on nerve fiber layer and optic nerve integrity were evaluated using immunohistochemistry, transmission electron microscopy and magnetic resonance imaging.

Results: During LHON disease progression, retinal ganglion cell axons are lost. Apoptotic cell bodies are seen in the nuclei of astrocytes or oligodendrocytes in the optic nerve, and there is thinning of the optic nerve and the nerve fiber layer of the retina. At 4 months after disease onset, XIAP gene therapy protects the nerve fiber layer and optic nerve architecture by preserving axon health. XIAP also decreases nuclear fragmentation in resident astrocytes or oligodendrocytes and decreases glial cell infiltration.

Conclusions: XIAP therapy improves optic nerve health and delays disease progression in LHON.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/iovs.61.8.49DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7425697PMC
July 2020

MiRNA-145-5p prevents differentiation of oligodendrocyte progenitor cells by regulating expression of myelin gene regulatory factor.

J Cell Physiol 2021 Feb 30;236(2):997-1012. Epub 2020 Jun 30.

Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Canada.

The roles of specific microRNAs (miRNA) in oligodendrocyte (OL) differentiation have been studied in depth. However, miRNAs in OL precursors and oligodendrocyte progenitor cells (OPCs) have been less extensively investigated. MiR-145-5p is highly expressed in OPCs relative to differentiating OLs, suggesting this miRNA may serve a function specifically in OPCs. Knockdown of miR-145-5p in primary OPCs led to spontaneous differentiation, as evidenced by an increased proportion of MAG cells, increased cell ramification, and upregulation of multiple myelin genes including MYRF, TPPP, and MAG, and OL cell cycle exit marker Cdkn1c. Supporting this transition to a differentiating state, proliferation was reduced in miR-145-5p knockdown OPCs. Further, knockdown of miR-145-5p in differentiating OLs showed enhanced differentiation, with increased branching, myelin membrane production, and myelin gene expression. We identified several OL-specific genes targeted by miR-145-5p that exhibited upregulation with miR-145-5p knockdown, including myelin gene regulatory factor (MYRF), that could be regulating the prodifferentiation phenotype in both miR-145 knockdown OPCs and OLs. Indeed, spontaneous differentiation with knockdown of miR-145-5p was fully rescued by concurrent knockdown of MYRF. However, proliferation rate was only partially rescued with MYRF knockdown, and overexpression of miR-145-5p in OPCs increased proliferation rate without affecting expression of already lowly expressed differentiation genes. Taken together, these data suggest that in OPCs miR-145-5p both prevents differentiation at least in part by preventing expression of MYRF and promotes proliferation via as-yet-unidentified mechanisms. These findings clarify the need for differential regulation of miR-145-5p between OPCs and OLs and may have further implications in demyelinating diseases such as multiple sclerosis where miR-145-5p is dysregulated.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jcp.29910DOI Listing
February 2021

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

EBioMedicine 2020 May 24;55:102750. Epub 2020 Apr 24.

Regenerative Medicine Program, Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario K1H 8L6, Canada; Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada; Centre for Neuromuscular Disease, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada; Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada; Department of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada. Electronic address:

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 Smn;SMN2, was generated by crossing Smn;SMN2 and Smn 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: Smn;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 Smn;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).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ebiom.2020.102750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184161PMC
May 2020

Blood Flow to the Spleen is Altered in a Mouse Model of Spinal Muscular Atrophy.

J Neuromuscul Dis 2020 ;7(3):315-322

Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.

Spinal muscular atrophy (SMA) is a neuromuscular disorder affecting young children. While pre-clinical models of SMA show small spleens, the same is not true in humans. Here, we show by doppler ultrasonography decreased splenic blood flow in Smn2B/- mice. Further, AAV9-SMN gene therapy does not rescue the distal ear and tail necrosis nor the spleen size in these mice, suggesting that the latter may be linked to a cardiovascular defect. Absence of smaller spleens in human patients is likely due to differences in presentation of defects in SMA between pre-clinical mouse models and human patients, particularly the susceptibility to cardiovascular issues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3233/JND-200493DOI Listing
January 2020

HSAN-VI: A spectrum disorder based on dystonin isoform expression.

Neurol Genet 2020 Feb 2;6(1):e389. Epub 2020 Jan 2.

Regenerative Medicine Program (A.L.-G., R.K.), Ottawa Hospital Research Institute; Department of Cellular and Molecular Medicine (A.L.-G., R.K.) and Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa; Department of Medicine (R.K.), University of Ottawa; and Centre for Neuromuscular Disease (R.K.), University of Ottawa, Canada.

Hereditary sensory and autonomic neuropathy (HSAN-VI) is a recessive genetic disorder that arises because of mutations in the human dystonin gene (, previously known as ). Although initial characterization of HSAN-VI reported it as a sensory neuropathy that was lethal in infancy, we now know of a number of heterozygous mutations in that result in milder forms of the disease. Akin to what we observe in the mouse model ( ), we believe that the heterogeneity of HSAN-VI can be attributed to a number of dystonin isoforms that the mutation affects. Lack of neuronal isoform dystonin-a2 is likely the universal determinant of HSAN-VI because all reported human cases are null for this isoform, as are all mouse alleles. Compensatory mechanisms by intact dystonin-a isoforms also likely play a role in regulating disease severity, although we have yet to determine what specific effect dystonin-a1 and dystonin-a3 have on the pathogenesis of HSAN-VI.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1212/NXG.0000000000000389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6975176PMC
February 2020

Characterization of gastrointestinal pathologies in the dystonia musculorum mouse model for hereditary sensory and autonomic neuropathy type VI.

Neurogastroenterol Motil 2020 04 9;32(4):e13773. Epub 2019 Dec 9.

Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada.

Background: Dystonia musculorum (Dst ) is a murine disease caused by recessive mutations in the dystonin (Dst) gene. Loss of dorsal root ganglion (DRG) sensory neurons, ataxia, and dystonic postures before death by postnatal day 18 (P18) is a hallmark feature. Recently we observed gas accumulation and discoloration in the small intestine and cecum in Dst mice by P15. The human disease resulting from dystonin loss-of-function, known as hereditary sensory and autonomic neuropathy type VI (HSAN-VI), has also been associated with gastrointestinal (GI) symptoms including chronic diarrhea and abdominal pain. As neuronal dystonin isoforms are expressed in the GI tract, we hypothesized that dystonin loss-of-function in Dst enteric nervous system (ENS) neurons resulted in neurodegeneration associated with the GI abnormalities.

Methods: We characterized the nature of the GI abnormalities observed in Dst mice through histological analysis of the gut, assessing the ENS for signs of neurodegeneration, evaluation of GI motility and absorption, and by profiling the microbiome.

Key Results: Though gut histology, ENS viability, and GI absorption were normal, slowed GI motility, thinning of the colon mucous layer, and reduced microbial richness/evenness were apparent in Dst mice by P15. Parasympathetic GI input showed signs of neurodegeneration, while sympathetic did not.

Conclusions & Inferences: Dst GI defects are not linked to ENS neurodegeneration, but are likely a result of an imbalance in autonomic control over the gut. Further characterization of HSAN-VI patient GI symptoms is necessary to determine potential treatments targeting symptom relief.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/nmo.13773DOI Listing
April 2020

Snf2h Drives Chromatin Remodeling to Prime Upper Layer Cortical Neuron Development.

Front Mol Neurosci 2019 17;12:243. Epub 2019 Oct 17.

Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada.

Alterations in the homeostasis of either cortical progenitor pool, namely the apically located radial glial (RG) cells or the basal intermediate progenitors (IPCs) can severely impair cortical neuron production. Such changes are reflected by microcephaly and are often associated with cognitive defects. Genes encoding epigenetic regulators are a frequent cause of intellectual disability and many have been shown to regulate progenitor cell growth, including our inactivation of the gene encoding Snf2l, which is one of two mammalian orthologs. Loss of the Snf2l protein resulted in dysregulation of Foxg1 and IPC proliferation leading to macrocephaly. Here we show that inactivation of the closely related gene encoding the Snf2h chromatin remodeler is necessary for embryonic IPC expansion and subsequent specification of callosal projection neurons. Telencephalon-specific cKO embryos have impaired cell cycle kinetics and increased cell death, resulting in fewer Tbr2+ and FoxG1+ IPCs by mid-neurogenesis. These deficits give rise to adult mice with a dramatic reduction in Satb2+ upper layer neurons, and partial agenesis of the corpus callosum. Mice survive into adulthood but molecularly display reduced expression of the clustered protocadherin genes that may further contribute to altered dendritic arborization and a hyperactive behavioral phenotype. Our studies provide novel insight into the developmental function of Snf2h-dependent chromatin remodeling processes during brain development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fnmol.2019.00243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6811508PMC
October 2019

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

Ann Clin Transl Neurol 2019 11 13;6(11):2340-2346. Epub 2019 Oct 13.

Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada, K1H 8L6.

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 Smn 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/acn3.50920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856606PMC
November 2019

Impaired kidney structure and function in spinal muscular atrophy.

Neurol Genet 2019 Oct 12;5(5):e353. Epub 2019 Aug 12.

Department of Neurology (F.C.N., J.J.S., A.W.M., P.N., A.J.J., R.Z., M.F., N.H., C.R.R.A., K.J.S.), Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA; Department of Pathology (I.R.), Massachusetts General Hospital, Boston, MA; Regenerative Medicine Program (M.-O.D., R.K.), Ottawa Hospital Research, Institute Ottawa, Ontario, Canada; Department of Cellular and Molecular Medicine (M.-O.D., R.K.), University of Ottawa, Canada; Centre for Neuromuscular Disease (M.-O.D., R.K.), University of Ottawa, Ottawa, Ontario, Canada; Division of Pediatric Nephrology (A.S.), Massachusetts General Hospital, Boston, MA; Department of Biology (N.H.), Federal University of São Carlos, Sorocaba, Sao Paulo, SP, Brazil; and Department of Medicine (R.K.), University of Ottawa, Ottawa, Ontario, Canada.

Objective: To determine changes in serum profiles and kidney tissues from patients with spinal muscular atrophy (SMA) type 1 compared with age- and sex-matched controls.

Methods: In this cohort study, we investigated renal structure and function in infants and children with SMA type 1 in comparison with age- and sex-matched controls.

Results: Patients with SMA had alterations in serum creatinine, cystatin C, sodium, glucose, and calcium concentrations, granular casts and crystals in urine, and nephrocalcinosis and fibrosis. Nephrotoxicity and polycystic kidney disease PCR arrays revealed multiple differentially expressed genes, and immunoblot analysis showed decreased calcium-sensing receptors and calbindin and increased insulin-like growth factor-binding proteins in kidneys from patients with SMA.

Conclusions: These findings demonstrate that patients with SMA type 1, in the absence of disease-modifying therapies, frequently manifest impaired renal function as a primary or secondary consequence of their disease. This study provides new insights into systemic contributions to SMA disease pathogenesis and the need to identify coadjuvant therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1212/NXG.0000000000000353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6705648PMC
October 2019

Inhibitory milieu at the multiple sclerosis lesion site and the challenges for remyelination.

Glia 2020 05 23;68(5):859-877. Epub 2019 Aug 23.

Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.

Regeneration of myelin, following injury, can occur within the central nervous system to reinstate proper axonal conductance and provide trophic support. Failure to do so renders the axons vulnerable, leading to eventual degeneration, and neuronal loss. Thus, it is essential to understand the mechanisms by which remyelination or failure to remyelinate occur, particularly in the context of demyelinating and neurodegenerative disorders. In multiple sclerosis, oligodendrocyte progenitor cells (OPCs) migrate to lesion sites to repair myelin. However, during disease progression, the ability of OPCs to participate in remyelination diminishes coincident with worsening of the symptoms. Remyelination is affected by a broad range of cues from intrinsic programming of OPCs and extrinsic local factors to the immune system and other systemic elements including diet and exercise. Here we review the literature on these diverse inhibitory factors and the challenges they pose to remyelination. Results spanning several disciplines from fundamental preclinical studies to knowledge gained in the clinic will be discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/glia.23711DOI Listing
May 2020

Abnormal fatty acid metabolism is a core component of spinal muscular atrophy.

Ann Clin Transl Neurol 2019 08 26;6(8):1519-1532. Epub 2019 Jul 26.

Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.

Objective: Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder leading to paralysis and subsequent death in young children. Initially considered a motor neuron disease, extra-neuronal involvement is increasingly recognized. The primary goal of this study was to investigate alterations in lipid metabolism in SMA patients and mouse models of the disease.

Methods: We analyzed clinical data collected from a large cohort of pediatric SMA type I-III patients as well as SMA type I liver necropsy data. In parallel, we performed histology, lipid analysis, and transcript profiling in mouse models of SMA.

Results: We identify an increased susceptibility to developing dyslipidemia in a cohort of 72 SMA patients and liver steatosis in pathological samples. Similarly, fatty acid metabolic abnormalities were present in all SMA mouse models studied. Specifically, Smn mice displayed elevated hepatic triglycerides and dyslipidemia, resembling non-alcoholic fatty liver disease (NAFLD). Interestingly, this phenotype appeared prior to denervation.

Interpretation: This work highlights metabolic abnormalities as an important feature of SMA, suggesting implementation of nutritional and screening guidelines in patients, as such defects are likely to increase metabolic distress and cardiovascular risk. This study emphasizes the need for a systemic therapeutic approach to ensure maximal benefits for all SMA patients throughout their life.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/acn3.50855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6689695PMC
August 2019

Pathologic Alterations in the Proteome of Synaptosomes from a Mouse Model of Spinal Muscular Atrophy.

J Proteome Res 2019 08 11;18(8):3042-3051. Epub 2019 Jul 11.

Regenerative Medicine Program , Ottawa Hospital Research Institute , Ottawa , Ontario K1H 8L6 , Canada.

Spinal muscular atrophy (SMA) is a human genetic disorder characterized by muscle weakness, muscle atrophy, and death of motor neurons. SMA is caused by mutations or deletions in a gene called (). is a housekeeping gene, but the most prominent pathologies in SMA are atrophy of myofibers and death of motor neurons. Further, degeneration of neuromuscular junctions, of synapses, and of axonal regions are features of SMA disease. Here, we have investigated the proteome dynamics of central synapses in P14 mice, a model of SMA. Label-free quantitative proteomics on isolated synaptosomes from spinal cords of these animals identified 2030 protein groups. Statistical data analysis revealed 65 specific alterations in the proteome of the central synapses at the early onset stage of disease. Functional analysis of the dysregulated proteins indicated a significant enrichment of proteins associated with mitochondrial dynamics, cholesterol biogenesis, and protein clearance. These pathways represent potential targets for therapy development with the goal of providing stability to the central synapses, thereby preserving neuronal integrity in the context of SMA disease. Data are available via ProteomeXchange with identifier PXD012850.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.9b00159DOI Listing
August 2019

Report on the 4th Ottawa International Conference on Neuromuscular Disease and Biology - September 5-7, 2017, Ottawa, Canada.

J Neuromuscul Dis 2018 ;5(4):539-552

Department of Medicine, The Ottawa Hospital and University of Ottawa, ON, Canada.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3233/JND-180353DOI Listing
January 2019

Surgical Artificial Insemination in Mice.

Cold Spring Harb Protoc 2018 09 4;2018(9). Epub 2018 Sep 4.

Artificial insemination can be achieved by directly adding sperm from a particular male into the oviduct of a female successfully bred with a vasectomized male by a surgical procedure. Those who are comfortable performing oviduct embryo transfers might find this approach much easier than delivering the sperm into the vagina. Multiple females can be inseminated with sperm from a single male to rescue the line, expand the line quickly, or generate relatively synchronous embryos.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/pdb.prot092734DOI Listing
September 2018

Dystonin-A3 upregulation is responsible for maintenance of tubulin acetylation in a less severe dystonia musculorum mouse model for hereditary sensory and autonomic neuropathy type VI.

Hum Mol Genet 2018 10;27(20):3598-3611

Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Canada.

Hereditary sensory and autonomic neuropathy type VI (HSAN-VI) is a recessive human disease that arises from mutations in the dystonin gene (DST; also known as Bullous pemphigoid antigen 1 gene). A milder form of HSAN-VI was recently described, resulting from loss of a single dystonin isoform (DST-A2). Similarly, mutations in the mouse dystonin gene (Dst) result in severe sensory neuropathy, dystonia musculorum (Dstdt). Two Dstdt alleles, Dstdt-Tg4 and Dstdt-27J, differ in the severity of disease. The less severe Dstdt-Tg4 mice have disrupted expression of Dst-A1 and -A2 isoforms, while the more severe Dstdt-27J allele affects Dst-A1, -A2 and -A3 isoforms. As dystonin is a cytoskeletal-linker protein, we evaluated microtubule network integrity within sensory neurons from Dstdt-Tg4 and Dstdt-27J mice. There is a significant reduction in tubulin acetylation in Dstdt-27J indicative of microtubule instability and severe microtubule disorganization within sensory axons. However, Dstdt-Tg4 mice have no change in tubulin acetylation, and microtubule organization was only mildly impaired. Thus, microtubule instability is not central to initiation of Dstdt pathogenesis, though it may contribute to disease severity. Maintenance of microtubule stability in Dstdt-Tg4 dorsal root ganglia could be attributed to an upregulation in Dst-A3 expression as a compensation for the absence of Dst-A1 and -A2 in Dstdt-Tg4 sensory neurons. Indeed, knockdown of Dst-A3 in these neurons resulted in a decrease in tubulin acetylation. These findings shed light on the possible compensatory role of dystonin isoforms within HSAN-VI, which might explain the heterogeneity in symptoms within the reported forms of the disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddy250DOI Listing
October 2018

Interventions Targeting Glucocorticoid-Krüppel-like Factor 15-Branched-Chain Amino Acid Signaling Improve Disease Phenotypes in Spinal Muscular Atrophy Mice.

EBioMedicine 2018 May 4;31:226-242. Epub 2018 May 4.

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom. Electronic address:

The circadian glucocorticoid-Krüppel-like factor 15-branched-chain amino acid (GC-KLF15-BCAA) signaling pathway is a key regulatory axis in muscle, whose imbalance has wide-reaching effects on metabolic homeostasis. Spinal muscular atrophy (SMA) is a neuromuscular disorder also characterized by intrinsic muscle pathologies, metabolic abnormalities and disrupted sleep patterns, which can influence or be influenced by circadian regulatory networks that control behavioral and metabolic rhythms. We therefore set out to investigate the contribution of the GC-KLF15-BCAA pathway in SMA pathophysiology of Taiwanese Smn;SMN2 and Smn mouse models. We thus uncover substantial dysregulation of GC-KLF15-BCAA diurnal rhythmicity in serum, skeletal muscle and metabolic tissues of SMA mice. Importantly, modulating the components of the GC-KLF15-BCAA pathway via pharmacological (prednisolone), genetic (muscle-specific Klf15 overexpression) and dietary (BCAA supplementation) interventions significantly improves disease phenotypes in SMA mice. Our study highlights the GC-KLF15-BCAA pathway as a contributor to SMA pathogenesis and provides several treatment avenues to alleviate peripheral manifestations of the disease. The therapeutic potential of targeting metabolic perturbations by diet and commercially available drugs could have a broader implementation across other neuromuscular and metabolic disorders characterized by altered GC-KLF15-BCAA signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ebiom.2018.04.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013932PMC
May 2018

Pathogenic commonalities between spinal muscular atrophy and amyotrophic lateral sclerosis: Converging roads to therapeutic development.

Eur J Med Genet 2018 Nov 5;61(11):685-698. Epub 2017 Dec 5.

The Institute for Neurosciences of Montpellier, Inserm UMR1051, Univ Montpellier, Saint Eloi Hospital, Montpellier, France. Electronic address:

Spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS) are the two most common motoneuron disorders, which share typical pathological hallmarks while remaining genetically distinct. Indeed, SMA is caused by deletions or mutations in the survival motor neuron 1 (SMN1) gene whilst ALS, albeit being mostly sporadic, can also be caused by mutations within genes, including superoxide dismutase 1 (SOD1), Fused in Sarcoma (FUS), TAR DNA-binding protein 43 (TDP-43) and chromosome 9 open reading frame 72 (C9ORF72). However, it has come to light that these two diseases may be more interlinked than previously thought. Indeed, it has recently been found that FUS directly interacts with an Smn-containing complex, mutant SOD1 perturbs Smn localization, Smn depletion aggravates disease progression of ALS mice, overexpression of SMN in ALS mice significantly improves their phenotype and lifespan, and duplications of SMN1 have been linked to sporadic ALS. Beyond genetic interactions, accumulating evidence further suggests that both diseases share common pathological identities such as intrinsic muscle defects, neuroinflammation, immune organ dysfunction, metabolic perturbations, defects in neuron excitability and selective motoneuron vulnerability. Identifying common molecular effectors that mediate shared pathologies in SMA and ALS would allow for the development of therapeutic strategies and targeted gene therapies that could potentially alleviate symptoms and be equally beneficial in both disorders. In the present review, we will examine our current knowledge of pathogenic commonalities between SMA and ALS, and discuss how furthering this understanding can lead to the establishment of novel therapeutic approaches with wide-reaching impact on multiple motoneuron diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejmg.2017.12.001DOI Listing
November 2018

Survival Motor Neuron Protein is Released from Cells in Exosomes: A Potential Biomarker for Spinal Muscular Atrophy.

Sci Rep 2017 10 24;7(1):13859. Epub 2017 Oct 24.

Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.

Spinal muscular atrophy (SMA) is caused by homozygous mutation of the survival motor neuron 1 (SMN1) gene. Disease severity inversely correlates to the amount of SMN protein produced from the homologous SMN2 gene. We show that SMN protein is naturally released in exosomes from all cell types examined. Fibroblasts from patients or a mouse model of SMA released exosomes containing reduced levels of SMN protein relative to normal controls. Cells overexpressing SMN protein released exosomes with dramatically elevated levels of SMN protein. We observed enhanced quantities of exosomes in the medium from SMN-depleted cells, and in serum from a mouse model of SMA and a patient with Type 3 SMA, suggesting that SMN-depletion causes a deregulation of exosome release or uptake. The quantity of SMN protein contained in the serum-derived exosomes correlated with the genotype of the animal, with progressively less protein in carrier and affected animals compared to wildtype mice. SMN protein was easily detectable in exosomes isolated from human serum, with a reduction in the amount of SMN protein in exosomes from a patient with Type 3 SMA compared to a normal control. Our results suggest that exosome-derived SMN protein may serve as an effective biomarker for SMA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-14313-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655039PMC
October 2017

Splicing arrays reveal novel RBM10 targets, including SMN2 pre-mRNA.

BMC Mol Biol 2017 07 20;18(1):19. Epub 2017 Jul 20.

Département de Microbiologie et d'infectiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada.

Background: RBM10 is an RNA binding protein involved in message stabilization and alternative splicing regulation. The objective of the research described herein was to identify novel targets of RBM10-regulated splicing. To accomplish this, we downregulated RBM10 in human cell lines, using small interfering RNAs, then monitored alternative splicing, using a reverse transcription-PCR screening platform.

Results: RBM10 knockdown (KD) provoked alterations in splicing events in 10-20% of the pre-mRNAs, most of which had not been previously identified as RBM10 targets. Hierarchical clustering of the genes affected by RBM10 KD revealed good conservation of alternative exon inclusion or exclusion across cell lines. Pathway annotation showed RAS signaling to be most affected by RBM10 KD. Of particular interest was the finding that splicing of SMN pre-mRNA, encoding the survival of motor neuron (SMN) protein, was influenced by RBM10 KD. Inhibition of RBM10 resulted in preferential expression of the full-length, exon 7 retaining, SMN transcript in four cancer cell lines and one normal skin fibroblast cell line. SMN protein is expressed from two genes, SMN1 and SMN2, but the SMN1 gene is homozygously disrupted in people with spinal muscular atrophy; as a consequence, all of the SMN that is expressed in people with this disease is from the SMN2 gene. Expression analyses using primary fibroblasts from control, carrier and spinal muscle atrophy donors demonstrated that RBM10 KD resulted in preferential expression of the full-length, exon 7 retaining, SMN2 transcript. At the protein level, upregulation of the full-length SMN2 was also observed. Re-expression of RBM10, in a stable RBM10 KD cancer cell line, correlated with a reversion of the KD effect, demonstrating specificity.

Conclusion: Our work has not only expanded the number of pre-mRNA targets for RBM10, but identified RBM10 as a novel regulator of SMN2 alternative inclusion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12867-017-0096-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5520337PMC
July 2017

New insights into SMA pathogenesis: immune dysfunction and neuroinflammation.

Ann Clin Transl Neurol 2017 07 18;4(7):522-530. Epub 2017 May 18.

Regenerative Medicine Program Ottawa Hospital Research Institute Ottawa Ontario K1H 8L6 Canada.

Spinal muscular atrophy (SMA) is a neuromuscular disorder characterized by motor neuron degeneration, although defects in multiple cell types and tissues have also been implicated. Three independent laboratories recently identified immune organ defects in SMA. We therefore propose a novel pathogenic mechanism contributory to SMA, resulting in higher susceptibility to infection and exacerbated disease progression caused by neuroinflammation. Overall, compromised immune function could significantly affect survival and quality of life of SMA patients. We highlight the recent findings in immune organ defects, their potential consequences on patients, our understanding of neuroinflammation in SMA, and new research hypotheses in SMA pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/acn3.423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5497530PMC
July 2017

Effect of genetic background on the phenotype of the Smn2B/- mouse model of spinal muscular atrophy.

Hum Mol Genet 2016 10;25(20):4494-4506

Department of Medicine, University of Ottawa, Ottawa, Ontario, Canada

Spinal muscular atrophy (SMA) is caused by mutations or deletions in the Survival Motor Neuron 1 (SMN1) gene in humans. Modifiers of the SMA symptoms have been identified and genetic background has a substantial effect in the phenotype and survival of the severe mouse model of SMA. Previously, we generated the less severe Smn2B/- mice on a mixed genetic background. To assess the phenotype of Smn deficiency on a pure genetic background, we produced Smn2B/2B congenic mice on either the C57BL/6 (BL6) or FVB strain background and characterized them at the 6th generation by breeding to Smn+/- mice. Smn2B/- mice from these crosses were evaluated for growth, survival, muscle atrophy, motor neuron loss, motor behaviour, and neuromuscular junction pathology. FVB Smn2B/- mice had a shorter life span than BL6 Smn2B/- mice (median of 19 days vs. 25 days). Similarly, all other defects assessed occurred at earlier stages in FVB Smn2B/-mice when compared to BL6 Smn2B/-mice. However, there were no differences in Smn protein levels in the spinal cords of these mice. Interestingly, levels of Plastin 3, a putative modifier of SMA, were significantly induced in spinal cords of BL6 Smn2B/- mice but not of FVB Smn2B/-mice. Our studies demonstrate that the phenotype in Smn2B/-mice is more severe in the FVB background than in the BL6 background, which could potentially be explained by the differential induction of genetic modifiers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddw278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409218PMC
October 2016

Immune dysregulation may contribute to disease pathogenesis in spinal muscular atrophy mice.

Hum Mol Genet 2017 02;26(4):801-819

Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada K1H 8L6.

Spinal muscular atrophy (SMA) has long been solely considered a neurodegenerative disorder. However, recent work has highlighted defects in many other cell types that could contribute to disease aetiology. Interestingly, the immune system has never been extensively studied in SMA. Defects in lymphoid organs could exacerbate disease progression by neuroinflammation or immunodeficiency. Smn depletion led to severe alterations in the thymus and spleen of two different mouse models of SMA. The spleen from Smn depleted mice was dramatically smaller at a very young age and its histological architecture was marked by mislocalization of immune cells in the Smn2B/- model mice. In comparison, the thymus was relatively spared in gross morphology but showed many histological alterations including cortex thinning in both mouse models at symptomatic ages. Thymocyte development was also impaired as evidenced by abnormal population frequencies in the Smn2B/- thymus. Cytokine profiling revealed major changes in different tissues of both mouse models. Consistent with our observations, we found that survival motor neuron (Smn) protein levels were relatively high in lymphoid organs compared to skeletal muscle and spinal cord during postnatal development in wild type mice. Genetic introduction of one copy of the human SMN2 transgene was enough to rescue splenic and thymic defects in Smn2B/- mice. Thus, Smn is required for the normal development of lymphoid organs, and altered immune function may contribute to SMA disease pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddw434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409095PMC
February 2017

Oligodendrocyte development and CNS myelination are unaffected in a mouse model of severe spinal muscular atrophy.

Hum Mol Genet 2017 01;26(2):282-292

Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.

The childhood neurodegenerative disease spinal muscular atrophy (SMA) is caused by loss-of-function mutations or deletions in the Survival Motor Neuron 1 (SMN1) gene resulting in insufficient levels of survival motor neuron (SMN) protein. Classically considered a motor neuron disease, increasing evidence now supports SMA as a multi-system disorder with phenotypes discovered in cortical neuron, astrocyte, and Schwann cell function within the nervous system. In this study, we sought to determine whether Smn was critical for oligodendrocyte (OL) development and central nervous system myelination. A mouse model of severe SMA was used to assess OL growth, migration, differentiation and myelination. All aspects of OL development and function studied were unaffected by Smn depletion. The tremendous impact of Smn depletion on a wide variety of other cell types renders the OL response unique. Further investigation of the OLs derived from SMA models may reveal disease modifiers or a compensatory mechanism allowing these cells to flourish despite the reduced levels of this multifunctional protein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddw385DOI Listing
January 2017

Voluntary Running Triggers VGF-Mediated Oligodendrogenesis to Prolong the Lifespan of Snf2h-Null Ataxic Mice.

Cell Rep 2016 10;17(3):862-875

Regenerative Medicine Program, Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, ON K1H 8L1, Canada; Department of Cellular and Molecular Medicine, University of Ottawa, ON K1N 6N5, Canada; Department of Biochemistry, Microbiology & Immunology, University of Ottawa, ON K1N 6N5, Canada. Electronic address:

Exercise has been argued to enhance cognitive function and slow progressive neurodegenerative disease. Although exercise promotes neurogenesis, oligodendrogenesis and adaptive myelination are also significant contributors to brain repair and brain health. Nonetheless, the molecular details underlying these effects remain poorly understood. Conditional ablation of the Snf2h gene impairs cerebellar development producing mice with poor motor function, progressive ataxia, and death between postnatal days 25-45. Here, we show that voluntary running induced an endogenous brain repair mechanism that resulted in a striking increase in hindbrain myelination and the long-term survival of Snf2h cKO mice. Further experiments identified the VGF growth factor as a major driver underlying this effect. VGF neuropeptides promote oligodendrogenesis in vitro, whereas Snf2h cKO mice treated with full-length VGF-encoding adenoviruses removed the requirement of exercise for survival. Together, these results suggest that VGF delivery could represent a therapeutic strategy for cerebellar ataxia and other pathologies of the CNS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2016.09.030DOI Listing
October 2016