SMN controls neuromuscular junction integrity through U7 snRNP

The neuromuscular junction (NMJ) is an essential synapse for animal survival whose loss is a key hallmark of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). While insights into the function of the causative genes implicate RNA dysregulation in NMJ pathogenesis, the RNA-mediated mechanisms controlling the biology of this specialized synapse that go awry in disease remain elusive. Here, we show that activity of the SMA-determining SMN protein in the assembly of U7 small nuclear ribonucleoprotein (snRNP), which functions in the 3'-end processing of replication-dependent histone mRNAs, is required for NMJ integrity. AAV9-mediated gene delivery of U7-specific Lsm10 and Lsm11 proteins selectively enhances U7 snRNP assembly, corrects histone mRNA processing defects, and rescues key structural and functional abnormalities of neuromuscular pathology in SMA mice - including NMJ denervation, reduced synaptic transmission, and skeletal muscle atrophy. Furthermore, U7 snRNP dysfunction induced by SMN deficiency drives selective loss of the synaptic organizing protein Agrin at NMJs innervating vulnerable axial muscles of SMA mice, revealing an unanticipated link between U7-dependent histone mRNA processing and motor neuron-derived expression of an essential factor for NMJ biology. Together, these findings establish a direct contribution of U7 snRNP dysfunction to the neuromuscular phenotype in SMA and the requirement of RNA-mediated histone gene regulation for maintaining functional synaptic connections between motor neurons and muscles.

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Molecular Mechanisms of Neurodegeneration in Spinal Muscular Atrophy

Journal of Experimental Neuroscience ◽

10.4137/jen.s33122 ◽

2016 ◽

Vol 10 ◽

pp. JEN.S33122 ◽

Cited By ~ 36

Author(s):

Saif Ahmad ◽

Kanchan Bhatia ◽

Annapoorna Kannan ◽

Laxman Gangwani

Keyword(s):

Spinal Muscular Atrophy ◽

Motor Neuron ◽

Motor Neurons ◽

Molecular Mechanisms ◽

Muscular Atrophy ◽

Survival Motor Neuron ◽

Skeletal Muscle Atrophy ◽

Spinal Motor Neurons ◽

Low Levels ◽

Smn Protein

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease with a high incidence and is the most common genetic cause of infant mortality. SMA is primarily characterized by degeneration of the spinal motor neurons that leads to skeletal muscle atrophy followed by symmetric limb paralysis, respiratory failure, and death. In humans, mutation of the Survival Motor Neuron 1 (SMN1) gene shifts the load of expression of SMN protein to the SMN2 gene that produces low levels of full-length SMN protein because of alternative splicing, which are sufficient for embryonic development and survival but result in SMA. The molecular mechanisms of the (a) regulation of SMN gene expression and (b) degeneration of motor neurons caused by low levels of SMN are unclear. However, some progress has been made in recent years that have provided new insights into understanding of the cellular and molecular basis of SMA pathogenesis. In this review, we have briefly summarized recent advances toward understanding of the molecular mechanisms of regulation of SMN levels and signaling mechanisms that mediate neurodegeneration in SMA.

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The Survival of Motor Neurons Protein Determines the Capacity for snRNP Assembly: Biochemical Deficiency in Spinal Muscular Atrophy

Molecular and Cellular Biology ◽

10.1128/mcb.25.13.5543-5551.2005 ◽

2005 ◽

Vol 25 (13) ◽

pp. 5543-5551 ◽

Cited By ~ 131

Author(s):

Lili Wan ◽

Daniel J. Battle ◽

Jeongsik Yong ◽

Amelie K. Gubitz ◽

Stephen J. Kolb ◽

...

Keyword(s):

Spinal Muscular Atrophy ◽

Motor Neurons ◽

Muscular Atrophy ◽

Protein Levels ◽

Small Nuclear Ribonucleoprotein ◽

Cell Extracts ◽

Smn Complex ◽

Survival Of Motor Neurons ◽

Smn Protein ◽

Nuclear Ribonucleoprotein

ABSTRACT Reduction of the survival of motor neurons (SMN) protein levels causes the motor neuron degenerative disease spinal muscular atrophy, the severity of which correlates with the extent of reduction in SMN. SMN, together with Gemins 2 to 7, forms a complex that functions in the assembly of small nuclear ribonucleoprotein particles (snRNPs). Complete depletion of the SMN complex from cell extracts abolishes snRNP assembly, the formation of heptameric Sm cores on snRNAs. However, what effect, if any, reduction of SMN protein levels, as occurs in spinal muscular atrophy patients, has on the capacity of cells to produce snRNPs is not known. To address this, we developed a sensitive and quantitative assay for snRNP assembly, the formation of high-salt- and heparin-resistant stable Sm cores, that is strictly dependent on the SMN complex. We show that the extent of Sm core assembly is directly proportional to the amount of SMN protein in cell extracts. Consistent with this, pulse-labeling experiments demonstrate a significant reduction in the rate of snRNP biogenesis in low-SMN cells. Furthermore, extracts of cells from spinal muscular atrophy patients have a lower capacity for snRNP assembly that corresponds directly to the reduced amount of SMN. Thus, SMN determines the capacity for snRNP biogenesis, and our findings provide evidence for a measurable deficiency in a biochemical activity in cells from patients with spinal muscular atrophy.

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Utilizing gene therapy methods to probe the genetic requirements to prevent spinal muscular atrophy.

10.32469/10355/57243 ◽

2016 ◽

Author(s):

◽

Madeline R. Miller

Keyword(s):

Neuromuscular Junction ◽

Spinal Muscular Atrophy ◽

Motor Neuron ◽

Motor Neurons ◽

Muscular Atrophy ◽

Survival Motor Neuron ◽

Downstream Effects ◽

Smn Protein ◽

Progressive Weakness

Spinal Muscular Atrophy is clinically recognized as a progressive weakness within the trunk and proximal limbs that will lead to breathing failure and death within infants. As a neurodegenerative genetic disease, SMA is caused by loss of motor neurons, which in turn is caused by low levels of the Survival Motor Neuron (SMN) protein. The mechanism by which a ubiquitously expressed protein such as SMN is able to cause the specific death of motor neurons is highly debated and of great interest. Work presented here focuses on understanding the biological requirements of SMN and its downstream effects on the neuromuscular junction. To this end we utilize viral based gene delivery as a powerful tool to assess the effects of genes of interest in vivo. Our findings contribute to the conversation regarding whether SMA is truly a "motor neuron" disease, suggesting that astrocytes play a meaningful role in staving off SMA. Further, we investigate the domains within SMN needed to maintain its function in a mammalian system. We take a novel and challenging approach to identify a minimal domain capable of maintaining function. Finally, we demonstrate the practical use of morophological analysis of the neuromuscular junction as a means to characterize SMA pathology.

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ZPR1 Is Essential for Survival and Is Required for Localization of the Survival Motor Neurons (SMN) Protein to Cajal Bodies

Molecular and Cellular Biology ◽

10.1128/mcb.25.7.2744-2756.2005 ◽

2005 ◽

Vol 25 (7) ◽

pp. 2744-2756 ◽

Cited By ~ 40

Author(s):

Laxman Gangwani ◽

Richard A. Flavell ◽

Roger J. Davis

Keyword(s):

Motor Neuron ◽

Motor Neurons ◽

Zinc Finger Protein ◽

Muscular Atrophy ◽

Cajal Bodies ◽

Survival Motor Neurons ◽

Small Nuclear Ribonucleoprotein ◽

Smn Protein ◽

Axonal Defects ◽

Interfering Rna

ABSTRACT Mutation of the survival motor neurons 1 (SMN1) gene causes motor neuron apoptosis and represents the major cause of spinal muscular atrophy in humans. Biochemical studies have established that the SMN protein plays an important role in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and that the SMN complex can interact with the zinc finger protein ZPR1. Here we report that targeted ablation of the Zpr1 gene in mice disrupts the subcellular localization of both SMN and spliceosomal snRNPs. Specifically, SMN localization to Cajal bodies and gems was not observed in cells derived from Zpr1−/− embryos and the amount of cytoplasmic snRNP detected in Zpr1 −/− embryos was reduced compared with that in wild-type embryos. We found that Zpr1 −/− mice die during early embryonic development, with reduced proliferation and increased apoptosis. These effects of Zpr1 gene disruption were confirmed and extended in studies of cultured motor neuron-like cells using small interfering RNA-mediated Zpr1 gene suppression; ZPR1 deficiency caused growth cone retraction, axonal defects, and apoptosis. Together, these data indicate that ZPR1 contributes to the regulation of SMN complexes and that it is essential for cell survival.

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SMN-deficiency disrupts SERCA2 expression and intracellular Ca2+ signaling in cardiomyocytes from SMA mice and patient-derived iPSCs

10.21203/rs.3.rs-24179/v1 ◽

2020 ◽

Author(s):

Guzal Khayrullina ◽

Kasey E Moritz ◽

James F. Schooley ◽

Naheed Fatima ◽

Coralie Viollet ◽

...

Keyword(s):

Motor Neurons ◽

Muscular Atrophy ◽

Functional Expression ◽

Cardiac Cells ◽

Survival Motor Neuron ◽

Skeletal Muscle Atrophy ◽

Intracellular Ca ◽

Model Mouse ◽

Smn Protein ◽

Disease Stages

Abstract Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by loss of alpha motor neurons and skeletal muscle atrophy. The disease is caused by mutations of the SMN1 gene that result in reduced functional expression of survival motor neuron (SMN) protein. SMN is ubiquitously expressed and there have been reports of cardiovascular dysfunction in the most severe SMA patients and animal models of the disease. In this study, we directly assessed the function of cardiomyocytes isolated from a severe SMA model mouse and cardiomyocytes generated from patient-derived IPSCs. Consistent with impaired cardiovascular function at the very early disease stages in mice, heart failure markers such as brain natriuretic peptide were significantly elevated. Functionally, cardiomyocyte relaxation kinetics were markedly slowed and the T50 for Ca2+ sequestration increased to 146 ± 4 msec in SMN-deficient cardiomyocytes from 126 ± 4 msec in wild type cells. Reducing SMN levels in cardiomyocytes from control patient IPSCs slowed calcium reuptake similar to SMA patent derived cardiac cells. Importantly, restoring SMN increased calcium reuptake rate. Taken together, these results indicate that SMN deficiency impairs cardiomyocyte function at least partially through intracellular Ca2+ cycling dysregulation.

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Mitochondrial defects in the respiratory complex I contribute to impaired translational initiation via ROS and energy homeostasis in SMA motor neurons

Acta Neuropathologica Communications ◽

10.1186/s40478-020-01101-6 ◽

2020 ◽

Vol 8 (1) ◽

Author(s):

Maximilian Paul Thelen ◽

Brunhilde Wirth ◽

Min Jeong Kye

Keyword(s):

Protein Synthesis ◽

Motor Neuron ◽

Motor Neurons ◽

Complex I ◽

Energy Homeostasis ◽

Muscular Atrophy ◽

Survival Motor Neuron ◽

Translational Initiation ◽

Protein Levels ◽

Smn Protein

AbstractSpinal muscular atrophy (SMA) is a neuromuscular disease characterized by loss of lower motor neurons, which leads to proximal muscle weakness and atrophy. SMA is caused by reduced survival motor neuron (SMN) protein levels due to biallelic deletions or mutations in the SMN1 gene. When SMN levels fall under a certain threshold, a plethora of cellular pathways are disturbed, including RNA processing, protein synthesis, metabolic defects, and mitochondrial function. Dysfunctional mitochondria can harm cells by decreased ATP production and increased oxidative stress due to elevated cellular levels of reactive oxygen species (ROS). Since neurons mainly produce energy via mitochondrial oxidative phosphorylation, restoring metabolic/oxidative homeostasis might rescue SMA pathology. Here, we report, based on proteome analysis, that SMA motor neurons show disturbed energy homeostasis due to dysfunction of mitochondrial complex I. This results in a lower basal ATP concentration and higher ROS production that causes an increase of protein carbonylation and impaired protein synthesis in SMA motor neurons. Counteracting these cellular impairments with pyruvate reduces elevated ROS levels, increases ATP and SMN protein levels in SMA motor neurons. Furthermore, we found that pyruvate-mediated SMN protein synthesis is mTOR-dependent. Most importantly, we showed that ROS regulates protein synthesis at the translational initiation step, which is impaired in SMA. As many neuropathies share pathological phenotypes such as dysfunctional mitochondria, excessive ROS, and impaired protein synthesis, our findings suggest new molecular interactions among these pathways. Additionally, counteracting these impairments by reducing ROS and increasing ATP might be beneficial for motor neuron survival in SMA patients.

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Concentrating pre-mRNA processing factors in the histone locus body facilitates efficient histone mRNA biogenesis

The Journal of Cell Biology ◽

10.1083/jcb.201504043 ◽

2016 ◽

Vol 213 (5) ◽

pp. 557-570 ◽

Cited By ~ 39

Author(s):

Deirdre C. Tatomer ◽

Esteban Terzo ◽

Kaitlin P. Curry ◽

Harmony Salzler ◽

Ivan Sabath ◽

...

Keyword(s):

Messenger Rna ◽

Mrna Processing ◽

Histone Mrna ◽

Histone Mrnas ◽

Assembly Factor ◽

U7 Snrnp ◽

Histone Locus Body ◽

Histone Locus ◽

Processing Factors

The histone locus body (HLB) assembles at replication-dependent histone genes and concentrates factors required for histone messenger RNA (mRNA) biosynthesis. FLASH (Flice-associated huge protein) and U7 small nuclear RNP (snRNP) are HLB components that participate in 3′ processing of the nonpolyadenylated histone mRNAs by recruiting the endonuclease CPSF-73 to histone pre-mRNA. Using transgenes to complement a FLASH mutant, we show that distinct domains of FLASH involved in U7 snRNP binding, histone pre-mRNA cleavage, and HLB localization are all required for proper FLASH function in vivo. By genetically manipulating HLB composition using mutations in FLASH, mutations in the HLB assembly factor Mxc, or depletion of the variant histone H2aV, we find that failure to concentrate FLASH and/or U7 snRNP in the HLB impairs histone pre-mRNA processing. This failure results in accumulation of small amounts of polyadenylated histone mRNA and nascent read-through transcripts at the histone locus. Thus, the HLB concentrates FLASH and U7 snRNP, promoting efficient histone mRNA biosynthesis and coupling 3′ end processing with transcription termination.

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SMN complexes of nucleus and cytoplasm: A proteomic study for SMA therapy

Translational Neuroscience ◽

10.2478/v10134-010-0027-6 ◽

2010 ◽

Vol 1 (4) ◽

Cited By ~ 2

Author(s):

Heidi Fuller ◽

Glenn Morris

Keyword(s):

Motor Neurons ◽

Muscular Atrophy ◽

Ubiquitin Proteasome System ◽

Neuromuscular Disorder ◽

Nuclear Extracts ◽

Smn Complex ◽

Proteomic Study ◽

Ubiquitin Proteasome ◽

Survival Of Motor Neurons ◽

Smn Protein

AbstractReduced levels of the survival of motor neurons protein (SMN), cause the inherited neuromuscular disorder, spinal muscular atrophy (SMA). The majority of therapeutic approaches to date have been focused on finding ways to increase expression of functional SMN protein, though stabilization of SMN protein may also be an important consideration. SMN interacts, directly or indirectly, stably or transiently, with a large number of other proteins, some of which contribute to SMN stability and may therefore be potential targets for SMA therapy. We recently characterized the nuclear SMN interactome using LC-MALDI-TOF/TOF analysis of anti-SMN pull-downs and identified myb-binding protein-1a (Mybbp1a) as a novel partner. In light of interest in cytoplasm-specific roles of the SMN complex, we have applied the same approach to characterise the cytoplasmic SMN interactome. We now show that SMN complexes from HeLa cytoplasmic extracts differ significantly from those found in nuclear extracts, with gemin5, importinbeta and annexin A2 easily detected only in the cytoplasmic extracts, whereas interaction of SMN with Mybbp1a appears to occur only in the nucleus. SMN is ubiquitinylated and we also found proteins of the ubiquitin-proteasome system associated with SMN in the cytoplasm.

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Functional analysis of the sea urchin U7 small nuclear RNA

Molecular and Cellular Biology ◽

10.1128/mcb.8.3.1076-1084.1988 ◽

1988 ◽

Vol 8 (3) ◽

pp. 1076-1084

Author(s):

G M Gilmartin ◽

F Schaufele ◽

G Schaffner ◽

M L Birnstiel

Keyword(s):

Sea Urchin ◽

Micrococcal Nuclease ◽

Small Nuclear Rna ◽

Histone Mrna ◽

Small Nuclear Ribonucleoprotein ◽

Nuclear Rna ◽

U7 Snrnp ◽

Structure Relationship ◽

Nuclear Ribonucleoprotein ◽

Sm Protein

U7 small nuclear RNA (snRNA) is an essential component of the RNA-processing machinery which generates the 3' end of mature histone mRNA in the sea urchin. The U7 small nuclear ribonucleoprotein particle (snRNP) is classified as a member of the Sm-type U snRNP family by virtue of its recognition by both anti-trimethylguanosine and anti-Sm antibodies. We analyzed the function-structure relationship of the U7 snRNP by mutagenesis experiments. These suggested that the U7 snRNP of the sea urchin is composed of three important domains. The first domain encompasses the 5'-terminal sequences, up to about nucleotides 7, which are accessible to micrococcal nuclease, while the remainder of the RNA is highly protected and hence presumably bound by proteins. This region contains the sequence complementarities between the U7 snRNA and the histone pre-mRNA which have previously been shown to be required for 3' processing (F. Schaufele, G. M. Gilmartin, W. Bannwarth, and M. L. Birnstiel, Nature [London] 323:777-781, 1986). Nucleotides 9 to 20 constitute a second domain which includes sequences for Sm protein binding. The complementarities between the U7 snRNA sequences in this region and the terminal palindrome of the histone mRNA appear to be fortuitous and play only a secondary, if any, role in 3' processing. The third domain is composed of the terminal palindrome of U7 snRNA, the secondary structure of which must be maintained for the U7 snRNP to function, but its sequence can be drastically altered without any observable effect on snRNP assembly or 3' processing.

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Circulating microRNAs as potential biomarkers and therapeutic targets in spinal muscular atrophy

Therapeutic Advances in Neurological Disorders ◽

10.1177/1756286420979954 ◽

2020 ◽

Vol 13 ◽

pp. 175628642097995

Author(s):

Tai-Heng Chen

Keyword(s):

Spinal Muscular Atrophy ◽

Motor Neurons ◽

Molecular Mechanisms ◽

Muscular Atrophy ◽

Therapeutic Targets ◽

Homozygous Deletion ◽

Survival Motor Neuron ◽

Novel Mirna ◽

Potential Applications ◽

Smn Protein

Spinal muscular atrophy (SMA), a leading genetic cause of infant death, is a neurodegenerative disease characterized by the selective loss of particular groups of motor neurons (MNs) in the anterior horn of the spinal cord with progressive muscle wasting. SMA is caused by a deficiency of the survival motor neuron (SMN) protein due to a homozygous deletion or mutation of the SMN1 gene. However, the molecular mechanisms whereby the SMN complex regulates MN functions are not fully elucidated. Emerging studies on SMA pathogenesis have turned the attention of researchers to RNA metabolism, given that increasingly identified SMN-associated modifiers are involved in both coding and non-coding RNA (ncRNA) processing. Among various ncRNAs, microRNAs (miRNAs) are the most studied in terms of regulation of posttranscriptional gene expression. Recently, the discovery that miRNAs are critical to MN function and survival led to the study of dysregulated miRNAs in SMA pathogenesis. Circulating miRNAs have drawn attention as a readily available biomarker due to their property of being clinically detectable in numerous human biofluids through non-invasive approaches. As there are recent promising findings from novel miRNA-based medicines, this article presents an extensive review of the most up-to-date studies connecting specific miRNAs to SMA pathogenesis and the potential applications of miRNAs as biomarkers and therapeutic targets for SMA.

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