Publications by authors named "Christian L Lorson"

86 Publications

Evolution, correlation, structural impact and dynamics of emerging SARS-CoV-2 variants.

Comput Struct Biotechnol J 2021 24;19:3799-3809. Epub 2021 Jun 24.

Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections remain unmanageable in some parts of the world. As with other RNA viruses, mutations in the SARS-CoV-2 gene have been continuously evolving. Recently, four variants have been identified, B.1.1.7, B.1.351, P.1 and CAL.20C. These variants appear to be more infectious and transmissible than the original Wuhan-Hu-1 virus. Using a combination of bioinformatics and structural analyses, we show that the new SARS-CoV-2 variants emerged in the background of an already known Spike protein mutation D614G together with another mutation P323L in the RNA polymerase of SARS-CoV-2. The phylogenetic analysis showed that the CAL.20C and B.1.351 shared one common ancestor, whereas the B.1.1.7 and P.1 shared a different ancestor. Structural comparisons did not show any significant difference between the wild-type and mutant ACE2/Spike complexes. Structural analysis indicated that the N501Y mutation may increase hydrophobic interactions at the ACE2/Spike interface. However, reported greater binding affinity of N501Y Spike with ACE2 does not seem to be entirely due to increased hydrophobic interactions, given that Spike mutation R417T in P.1 or K417N in B.1.351 results in the loss of a salt-bridge interaction between ACE2 and S-RBD. The calculated change in free energy did not provide a clear trend of S protein stability of mutations in the variants. As expected, we show that the CAL.20C generally migrated from the west coast to the east coast of the USA. Taken together, the analyses suggest that the evolution of variants and their infectivity is complex and may depend upon many factors.
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http://dx.doi.org/10.1016/j.csbj.2021.06.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8225291PMC
June 2021

Survival motor neuron deficiency slows myoblast fusion through reduced myomaker and myomixer expression.

J Cachexia Sarcopenia Muscle 2021 Jun 11. Epub 2021 Jun 11.

Department of Anatomy, Physiology, and Genetics, Uniformed Services University of the Health Sciences, F. Edward Hébert School of Medicine, Bethesda, MD, USA.

Background: Spinal muscular atrophy is an inherited neurodegenerative disease caused by insufficient levels of the survival motor neuron (SMN) protein. Recently approved treatments aimed at increasing SMN protein levels have dramatically improved patient survival and have altered the disease landscape. While restoring SMN levels slows motor neuron loss, many patients continue to have smaller muscles and do not achieve normal motor milestones. While timing of treatment is important, it remains unclear why SMN restoration is insufficient to fully restore muscle size and function. We and others have shown that SMN-deficient muscle precursor cells fail to efficiently fuse into myotubes. However, the role of SMN in myoblast fusion is not known.

Methods: In this study, we show that SMN-deficient myoblasts readily fuse with wild-type myoblasts, demonstrating fusion competency. Conditioned media from wild type differentiating myoblasts do not rescue the fusion deficit of SMN-deficient cells, suggesting that compromised fusion may primarily be a result of altered membrane dynamics at the cell surface. Transcriptome profiling of skeletal muscle from SMN-deficient mice revealed altered expression of cell surface fusion molecules. Finally, using cell and mouse models, we investigate if myoblast fusion can be rescued in SMN-deficient myoblast and improve the muscle pathology in SMA mice.

Results: We found reduced expression of the muscle fusion proteins myomaker (P = 0.0060) and myomixer (P = 0.0051) in the muscle of SMA mice. Suppressing SMN expression in C2C12 myoblast cells reduces expression of myomaker (35% reduction; P < 0.0001) and myomixer, also known as myomerger and minion, (30% reduction; P < 0.0001) and restoring SMN levels only partially restores myomaker and myomixer expression. Ectopic expression of myomixer improves myofibre number (55% increase; P = 0.0006) and motor function (35% decrease in righting time; P = 0.0089) in SMA model mice and enhances motor function (82% decrease in righting time; P < 0.0001) and extends survival (28% increase; P < 0.01) when administered in combination with an antisense oligonucleotide that increases SMN protein levels.

Conclusions: Here, we identified reduced expression of muscle fusion proteins as a key factor in the fusion deficits of SMN-deficient myoblasts. This discovery provides a novel target to improve SMA muscle pathology and motor function, which in combination with SMN increasing therapy could enhance clinical outcomes for SMA patients.
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http://dx.doi.org/10.1002/jcsm.12740DOI Listing
June 2021

Discovery and in-vitro evaluation of potent SARS-CoV-2 entry inhibitors.

bioRxiv 2021 Apr 2. Epub 2021 Apr 2.

SARS-CoV-2 infection initiates with the attachment of spike protein to the ACE2 receptor. While vaccines have been developed, no SARS-CoV-2 specific small molecule inhibitors have been approved. Herein, utilizing the crystal structure of the ACE2/Spike receptor binding domain (S-RBD) complex in computer-aided drug design (CADD) approach, we docked ∼8 million compounds within the pockets residing at S-RBD/ACE2 interface. Five best hits depending on the docking score, were selected and tested for their efficacy to block SARS-CoV-2 replication. Of these, two compounds (MU-UNMC-1 and MU-UNMC-2) blocked SARS-CoV-2 replication at sub-micromolar IC in human bronchial epithelial cells (UNCN1T) and Vero cells. Furthermore, MU-UNMC-2 was highly potent in blocking the virus entry by using pseudoviral particles expressing SARS-CoV-2 spike. Finally, we found that MU-UNMC-2 is highly synergistic with remdesivir (RDV), suggesting that minimal amounts are needed when used in combination with RDV, and has the potential to develop as a potential entry inhibitor for COVID-19.
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http://dx.doi.org/10.1101/2021.04.02.438204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8020965PMC
April 2021

Factors Associated with Emerging and Re-emerging of SARS-CoV-2 Variants.

bioRxiv 2021 Mar 24. Epub 2021 Mar 24.

Global spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has triggered unprecedented scientific efforts, as well as containment and treatment measures. Despite these efforts, SARS-CoV-2 infections remain unmanageable in some parts of the world. Due to inherent mutability of RNA viruses, it is not surprising that the SARS-CoV-2 genome has been continuously evolving since its emergence. Recently, four functionally distinct variants, B.1.1.7, B.1.351, P.1 and CAL.20C, have been identified, and they appear to more infectious and transmissible than the original (Wuhan-Hu-1) virus. Here we provide evidence based upon a combination of bioinformatics and structural approaches that can explain the higher infectivity of the new variants. Our results show that the greater infectivity of SARS-CoV-2 than SARS-CoV can be attributed to a combination of several factors, including alternate receptors. Additionally, we show that new SARS-CoV-2 variants emerged in the background of D614G in Spike protein and P323L in RNA polymerase. The correlation analyses showed that all mutations in specific variants did not evolve simultaneously. Instead, some mutations evolved most likely to compensate for the viral fitness.
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http://dx.doi.org/10.1101/2021.03.24.436850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010727PMC
March 2021

Coronavirus helicases: attractive and unique targets of antiviral drug-development and therapeutic patents.

Expert Opin Ther Pat 2021 Apr 21;31(4):339-350. Epub 2021 Apr 21.

Bond Life Sciences Center, University of Missouri, Columbia, MO, USA.

: Coronaviruses encode a helicase that is essential for viral replication and represents an excellent antiviral target. However, only a few coronavirus helicase inhibitors have been patented. These patents include drug-like compound SSYA10-001, aryl diketo acids (ADK), and dihydroxychromones. Additionally, adamantane-derived bananins, natural flavonoids, one acrylamide derivative [(E)-3-(furan-2-yl)-N-(4-sulfamoylphenyl)acrylamide], a purine derivative (7-ethyl-8-mercapto-3-methyl-3,7-dihydro-1 H-purine-2,6-dione), and a few bismuth complexes. The IC of patented inhibitors ranges between 0.82 μM and 8.95 μM, depending upon the assays used. Considering the urgency of clinical interventions against Coronavirus Disease-19 (COVID-19), it is important to consider developing antiviral portfolios consisting of small molecules.: This review examines coronavirus helicases as antiviral targets, and the potential of previously patented and experimental compounds to inhibit the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) helicase.: Small molecule coronavirus helicase inhibitors represent attractive pharmacological modalities for the treatment of coronaviruses such as SARS-CoV and SARS-CoV-2. Rightfully so, the current emphasis is focused upon the development of vaccines. However, vaccines may not work for everyone and broad-based adoption of vaccinations is an increasingly challenging societal endeavor. Therefore, it is important to develop additional pharmacological antivirals against the highly conserved coronavirus helicases to broadly protect against this and subsequent coronavirus epidemics.
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http://dx.doi.org/10.1080/13543776.2021.1884224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8074651PMC
April 2021

Short-duration splice promoting compound enables a tunable mouse model of spinal muscular atrophy.

Life Sci Alliance 2021 01 24;4(1). Epub 2020 Nov 24.

Department of Dermatology, Indiana University School of Medicine, Indianapolis, IN, USA

Spinal muscular atrophy (SMA) is a motor neuron disease and the leading genetic cause of infant mortality. SMA results from insufficient survival motor neuron (SMN) protein due to alternative splicing. Antisense oligonucleotides, gene therapy and splicing modifiers recently received FDA approval. Although severe SMA transgenic mouse models have been beneficial for testing therapeutic efficacy, models mimicking milder cases that manifest post-infancy have proven challenging to develop. We established a titratable model of mild and moderate SMA using the splicing compound NVS-SM2. Administration for 30 d prevented development of the SMA phenotype in severe SMA mice, which typically show rapid weakness and succumb by postnatal day 11. Furthermore, administration at day eight resulted in phenotypic recovery. Remarkably, acute dosing limited to the first 3 d of life significantly enhanced survival in two severe SMA mice models, easing the burden on neonates and demonstrating the compound as suitable for evaluation of follow-on therapies without potential drug-drug interactions. This pharmacologically tunable SMA model represents a useful tool to investigate cellular and molecular pathogenesis at different stages of disease.
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http://dx.doi.org/10.26508/lsa.202000889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7723287PMC
January 2021

Infectivity of SARS-CoV-2: there Is Something More than D614G?

J Neuroimmune Pharmacol 2020 12 15;15(4):574-577. Epub 2020 Sep 15.

Bond Life Sciences Center, University of Missouri, Columbia, MO, 65211, USA.

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http://dx.doi.org/10.1007/s11481-020-09954-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7490321PMC
December 2020

AAV9-DOK7 gene therapy reduces disease severity in Smn SMA model mice.

Biochem Biophys Res Commun 2020 09 30;530(1):107-114. Epub 2020 Jul 30.

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA; Bond Life Sciences Center, University of Missouri, Columbia, MO, 65211, USA. Electronic address:

Spinal Muscular Atrophy (SMA) is an autosomal recessive neuromuscular disease caused by deletions or mutations in the survival motor neuron (SMN1) gene. An important hallmark of disease progression is the pathology of neuromuscular junctions (NMJs). Affected NMJs in the SMA context exhibit delayed maturation, impaired synaptic transmission, and loss of contact between motor neurons and skeletal muscle. Protection and maintenance of NMJs remains a focal point of therapeutic strategies to treat SMA, and the recent implication of the NMJ-organizer Agrin in SMA pathology suggests additional NMJ organizing molecules may contribute. DOK7 is an NMJ organizer that functions downstream of Agrin. The potential of DOK7 as a putative therapeutic target was demonstrated by adeno-associated virus (AAV)-mediated gene therapy delivery of DOK7 in Amyotrophic Lateral Sclerosis (ALS) and Emery Dreyefuss Muscular Dystrophy (EDMD). To assess the potential of DOK7 as a disease modifier of SMA, we administered AAV-DOK7 to an intermediate mouse model of SMA. AAV9-DOK7 treatment conferred improvements in NMJ architecture and reduced muscle fiber atrophy. Additionally, these improvements resulted in a subtle reduction in phenotypic severity, evidenced by improved grip strength and an extension in survival. These findings reveal DOK7 is a novel modifier of SMA.
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http://dx.doi.org/10.1016/j.bbrc.2020.07.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7453709PMC
September 2020

Minor snRNA gene delivery improves the loss of proprioceptive synapses on SMA motor neurons.

JCI Insight 2020 06 18;5(12). Epub 2020 Jun 18.

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri, USA.

Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder caused by reduced expression of the survival motor neuron (SMN) protein. SMN has key functions in multiple RNA pathways, including the biogenesis of small nuclear ribonucleoproteins that are essential components of both major (U2-dependent) and minor (U12-dependent) spliceosomes. Here we investigated the specific contribution of U12 splicing dysfunction to SMA pathology through selective restoration of this RNA pathway in mouse models of varying phenotypic severity. We show that virus-mediated delivery of minor snRNA genes specifically improves select U12 splicing defects induced by SMN deficiency in cultured mammalian cells, as well as in the spinal cord and dorsal root ganglia of SMA mice without increasing SMN expression. This approach resulted in a moderate amelioration of several parameters of the disease phenotype in SMA mice, including survival, weight gain, and motor function. Importantly, minor snRNA gene delivery improved aberrant splicing of the U12 intron-containing gene Stasimon and rescued the severe loss of proprioceptive sensory synapses on SMA motor neurons, which are early signatures of motor circuit dysfunction in mouse models. Taken together, these findings establish the direct contribution of U12 splicing dysfunction to synaptic deafferentation and motor circuit pathology in SMA.
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http://dx.doi.org/10.1172/jci.insight.130574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406293PMC
June 2020

Development of a novel severe mouse model of spinal muscular atrophy with respiratory distress type 1: FVB-nmd.

Biochem Biophys Res Commun 2019 12 8;520(2):341-346. Epub 2019 Oct 8.

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA; Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO, USA; Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO, USA. Electronic address:

Spinal Muscular Atrophy with Respiratory Distress type 1 (SMARD1) is an autosomal recessive disease that develops early during infancy. The gene responsible for disease development is immunoglobulin helicase μ-binding protein 2 (IGHMBP2). IGHMBP2 is a ubiquitously expressed gene but its mutation results in the loss of alpha-motor neurons and subsequent muscle atrophy initially of distal muscles. The current SMARD1 mouse model arose from a spontaneous mutation originally referred to as neuromuscular degeneration (nmd). The nmd mice have the C57BL/6 genetic background and contain an A to G mutation in intron 4 of the endogenous Ighmbp2 gene. This mutation causes aberrant splicing, resulting in only 20-25% of full-length functional protein. Several congenital conditions including hydrocephalus are common in the C57BL/6 background, consistent with our previous observations when developing a gene therapy approach for SMARD1. Additionally, a modifier allele exists on chromosome 13 in nmd mice that can partially suppress the phenotype, resulting in some animals that have extended life spans (up to 200 days). To eliminate the intrinsic complications of the C57BL/6 background and the variation in survival due to the genetic modifier effect, we created a new SMARD1 mouse model that contains the same intron 4 mutation in Ighmbp2 as nmd mice but is now on a FVB congenic background. FVB-nmd are consistently more severe than the original nmd mice with respect to survival, weigh and motor function. The relatively short life span (18-21 days) of FVB-nmd mice allows us to monitor therapeutic efficacy for a variety of novel therapeutics in a timely manner without the complication of the small percentage of longer-lived animals that were observed in our colony of nmd mice.
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http://dx.doi.org/10.1016/j.bbrc.2019.10.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936219PMC
December 2019

Functional characterization of SMN evolution in mouse models of SMA.

Sci Rep 2019 07 1;9(1):9472. Epub 2019 Jul 1.

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO, 65211, USA.

Spinal Muscular Atrophy (SMA) is a monogenic neurodegenerative disorder and the leading genetic cause of infantile mortality. While several functions have been ascribed to the SMN (survival motor neuron) protein, their specific contribution to the disease has yet to be fully elucidated. We hypothesized that some, but not all, SMN homologues would rescue the SMA phenotype in mouse models, thereby identifying disease-relevant domains. Using AAV9 to deliver Smn homologs to SMA mice, we identified a conservation threshold that marks the boundary at which homologs can rescue the SMA phenotype. Smn from Danio rerio and Xenopus laevis significantly prevent disease, whereas Smn from Drosophila melanogaster, Caenorhabditis elegans, and Schizosaccharomyces pombe was significantly less efficacious. This phenotypic rescue correlated with correction of RNA processing defects induced by SMN deficiency and neuromuscular junction pathology. Based upon the sequence conservation in the rescuing homologs, a minimal SMN construct was designed consisting of exons 2, 3, and 6, which showed a partial rescue of the SMA phenotype. While a significant extension in survival was observed, the absence of a complete rescue suggests that while the core conserved region is essential, additional sequences contribute to the overall ability of the SMN protein to rescue disease pathology.
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http://dx.doi.org/10.1038/s41598-019-45822-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6603021PMC
July 2019

Muscle fiber-type selective propensity to pathology in the nmd mouse model of SMARD1.

Biochem Biophys Res Commun 2019 08 28;516(1):313-319. Epub 2019 Jun 28.

Bond Life Sciences Center, University of Missouri, Columbia, MO, 65211, USA; Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO, 65211, USA. Electronic address:

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an autosomal recessive disease that causes distal limb muscle atrophy, due to motor neuron degeneration. Similar to other motor neuron diseases, SMARD1 shows differential vulnerability to denervation in various muscle groups, which is recapitulated in the nmd mouse, a model of SMARD1. In multiple neurodegenerative disease models, transcriptomic analysis has identified differentially expressed genes between vulnerable motor neuron populations, but the mechanism leading to susceptibility is largely unknown. To investigate if denervation vulnerability is linked to intrinsic muscle properties, we analyzed muscle fiber-type composition in muscles from motor units that show different degrees of denervation in nmd mice: gastrocnemius, tibialis anterior (TA), and extensor digitorum longus (EDL). Our results revealed that denervation vulnerability correlated with atrophy and loss of MyHC-IIb and MyHC-IIx muscle fiber types. Interestingly, increased vulnerability also correlated with an increased abundance of MyHC-I and MyHC-IIa muscle fibers. These results indicated that MyHC-IIx muscle fibers are the most vulnerable to denervation, followed by MyHC-IIb muscle fibers. Moreover, our data indicate that type MyHC-IIa and MyHC-IIb muscle fibers show resistance to denervation and compensate for the loss of MyHC-IIx and MyHC-IIb muscle fibers in the most vulnerable muscles. Taken together these results provide a basis for the selective vulnerability to denervation of specific muscles in nmd mice and identifies new targets for potential therapeutic intervention.
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http://dx.doi.org/10.1016/j.bbrc.2019.06.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662199PMC
August 2019

AAV9-mediated delivery of miR-23a reduces disease severity in Smn2B/-SMA model mice.

Hum Mol Genet 2019 10;28(19):3199-3210

Department of Veterinary Pathobiology, College of Veterinary Medicine and Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by deletions or mutations in survival motor neuron 1 (SMN1). The molecular mechanisms underlying motor neuron degeneration in SMA remain elusive, as global cellular dysfunction obscures the identification and characterization of disease-relevant pathways and potential therapeutic targets. Recent reports have implicated microRNA (miRNA) dysregulation as a potential contributor to the pathological mechanism in SMA. To characterize miRNAs that are differentially regulated in SMA, we profiled miRNA levels in SMA induced pluripotent stem cell (iPSC)-derived motor neurons. From this array, miR-23a downregulation was identified selectively in SMA motor neurons, consistent with previous reports where miR-23a functioned in neuroprotective and muscle atrophy-antagonizing roles. Reintroduction of miR-23a expression in SMA patient iPSC-derived motor neurons protected against degeneration, suggesting a potential miR-23a-specific disease-modifying effect. To assess this activity in vivo, miR-23a was expressed using a self-complementary adeno-associated virus serotype 9 (scAAV9) viral vector in the Smn2B/- SMA mouse model. scAAV9-miR-23a significantly reduced the pathology in SMA mice, including increased motor neuron size, reduced neuromuscular junction pathology, increased muscle fiber area, and extended survival. These experiments demonstrate that miR-23a is a novel protective modifier of SMA, warranting further characterization of miRNA dysfunction in SMA.
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http://dx.doi.org/10.1093/hmg/ddz142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6859438PMC
October 2019

A Direct Comparison of IV and ICV Delivery Methods for Gene Replacement Therapy in a Mouse Model of SMARD1.

Mol Ther Methods Clin Dev 2018 Sep 17;10:348-360. Epub 2018 Aug 17.

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an infantile autosomal recessive disease caused by the loss of the ubiquitously expressed gene. SMARD1 causes degeneration of alpha-motor neurons, resulting in distal muscle weakness, diaphragm paralysis, and respiratory malfunction. We have reported that delivery of a low dose of AAV9- to the CNS results in a significant rescue of the SMARD1 mouse model (). To examine how a delivery route can impact efficacy, a direct comparison of intravenous (IV) and intracerebroventricular (ICV) delivery of AAV9- was performed. Using a low-dose, both IV and ICV delivery routes led to a significant extension in survival and increased body weight. Conversely, only ICV-treated animals demonstrated improvements in the hindlimb muscle, neuromuscular junction, and motor function. The hindlimb phenotype of IV-treated mice resembled the untreated mice. We investigated whether the increased survival of IV-treated mice was the result of a positive impact on the cardiac function. Our results revealed that cardiac function and pathology were similarly improved in IV- and ICV-treated mice. We concluded that while IV delivery of a low dose does not improve the hindlimb phenotype and motor function, partial restoration of cardiac performance is sufficient to significantly extend survival.
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http://dx.doi.org/10.1016/j.omtm.2018.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6127875PMC
September 2018

Selective vulnerability in neuronal populations in nmd/SMARD1 mice.

Hum Mol Genet 2018 02;27(4):679-690

Department of Veterinary Pathobiology, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an autosomal recessive motor neuron disease causing distal limb muscle atrophy that progresses proximally and is accompanied by diaphragmatic paralysis. Neuromuscular junction (NMJ) alterations have been reported in muscles of SMARD1 model mice, known as nmd mice, with varying degrees of severity, suggesting that different muscles are specifically and selectively resistant or susceptible to denervation. To evaluate the extent of NMJ pathology in a broad range of muscles, a panel of axial and appendicular muscles were isolated and immunostained from nmd mice. These analyses revealed that selective distal appendage muscles were highly vulnerable to denervation. Susceptibility to pathology was not limited to NMJ alterations, but included defects in myelination within those neurons innervating susceptible muscles. Interestingly, end plate fragmentation was present within all muscles independent of the extent of NMJ alterations, suggesting that end plate fragmentation is an early hallmark of SMARD1 pathogenesis. Expressing the full-length IGHMBP2 cDNA using an adeno-associated virus (AAV9) significantly decreased all aspects of muscle and nerve disease pathology. These results shed new light onto the pathogenesis of SMARD1 by identifying specific motor units that are resistant and susceptible to neurodegeneration in an important model of SMARD1.
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http://dx.doi.org/10.1093/hmg/ddx434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886155PMC
February 2018

Self-oligomerization regulates stability of survival motor neuron protein isoforms by sequestering an SCF degron.

Mol Biol Cell 2018 01 22;29(2):96-110. Epub 2017 Nov 22.

Curriculum in Genetics and Molecular Biology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human  Expression of a duplicate gene () primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although  exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a  model system and label-free proteomics to identify the SCF ubiquitin E3 ligase complex as a novel SMN binding partner. SCF interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCF binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers.
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http://dx.doi.org/10.1091/mbc.E17-11-0627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909936PMC
January 2018

Optimization of a series of heterocycles as survival motor neuron gene transcription enhancers.

Bioorg Med Chem Lett 2017 12 26;27(23):5144-5148. Epub 2017 Oct 26.

Laboratory for Drug Discovery in Neurodegeneration, Brigham and Women's Hospital and Harvard Medical School, 65 Landsdowne Street, Cambridge, MA, USA. Electronic address:

Spinal muscular atrophy (SMA) is a neurodegenerative disorder that results from mutations in the SMN1 gene, leading to survival motor neuron (SMN) protein deficiency. One therapeutic strategy for SMA is to identify compounds that enhance the expression of the SMN2 gene, which normally only is a minor contributor to functional SMN protein production, but which is unaffected in SMA. A recent high-throughput screening campaign identified a 3,4-dihydro-4-phenyl-2(1H)-quinolinone derivative (2) that increases the expression of SMN2 by 2-fold with an EC = 8.3 µM. A structure-activity relationship (SAR) study revealed that the array of tolerated substituents, on either the benzo portion of the quinolinone or the 4-phenyl, was very narrow. However, the lactam ring of the quinolinone was more amenable to modifications. For example, the quinazolinone (9a) and the benzoxazepin-2(3H)-one (19) demonstrated improved potency and efficacy for increase in SMN2 expression as compared to 2.
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http://dx.doi.org/10.1016/j.bmcl.2017.10.066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701662PMC
December 2017

Optimization of -Splicing for Huntington's Disease RNA Therapy.

Front Neurosci 2017 10;11:544. Epub 2017 Oct 10.

Cedars-Sinai Medical Center, Board of Governors Regenerative Medicine Institute, Los Angeles, CA, United States.

Huntington's disease (HD) is a devastating neurodegenerative disorder caused by a polyglutamine (polyQ) expansion in exon 1 of the () gene. We have previously demonstrated that spliceosome-mediated -splicing is a viable molecular strategy to specifically reduce and repair mutant HTT (mtHTT). Here, the targeted tethering efficacy of the pre-mRNA -splicing modules (PTM) in HTT was optimized. Various PTMs that targeted the 3' end of HTT intron 1 or the intron 1 branch point were shown -splice into an HTT mini-gene, as well as the endogenous HTT pre-mRNA. PTMs that specifically target the endogenous intron 1 branch point increased the -splicing efficacy from 1-5 to 10-15%. Furthermore, lentiviral expression of PTMs in a human HD patient iPSC-derived neural culture significantly reversed two previously established polyQ-length dependent phenotypes. These results suggest that pre-mRNA repair of mtHTT could hold therapeutic benefit and it demonstrates an alternative platform to correct the mRNA product produced by the mt allele in the context of HD.
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http://dx.doi.org/10.3389/fnins.2017.00544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5641306PMC
October 2017

Astrocyte-produced miR-146a as a mediator of motor neuron loss in spinal muscular atrophy.

Hum Mol Genet 2017 09;26(17):3409-3420

Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, 53226 WI, USA.

Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, is caused by the loss of the survival motor neuron-1 (SMN1) gene, which leads to motor neuron loss, muscle atrophy, respiratory distress, and death. Motor neurons exhibit the most profound loss, but the mechanisms underlying disease pathogenesis are not fully understood. Recent evidence suggests that motor neuron extrinsic influences, such as those arising from astrocytes, contribute to motor neuron malfunction and loss. Here we investigated both loss-of-function and toxic gain-of-function astrocyte mechanisms that could play a role in SMA pathology. We had previously found that glial derived neurotrophic factor (GDNF) is reduced in SMA astrocytes. However, reduced GDNF expression does not play a major role in SMA pathology as viral-mediated GDNF re-expression did not improve astrocyte function or motor neuron loss. In contrast, we found that SMA astrocytes increased microRNA (miR) production and secretion compared to control astrocytes, suggesting potential toxic gain-of-function properties. Specifically, we found that miR-146a was significantly upregulated in SMA induced pluripotent stem cell (iPSC)-derived astrocytes and SMNΔ7 mouse spinal cord. Moreover, increased miR-146a was sufficient to induce motor neuron loss in vitro, whereas miR-146a inhibition prevented SMA astrocyte-induced motor neuron loss. Together, these data indicate that altered astrocyte production of miR-146a may be a contributing factor in astrocyte-mediated SMA pathology.
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http://dx.doi.org/10.1093/hmg/ddx230DOI Listing
September 2017

Analysis of Azithromycin Monohydrate as a Single or a Combinatorial Therapy in a Mouse Model of Severe Spinal Muscular Atrophy.

J Neuromuscul Dis 2017;4(3):237-249

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.

Background: Spinal muscular atrophy (SMA) is a neurodegenerative autosomal recessive disorder characterized by the loss of α-motor neurons. A variety of molecular pathways are being investigated to elevate SMN protein expression in SMA models and in the clinic. One of these approaches involves stabilizing the SMNΔ7 protein by inducing translational read-through. Previous studies have demonstrated that functionality and stability are partially restored to the otherwise unstable SMNΔ7 by the addition of non-specific C-terminal peptide sequences, or by inducing a similar molecular event through the use of read-through inducing compounds such as aminoglycosides.

Objective: The objective was to determine the efficacy of the macrolide Azithromycin (AZM), an FDA approved read-through-inducing compound, in the well-established severe mouse model of SMA.

Methods: Initially, dosing regimen following ICV administrations of AZM at different post-natal days and concentrations was determined by their impact on SMN levels in disease-relevant tissues. Selected dose was then tested for phenotypic parameters changes as compared to the appropriate controls and in conjugation to another therapy.

Results: AZM increases SMN protein in disease relevant tissues, however, this did not translate into similar improvements in the SMA phenotype in a severe mouse model of SMA. Co-administration of AZM and a previously developed antisense oligonucleotide that increases SMN2 splicing, resulted in an improvement in the SMA phenotype beyond either AZM or ASO alone, including a highly significant extension in survival with improvement in body weight and movement.

Conclusions: It is important to explore various approaches for SMA therapeutics, hence compounds that specifically induce SMNΔ7 read-through, without having prohibitive toxicity, may provide an alternative platform for a combinatorial treatment. Here we established that AZM activity at a low dose can increase SMN protein in disease-relevant animal model and can impact disease severity.
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http://dx.doi.org/10.3233/JND-170230DOI Listing
April 2018

Discovery of a Small Molecule Probe That Post-Translationally Stabilizes the Survival Motor Neuron Protein for the Treatment of Spinal Muscular Atrophy.

J Med Chem 2017 06 19;60(11):4594-4610. Epub 2017 May 19.

Laboratory for Drug Discovery in Neurodegeneration, Brigham & Women's Hospital and Harvard Medical School , 65 Landsdowne Street, Cambridge, Massachusetts 02139, United States.

Spinal muscular atrophy (SMA) is the leading genetic cause of infant death. We previously developed a high-throughput assay that employs an SMN2-luciferase reporter allowing identification of compounds that act transcriptionally, enhance exon recognition, or stabilize the SMN protein. We describe optimization and characterization of an analog suitable for in vivo testing. Initially, we identified analog 4m that had good in vitro properties but low plasma and brain exposure in a mouse PK experiment due to short plasma stability; this was overcome by reversing the amide bond and changing the heterocycle. Thiazole 27 showed excellent in vitro properties and a promising mouse PK profile, making it suitable for in vivo testing. This series post-translationally stabilizes the SMN protein, unrelated to global proteasome or autophagy inhibition, revealing a novel therapeutic mechanism that should complement other modalities for treatment of SMA.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5920559PMC
June 2017

Comparison of independent screens on differentially vulnerable motor neurons reveals alpha-synuclein as a common modifier in motor neuron diseases.

PLoS Genet 2017 03 31;13(3):e1006680. Epub 2017 Mar 31.

Centre for Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom.

The term "motor neuron disease" encompasses a spectrum of disorders in which motor neurons are the primary pathological target. However, in both patients and animal models of these diseases, not all motor neurons are equally vulnerable, in that while some motor neurons are lost very early in disease, others remain comparatively intact, even at late stages. This creates a valuable system to investigate the factors that regulate motor neuron vulnerability. In this study, we aim to use this experimental paradigm to identify potential transcriptional modifiers. We have compared the transcriptome of motor neurons from healthy wild-type mice, which are differentially vulnerable in the childhood motor neuron disease Spinal Muscular Atrophy (SMA), and have identified 910 transcriptional changes. We have compared this data set with published microarray data sets on other differentially vulnerable motor neurons. These neurons were differentially vulnerable in the adult onset motor neuron disease Amyotrophic Lateral Sclerosis (ALS), but the screen was performed on the equivalent population of neurons from neurologically normal human, rat and mouse. This cross species comparison has generated a refined list of differentially expressed genes, including CELF5, Col5a2, PGEMN1, SNCA, Stmn1 and HOXa5, alongside a further enrichment for synaptic and axonal transcripts. As an in vivo validation, we demonstrate that the manipulation of a significant number of these transcripts can modify the neurodegenerative phenotype observed in a Drosophila line carrying an ALS causing mutation. Finally, we demonstrate that vector-mediated expression of alpha-synuclein (SNCA), a transcript decreased in selectively vulnerable motor neurons in all four screens, can extend life span, increase weight and decrease neuromuscular junction pathology in a mouse model of SMA. In summary, we have combined multiple data sets to identify transcripts, which are strong candidates for being phenotypic modifiers, and demonstrated SNCA is a modifier of pathology in motor neuron disease.
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http://dx.doi.org/10.1371/journal.pgen.1006680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5391970PMC
March 2017

Plastin-3 extends survival and reduces severity in mouse models of spinal muscular atrophy.

JCI Insight 2017 03 9;2(5):e89970. Epub 2017 Mar 9.

Molecular Pathogeneses and Therapeutics Program.

Spinal muscular atrophy (SMA) is a leading genetic cause of infantile death and is caused by the loss of survival motor neuron-1 (). Importantly, a nearly identical gene is present called ; however, the majority of -derived transcripts are alternatively spliced and encode a truncated, dysfunctional protein. Recently, several compounds designed to increase SMN protein have entered clinical trials, including antisense oligonucleotides (ASOs), traditional small molecules, and gene therapy. Expanding beyond SMN-centric therapeutics is important, as it is likely that the breadth of the patient spectrum and the inherent complexity of the disease will be difficult to address with a single therapeutic strategy. Several SMN-independent pathways that could impinge upon the SMA phenotype have been examined with varied success. To identify disease-modifying pathways that could serve as stand-alone therapeutic targets or could be used in combination with an SMN-inducing compound, we investigated adeno-associated virus-mediated (AAV-mediated) gene therapy using plastin-3 (). Here, we report that AAV9- extends survival in an intermediate model of SMA mice as well as in a pharmacologically induced model of SMA using a splice-switching ASO that increases SMN production. coadministration improves the phenotype beyond the ASO, demonstrating the potential utility of combinatorial therapeutics in SMA that target SMN-independent and SMN-dependent pathways.
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http://dx.doi.org/10.1172/jci.insight.89970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333955PMC
March 2017

SMN deficiency negatively impacts red pulp macrophages and spleen development in mouse models of spinal muscular atrophy.

Hum Mol Genet 2017 03;26(5):932-941

Molecular Pathogeneses and Therapeutics Program.

Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease that is the leading genetic cause of infantile death. It is caused by a severe deficiency of the ubiquitously expressed Survival Motor Neuron (SMN) protein. SMA is characterized by α-lower motor neuron loss and muscle atrophy, however, there is a growing list of tissues impacted by a SMN deficiency beyond motor neurons. The non-neuronal defects are observed in the most severe Type I SMA patients and most of the widely used SMA mouse models, however, as effective therapeutics are developed, it is unclear whether additional symptoms will be uncovered in longer lived patients. Recently, the immune system and inflammation has been identified as a contributor to neurodegenerative diseases such as ALS. To determine whether the immune system is comprised in SMA, we analyzed the spleen and immunological components in SMA mice. In this report, we identify: a significant reduction in spleen size in multiple SMA mouse models and a pathological reduction in red pulp and extramedullary hematopoiesis. Additionally, red pulp macrophages, a discrete subset of yolk sac-derived macrophages, were found to be altered in SMA spleens even in pre-symptomatic post-natal day 2 animals. These cells, which are involved in iron metabolism and the phagocytosis of erythrocytes and blood-borne pathogens are significantly reduced prior to the development of the neurodegenerative hallmarks of SMA, implying a differential role of SMN in myeloid cell ontogeny. Collectively, these results demonstrate that SMN deficiency impacts spleen development and suggests a potential role for immunological development in SMA.
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http://dx.doi.org/10.1093/hmg/ddx008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6075362PMC
March 2017

Retraction notice: the SMN structure reveals its crucial role in snRNP assembly.

Hum Mol Genet 2016 12;25(24):5516

Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ, USA.

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http://dx.doi.org/10.1093/hmg/ddw355DOI Listing
December 2016

Optimization of Morpholino Antisense Oligonucleotides Targeting the Intronic Repressor Element1 in Spinal Muscular Atrophy.

Mol Ther 2016 09 9;24(9):1592-601. Epub 2016 Jul 9.

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri, USA.

Loss of Survival Motor Neuron-1 (SMN1) causes Spinal Muscular Atrophy, a devastating neurodegenerative disease. SMN2 is a nearly identical copy gene; however SMN2 cannot prevent disease development in the absence of SMN1 since the majority of SMN2-derived transcripts are alternatively spliced, encoding a truncated, unstable protein lacking exon 7. Nevertheless, SMN2 retains the ability to produce low levels of functional protein. Previously we have described a splice-switching Morpholino antisense oligonucleotide (ASO) sequence that targets a potent intronic repressor, Element1 (E1), located upstream of SMN2 exon 7. In this study, we have assessed a novel panel of Morpholino ASOs with the goal of optimizing E1 ASO activity. Screening for efficacy in the SMNΔ7 mouse model, a single ASO variant was more active in vivo compared with the original E1(MO)-ASO. Sequence variant eleven (E1(MOv11)) consistently showed greater efficacy by increasing the lifespan of severe Spinal Muscular Atrophy mice after a single intracerebroventricular injection in the central nervous system, exhibited a strong dose-response across an order of magnitude, and demonstrated excellent target engagement by partially reversing the pathogenic SMN2 splicing event. We conclude that Morpholino modified ASOs are effective in modifying SMN2 splicing and have the potential for future Spinal Muscular Atrophy clinical applications.
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http://dx.doi.org/10.1038/mt.2016.145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5113110PMC
September 2016

Rescue of a Mouse Model of Spinal Muscular Atrophy With Respiratory Distress Type 1 by AAV9-IGHMBP2 Is Dose Dependent.

Mol Ther 2016 05 10;24(5):855-66. Epub 2016 Feb 10.

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri, USA.

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an autosomal recessive disease occurring during childhood. The gene responsible for disease development is a ubiquitously expressed protein, IGHMBP2. Mutations in IGHMBP2 result in the loss of α-motor neurons leading to muscle atrophy in the distal limbs accompanied by respiratory complications. Although genetically and clinically distinct, proximal SMA is also caused by the loss of a ubiquitously expressed gene (SMN). Significant preclinical success has been achieved in proximal SMA using viral-based gene replacement strategies. We leveraged the technologies employed in SMA to demonstrate gene replacement efficacy in an SMARD1 animal model. Intracerebroventricular (ICV) injection of single-stranded AAV9 expressing the full-length cDNA of IGHMBP2 in a low dose led to a significant level of rescue in treated SMARD1 animals. Consistent with drastically increased survival, weight gain, and strength, the rescued animals demonstrated a significant improvement in muscle, NMJ, motor neurons, and axonal pathology. In addition, increased levels of IGHMBP2 in lumbar motor neurons verified the efficacy of the virus to transduce the target tissues. Our results indicate that AAV9-based gene replacement is a viable strategy for SMARD1, although dosing effects and potential negative impacts of high dose and ICV injection should be thoroughly investigated.
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http://dx.doi.org/10.1038/mt.2016.33DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4881770PMC
May 2016

Placental development in a mouse model of spinal muscular atrophy.

Biochem Biophys Res Commun 2016 Jan 31;470(1):82-87. Epub 2015 Dec 31.

Division of Biological Sciences, University of Missouri, Columbia, MO, USA; Department of Obstetrics, Gynecology and Women's Health, University of Missouri, Columbia, MO, USA. Electronic address:

Spinal Muscular Atrophy (SMA) is an autosomal recessive disorder, leading to fatal loss of motor neurons. It is caused by loss of function of the SMN gene, which is expressed throughout the body, and there is increasing evidence of dysfunction in non-neuronal tissues. Birthweight is one of most powerful prognostic factors for infants born with SMA, and intrauterine growth restriction is common. In the SMNΔ7 mouse model of SMA, pups with the disease lived 25% longer when their mothers were fed a higher fat, "breeder" diet. The placenta is responsible for transport of nutrients from mother to fetus, and is a major determinant of fetal growth. Thus, the present study tested the hypothesis that placental development is impaired in SMNΔ7 conceptuses. Detailed morphological characterization revealed no defects in SMNΔ7 placental development, and expression of key transcription factors regulating mouse placental development was unaffected. The intrauterine growth restriction observed in SMA infants likely does not result from impaired placental development.
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http://dx.doi.org/10.1016/j.bbrc.2015.12.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4755490PMC
January 2016

Astrocytes influence the severity of spinal muscular atrophy.

Hum Mol Genet 2015 Jul 24;24(14):4094-102. Epub 2015 Apr 24.

Department of Veterinary Pathobiology and Department of Molecular Microbiology and Immunology, University of Missouri, Bond Life Sciences Center, Columbia, MO 65211, USA,

Systemically low levels of survival motor neuron-1 (SMN1) protein cause spinal muscular atrophy (SMA). α-Motor neurons of the spinal cord are considered particularly vulnerable in this genetic disorder and their dysfunction and loss cause progressive muscle weakness, paralysis and eventually premature death of afflicted individuals. Historically, SMA was therefore considered a motor neuron-autonomous disease. However, depletion of SMN in motor neurons of normal mice elicited only a very mild phenotype. Conversely, restoration of SMN to motor neurons in an SMA mouse model had only modest effects on the SMA phenotype and survival. Collectively, these results suggested that additional cell types contribute to the pathogenesis of SMA, and understanding the non-autonomous requirements is crucial for developing effective therapies. Astrocytes are critical for regulating synapse formation and function as well as metabolic support for neurons. We hypothesized that astrocyte functions are disrupted in SMA, exacerbating disease progression. Using viral-based restoration of SMN specifically to astrocytes, survival in severe and intermediate SMA mice was observed. In addition, neuromuscular circuitry was improved. Astrogliosis was prominent in end-stage SMA mice and in post-mortem patient spinal cords. Increased expression of proinflammatory cytokines was partially normalized in treated mice, suggesting that astrocytes contribute to the pathogenesis of SMA.
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http://dx.doi.org/10.1093/hmg/ddv148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007659PMC
July 2015

The SMN structure reveals its crucial role in snRNP assembly.

Hum Mol Genet 2015 Apr 5;24(8):2138-46. Epub 2015 Jan 5.

Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287, USA and

The spliceosome plays a fundamental role in RNA metabolism by facilitating pre-RNA splicing. To understand how this essential complex is formed, we have used protein crystallography to determine the first complete structures of the key assembler protein, SMN, and the truncated isoform, SMNΔ7, which is found in patients with the disease spinal muscular atrophy (SMA). Comparison of the structures of SMN and SMNΔ7 shows many similar features, including the presence of two Tudor domains, but significant differences are observed in the C-terminal domain, including 12 additional amino acid residues encoded by exon 7 in SMN compared with SMNΔ7. Mapping of missense point mutations found in some SMA patients reveals clustering around three spatial locations, with the largest cluster found in the C-terminal domain. We propose a structural model of SMN binding with the Gemin2 protein and a heptameric Sm ring, revealing a critical assembly role of the residues 260-294, with the differences at the C-terminus of SMNΔ7 compared with SMN likely leading to loss of small nuclear ribonucleoprotein (snRNP) assembly. The SMN complex is proposed to form a dimer driven by formation of a glycine zipper involving α helix formed by amino acid residues 263-294. These results explain how structural changes of SMN give rise to loss of SMN-mediated snRNP assembly and support the hypothesis that this loss results in atrophy of neurons in SMA.
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http://dx.doi.org/10.1093/hmg/ddu734DOI Listing
April 2015
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