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Motor neuron diseases (MNDs) are neuromuscular disorders where the spinal motor neurons-either the cell bodies themselves or their axons-are the primary cells affected. To date, there are 120 different genes that are lost or mutated in pediatric-onset MNDs. Most of these childhood-onset disorders, aside from spinal muscular atrophy (SMA), lack viable therapeutic options. Previous research onMNDs has focused on understanding the pathobiology of a single, specific gene mutation and targeting therapies to that pathobiology. This reductionist approach has yielded therapeutic options for a specific disorder, in this case SMA. Unfortunately, therapies specific for SMA have not been effective against other pediatric-onset MNDs. Pursuing the same approach for the other defined MNDs would require development of at least 120 independent treatments raising feasibility issues. We propose an alternative to this this type of reductionist approach by conceptualizing MNDs in a complex adaptive systems framework that will allow identification of common molecular and cellular pathways which form biological networks that are adversely affected in early-onset MNDs and thus MNDs with similar phenotypes despite diverse genotypes. This systems biology approach highlights the complexity and self-organization of the motor system as well as the ways in which it can be affected by these genetic disorders. Using this integrated approach to understand early-onset MNDs, we would be better poised to expand the therapeutic repertoire for multiple MNDs. [ABSTRACT FROM AUTHOR]

Simple Summary: Transposable elements are DNA sequences that can move throughout the genome. They play essential roles in gene regulation and function. Spinal muscular atrophy (SMA) is a leading genetic cause of infant mortality worldwide. Since transposable elements have been linked to other genetic diseases, we examined the genomes from SMA patients as well as healthy genomes for the presence of transposable elements. We identified distinct transposable elements that may impact gene expression by affecting promoter activity or transcriptional termination of the SMN genes. These elements within the SMA genes may play key roles in understanding this early-onset neurodegenerative disease as well as how transposable elements can impact gene expression. Understanding the roles of transposable elements in SMA may provide key insights into other neurodegenerative diseases. Transposable elements (TEs) are interspersed repetitive and mobile DNA sequences within the genome. Better tools for evaluating TE-derived sequences have provided insights into the contribution of TEs to human development and disease. Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease that is caused by deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene but retention of its nearly perfect orthologue SMN2. Both genes are highly enriched in TEs. To establish a link between TEs and SMA, we conducted a comprehensive, in silico analysis of TE insertions within the SMN1/2 loci of SMA, carrier and healthy genomes. We found an Alu insertion in the promoter region and one L1 element in the 3′UTR that may play an important role in alternative promoter as well as in alternative transcriptional termination. Additionally, several intronic Alu repeats may influence alternative splicing via RNA circularization and causes the presence of new alternative exons. These Alu repeats present throughout the genes are also prone to recombination events that could lead to SMN1 exons deletions and, ultimately, SMA. TE characterization of the SMA genomic region could provide for a better understanding of the implications of TEs on human disease and genomic evolution. [ABSTRACT FROM AUTHOR]

C5-substituted 2,4-diaminoquinazolines (2,4-DAQs) ameliorate disease severity in SMA mice. It is uncertain, however, that these compounds increase SMN protein levels in vivo even though they were identified as activators of the SMN2 promoter. These compounds also regulate the expression of other transcripts in neuroblastoma cells. In this study, we investigate the mechanism by which the 2,4-DAQs regulate the expression of SMN2 as well as other targets. D156844, D158872, D157161 and D157495 (RG3039) increased SMN2 promoter-driven reporter gene activity by at least 3-fold in NSC-34 cells. These compounds, however, did not significantly increase SMN2 mRNA levels in type II SMA fibroblasts nor in NSC-34 cells, although there was a trend for these compounds increasing SMN protein in SMA fibroblasts. The number of SMN-containing gems was increased in SMA fibroblasts in response to 2,4-DAQ treatment in a dose-dependent manner. ATOH7 mRNA levels were significantly lower in type II SMA fibroblasts. 2,4-DAQs significantly increased ATOH7, DRNT1 and DRTN2 transcript levels in type II SMA fibroblasts and restored ATOH7 levels to those observed in healthy fibroblasts. These compounds also increase Atoh7 mRNA expression in NSC-34 cells. In conclusion, 2,4-DAQs regulate SMN2 by increasing protein levels and gem localization. They also increase ATOH7, DRNT1 and DRNT2 transcript levels. This study reveals that the protective effects of 2,4-DAQs in SMA may be independent of SMN2 gene regulation. These compounds could be used in concert with a proven SMN2 inducer to develop a multi-faceted approach to treating SMA. [ABSTRACT FROM AUTHOR]

Proximal spinal muscular atrophy ( SMA) is an early-onset motor neuron disease characterized by loss of α-motor neurons and associated muscle atrophy. SMA is caused by deletion or other disabling mutation of survival motor neuron 1 ( SMN1). In the human genome, a large duplication of the SMN-containing region gives rise to a second copy of this gene ( SMN2) that is distinguishable by a single nucleotide change in exon 7. Within the SMA population, there is substantial variation in SMN2 copy number; in general, those individuals with SMA who have a high SMN2 copy number have a milder disease. Because SMN2 functions as a disease modifier, its accurate copy number determination may have clinical relevance. In this study, we describe the development of an assay to assess SMN1 and SMN2 copy numbers in DNA samples using an array-based digital PCR ( dPCR) system. This dPCR assay can accurately and reliably measure the number of SMN1 and SMN2 copies in DNA samples. In a cohort of SMA patient-derived cell lines, the assay confirmed a strong inverse correlation between SMN2 copy number and disease severity. Array dPCR is a practical technique to determine, accurately and reliably, SMN1 and SMN2 copy numbers from SMA samples. [ABSTRACT FROM AUTHOR]

Spinal muscular atrophy (SMA), a leading genetic cause of infant death worldwide, is an autosomal recessive disorder caused by the loss of SMN1 (survival motor neuron 1), which encodes the protein SMN. The loss of SMN1 causes a deficiency in SMN protein levels leading to motor neuron cell death in the anterior horn of the spinal cord. SMN2, however, can also produce some functional SMN to partially compensate for loss of SMN1 in SMA suggesting increasing transcription of SMN2 as a potential therapy to treat patients with SMA. A cAMP response element was identified on the SMN2 promoter, implicating cAMP activation as a step in the transcription of SMN2. Therefore, we investigated the effects of modulating the cAMP signaling cascade on SMN production in vitro and in silico. SMA patient fibroblasts were treated with the cAMP signaling modulators rolipram, salbutamol, dbcAMP, epinephrine and forskolin. All of the modulators tested were able to increase gem formation, a marker for SMN protein in the nucleus, in a dose-dependent manner. We then derived two possible mathematical models simulating the regulation of SMN2 expression by cAMP signaling. Both models fit well with our experimental data. In silico treatment of SMA fibroblasts simultaneously with two different cAMP modulators resulted in an additive increase in gem formation. This study shows how a systems biology approach can be used to develop potential therapeutic targets for treating SMA. [ABSTRACT FROM AUTHOR]

Proximal spinal muscular atrophy (SMA) is an early onset, autosomal recessive motor neuron disease caused by loss of or mutation in SMN1 (survival motor neuron 1). Despite understanding the genetic basis underlying this disease, it is still not known why motor neurons (MNs) are selectively affected by the loss of the ubiquitously expressed SMN protein. Using a mouse embryonic stem cell (mESC) model for severe SMA, the RNA transcript profiles (transcriptomes) between control and severe SMA (SMN2+/+;mSmn−/−) mESC-derived MNs were compared in this study using massively parallel RNA sequencing (RNA-Seq). The MN differentiation efficiencies between control and severe SMA mESCs were similar. RNA-Seq analysis identified 3,094 upregulated and 6,964 downregulated transcripts in SMA mESC-derived MNs when compared against control cells. Pathway and network analysis of the differentially expressed RNA transcripts showed that pluripotency and cell proliferation transcripts were significantly increased in SMA MNs while transcripts related to neuronal development and activity were reduced. The differential expression of selected transcripts such as Crabp1, Crabp2 and Nkx2.2 was validated in a second mESC model for SMA as well as in the spinal cords of low copy SMN2 severe SMA mice. Furthermore, the levels of these selected transcripts were restored in high copy SMN2 rescue mouse spinal cords when compared against low copy SMN2 severe SMA mice. These findings suggest that SMN deficiency affects processes critical for normal development and maintenance of MNs. [ABSTRACT FROM AUTHOR]

In the present study, we investigated the role of membrane cholesterol in the function of glutamate transporters. Depletion of membrane cholesterol by methylf-β-cyclodextrin resulted in reduced Na+-dependent glutamate uptake in primary cortical cultures. Glial glutamate transporter EAAT2-mediated uptake was more sensitive to this effect. Cell surface biotinylation and immunostaining experiments revealed that the loss of cholesterol significantly altered the trafficking of EAAT2 to the plasma membrane as well as their membrane distribution. These effects were also observed in neuronal glutamate transporter EAAT3 but to a lesser extent. Furthermore, the treatment of mouse brain plasma membrane vesicles with methyl-β-cyclodextrin resulted in a significant reduction in glutamate uptake, suggesting that cholesterol depletion has a direct effect on the function of the glutamate transporters. Plasma membrane cholesterol is localized within discreet microdomains known as lipid rafts. Analyses of purified lipid raft microdomains revealed that a large portion of total EAAT2 and a minor portion of total EAAT1, EAAT3, and EAAT4 were associated with lipid rafts. Artificial aggregation of lipid rafts in vivo resulted in the formation of larger EAAT2-immunoreactive clusters on the cell surface. The purified lipid raft-associated fractions were capable of Na+-dependent glutamate uptake. Our data suggest that the glutamate transporters, especially EAAT2, are associated with cholesterol-rich lipid raft microdomains of the plasma membrane and that the association with these cholesterol-rich microdomains is important for excitatory amino acid transporter localization and function. [ABSTRACT FROM AUTHOR]

Spinal muscular atrophy (SMA) is a leading genetic cause of infant death worldwide that is characterized by loss of spinal motor neurons leading to muscle weakness and atrophy. SMA results from the loss of survival motor neuron 1 (SMN1) gene but retention of its paralog SMN2. The copy numbers of SMN1 and SMN2 are variable within the human population with SMN2 copy number inversely correlating with SMA severity. Current therapeutic options for SMA focus on increasing SMN2 expression and alternative splicing so as to increase the amount of SMN protein. Recent work has demonstrated that not all SMN2, or SMN1, genes are equivalent and there is a high degree of genomic heterogeneity with respect to the SMN genes. Because SMA is now an actionable disease with SMN2 being the primary target, it is imperative to have a comprehensive understanding of this genomic heterogeneity with respect to hybrid SMN1–SMN2 genes generated by gene conversion events as well as partial deletions of the SMN genes. This review will describe this genetic heterogeneity in SMA and its impact on disease phenotype as well as therapeutic efficacy. [ABSTRACT FROM AUTHOR]

Abnormal splicing of astroglial glutamate transporter EAAT2 mRNA has been suggested to account for the loss of EAAT2 protein in amyotrophic lateral sclerosis (ALS) and Alzheimer''s disease (AD). We have identified several clones of human U251 glioma cells which express varying amounts of aberrantly spliced EAAT2 mRNA; these clones do not express any detectable EAAT2 protein. When the wild-type EAAT2 cDNA was expressed in each of these clones, we found that the amount of EAAT2 protein inversely correlated with the levels of endogenous aberrant EAAT2 mRNA. We also observed that ectopic expression of normal EAAT2 protein is toxic to U251 cells as well as to undifferentiated primary astrocytes. We conclude that expression of aberrant EAAT2 mRNA may be one possible mechanism to repress normal EAAT2 protein expression. The implication of this study for the mechanisms of EAAT2 protein loss in ALS and AD is discussed. [Copyright &y& Elsevier]

Cachexia is a debilitating condition characterized by extreme skeletal muscle wasting that contributes significantly to morbidity and mortality. Efforts to elucidate the underlying mechanisms of muscle loss have predominantly focused on events intrinsic to the myofiber. In contrast, less regard has been given to potential contributory factors outside the fiber within the muscle microenvironment. In tumor-bearing mice and patients with pancreatic cancer, we found that cachexia was associated with a type of muscle damage resulting in activation of both satellite and nonsatellite muscle progenitor cells. These muscle progenitors committed to a myogenic program, but were inhibited from completing differentiation by an event linked with persistent expression of the self-renewing factor Pax7. Overexpression of Pax7 was sufficient to induce atrophy in normal muscle, while under tumor conditions, the reduction of Pax7 or exogenous addition of its downstream target, MyoD, reversed wasting by restoring cell differentiation and fusion with injured fibers. Furthermore, Pax7 was induced by serum factors from cachectic mice and patients, in an NF-κB-dependent manner, both in vitro and in vivo. Together, these results suggest that Pax7 responds to NF-κB by impairing the regenerative capacity of myogenic cells in the muscle microenvironment to drive muscle wasting in cancer. [ABSTRACT FROM AUTHOR]

Glutamate is the major excitatory neurotransmitter in the central nervous system. Its activity is carefully modulated in the synaptic cleft by glutamate transporters. The glial glutamate transporter EAAT2 is the main mediator of glutamate clearance. Reduced EAAT2 function could lead to accumulation of extracellular glutamate, resulting in a form of cell death known as excitotoxicity. In amyotrophic lateral sclerosis and Alzheimer disease, EAAT2 protein levels are significantly decreased in affected areas. EAAT2 mRNA levels, however, remain constant, indicating that alterations in EAAT2 expression are due to disturbances at the post-transcriptional level. In the present study, we found that some EAAT2 transcripts contained 5′-untranslated regions (5′-UTRs) greater than 300 nucleotides. The mRNAs that bear long 5′-UTRs are often regulated at the translational level. We tested this possibility initially in a primary astrocyte line that constantly expressed an EAAT2 transcript containing the 565-nt 5′-UTR and found that translation of this transcript was regulated by many extracellular factors, including corticosterone and retinol. Moreover, many disease-associated insults affected the efficiency of translation of this transcript. Importantly, this translational regulation of EAAT2 occurred in vivo (i.e. both in primary cortical neurons-astrocytes mixed cultures and in mice). These results indicate that expression of EAAT2 protein is highly regulated at the translational level and also suggest that translational regulation may play an important role in the differential EAAT2 protein expression under normal and disease conditions. [ABSTRACT FROM AUTHOR]

Spinal muscular atrophy (SMA) is an autosomal recessive, pediatric-onset disorder caused by the loss of spinal motor neurons, thereby leading to muscle atrophy. SMA is caused by the loss of or mutations in the survival motor neuron 1 ( SMN1 ) gene. SMN1 is duplicated in humans to give rise to the paralogous survival motor neuron 2 ( SMN2 ) gene. This paralog is nearly identical except for a cytosine to thymine transition within an exonic splicing enhancer element within exon 7. As a result, the majority of SMN2 transcripts lack exon 7 (SMNΔ7), which produces a truncated and unstable SMN protein. Since SMN2 copy number is inversely related to disease severity, it is a well established target for SMA therapeutics development. 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of sodium/proton exchangers (NHEs), has previously been shown to increase exon 7 inclusion and SMN protein levels in SMA cells. In this study, NHE inhibitors were evaluated for their ability to modulate SMN2 expression. EIPA as well as 5-(N,N-hexamethylene)amiloride (HMA) increase exon 7 inclusion in SMN2 splicing reporter lines as well as in SMA fibroblasts. The EIPA-induced exon 7 inclusion occurs via a unique mechanism that does not involve previously identified splicing factors. Transcriptome analysis identified novel targets, including TIA1 and FABP3 , for further characterization. EIPA and HMA are more selective at inhibiting the NHE5 isoform, which is expressed in fibroblasts as well as in neuronal cells. These results show that NHE5 inhibition increases SMN2 expression and may be a novel target for therapeutics development. SIGNIFICANCE STATEMENT: This study demonstrates a molecular mechanism by which inhibitors of the sodium-protein exchanger increase the alternative splicing of SMN2 in spinal muscular atrophy cells. NHE5 selective inhibitors increase the inclusion of full-length SMN2 mRNAs by targeting TIA1 and FABP3 expression, which is distinct from other small molecule regulators of SMN2 alternative splicing. This study provides a novel means to increase full-length SMN2 expression and a novel target for therapeutics development., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Purpose: Spinal muscular atrophy (SMA), caused by loss of the SMN1 gene, is a leading cause of early childhood death. Due to the near identical sequences of SMN1 and SMN2, analysis of this region is challenging. Population-wide SMA screening to quantify the SMN1 copy number (CN) is recommended by the American College of Medical Genetics and Genomics., Methods: We developed a method that accurately identifies the CN of SMN1 and SMN2 using genome sequencing (GS) data by analyzing read depth and eight informative reference genome differences between SMN1/2., Results: We characterized SMN1/2 in 12,747 genomes, identified 1568 samples with SMN1 gains or losses and 6615 samples with SMN2 gains or losses, and calculated a pan-ethnic carrier frequency of 2%, consistent with previous studies. Additionally, 99.8% of our SMN1 and 99.7% of SMN2 CN calls agreed with orthogonal methods, with a recall of 100% for SMA and 97.8% for carriers, and a precision of 100% for both SMA and carriers., Conclusion: This SMN copy-number caller can be used to identify both carrier and affected status of SMA, enabling SMA testing to be offered as a comprehensive test in neonatal care and an accurate carrier screening tool in GS sequencing projects.

Systems biology uses a combination of experimental and mathematical approaches to investigate the complex and dynamic interactions with a given system or biological process. Systems biology integrates genetics, signal transduction, biochemistry and cell biology with mathematical modeling. It can be used to identify novel pathways implicated in diseases as well as to understand the mechanisms by which a specific gene is regulated. This review describes the development of mathematical models for the regulation of an endogenous modifier gene, SMN2, in spinal muscular atrophy-an early-onset motor neuron disease that is a leading genetic cause of infant mortality worldwide-by cAMP signaling. These mathematical models not only can aid in understanding how SMN2 expression is regulated but they can also be used to examine the best ways to manipulate cAMP signaling to maximally increase SMN2 expression. These models will lead to the development of therapeutic strategies for treating SMA. This systems biology approach can also be applied to other neurological diseases, particularly those in which a disease-causing gene or a modifier gene has been identified.

Fibroblasts and lymphoblastoid cell lines (LCLs) derived from individuals with spinal muscular atrophy (SMA) have been and continue to be essential for translational SMA research. Authentication of cell lines helps ensure reproducibility and rigor in biomedical research. This quality control measure identifies mislabeling or cross-contamination of cell lines and prevents misinterpretation of data. Unfortunately, authentication of SMA cell lines used in various studies has not been possible because of a lack of a reference. In this study, we provide said reference so that SMA cell lines can be subsequently authenticated. We use short tandem repeat (STR) profiling and digital PCR (dPCR), which quantifies SMN1 and SMN2 copy numbers, to generate molecular identity codes for fibroblasts and LCLs that are commonly used in SMA research. Using these molecular identity codes, we clarify the familial relationships within a set of fibroblasts commonly used in SMA research. This study presents the first cell line reference set for the SMA research community and demonstrates its usefulness for re-identification and authentication of lines commonly used as in vitro models for future studies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Spinal muscular atrophy (SMA) is an early-onset motor neuron disease that leads to loss of muscle function. Butyrate (BA)-based compounds markedly improve the survival and motor phenotype of SMA mice. In this study, we examine the protective effects of the BA prodrug pivaloyloxymethyl butyrate (AN9) on the survival of SMNΔ7 SMA mice. Oral administration of AN9 beginning at PND04 almost doubled the average lifespan of SMNΔ7 SMA mice. AN9 treatment also increased the growth rate of SMNΔ7 SMA mice when compared to vehicle-treated SMNΔ7 SMA mice. In conclusion, BA prodrugs like AN9 have ameliorative effects on SMNΔ7 SMA mice., Competing Interests: AUTHORS CONFLICTS OF INTEREST The authors have no conflict of interest to report.

Topoisomerase 1 (TOP1) poisons like camptothecin (CPT) are currently used in cancer chemotherapy but these compounds can have damaging, off-target effects on neurons leading to cognitive, sensory and motor deficits. To understand the molecular basis for the enhanced sensitivity of neurons to CPT, we examined the effects of compounds that inhibit TOP1-CPT, actinomycin D (ActD) and β-lapachone (β-Lap)-on primary cultured rat motor (MN) and cortical (CN) neurons as well as fibroblasts. Neuronal cells expressed higher levels of Top1 mRNA than fibroblasts but transcript levels are reduced in all cell types after treatment with CPT. Microarray analysis was performed to identify differentially regulated transcripts in MNs in response to a brief exposure to CPT. Pathway analysis of the differentially expressed transcripts revealed activation of ERK and JNK signaling cascades in CPT-treated MNs. Immediate-early genes like Fos, Egr-1 and Gadd45b were upregulated in CPT-treated MNs. Fos mRNA levels were elevated in all cell types treated with CPT; Egr-1, Gadd45b and Dyrk3 transcript levels, however, increased in CPT-treated MNs and CNs but decreased in CPT-treated fibroblasts. These transcripts may represent new targets for the development of therapeutic agents that mitigate the off-target effects of chemotherapy on the nervous system., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Early-onset rare diseases have a strong impact on child healthcare even though the incidence of each of these diseases is relatively low. In order to better manage the care of these children, it is imperative to quickly diagnose the molecular bases for these disorders as well as to develop technologies with prognostic potential. Digital PCR (dPCR) is well suited for this role by providing an absolute quantification of the target DNA within a sample. This review illustrates how dPCR can be used to identify genes associated with pediatric-onset disorders, to identify copy number status of important disease-causing genes and variants and to quantify modifier genes. It is also a powerful technology to track changes in genomic biomarkers with disease progression. Based on its capability to accurately and reliably detect genomic alterations with high sensitivity and a large dynamic detection range, dPCR has the potential to become the tool of choice for the verification of pediatric disease-associated mutations identified by next generation sequencing, copy number determination and noninvasive prenatal screening.

Proximal spinal muscular atrophy (SMA) is a childhood-onset degenerative disease resulting from the selective loss of motor neurons in the spinal cord. SMA is caused by the loss of SMN1 (survival motor neuron 1) but retention of SMN2. The number of copies of SMN2 modifies disease severity in SMA patients as well as in mouse models, making SMN2 a target for therapeutics development. Sodium butyrate (BA) and its analog (4PBA) have been shown to increase SMN2 expression in SMA cultured cells. In this study, we examined the effects of BA, 4PBA as well as two BA prodrugs-glyceryl tributyrate (BA3G) and VX563-on the phenotype of SMNΔ7 SMA mice. Treatment with 4PBA, BA3G and VX563 but not BA beginning at PND04 significantly improved the lifespan and delayed disease end stage, with administration of VX563 also improving the growth rate of these mice. 4PBA and VX563 improved the motor phenotype of SMNΔ7 SMA mice and prevented spinal motor neuron loss. Interestingly, neither 4PBA nor VX563 had an effect on SMN expression in the spinal cords of treated SMNΔ7 SMA mice; however, they inhibited histone deacetylase (HDAC) activity and restored the normal phosphorylation states of Akt and glycogen synthase kinase 3β, both of which are altered by SMN deficiency in vivo. These observations show that BA-based compounds with favorable pharmacokinetics ameliorate SMA pathology possibly by modulating HDAC and Akt signaling., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Proximal spinal muscular atrophy (SMA), a leading genetic cause of infant death worldwide, is an early-onset, autosomal recessive neurodegenerative disease characterized by the loss of spinal α-motor neurons. This loss of α-motor neurons is associated with muscle weakness and atrophy. SMA can be classified into five clinical grades based on age of onset and severity of the disease. Regardless of clinical grade, proximal SMA results from the loss or mutation of SMN1 (survival motor neuron 1) on chromosome 5q13. In humans a large tandem chromosomal duplication has lead to a second copy of the SMN gene locus known as SMN2. SMN2 is distinguishable from SMN1 by a single nucleotide difference that disrupts an exonic splice enhancer in exon 7. As a result, most of SMN2 mRNAs lack exon 7 (SMNΔ7) and produce a protein that is both unstable and less than fully functional. Although only 10-20% of the SMN2 gene product is fully functional, increased genomic copies of SMN2 inversely correlates with disease severity among individuals with SMA. Because SMN2 copy number influences disease severity in SMA, there is prognostic value in accurate measurement of SMN2 copy number from patients being evaluated for SMA. This prognostic value is especially important given that SMN2 copy number is now being used as an inclusion criterion for SMA clinical trials. In addition to SMA, copy number variations (CNVs) in the SMN genes can affect the clinical severity of other neurological disorders including amyotrophic lateral sclerosis (ALS) and progressive muscular atrophy (PMA). This review will discuss how SMN1 and SMN2 CNVs are detected and why accurate measurement of SMN1 and SMN2 copy numbers is relevant for SMA and other neurodegenerative diseases.

Spinal muscular atrophy (SMA), a leading genetic cause of pediatric death in the world, is an early-onset disease affecting the motor neurons in the anterior horn of the spinal cord. This degeneration of motor neurons leads to loss of muscle function. At the molecular level, SMA results from the loss of or mutation in the survival motor neuron 1 (SMN1) gene. The number of copies of the nearly duplicated gene SMN2 modulates the disease severity in humans as well as in transgenic mouse models for SMA. Most preclinical therapeutic trials focus on identifying ways to increase SMN2 expression and to alter its splicing. Other therapeutic strategies have investigated compounds which protect affected motor neurons and their target muscles in an SMN-independent manner. In the present study, the effect of a combination regimen of the SMN2 inducer D156844 and the protectant follistatin on the disease progression and survival was measured in the SMNΔ7 SMA mouse model. The D156844/follistatin combination treatment improved the survival of, delayed the end stage of disease in and ameliorated the growth rate of SMNΔ7 SMA mice better than follistatin treatment alone. The D156844/follistatin combination treatment, however, did not provide additional benefit over D156844 alone with respect to survival and disease end stage even though it provided some additional therapeutic benefit over D156844 alone with respect to motor phenotype., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Spinal muscular atrophy (SMA) is an early-onset motor neuron disease characterized by loss of spinal motor neurons which leads to skeletal muscle atrophy. Proximal SMA results from the loss or mutation of the survival motor neuron (SMN) gene. In humans, the SMN gene is duplicated to produce two nearly identical genes, SMN1 and SMN2. SMN1 is lost in SMA but SMN2 is retained; in fact, the number of SMN2 copies correlates with disease severity. The SMN2 inducer D156844 increases the survival and improves phenotype of SMN∆7 SMA mice. Maternal diet also modifies the survival and phenotype of these mice. In this study, we show the effect of maternal diet on the protective effects of D156844 in SMN∆7 SMA mice. SMA mice maintained on the PicoLab20 Mouse diet survived longer when treated with D156844; the effect of diet was additive to the effect of D156844 on these mice. Brain levels of D156844 were higher in neonatal mice maintained on the PicoLab20 diet than those on the Harlan-Teklad 22/5 diet. SMN protein levels in the spinal cord were modestly elevated in D156844-treated, PicoLab20-maintained SMA mice. These data show that maternal diet does influence the responsiveness of D156844 in neonatal SMN∆7 SMA mice., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Proximal spinal muscular atrophy (SMA), one of the most common genetic causes of infant death, results from the selective loss of motor neurons in the spinal cord. SMA is a consequence of low levels of survival motor neuron (SMN) protein. In humans, the SMN gene is duplicated; SMA results from the loss of SMN1 but SMN2 remains intact. SMA severity is related to the copy number of SMN2. Compounds which increase the expression of SMN2 could, therefore, be potential therapeutics for SMA. Ultrahigh-throughput screening recently identified substituted quinazolines as potent SMN2 inducers. A series of C5-quinazoline derivatives were tested for their ability to increase SMN expression in vivo. Oral administration of three compounds (D152344, D153249 and D156844) to neonatal mice resulted in a dose-dependent increase in Smn promoter activity in the central nervous system. We then examined the effect of these compounds on the progression of disease in SMN lacking exon 7 (SMNDelta7) SMA mice. Oral administration of D156844 significantly increased the mean lifespan of SMNDelta7 SMA mice by approximately 21-30% when given prior to motor neuron loss. In summary, the C5-quinazoline derivative D156844 increases SMN expression in neonatal mouse neural tissues, delays motor neuron loss at PND11 and ameliorates the motor phenotype of SMNDelta7 SMA mice.

Proximal spinal muscular atrophy (SMA) is a leading genetic cause of infant death. Patients with SMA lose alpha-motor neurons in the ventral horn of the spinal cord which leads to skeletal muscle weakness and atrophy. SMA is the result of reduction in Survival Motor Neuron (SMN) expression. Transgenic mouse models of SMA have been generated and are extremely useful in understanding the mechanisms of motor neuron degeneration in SMA and in developing new therapeutic candidates for SMA patients. Several research groups have reported varying average lifespans of SMNDelta7 SMA mice (SMN2(+/+);SMNDelta7(+/+);mSmn(-/-)), the most commonly used mouse model for preclinical therapeutic candidate testing. One environmental factor that varied between research groups was maternal diet. In this study, we compared the effects of two different commercially available rodent chows (PicoLab20 Mouse diet and Harlan-Teklad 22/5 diet) on the survival and motor phenotype of the SMNDelta7 mouse model of SMA. Specifically, the PicoLab20 diet significantly extends the average lifespan of the SMNDelta7 SMA mice by approximately 25% and improved the motor phenotype as compared to the Harlan diet. These findings indicate that maternal diet alone can have considerable impact on the SMA phenotype., (Copyright 2009 Elsevier Inc. All rights reserved.)

Spinal muscular atrophy (SMA), the leading genetic cause of infant death results from loss of spinal motor neurons causing atrophy of skeletal muscle. SMA is caused by loss of the Survival Motor Neuron 1 (SMN1) gene, however, an identically coding gene called SMN2 is retained, but is alternatively spliced to produce approximately 90% truncated protein. Most SMA translational and preclinical drug development has relied on the use of SMA mice to determine changes in SMN protein levels. However, the SMA mouse models are relatively severe and analysis of SMN-inducing compounds is confounded by the early mortality of these animals. An antibody that could detect SMN protein on a Smn background could circumvent this limitation and allow unaffected, heterozygous animals to be examined. Here we describe the generation and characterization of a monoclonal anti-SMN antibody, 4F11, which specifically recognizes human SMN protein. 4F11 detects SMN (human) but not native Smn (mouse) protein in SMN2 transgenic mice and in SMA cell lines. We demonstrate the feasibility of using 4F11 to detect changes in SMN2-derived SMN protein in SMA patient fibroblasts and in healthy SMN2 transgenic mice. This antibody is, therefore, an excellent tool for examining SMN2-inducing therapeutics in patient cells as well as in transgenic mice.

Proximal spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by death of motor neurons in the spinal cord that is caused by deletion and/or mutation of the survival motor neuron gene ( SMN1). Adjacent to SMN1 are a variable number of copies of the SMN2 gene. The two genes essentially differ by a single nucleotide, which causes the majority of the RNA transcripts from SMN2 to lack exon 7. Although both SMN1 and SMN2 encode the same Smn protein amino acid sequence, the loss of SMN1 and incorrect splicing of SMN2 have the consequence that Smn protein levels are insufficient for the survival of motor neurons. The therapeutic goal of our medicinal chemistry effort was to identify small-molecule activators of the SMN2 promoter that, by up-regulating gene transcription, would produce greater quantities of full-length Smn protein. Our initial medicinal chemistry effort explored a series of C5 substituted benzyl ether based 2,4-diaminoquinazoline derivatives that were found to be potent activators of the SMN2 promoter; however, inhibition of DHFR was shown to be an off-target activity that was linked to ATP depletion. We used a structure-guided approach to overcome DHFR inhibition while retaining SMN2 promoter activation. A lead compound 11a was identified as having high potency (EC50 = 4 nM) and 2.3-fold induction of the SMN2 promoter. Compound 11a possessed desirable pharmaceutical properties, including excellent brain exposure and long brain half-life following oral dosing to mice. The piperidine compound 11a up-regulated expression of the mouse SMN gene in NSC-34 cells, a mouse motor neuron hybrid cell line. In type 1 SMA patient fibroblasts, compound 11a induced Smn in a dose-dependent manner when analyzed by immunoblotting and increased the number of intranuclear particles called gems. The compound restored gems numbers in type I SMA patient fibroblasts to levels near unaffected genetic carriers of SMA.

Alternative splicing emerges as one of the most important mechanisms to generate transcript diversity. It is regulated by the formation of protein complexes on pre-mRNA. We demonstrate that protein phosphatase 1 (PP1) binds to the splicing factor transformer2-beta1 (tra2-beta1) via a phylogenetically conserved RVDF sequence located on the RNA recognition motif (RRM) of tra2-beta1. PP1 binds directly to tra2-beta1 and dephosphorylates it, which regulates the interaction between tra2-beta1 and other proteins. Eight other proteins, including SF2/ASF and SRp30c, contain an evolutionary conserved PP1 docking motif in the beta-4 strand of their RRMs indicating that binding to PP1 is a new function of some RRMs. Reducing PP1 activity promotes usage of numerous alternative exons, demonstrating a role of PP1 activity in splice site selection. PP1 inhibition promotes inclusion of the survival of motoneuron 2 exon 7 in a mouse model expressing the human gene. This suggests that reducing PP1 activity could be a new therapeutic principle to treat spinal muscular atrophy and other diseases caused by missplicing events. Our data indicate that the binding of PP1 to evolutionary conserved motifs in several RRMs is the link between known signal transduction pathways regulating PP1 activity and pre-mRNA processing.

Spinal muscular atrophy (SMA) is a motor neuron disease caused by reduced levels of the survival motor neuron (SMN) protein. SMN together with Gemins2-8 and unrip proteins form a macromolecular complex that functions in the assembly of small nuclear ribonucleoproteins (snRNPs) of both the major and the minor splicing pathways. It is not known whether the levels of spliceosomal snRNPs are decreased in SMA. Here we analyzed the consequence of SMN deficiency on snRNP metabolism in the spinal cord of mouse models of SMA with differing phenotypic severities. We demonstrate that the expression of a subset of Gemin proteins and snRNP assembly activity are dramatically reduced in the spinal cord of severe SMA mice. Comparative analysis of different tissues highlights a similar decrease in SMN levels and a strong impairment of snRNP assembly in tissues of severe SMA mice, although the defect appears smaller in kidney than in neural tissue. We further show that the extent of reduction in both Gemin proteins expression and snRNP assembly activity in the spinal cord of SMA mice correlates with disease severity. Remarkably, defective SMN complex function in snRNP assembly causes a significant decrease in the levels of a subset of snRNPs and preferentially affects the accumulation of U11 snRNP--a component of the minor spliceosome--in tissues of severe SMA mice. Thus, impairment of a ubiquitous function of SMN changes the snRNP profile of SMA tissues by unevenly altering the normal proportion of endogenous snRNPs. These findings are consistent with the hypothesis that SMN deficiency affects the splicing machinery and in particular the minor splicing pathway of a rare class of introns in SMA.

Spinal muscular atrophy (SMA) is a recessive motor neuron disease that affects motor neurons in the anterior horn of the spinal cord. SMA results from the reduction of SMN (survival motor neuron) protein. Even though SMN is ubiquitously expressed, motor neurons are more sensitive to the reduction in SMN than other cell types. We have previously generated mouse models of SMA with varying degrees of clinical severity. So as to more clearly understand the pathogenesis of motor neuron degeneration in SMA, we have characterized the phenotype of the SMNDelta7 SMA mouse which normally lives for 13.6+/-0.7 days. These mice are smaller than their non-SMA littermates and begin to lose body mass at 10.4+/-0.4 days. SMNDelta7 SMA mice exhibit impaired responses to surface righting, negative geotaxis and cliff aversion but not to tactile stimulation. Spontaneous motor activity and grip strength are also significantly impaired in SMNDelta7 SMA mice. In summary, we have demonstrated an impairment of neonatal motor responses in SMNDelta7 SMA mice. This phenotype characterization could be used to assess the effectiveness of potential therapies for SMA.

Spinal muscular atrophy (SMA) is a devastating motor neuron disease that is one of the leading genetic causes of infant mortality. Currently, there is no cure for SMA. Mouse models that genetically and phenotypically resemble SMA have been generated and have the potential to be used for the discovery of novel therapeutics. Oral administration is a commonly used mode of drug delivery in humans as well as in rodents. Unfortunately, there is no method of drug delivery that can accurately and reliably deliver drug compounds orally to neonatal mice. In this report, we describe a novel method to orally administer compounds to neonatal SMA mice. Oral delivery to neonatal mice, usually starting at postnatal day 4 (PND04), is both rapid and safe to the pup. Oral delivery of two different commonly used vehicle formulations, distilled water and 2-hydroxypropyl-beta-cyclodextrin, does not affect the survival of SMA mice. After oral delivery for 3 days, 5-bromo-2'-deoxyuridine could be detected in the kidneys, brains and spinal cords of treated non-SMA as well as SMA neonatal pups. In conclusion, we have developed a method by which drugs can be safely and reliably administered orally to neural targets of neonatal mice. This approach offers a simple and rapid means by which potential therapeutics for SMA can be identified.

Glutamate is the major excitatory neurotransmitter in the central nervous system. Its activity is carefully modulated in the synaptic cleft by glutamate transporters. The glial glutamate transporter EAAT2 is the main mediator of glutamate clearance. Reduced EAAT2 function could lead to accumulation of extracellular glutamate, resulting in a form of cell death known as excitotoxicity. In amyotrophic lateral sclerosis and Alzheimer disease, EAAT2 protein levels are significantly decreased in affected areas. EAAT2 mRNA levels, however, remain constant, indicating that alterations in EAAT2 expression are due to disturbances at the post-transcriptional level. In the present study, we found that some EAAT2 transcripts contained 5'-untranslated regions (5'-UTRs) greater than 300 nucleotides. The mRNAs that bear long 5'-UTRs are often regulated at the translational level. We tested this possibility initially in a primary astrocyte line that constantly expressed an EAAT2 transcript containing the 565-nt 5'-UTR and found that translation of this transcript was regulated by many extracellular factors, including corticosterone and retinol. Moreover, many disease-associated insults affected the efficiency of translation of this transcript. Importantly, this translational regulation of EAAT2 occurred in vivo (i.e. both in primary cortical neurons-astrocytes mixed cultures and in mice). These results indicate that expression of EAAT2 protein is highly regulated at the translational level and also suggest that translational regulation may play an important role in the differential EAAT2 protein expression under normal and disease conditions.

Cachexia contributes to nearly a third of all cancer deaths, yet the mechanisms underlying skeletal muscle wasting in this syndrome remain poorly defined. We report that tumor-induced alterations in the muscular dystrophy-associated dystrophin glycoprotein complex (DGC) represent a key early event in cachexia. Muscles from tumor-bearing mice exhibited membrane abnormalities accompanied by reduced levels of dystrophin and increased glycosylation on DGC proteins. Wasting was accentuated in tumor mdx mice lacking a DGC but spared in dystrophin transgenic mice that blocked induction of muscle E3 ubiquitin ligases. Furthermore, DGC deregulation correlated positively with cachexia in patients with gastrointestinal cancers. Based on these results, we propose that, similar to muscular dystrophy, DGC dysfunction plays a critical role in cancer-induced wasting.

The glial glutamate transporter EAAT2 is primarily responsible for clearance of glutamate from the synaptic cleft and loss of EAAT2 has been previously reported in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. The loss of functional EAAT2 could lead to the accumulation of extracellular glutamate, resulting in cell death known as excitotoxicity. However, it is still unknown whether it is a primary cause in the cascade leading to neuron degeneration or a secondary event to cell death. The goals of this study were to generate transgenic mice overexpressing EAAT2 and then to cross these mice with the ALS-associated mutant SOD1(G93A) mice to investigate whether supplementation of the loss of EAAT2 would delay or rescue the disease progression. We show that the amount of EAAT2 protein and the associated Na+-dependent glutamate uptake was increased about 2-fold in our EAAT2 transgenic mice. The transgenic EAAT2 protein was properly localized to the cell surface on the plasma membrane. Increased EAAT2 expression protects neurons from L-glutamate induced cytotoxicity and cell death in vitro. Furthermore, our EAAT2/G93A double transgenic mice showed a statistically significant (14 days) delay in grip strength decline but not in the onset of paralysis, body weight decline or life span when compared with G93A littermates. Moreover, a delay in the loss of motor neurons and their axonal morphologies as well as other events including caspase-3 activation and SOD1 aggregation were also observed. These results suggest that the loss of EAAT2 may contribute to, but does not cause, motor neuron degeneration in ALS.

The Na+-dependent glutamate transporter EAAT3 facilitates glutamate uptake into neurons as well as many other cell types. GTRAP3-18 (JWA, Arl6ip5) is a novel protein that interacts with EAAT3 and negatively modulates EAAT3-mediated glutamate uptake. Previous studies suggest that retinoic acid (RA) decreases Na+-dependent glutamate uptake and increases GTRAP3-18 protein expression. However, the RA used in those studies was complexed with methyl-beta-cyclodextrin (MebetaCD). In the present study we found that MebetaCD, but not RA, significantly reduced Na+-dependent EAAT3-mediated [3H]glutamate uptake in human embryonic kidney 293 (HEK293) cells. MebetaCD also significantly increased GTRAP3-18 protein expression in HEK293 cells as well as in rat hypothalamic neuron cultures. Intracerebroventricular administration of MebetaCD to the mouse brain resulted in a significant increase in GTRAP3-18 immunoreactivity in the hippocampus and cerebral cortex. In conclusion, we have shown that MebetaCD reduces EAAT3-mediated glutamate uptake and induces the expression of GTRAP3-18 protein.

Glutamate is an important amino acid implicated in energy metabolism, protein biosynthesis and neurotransmission. The Na(+)-dependent high-affinity excitatory amino acid transporter EAAT3 (EAAC1) facilitates glutamate uptake into most cells. Recently, a novel rat EAAT3-interacting protein called GTRAP3-18 has been identified by a yeast two-hybrid screening. GTRAP3-18 functions as a negative modulator of EAAT3-mediated glutamate transport. In order to further understand the function and regulation of GTRAP3-18, we cloned the mouse orthologue to GTRAP3-18 and determined its gene structure and its expression pattern. GTRAP3-18 encodes a 188-residue hydrophobic protein whose sequence is highly conserved amongst vertebrates. Mouse and human GTRAP3-18 genes contain three exons separated by two introns. The GTRAP3-18 gene is found on mouse chromosome 6D3 and on human chromosome 3p14, a susceptibility locus for cancer and epilepsy. GTRAP3-18 protein and RNA were found both in neuronal rich regions of the brain and in non-neuronal tissues such as the kidney, heart and skeletal muscle. Mouse GTRAP3-18 inhibited EAAT3-mediated glutamate transport in a dose-dependent manner. These studies show that GTRAP3-18 is a ubiquitously expressed protein that functions as a negative regulator of EAAT3 function.