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Spinal muscular atrophy (SMA) is a motor neuron disease caused by insufficient levels of the survival motor neuron (SMN) protein. One of the most prominent pathological characteristics of SMA involves defects of the neuromuscular junction (NMJ), such as denervation and reduced clustering of acetylcholine receptors (AChRs). Recent studies suggest that upregulation of agrin, a crucial NMJ organizer promoting AChR clustering, can improve NMJ innervation and reduce muscle atrophy in the delta7 mouse model of SMA. To test whether the muscle-specific kinase (MuSK), part of the agrin receptor complex, also plays a beneficial role in SMA, we treated the delta7 SMA mice with an agonist antibody to MuSK. MuSK agonist antibody #13, which binds to the NMJ, significantly improved innervation and synaptic efficacy in denervation-vulnerable muscles. MuSK agonist antibody #13 also significantly increased the muscle cross-sectional area and myofiber numbers in these denervation-vulnerable muscles but not in denervation-resistant muscles. Although MuSK agonist antibody #13 did not affect the body weight, our study suggests that preservation of NMJ innervation by the activation of MuSK may serve as a complementary therapy to SMN-enhancing drugs to maximize the therapeutic effectiveness for all types of SMA patients.

Aminoacyl-RNA synthetases (aaRSs) are among the key enzymes of protein biosynthesis. They are responsible for conducting the first step in the protein biosynthesis, namely attaching amino acids to the corresponding tRNA molecules both in cytoplasm and mitochondria. More and more research demonstrates that mutations in the genes encoding aaRSs lead to the development of various neurodegenerative diseases, such as incurable Charcot-Marie-Tooth disease (CMT) and distal spinal muscular atrophy. Some mutations result in the loss of tRNA aminoacylation activity, while other mutants retain their classical enzyme activity. In the latter case, disease manifestations are associated with additional neuron-specific functions of aaRSs. At present, seven aaRSs (GlyRS, TyrRS, AlaRS, HisRS, TrpRS, MetRS, and LysRS) are known to be involved in the CMT etiology with glycyl-tRNA synthetase (GlyRS) being the most studied of them.

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by loss of the survival motor neuron 1 (SMN1) gene. SMA is characterized by the degeneration of spinal cord motoneurons (MNs), progressive skeletal muscle atrophy, and weakness. The cellular and molecular mechanisms causing MN loss of function are only partially known. Recent advances in SMA research postulate the role of calpain protease regulating survival motor neuron (SMN) protein and the positive effect on SMA phenotype of treatment with calpain inhibitors. We analyzed the level of calpain pathway members in mice and human cellular SMA models. Results indicate an increase of calpain activity in SMN-reduced MNs. Spinal cord analysis of SMA mice treated with calpeptin, a calpain inhibitor, showed an increase of SMN, calpain, and its endogenous inhibitor calpastatin in MNs. Finally, in vitro calpeptin treatment prevented microtubule-associated protein 1A/1B-light chain 3 (LC3) increase in MNs neurites, indicating that calpain inhibition may reduce autophagosome accumulation in neuron prolongations, but not in soma. Thus, our results show that calpain activity is increased in SMA MNs and its inhibition may have a beneficial effect on SMA phenotype through the increase of SMN in spinal cord MNs.

Background: Distal motor neuropathies with a genetic origin have a heterogeneous clinical presentation with overlapping features affecting distal nerves and including spinal muscular atrophies and amyotrophic lateral sclerosis. This indicates that their genetic background is heterogeneous., Patient and Methods: In this work, we have identified and characterized the genetic and molecular base of a patient with a distal sensorimotor neuropathy of unknown origin. For this study, we performed whole-exome sequencing, molecular modelling, cloning and expression of mutant gene, and biochemical and cell biology analysis of the mutant protein., Results: A novel homozygous recessive mutation in the human VRK1 gene, coding for a chromatin kinase, causing a substitution (c.637T > C; p.Tyr213His) in exon 8, was detected in a patient presenting since childhood a progressive distal sensorimotor neuropathy and spinal muscular atrophy syndrome, with normal intellectual development. Molecular modelling predicted this mutant VRK1 has altered the kinase activation loop by disrupting its interaction with the C-terminal regulatory region. The p.Y213H mutant protein has a reduced kinase activity with different substrates, including histones H3 and H2AX, proteins involved in DNA damage responses, such as p53 and 53BP1, and coilin, the scaffold for Cajal bodies. The mutant VRK1(Y213H) protein is unable to rescue the formation of Cajal bodies assembled on coilin, in the absence of wild-type VRK1., Conclusion: The VRK1(Y213H) mutant protein alters the activation loop, impairs the kinase activity of VRK1 causing a functional insufficiency that impairs the formation of Cajal bodies assembled on coilin, a protein that regulates SMN1 and Cajal body formation., (© 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Acid ceramidase (aCDase, ASAH1) hydrolyzes lysosomal membrane ceramide into sphingosine, the backbone of all sphingolipids, to regulate many cellular processes. Abnormal function of aCDase leads to Farber disease, spinal muscular atrophy with progressive myoclonic epilepsy, and is associated with Alzheimer's, diabetes, and cancer. Here, we present crystal structures of mammalian aCDases in both proenzyme and autocleaved forms. In the proenzyme, the catalytic center is buried and protected from solvent. Autocleavage triggers a conformational change exposing a hydrophobic channel leading to the active site. Substrate modeling suggests distinct catalytic mechanisms for substrate hydrolysis versus autocleavage. A hydrophobic surface surrounding the substrate binding channel appears to be a site of membrane attachment where the enzyme accepts substrates facilitated by the accessory protein, saposin-D. Structural mapping of disease mutations reveals that most would destabilize the protein fold. These results will inform the rational design of aCDase inhibitors and recombinant aCDase for disease therapeutics.

Spinal Muscular Atrophy (SMA) is a motoneuron disease caused by low levels of functional survival of motoneuron protein (SMN). Molecular disease mechanisms downstream of functional SMN loss are still largely unknown. Previous studies suggested an involvement of Rho kinase (ROCK) as well as the extracellular signal-regulated kinases (ERK) pathways in the pathomechanism. Both pathways are bi-directionally linked and inhibit each other. Thus, we hypothesize that both pathways regulate SMA pathophysiology in vivo in a combined manner rather than acting separately. Here, we applied the repurposed drugs, selumetinib, an ERK inhibitor, and the ROCK inhibitor fasudil to severe SMA mice. Thereby, separately applied inhibitors as well as a combination enabled us to explore the impact of the ROCK-ERK signaling network on SMA pathophysiology. ROCK inhibition specifically ameliorated the phenotype of selumetinib-treated SMA mice demonstrating an efficient ROCK to ERK crosstalk relevant for the SMA pathophysiology. However, ERK inhibition alone aggravated the condition of SMA mice and reduced the number of motoneurons indicating a compensatory hyper-activation of ERK in motoneurons. Taken together, we identified a regulatory network acting downstream of SMN depletion and upstream of the SMA pathophysiology thus being a future treatment target in combination with SMN dependent strategies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

C5-substituted 2,4-diaminoquinazoline inhibitors of the decapping scavenger enzyme DcpS (DAQ-DcpSi) have been developed for the treatment of spinal muscular atrophy (SMA), which is caused by genetic deficiency in the Survival Motor Neuron (SMN) protein. These compounds are claimed to act as SMN2 transcriptional activators but data underlying that claim are equivocal. In addition it is unclear whether the claimed effects on SMN2 are a direct consequence of DcpS inhibitor or might be a consequence of lysosomotropism, which is known to be neuroprotective. DAQ-DcpSi effects were characterized in cells in vitro utilizing DcpS knockdown and 7-methyl analogues as probes for DcpS vs non-DcpS-mediated effects. We also performed analysis of Smn transcript levels, RNA-Seq analysis of the transcriptome and SMN protein in order to identify affected pathways underlying the therapeutic effect, and studied lysosomotropic and non-lysosomotropic DAQ-DCpSi effects in 2B/- SMA mice. Treatment of cells caused modest and transient SMN2 mRNA increases with either no change or a decrease in SMNΔ7 and no change in SMN1 transcripts or SMN protein. RNA-Seq analysis of DAQ-DcpSi-treated N2a cells revealed significant changes in expression (both up and down) of approximately 2,000 genes across a broad range of pathways. Treatment of 2B/- SMA mice with both lysomotropic and non-lysosomotropic DAQ-DcpSi compounds had similar effects on disease phenotype indicating that the therapeutic mechanism of action is not a consequence of lysosomotropism. In striking contrast to the findings in vitro, Smn transcripts were robustly changed in tissues but there was no increase in SMN protein levels in spinal cord. We conclude that DAQ-DcpSi have reproducible benefit in SMA mice and a broad spectrum of biological effects in vitro and in vivo, but these are complex, context specific, and not the result of simple SMN2 transcriptional activation.

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease characterized by motor neurons degeneration and muscular atrophy. There is no effective SMA treatment. Loganin is a botanical candidate with anti-inflammatory, anti-oxidant, glucose-lowering and anti-diabetic nephropathy activities. The aim of this study is to investigate the potential protective effects of loganin on SMA using two cellular models, SMN-deficient NSC34 cells and SMA patient fibroblasts, and an animal disease model, SMAΔ7 mice. In SMN-deficient NSC34 cells, loganin increased cell viability, neurite length, and expressions of SMN, Gemin2, SMN-Gemin2 complex, p-Akt, p-GSK-3β, p-CREB, BDNF and Bcl-2. However, both AG1024 (IGF-1 R antagonist) and IGF-1 R siRNA attenuated the protective effects of loganin on SMN level and cell viability in SMN-deficient NSC34 cells. In SMA patient fibroblasts, loganin up-regulated levels of SMN, FL-SMN2, and Gemins, increased numbers of SMN-containing nuclear gems, modulated splicing factors, and up-regulated p-Akt. Furthermore, in the brain, spinal cord and gastrocnemius muscle of SMAΔ7 mice, loganin up-regulated the expressions of SMN and p-Akt. Results from righting reflex and hind-limb suspension tests indicated loganin improved muscle strength of SMAΔ7 mice; moreover, loganin activated Akt/mTOR signal and inhibited atrogin-1/MuRF-1 signal in gastrocnemius muscle of SMAΔ7 mice. Loganin also increased body weight, but the average lifespan of loganin (20mg/kg/day)-treated SMA mice was 16.80±0.73 days, while saline-treated SMA mice was 10.91±0.96 days. In conclusion, the present results demonstrate that loganin provides benefits to SMA therapeutics via improving SMN restoration, muscle strength and body weight. IGF-1 plays an important role in loganin neuroprotection. Loganin can be therefore a valuable complementary candidate for treatment of neuromuscular diseases via regulation of muscle protein synthesis and neuroprotection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

To broaden the scope of existing methods based on (19)F nucleotide labeling, we developed a new method for the synthesis of fluorophosphate (oligo)nucleotide analogues containing an O to F substitution at the terminal position of the (oligo)phosphate moiety and evaluated them as tools for (19)F NMR studies. Using three efficient and comprehensive synthetic approaches based on phosphorimidazolide chemistry and tetra-n-butylammonium fluoride, fluoromonophosphate, or fluorophosphate imidazolide as fluorine sources, we prepared over 30 fluorophosphate-containing nucleotides, varying in nucleobase type (A, G, C, U, m(7)G), phosphate chain length (from mono to tetra), and presence of additional phosphate modifications (thio, borano, imido, methylene). Using fluorophosphate imidazolide as fluorophosphorylating reagent for 5'-phosphorylated oligos we also synthesized oligonucleotide 5'-(2-fluorodiphosphates), which are potentially useful as (19)F NMR hybridization probes. The compounds were characterized by (19)F NMR and evaluated as (19)F NMR molecular probes. We found that fluorophosphate nucleotide analogues can be used to monitor activity of enzymes with various specificities and metal ion requirements, including human DcpS enzyme, a therapeutic target for spinal muscular atrophy. The compounds can also serve as reporter ligands for protein binding studies, as exemplified by studying interaction of fluorophosphate mRNA cap analogues with eukaryotic translation initiation factor (eIF4E).

Aim: Levels of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) are robustly increased in spinal muscular atrophy (SMA) patient fibroblasts and mouse models. We therefore wanted to establish whether changes in UCHL1 contribute directly to disease pathogenesis, and to assess whether pharmacological inhibition of UCHL1 represents a viable therapeutic option for SMA., Methods: SMA mice and control littermates received a pharmacological UCHL1 inhibitor (LDN-57444) or DMSO vehicle. Survival and weight were monitored daily, a righting test of motor performance was performed, and motor neurone loss, muscle fibre atrophy and neuromuscular junction pathology were all quantified. Ubiquitin-like modifier activating enzyme 1 (Uba1) was then pharmacologically inhibited in neurones in vitro to examine the relationship between Uba1 levels and UCHL1 in SMA., Results: Pharmacological inhibition of UCHL1 failed to improve survival, motor symptoms or neuromuscular pathology in SMA mice and actually precipitated the onset of weight loss. LDN-57444 treatment significantly decreased spinal cord mono-ubiquitin levels, further exacerbating ubiquitination defects in SMA mice. Pharmacological inhibition of Uba1, levels of which are robustly reduced in SMA, was sufficient to induce accumulation of UCHL1 in primary neuronal cultures., Conclusion: Pharmacological inhibition of UCHL1 exacerbates rather than ameliorates disease symptoms in a mouse model of SMA. Thus, pharmacological inhibition of UCHL1 is not a viable therapeutic target for SMA. Moreover, increased levels of UCHL1 in SMA likely represent a downstream consequence of decreased Uba1 levels, indicative of an attempted supportive compensatory response to defects in ubiquitin homeostasis caused by low levels of SMN protein., (© 2014 British Neuropathological Society.)

Among hereditary diseases, the group of motor neuron diseases (MNDs) includes some of the most devastating and rapidly progressive lethal conditions. Although degeneration of motor neurons is common to all of them, the phenotypic spectrum of MNDs is relatively broad and ranges from perinatal conditions like spinal muscular atrophy (SMA) to adult-onset diseases such as amyotrophic lateral sclerosis (ALS). While the understanding of the pathology of the diseases is constantly growing, the development of therapeutic approaches lags behind. In fact, there is no approved therapy for MNDs available at the moment. Recent findings demonstrated the existence of some patterns that are shared by several MNDs such as transcriptional dysregulation. In addition, conditions like SMA or certain types of Charcot-Marie-Tooth disease provide some defined targets which may be amenable to therapeutic approaches. Consequently, counteracting this dysregulation may be a valuable therapeutic option and ameliorate disease progression in MND patients. The feasibility of such an approach has been proven during the past years by the epigenetic treatment of various neoplastic entities with histone deacetylase inhibitors (HDACi). On these grounds, also epigenetic therapy of MNDs has become a promising option. So far, several HDACi have been tested in vitro and in animal models and some proceeded further and were evaluated in clinical trials. This review will summarize the advances of HDACi in MNDs and will give a perspective where the road will lead us.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease caused by mutations in the survival of motor neuron 1 (SMN1) gene and deficient expression of the ubiquitously expressed SMN protein. Pathologically, SMA is characterized by motor neuron loss and severe muscle atrophy. During muscle atrophy, the E3 ligase atrogenes, atrogin-1 and muscle ring finger 1 (MuRF1), mediate muscle protein breakdown through the ubiquitin proteasome system. Atrogene expression can be induced by various upstream regulators. During acute denervation, they are activated by myogenin, which is in turn regulated by histone deacetylases 4 and 5. Here we show that atrogenes are induced in SMA model mice and in SMA patient muscle in association with increased myogenin and histone deacetylase-4 (HDAC4) expression. This activation during both acute denervation and SMA disease progression is suppressed by treatment with a histone deacetylase inhibitor; however, this treatment has no effect when atrogene induction occurs independently of myogenin. These results indicate that myogenin-dependent atrogene induction is amenable to pharmacological intervention with histone deacetylase inhibitors and help to explain the beneficial effects of these agents on SMA and other denervating diseases.

Spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS) are among the most common motor neuron diseases to afflict the human population. A deficiency of the survival of motor neuron (SMN) protein causes SMA and is also reported to be an exacerbating factor in the development of ALS. However, pathways linking the two diseases have yet to be defined and it is not clear precisely how the pathology of ALS is aggravated by reduced SMN or whether mutant proteins underlying familial forms of ALS interfere with SMN-related biochemical pathways to exacerbate the neurodegenerative process. In this study, we show that mutant superoxide dismutase-1 (SOD1), a cause of familial ALS, profoundly alters the sub-cellular localization of the SMN protein, preventing the formation of nuclear 'gems' by disrupting the recruitment of the protein to Cajal bodies. Overexpressing the SMN protein in mutant SOD1 mice, a model of familial ALS, alleviates this phenomenon, most likely in a cell-autonomous manner, and significantly mitigates the loss of motor neurons in the spinal cord and in culture dishes. In the mice, the onset of the neuromuscular phenotype is delayed and motor function enhanced, suggestive of a therapeutic benefit for ALS patients treated with agents that augment the SMN protein. Nevertheless, this finding is tempered by an inability to prolong survival, a limitation most likely imposed by the inexorable denervation that characterizes ALS and eventually disrupts the neuromuscular synapses even in the presence of increased SMN.

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, neurodegenerative disorder caused by the degeneration of motor neurons in the CNS, which results in complete paralysis of skeletal muscles. Recent experimental studies have suggested that the disease could initiate in skeletal muscle, rather than in the motor neurons. To establish the timeframe of motor neuron degeneration in relation to muscle atrophy in motor neuron disease, we have used MRI to monitor changes throughout disease in brain and skeletal muscle of G93A-SOD1 mice, a purported model of ALS. Longitudinal MRI examination of the same animals indicated that muscle volume in the G93A-SOD1 mice was significantly reduced from as early as week 8 of life, 4 weeks prior to clinical onset. Progressive muscle atrophy from week 8 onwards was confirmed by histological analysis. In contrast, brain MRI indicated that neurodegeneration occurs later in G93A-SOD1 mice, with hyperintensity MRI signals detected only at weeks 10-18. Neurodegenerative changes were observed only in the motor nuclei areas of the brainstem; MRI changes indicative of neurodegeneration were not detected in the motor cortex where first motor neurons originate, even at the late disease stage. This longitudinal MRI study establishes unequivocally that, in the experimental murine model of ALS, muscle degeneration occurs before any evidence of neurodegeneration and clinical signs, supporting the postulate that motor neuron disease can initiate from muscle damage and result from retrograde dying-back of the motor neurons., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Spinal muscular atrophy (SMA) is an inherited disease resulting in the highest mortality of children under the age of two. SMA is caused by mutations or deletions in the survival motor neuron 1 (SMN1) gene, leading to aberrant neuromuscular junction (NMJ) development and the loss of spinal cord alpha-motor neurons. Here, we show that Smn depletion leads to increased activation of RhoA, a major regulator of actin dynamics, in the spinal cord of an intermediate SMA mouse model. Treating these mice with Y-27632, which inhibits ROCK, a direct downstream effector of RhoA, dramatically improves their survival. This lifespan rescue is independent of Smn expression and is accompanied by an improvement in the maturation of the NMJs and an increase in muscle fiber size in the SMA mice. Our study presents evidence linking disruption of actin cytoskeletal dynamics to SMA pathogenesis and, for the first time, identifies RhoA effectors as viable targets for therapeutic intervention in the disease.

Histone deacetylases (HDACs) play a key role in homeostasis of protein acetylation in histones and other proteins and in regulating fundamental cellular activities such as transcription. A wide range of brain disorders are associated with imbalances in protein acetylation levels and transcriptional dysfunctions. Treatment with various HDAC inhibitors can correct these deficiencies and has emerged as a promising new strategy for therapeutic intervention in neurodegenerative disease. Here, we review and discuss intriguing recent developments in the use of HDAC inhibitors to combat neurodegenerative conditions in cellular and disease models. HDAC inhibitors have neuroprotective, neurotrophic and anti-inflammatory properties; improvements in neurological performance, learning/memory and other disease phenotypes are frequently seen in these models. We discuss the targets and mechanisms underlying these effects of HDAC inhibition and comment on the potential for some HDAC inhibitors to prove clinically effective in the treatment of neurodegenerative disorders.

Distal spinal muscular atrophy type 1 (DSMA1) is an autosomal recessive disease that is clinically characterized by distal limb weakness and respiratory distress. In this disease, the degeneration of alpha-motoneurons is caused by mutations in the immunoglobulin mu-binding protein 2 (IGHMBP2). This protein has been implicated in DNA replication, pre-mRNA splicing and transcription, but its precise function in all these processes has remained elusive. We have purified catalytically active recombinant IGHMBP2, which has enabled us to assess its enzymatic properties and to identify its cellular targets. Our data reveal that IGHMBP2 is an ATP-dependent 5' --> 3' helicase, which unwinds RNA and DNA duplices in vitro. Importantly, this helicase localizes predominantly to the cytoplasm of neuronal and non-neuronal cells and associates with ribosomes. DSMA1-causing amino acid substitutions in IGHMBP2 do not affect ribosome binding yet severely impair ATPase and helicase activity. We propose that IGHMBP2 is functionally linked to translation, and that mutations in its helicase domain interfere with this function in DSMA1 patients.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed, essential enzymes responsible for performing the first step of protein synthesis. Specifically, ARSs attach amino acids to their cognate tRNA molecules in the cytoplasm and mitochondria. Recent studies have demonstrated that mutations in genes encoding ARSs can result in neurodegeneration, raising many questions about the role of these enzymes (and protein synthesis in general) in neuronal function. In this review, we summarize the current knowledge of genetic diseases that are associated with mutations in ARS-encoding genes, discuss the potential pathogenic mechanisms underlying these disorders, and point to likely areas of future research that will advance our understanding about the role of ARSs in genetic diseases.

Neurodegenerative disease strikes millions worldwide and there is mounting evidence suggesting that underlying the onset and progression of these debilitating diseases is inappropriate neuronal apoptosis. Recent reports have implicated a family of proteins known as histone deacetylases (HDACs) in various neuronal processes including the neuronal death program. Initial headway in this field has been made largely through the use of broad-spectrum HDAC inhibitors. In fact, pharmacological inhibition of HDAC activity has been shown to protect neurons in several models of neurodegeneration. The observation that HDAC inhibitors can have opposing effects in different paradigms of neurodegeneration suggests that individual members of the HDAC protein family may play distinct roles that could depend on the specific cell type under study. The purpose of this review is to detail work involving the use of HDAC inhibitors within the context of neurodegeneration and examine the roles of individual HDAC members in the nervous system with specific focus on neuronal cell death.

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron degenerative disease whose etiology and pathogenesis remain poorly understood. Most cases of ALS ( approximately 90%) are sporadic (SALS), occurring in the absence of genetic associations. Approximately 20% of familial ALS (FALS) cases are due to known mutations in the copper, zinc superoxide dismutase (SOD1) gene. Molecular evidence for a common pathogenesis of SALS and FALS has remained elusive. Here we use covalent chemical modification to reveal an attribute of spinal cord SOD1 common to both SOD1-linked FALS and SALS, but not present in normal or disease-affected tissues from other neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases and spinal muscular atrophy, a non-ALS motor neuron disease. Biotinylation reveals a 32-kDa, covalently cross-linked SOD1-containing protein species produced not only in FALS caused by SOD1 mutation, but also in SALS. These studies use chemical modification as a novel tool for the detection of a disease-associated biomarker. Our results identify a shared molecular event involving a known target gene and suggest a common step in the pathogenesis between SALS and FALS.

Dominant mutations in the ubiquitous enzyme glycyl-tRNA synthetase (GlyRS), including S581L, lead to motor nerve degeneration. We have determined crystal structures of wildtype and S581L-mutant human GlyRS. The S581L mutation is approximately 50A from the active site, and yet gives reduced aminoacylation activity. The overall structures of wildtype and S581L-GlyRS, including the active site, are very similar. However, residues 567-575 of the anticodon-binding domain shift position and in turn could indirectly affect glycine binding via the tRNA or alternatively inhibit conformational changes. Reduced enzyme activity may underlie neuronal degeneration, although a dominant-negative effect is more likely in this autosomal dominant disorder.

The bovine form of the autosomal recessive neurodegenerative disease spinal muscular atrophy (SMA) shows striking similarity to the human form of the disease. It has, however, been mapped to a genomic region not harboring the bovine orthologue of the SMN gene, mutation of which causes human SMA. After refinement of the mapping results we analyzed positional and functional candidate genes. One of three candidate genes, FVT1, encoding 3-ketodihydrosphingosine reductase, which catalyzes a crucial step in the glycosphingolipid metabolism, showed a G-to-A missense mutation that changes Ala-175 to Thr. The identified mutation is limited to SMA-affected animals and carriers and always appears in context of the founder haplotype. The Ala variant found in healthy animals showed the expected 3-ketodihydrosphingosine reductase activity in an in vitro enzyme assay. Importantly, the Thr variant found in SMA animals showed no detectable activity. Surprisingly, in an in vivo assay the mutated gene complements the growth defect of a homologous yeast knockout strain as well as the healthy variant. This finding explains the viability of affected newborn calves and the later neuron-specific onset of the disease, which might be due to the high sensitivity of these neurons to changes in housekeeping functions. Taken together, the described mutation in FVT1 is a strong candidate for causality of SMA in cattle. This result provides an animal model for understanding the underlying mechanisms of the development of SMA and will allow efficient selection against the disease in cattle.

X-linked spinal and bulbar muscular atrophy (SBMA or Kennedy's disease) has a variable prognosis. Most male carriers are affected by their fourth or fifth decade of life, while some remain asymptomatic lifelong. Elevations of serum creatine kinase are well known to occur in clinically manifesting SBMA patients. Elevations prior to the onset of the clinical syndrome have not been reported. Here we report two cases of SBMA presenting with 'idiopathic' elevations of serum transaminases and creatine kinase a decade in advance of their symptomatic onset. These cases emphasize the need to consider SBMA and genetic testing for the androgen receptor trinucleotide CAG expansion in males otherwise healthy with 'idiopathic' elevated creatinine kinase.

We investigated the expression of choline acetyltransferase, a specific marker for cholinergic neurons, in control and spinal muscular atrophy fetuses and newborns. By immunoblot we observed at 12 and 15 weeks a similar pattern of choline acetyltransferase expression in spinal muscular atrophy with respect to controls, although at 22 weeks this expression was reduced, probably due to a smaller number of motor neurons in the spinal muscular atrophy spinal cord. By immunohistochemistry, the counting of positive and negative motor neurons for choline acetyltransferase immunostaining in control and spinal muscular atrophy fetuses showed a similar proportion at all stages analyzed. The choline acetyltransferase-negative motor neurons were of similar appearance in both groups. After birth, chromatolytic motor neurons were detected in spinal muscular atrophy, all of which were choline acetyltransferase-negative. Our results in spinal muscular atrophy fetuses indicate that choline acetyltransferase immunostaining does not identify early events in neuronal pathogenesis and suggest that the spinal muscular atrophy surviving motor neurons may not be dysfunctional during this period. Furthermore, spinal muscular atrophy choline acetyltransferase-negative motor neurons showed detectable pathological changes only after birth, indicating that choline acetyltransferase is a late marker for motor neuron degeneration and not a primary contributing factor in this process.

Homozygous deletion or mutation in the survival motor neuron (SMN)1 gene causes proximal spinal muscular atrophy (SMA), whereas SMN2 acts as a modifying gene that can influence the severity of SMA. It has been suggested that restoration of the SMN protein level in neuronal cells may prevent cell loss and may be helpful for treatment of SMA. Recent studies indicate that the ubiquitin/proteasome pathway is a major system for proteolysis of intracellular proteins. In this study, we investigate whether SMN protein is degraded via the ubiquitin/proteasome pathway. Primary fibroblasts were established from the skin biopsies of SMA patients and the effect of a proteasome inhibitor MG132 and lysosome inhibitor NH(4)Cl on SMN protein level was examined. We found that MG132, but not NH(4)Cl, significantly increased the amount and nuclear accumulation of SMN protein in SMA patient's fibroblasts. Immunoprecipitation/western blot analysis indicated that SMN protein was ubiquitinated in cells. In vitro protein ubiquitination assay also demonstrated that SMN protein could be conjugated with ubiquitin. Taken together, we have provided clear evidences that degradation of SMN protein is mediated via the ubiquitin/proteasome pathway and suggest that proteasome inhibitors may up-regulate SMN protein level and may be useful for the treatment of SMA.

Spinal muscular atrophy (SMA) is a neuromuscular disorder in childhood leading to a dramatic loss of muscle strength. Functional investigations with high-resolution polarography and enzyme measurements of the respiratory chain revealed lowered activities in muscle tissue of SMA patients. To gain a better understanding of this low energy supply we analyzed the amount of mitochondrial DNA (mtDNA) in skeletal muscle of 20 unrelated children with genetically proven SMA and 31 controls. Quantitative Southern blot analysis revealed a severe and homogeneous decrease in the content of muscle mtDNA in relation to nuclear DNA in SMA patients (90.3+/-7.8%), whereas by immunofluorescence no decrease in the number of mitochondria was detected. In addition, a two- to threefold reduction of the nuclear-encoded complex II (succinate dehydrogenase) activity was detected in SMA muscle tissue. Western blot analysis showed a significant reduction of both mitochondrial- and nuclear-encoded cytochrome c oxidase subunits. Our results indicate that mtDNA depletion in SMA is a consequence of severe atrophy, and has to be differentiated by measurement of complex II from an isolated reduction of mtDNA as found in patients with mitochondriocytopathies and the so-called mtDNA depletion syndrome.

Five affected siblings were referred with a probable diagnosis of proximal adult-type spinal muscular atrophy (SMA) based on lower motor neuron signs (muscle weakness and atrophy, hypotony, hypoactive or absent reflexes, and fasciculations), normal or borderline serum creatine kinase levels, and a neurogenic pattern on electromyography, compatible with motor neuron disease, in one patient. No exon 7-8 deletion in the survival motor neuron (SMN) gene was found. Linkage analysis excluded the SMN and all known autosomal recessive limb girdle muscular dystrophy loci, with the exception of LGMD-2A. A homozygous R769Q mutation in the calpain-3 gene and absence of muscle calpain-3 protein confirmed a calpainopathy. This family suggests that the clinical spectrum of calpainopathy might be broader and that this diagnosis might be considered in patients with an atypical motor neuron disease.

Background: The mitochondrial DNA (mtDNA) depletion syndrome (MDS) is an autosomal recessive disorder of early childhood characterized by decreased mtDNA copy number in affected tissues. Recently, MDS has been linked to mutations in two genes involved in deoxyribonucleotide (dNTP) metabolism: thymidine kinase 2 (TK2) and deoxy-guanosine kinase (dGK). Mutations in TK2 have been associated with the myopathic form of MDS, and mutations in dGK with the hepatoencephalopathic form., Objectives: To further characterize the frequency and clinical spectrum of these mutations, the authors screened 20 patients with myopathic MDS., Results: No patient had dGK gene mutations, but four patients from two families had TK2 mutations. Two siblings were compound heterozygous for a previously reported H90N mutation and a novel T77M mutation. The other siblings harbored a homozygous I22M mutation, and one of them had evidence of lower motor neuron disease. The pathogenicity of these mutations was confirmed by reduced TK2 activity in muscle (28% to 37% of controls)., Conclusions: These results show that the clinical expression of TK2 mutations is not limited to myopathy and that the myopathic form of MDS is genetically heterogeneous.

Mutations in Cu/Zn superoxide dismutase (SOD1), a major cytosolic antioxidant enzyme in eukaryotic cells, have been reported in approximately 20% of familial amyotrophic lateral sclerosis (FALS) patients. Hereditary canine spinal muscular atrophy (HCSMA), a fatal inherited motor neuron disease in Brittany spaniels, shares many clinical and pathological features with human motor neuron disease, including FALS. The SOD1 coding region has been sequenced and cloned from several animal species, but not from the dog. We have mapped the chromosomal location, sequenced, and characterized the canine SOD1 gene. Extending this analysis, we have evaluated SOD1 as a candidate for HCSMA. The 462 bp SOD1 coding region in the dog encodes 153 amino acid residues and exhibits more than 83% and 79% sequence identity to other mammalian homologues at both the nucleotide and amino acid levels, respectively. The canine SOD1 gene maps to CFA31 close to syntenic group 13 on the radiation hybrid (RH) map in the vicinity of sodium myo/inositol transporter (SMIT) gene. The human orthologous SOD1 and SMIT genes have been localized on HSA 21q22.1 and HSA 21q21, respectively, confirming the conservation of synteny between dog syntenic group 13 and HSA 21. Direct sequencing of SOD1 cDNA from six dogs with HCSMA revealed no mutations. Northern analysis indicated no differences in steady-state levels of SOD1 mRNA.

The mitochondrial function in skeletal muscle was investigated in skeletal muscle biopsies of 26 patients with sporadic amyotrophic lateral sclerosis (ALS) and compared with investigations of 28 age-matched control muscle samples and biopsies of 6 patients with spinal muscular atrophy (SMA) and two patients with Tay-Sachs disease. In comparison to the control, SMA and Tay-Sachs biopsies, we observed in the ALS samples a significant about two-fold lower activity of complex I of mitochondrial respiratory chain. To visualise the distribution of the mitochondrial defect in skeletal muscle fibers we applied confocal laser-scanning microscopy and video fluorescence microscopy of NAD(P)H and fluorescent flavoproteins. The redox change of mitochondrial NAD(P)H and flavoproteins on addition of mitochondrial substrates, ADP, or cyanide were determined by measurement of fluorescence intensities with dual-photon UV-excitation and single-photon blue excitation. In skeletal muscle fibers of ALS patients with abnormalities of mitochondrial DNA (multiple deletions, n=1, or lower mtDNA levels, n=14) we observed a heterogeneous distribution of the mitochondrial defects among individual fibers and even within single fibers. In some patients (n=3) a mitochondrial defect was also detectable in cultivated skin fibroblasts. These findings support the viewpoint that the observed impairment of mitochondrial function in muscle of certain ALS patients is caused by an intrinsic mitochondrial defect which may be of pathophysiological significance in the etiology of this neurodegenerative disease.

Proteolytic enzyme expression was studied by matrix metalloproteinases (MMP) immunoreactivity (-IR) in muscle biopsies from patients with amyotrophic lateral sclerosis (ALS), spinal muscle atrophy (SMA) and chronic axonal neuropathies (CANP). In normal muscle MMP-2-, MMP-7-, and MMP-9-IR was localized at neuromuscular junctions, in vessels and nerve branches. ALS biopsies of clinically non-affected muscles revealed neither MMP-2, -7-IR nor MMP-9-IR in atrophied myofibers. ALS-affected biopsies revealed MMP-9-IR, and to lesser extent MMP-2- and MMP-7-IR at normal sized and atrophied myofibers. Biopsies of SMA showed MMP-9- and MMP-7-IR at normal sized and atrophic myofibers, while MMP-2-IR was undetectable. In CANP MMP-9-IR was found at normal sized and atrophied myofibers. Distinct expression patterns of MMPs may thus reflect different stages of muscle denervation atrophy.

X-linked spinal and bulbar muscular atrophy (SBMA), Kennedy's disease, is a degenerative disease of the motor neurons that is associated with an increase in the number of CAG repeats encoding a polyglutamine stretch within the androgen receptor (AR). Recent work has demonstrated that the gene products associated with open reading frame triplet repeat expansions may be substrates for the cysteine protease cell death executioners, the caspases. However, the role that caspase cleavage plays in the cytotoxicity associated with expression of the disease-associated alleles is unknown. Here, we report the first conclusive evidence that caspase cleavage is a critical step in cytotoxicity; the expression of the AR with an expanded polyglutamine stretch enhances its ability to induce apoptosis when compared with the normal AR. The AR is cleaved by a caspase-3 subfamily protease at Asp146, and this cleavage is increased during apoptosis. Cleavage of the AR at Asp146 is critical for the induction of apoptosis by AR, as mutation of the cleavage site blocks the ability of the AR to induce cell death. Further, mutation of the caspase cleavage site at Asp146 blocks the ability of the SBMA AR to form perinuclear aggregates. These studies define a fundamental role for caspase cleavage in the induction of neural cell death by proteins displaying expanded polyglutamine tracts, and therefore suggest a strategy that may be useful to treat neurodegenerative diseases associated with polyglutamine repeat expansions.

Hereditary canine spinal muscular atrophy (HCSMA) is a dominantly inherited motor neuron disease in Brittany spaniels that is clinically characterized by progressive muscle weakness leading to paralysis. Histopathologically, degeneration is confined to motor neurons with accumulation of phosphorylated neurofilaments in axonal internodes. Cyclin-dependent kinase 5 (CDK5), a kinase related to the cell cycle kinase cdc2, phosphorylates neurofilaments and regulates neurofilament dynamics. We examined CDK5 activity, protein levels, and cellular immunoreactivity in nervous tissue from dogs with HCSMA, from closely age-matched controls and from dogs with other neurological diseases. On immunoblot analysis, CDK5 protein levels were increased in the HCSMA dogs (by approximately 1.5-fold in both the cytosolic and the particulate fractions). CDK5 activity was significantly increased (by approximately 3-fold) in the particulate fractions in the HCSMA dogs compared to all controls. The finding that CDK5 activity was increased in the young HCSMA homozygotes with the accelerated form of the disease, who do not show axonal swellings histologically, suggests that alterations in CDK5 occurs early in the pathogenesis, prior to the development of significant neurofilament pathology. Immunocytochemically, there was strong CDK5 staining of the nuclei, cytoplasm and axonal processes of the motor neurons in both control dogs and dogs with HCSMA. Further immunocytochemical studies demonstrated CDK5 staining where neurofilaments accumulated, in axonal swellings in the dogs with HCSMA. Our observations suggest phosphorylation-dependent events mediated by CDK5 occur in canine motor neuron disease.

In this study we examined the deletion of the SMN and NAIP genes in 14 Saudi families (16 patients and 38 relatives of the patients, including parents and siblings) and six healthy Saudi volunteers. The homozygous deletions of exons 7 and 8 of the telomeric SMN gene and exon 5 of the NAIP gene were found in seven out of eight spinal muscular atrophy (SMA) type-I patients. In seven SMA type-II patients, exons 7 and 8 of telomeric SMN were deleted in six cases and exon 5 of NAIP was deleted in three cases. Three patients with SMA diagnosis did not show either of the above deletions. All control Saudi volunteers and all but two family members of the patients had both normal SMN and NAIP genes. Our results show that the incidence of NAIP deletion is higher in the more severe SMA cases and the dual deletions of the SMN and NAIP genes are more common in Saudi SMA type-I patients compared to patients of other ethnic groups.

Mild to moderately elevated creatine kinase (CK) activity is a frequent biochemical finding in proximal spinal muscular atrophy (SMA). In a collaborative study on all types of childhood and juvenile onset SMA, we analysed the CK activity of 504 SMA patients (138 type I, 127 type II, 144 type IIIa, and 95 type IIIb patients). Under the assumption of a lognormal distribution of CK activity as the most appropriate statistical model, CK levels were transformed into logarithms and compared by standard deviation scores = CK-SDS (log). CK activity was statistically different between early and later onset SMA: in SMA I and II, about one-third of patients showed CK-SDS (log) >2 SD, the analysis of the means did not show significant differences. In SMA III, CK-SDS (log) was significantly higher (p < 0.01) than in the two other groups, which was most pronounced in SMA IIIb. More than 90% of SMA IIIb patients showed CK-SDS (log) values >2 vs. 57% in SMA IIIa. As similar values were obtained for a subgroup of 100 patients in whom the diagnosis of autosomal recessive SMA was confirmed by a deletion of the telomeric copy of the survival motor neuron gene, our results can be considered representative for SMA I-III. There was no correlation between CK level and disease duration. The fact that patients were ambulatory or chair-bound had no influence on CK activity in type III SMA. There was no sex influence in SMA I, II and IIIa. The observed higher male values in the group SMA IIIb are most likely the result of a lack of female patients with onset after puberty.

We propose a classification system for spinal muscular atrophies (SMA) based on the distribution of clinical signs, paresis and atrophy, as well as on the location of the responsible gene and the resulting enzyme deficiency, whenever these are known. This highly practical classification system encompasses three large SMA groups, as follows. A) Generalized forms, many of which are hereditary, are generally transmitted in a recessive autosomal manner. The course of disease is more severe when symptoms manifest early. Patients whose symptoms first occur after the first year of life often reach adolescence and even adulthood, confirming a highly apparent congruence of intermediate and pseudomyopathic juvenile forms. The same genetic defect, deletion in the 5q11-13.3 locus, that is responsible for acute infantile SMA has been demonstrated in both the aforementioned forms. B) Focal forms are restricted and often isolated cases; when they are hereditary, the genetic profile is highly heterogeneous. Though the disease will not necessarily evolve, it may progress to a generalized form. Focal forms may be symetric, assymetric, spinal-bulbar or multisegmental. The genetic abnormality has been identified for only some forms, such as chronic bulbar-spinal amyotrophy linked to the X-chromosome, at whose location, Xq11, the androgenic receptor is found. C) Amyotrophic lateral sclerosis (ALS) manifests clinically in a variety of ways and may be isolated, familial, juvenile or associated. Familial ALS is related to a gene defect in the 21q22.1 location that codifies for the superoxide dismutase enzyme. One juvenile form of ALS is related to a defect in the 2q33-35 chromosome. Any type of SMA can be related to degenerative neuronal disease of the central nervous system, especially juvenile ALS with generalized SMA, although such a link is at present merely an attractive hypothesis. Specific bibliographic references are given for each SMA form. Figures are provided to illustrate most of the SMA forms included in this classification system, the patients being at this time older adolescents and adults whose disease has been in evidence over many years.

The disease locus for the clinically heterogeneous childhood spinal muscular atrophies (SMA) maps to the chromosome 5 subregion, 5q11.2-13.3. The beta-subunit of beta-D-N-acetylhexosaminidase (hexosaminidase) (EC 3.2.1.52) (Hex B) maps to the same region, and the protein required for substrate recognition by this enzyme, GM2-activator protein, likewise maps to chromosome 5. We have investigated the possibility of allelic variation among some forms of SMA and hexosaminidase deficiency. Recombination between the Hex B and SMA loci eliminates this enzyme as a candidate site for defects causing the illness. Furthermore, we show that, despite previous evidence to the contrary, the GM2-activator locus does not map to chromosome 5, thereby eliminating it as a candidate gene for SMA.

The medical records of 31 patients (19 male and 12 female) with clinical and electrophysiologic features of Wohlfart-Kugelberg-Welander syndrome were reviewed. The reported age at onset ranged from less than one year to 46 years, and the age at diagnosis ranged from three to 66 years. Proximal muscle weakness, especially of the lower extremities, and muscular atrophy were the predominant clinical features. Elevated serum creatine kinase levels were noted in four female and 12 male patients, and the degree of elevation was higher in the male patients (up to 32 times the upper limit of normal) than in the female patients. On initial evaluation, two patients were wheelchair-bound, whereas the others were ambulatory. On follow-up evaluation three to 32 years later (mean, 15.5 years), 11 patients used wheelchairs, although only three were wheelchair-bound. The disease followed a steady, slowly progressive course. The outcome of ambulatory status did not correlate with the initial creatine kinase determination.

Morphological histochemical and ultrastructural examination of the ring fibers in one case of myotonic dystrophy and one case of mitochondrial myopathy provides evidence that the ring fibers develop in the course of fiber splitting and reinnervation of muscle fiber fragments. The reinnervation may have been due to a neurogenic lesion coexisting in both cases with the myopathic picture. The histologically different type of annular myofibrils observed in one case of cap disease is probably due to delayed embryonic development and abnormal relations between the myofibrils and elements of the cytoskeleton.

Lipid peroxidation (LP) and superoxide dismutase (SOD) activity were determined in erythrocytes and skeletal muscle obtained from patients with limb-girdle and facioscapulohumeral muscular dystrophies, neurogenic atrophies and from age-matched control subjects. Neither lipid peroxidation nor SOD activity in erythrocytes of patients differed from control values. SOD activity and LP in muscle specimens were also normal in types of neurogenic atrophy. Lipid peroxidation in the muscle from patients with adult types of muscular dystrophy had a tendency to be increased. The values were widely scattered, the highest being obtained in the older patients with long duration of disease.

Serum acetylcholinesterase (AChE) and pseudocholinesterase (ChE) activity in infantile and juvenile spinal muscular atrophy (SMA) was determined. The total AChE activity was either normal or decreased in the childhood SMA (Type 1), the other SMA groups and disease controls (ALS, X-linked SMA). In the majority of SMA Type 1 cases (6/7 tested) an absence of the asymmetric A12 form was found. This was accompanied by changes in the other asymmetric and globular forms. The latter was, however, not specific for SMA Type 1 cases. The ChE activity was increased in the majority of SMA cases as well as disease controls. The asymmetric A12 ChE form was increased in all SMA Type 3 cases, the values of this form in SMA Type 1 was variable. A change in the ChE globular forms in SMA Type 1 and SMA Type 2 was a frequent finding. It is suggested that the absence of the asymmetric A12 AChE form in SMA Type 1 arises because of muscle cell immaturity and undeveloped muscle-nerve interactions. The reason of ChE changes is obscure.