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Mutations within immunoglobulin mu DNA binding protein (IGHMBP2), an RNA-DNA helicase, result in SMA with respiratory distress type I (SMARD1) and Charcot Marie Tooth type 2S (CMT2S). The underlying biochemical mechanism of IGHMBP2 is unknown as well as the functional significance of IGHMBP2 mutations in disease severity. Here we report the biochemical mechanisms of IGHMBP2 disease-causing mutations D565N and H924Y, and their potential impact on therapeutic strategies. The IGHMBP2-D565N mutation has been identified in SMARD1 patients, while the IGHMBP2-H924Y mutation has been identified in CMT2S patients. For the first time, we demonstrate a correlation between the altered IGHMBP2 biochemical activity associated with the D565N and H924Y mutations and disease severity and pathology in patients and our Ighmbp2 mouse models. We show that IGHMBP2 mutations that alter the association with activator of basal transcription (ABT1) impact the ATPase and helicase activities of IGHMBP2 and the association with the 47S pre-rRNA 5' external transcribed spacer. We demonstrate that the D565N mutation impairs IGHMBP2 ATPase and helicase activities consistent with disease pathology. The H924Y mutation alters IGHMBP2 activity to a lesser extent while maintaining association with ABT1. In the context of the compound heterozygous patient, we demonstrate that the total biochemical activity associated with IGHMBP2-D565N and IGHMBP2-H924Y proteins is improved over IGHMBP2-D565N alone. Importantly, we demonstrate that the efficacy of therapeutic applications may vary based on the underlying IGHMBP2 mutations and the relative biochemical activity of the mutant IGHMBP2 protein., Competing Interests: Declaration of competing interest CL is co-founder and chief scientific officer of Shift Pharmaceuticals. CLL has received more than $10,000 in income per annum from Shift Pharmaceuticals. Research in the CLL and MAL labs have been supported by subawards from Shift Pharmaceuticals (as part of grants from the DOD, CMT Research Foundation and the NIH). CLL and MU share patents on novel compounds licensed by Shift Pharmaceuticals and planned patents for additional novel compounds. KS is chief scientific officer for Sanctum Therapeutics Corporation. KS has received more than $10,000 in income per annum from Sanctum Therapeutics Corporation. KS and MU share patents on novel compounds licensed by Sanctum Therapeutics Corporation and planned patents for additional novel compounds. MAL is associated with Shift by family relation., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Spinal Muscular Atrophy (SMA) is an autosomal recessive neuromuscular disease caused by deletions or mutations in the survival motor neuron (SMN1) gene. An important hallmark of disease progression is the pathology of neuromuscular junctions (NMJs). Affected NMJs in the SMA context exhibit delayed maturation, impaired synaptic transmission, and loss of contact between motor neurons and skeletal muscle. Protection and maintenance of NMJs remains a focal point of therapeutic strategies to treat SMA, and the recent implication of the NMJ-organizer Agrin in SMA pathology suggests additional NMJ organizing molecules may contribute. DOK7 is an NMJ organizer that functions downstream of Agrin. The potential of DOK7 as a putative therapeutic target was demonstrated by adeno-associated virus (AAV)-mediated gene therapy delivery of DOK7 in Amyotrophic Lateral Sclerosis (ALS) and Emery Dreyefuss Muscular Dystrophy (EDMD). To assess the potential of DOK7 as a disease modifier of SMA, we administered AAV-DOK7 to an intermediate mouse model of SMA. AAV9-DOK7 treatment conferred improvements in NMJ architecture and reduced muscle fiber atrophy. Additionally, these improvements resulted in a subtle reduction in phenotypic severity, evidenced by improved grip strength and an extension in survival. These findings reveal DOK7 is a novel modifier of SMA., Competing Interests: Declaration of competing interest C.L.L. is the co-founder and Chief Scientific Officer of Shift Pharmaceuticals., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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

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

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

Spinal Muscular Atrophy (SMA), an autosomal recessive neuromuscular disorder, is the leading genetic cause of infant mortality. SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1). SMA, however, is not due to complete absence of SMN, rather a low level of functional full-length SMN is produced by a nearly identical copy gene called SMN2. Despite SMN's ubiquitous expression, motor neurons are preferentially affected by low SMN levels. Recently gene replacement strategies have shown tremendous promise in animal models of SMA. In this study, we used self-complementary Adeno Associated Virus (scAAV) expressing full-length SMN cDNA to compare two different routes of viral delivery in a severe SMA mouse model. This was accomplished by injecting scAAV9-SMN vector intravenously (IV) or intracerebroventricularly (ICV) into SMA mice. Both routes of delivery resulted in a significant increase in lifespan and weight compared to untreated mice with a subpopulation of mice surviving more than 200days. However, the ICV injected mice gained significantly more weight than their IV treated counterparts. Likewise, survival analysis showed that ICV treated mice displayed fewer early deaths than IV treated animals. Collectively, this report demonstrates that route of delivery is a crucial component of gene therapy treatment for SMA., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.)

The Ewing Sarcoma (EWS) protein is a ubiquitously expressed RNA processing factor that localises predominantly to the nucleus. However, the mechanism through which EWS enters the nucleus remains unclear, with differing reports identifying three separate import signals within the EWS protein. Here we have utilized a panel of truncated EWS proteins to clarify the reported nuclear localisation signals. We describe three C-terminal domains that are important for efficient EWS nuclear localization: (1) the third RGG-motif; (2) the last 10 amino acids (known as the PY-import motif); and (3) the zinc-finger motif. Although these three domains are involved in nuclear import, they are not independently capable of driving the efficient import of a GFP-moiety. However, collectively they form a complex tripartite signal that efficiently drives GFP-import into the nucleus. This study helps clarify the EWS import signal, and the identification of the involvement of both the RGG- and zinc-finger motifs has wide reaching implications.

Gemin4 is a ubiquitously expressed multifunctional protein that is involved in U snRNP assembly, apoptosis, nuclear/cytoplasmic transportation, transcription, and RNAi pathways. Gemin4 is one of the core components of the Gemin-complex, which also contains survival motor neuron (SMN), the seven Gemin proteins (Gemin2-8), and Unrip. Mutations in the SMN1 gene cause the autosomal recessive disorder spinal muscular atrophy (SMA). Although the functions assigned to Gemin4 predominantly occur in the nucleus, the mechanisms that mediate the nuclear import of Gemin4 remain unclear. Here, using a novel panel of Gemin4 constructs we identify a canonical nuclear import sequence (NLS) in the N-terminus of Gemin4. The Gemin4 NLS is necessary and independently sufficient to mediate nuclear import of Gemin4. This is the first functional NLS identified within the SMN-Gemin complex.

Spinal muscular atrophy (SMA) is a severe neuromuscular disease characterized by loss of spinal alpha-motor neurons, resulting in the paralysis of skeletal muscle. SMA is caused by deficiency of survival motor neuron (SMN) protein levels. Recent evidence has highlighted an axon-specific role for SMN protein, raising the possibility that axon degeneration may be an early event in SMA pathogenesis. The Wallerian degeneration slow (Wld(s)) gene is a spontaneous dominant mutation in mice that delays axon degeneration by approximately 2-3 weeks. We set out to examine the effect of Wld(s) on the phenotype of a mouse model of SMA. We found that Wld(s) does not alter the SMA phenotype, indicating that Wallerian degeneration does not directly contribute to the pathogenesis of SMA development.