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Mutations in the RNA binding protein FUS cause amyotrophic lateral sclerosis (ALS), a fatal adult motor neuron disease. Decreased expression of SMN causes the fatal childhood motor neuron disorder spinal muscular atrophy (SMA). The SMN complex localizes in both the cytoplasm and nuclear Gems, and loss of Gems is a cellular hallmark of fibroblasts i...
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Spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS) are severe nervous system diseases characterized by the degeneration of lower motor neurons. They share a number of additional pathological, cellular, and genetic parallels suggesting that mechanistic and clinical insights into one disorder may have value for the other. While the...
Spinal muscular atrophy (SMA) is a severe and clinically-heterogeneous motor neuron disease caused, in most cases, by a homozygous mutation in the SMN1 gene. Regarding the age of onset and motor involvement, at least four distinct clinical phenotypes have been recognized. This clinical variability is, in part, related to the SMN2 copy number. By no...
Dysregulated miRNA expression and mutation of genes involved in miRNA biogenesis have been reported in motor neuron diseases including spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Therefore, identifying molecular mechanisms governing miRNA expression is important to understand these diseases. Here, we report that expressio...
Citations
... Then, cationic dyes can have strong electrostatic interactions with these groups [24]. Also, GO reveals good biocompatibility [25]. In addition, the preparation of GO is easy and provides the potential for the synthesis of low-cost and large-scale productions. ...
In the present study, a quaternary composite consisting of α-ketoglutaric acid, chitosan, polyaniline, and graphene oxide (α-CTS-PANI-GO) was synthesized and characterized by Scanning Electron Microscope, Fourier Transform Infrared Spectroscopy, and Raman spectroscopy. The adsorption of dye on the film was proved by the X-ray diffraction analysis method. According to the increase in the intensity of the peak before and after adsorption, it showed the presence of Methylene Blue (MB) and Acid Orange 7 (AO7). Also, the appearance of peaks related to sulfur in Energy dispersive X-ray (EDX) and EDX elemental mapping, and X-ray photoelectron spectroscopy data after the adsorption process proved the presence of dyes compounds and their adsorption by the synthetic film. The simultaneous removal properties of α-CTS-PANI-GO were evaluated in the removal of cationic and anionic dyes, MB and AO7. Using the Central Composite Decomposition, the effect of different variables such as pH, temperature, adsorbent dose, and time on AO7 and MB removal was studied. The regeneration of α-CTS-PANI-GO film was studied and the zero point charge of the synthesized film was also determined. Various isotherm models, such as Langmuir and Freundlich, were investigated, and the processing of experimental equilibrium data confirmed the suitability and application of the Langmuir model. Analysis of experimental adsorption data using various kinetic models, such as pseudo-first and second-order models, shows the applicability of the first-order equation. The results showed that in the optimal conditions, the removal percentage was about 95% for AO7 and 80% for MB, which indicates the successful performance of the synthetic film compared to the method.
... Thus, SMA and ALS are not only connected by pathogenic commonalities [26], but also appear to both originate from alterations in RBP-mediated regulatory mechanisms. Further strengthening the possibility that these two MNDs may be molecularly connected, recent studies have suggested that SMN, FUS and TDP-43 belong to common molecular complexes and also exhibit functional interactions [27][28][29][30][31][32][33]. Together, these results have raised the hypothesis that SMN, FUS and TDP-43 may control common transcriptional and/or posttranscriptional regulatory steps. ...
Background
Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS) share phenotypic and molecular commonalities, including the fact that they can be caused by mutations in ubiquitous proteins involved in RNA metabolism, namely SMN, TDP-43 and FUS. Although this suggests the existence of common disease mechanisms, there is currently no model to explain the resulting motor neuron dysfunction. In this work we generated a parallel set of Drosophila models for adult-onset RNAi and tagged neuronal expression of the fly orthologues of the three human proteins, named Smn, TBPH and Caz, respectively. We profiled nuclear and cytoplasmic bound mRNAs using a RIP-seq approach and characterized the transcriptome of the RNAi models by RNA-seq. To unravel the mechanisms underlying the common functional impact of these proteins on neuronal cells, we devised a computational approach based on the construction of a tissue-specific library of protein functional modules, selected by an overall impact score measuring the estimated extent of perturbation caused by each gene knockdown.
Results
Transcriptome analysis revealed that the three proteins do not bind to the same RNA molecules and that only a limited set of functionally unrelated transcripts is commonly affected by their knock-down. However, through our integrative approach we were able to identify a concerted effect on protein functional modules, albeit acting through distinct targets. Most strikingly, functional annotation revealed that these modules are involved in critical cellular pathways for motor neurons, including neuromuscular junction function. Furthermore, selected modules were found to be significantly enriched in orthologues of human neuronal disease genes.
Conclusions
The results presented here show that SMA and ALS disease-associated genes linked to RNA metabolism functionally converge on neuronal protein complexes, providing a new hypothesis to explain the common motor neuron phenotype. The functional modules identified represent promising biomarkers and therapeutic targets, namely given their alteration in asymptomatic settings.
... The GAPDH and TDP-43 antibodies were from Proteintech (Cat No. 60004-1-Ig and 10782-2-AP, respectively). The FUS antibody was described previously (27). The HLA-DR antibody was from Abcam (ab20181). ...
Mutations in RNA/DNA-binding proteins cause amyotrophic lateral sclerosis (ALS), but the underlying disease mechanisms remain unclear. Here, we report that a set of ALS-associated proteins, namely FUS, EWSR1, TAF15, and MATR3, impact the expression of genes encoding the major histocompatibility complex II (MHC II) antigen presentation pathway. Both subunits of the MHC II heterodimer, HLA-DR, are down-regulated in ALS gene knockouts/knockdown in HeLa and human microglial cells, due to loss of the MHC II transcription factor CIITA. Importantly, hematopoietic progenitor cells (HPCs) derived from human embryonic stem cells bearing the FUS R495X mutation and HPCs derived from C9ORF72 ALS patient induced pluripotent stem cells also exhibit disrupted MHC II expression. Given that HPCs give rise to numerous immune cells, our data raise the possibility that loss of the MHC II pathway results in global failure of the immune system to protect motor neurons from damage that leads to ALS.
... Pre-mRNA splicing, CBs and SMN-containing Gem structures are all commonly disrupted in ALS and spinal muscular atrophy (SMA) 44,[52][53][54] . In light of this, and the association of hnRNPUL1 with other ALScausing proteins, C9orf72 repeats and its prion-like characteristics, we screened two cohorts of familial (n=1,022, ALS variant server) and sporadic (n=4,366) 55 ALS patients for pathogenic coding mutations within hnRNPUL1. ...
Integrator cleaves nascent RNA, triggering RNA polymerase II transcription termination, but how cleavage is regulated is poorly understood. Here we show hnRNPUL1 ensures efficient Integrator-mediated cleavage of nascent RNA downstream of snRNA genes and, in the case of U2 snRNA, binds a terminal stem-loop involved in this process. In the nucleoplasm, hnRNPUL1 binds U4 snRNA and SART3 and enables efficient reformation of the U4:U6 di-snRNP for further rounds of pre-mRNA splicing. Sustained hnRNPUL1 loss leads to reduced levels of snRNAs, defects in histone mRNA 3′ end processing and loss of Cajal bodies. hnRNPUL1 binds RNA through multiple domains, including a globular central domain comprising tightly juxtaposed SPRY and dead polynucleotide kinase folds. This latter fold allows binding to 5′-monophosphorylated RNAs in a mutually exclusive manner with ATP binding and functions as an XRN2 antagonist when overexpressed. We identify a cohort of amyotrophic lateral sclerosis patients harbouring disruptive mutations in hnRNPUL1. SMN loss in spinal muscular atrophy and hnRNPUL1 loss both disrupt snRNP biogenesis, leading to motor neuron death, suggesting a common aetiology.
... Recently, it was shown that mutant FUS aberrantly contacts U1 snRNA and irreversibly sequesters small nuclear RNP (snRNP) assembly intermediates by promoting the maturation of stress granules into pathological FUS aggregates (104). There is also evidence that mutant FUS interacts with the SMN factor and sequesters it into FUS cytoplasmic condensates, leading to a loss of splicing activity (20,76,105). Even though we were able to identify several factors that probably contribute to the alterations in AS during the pathology, the observed complex changes in AS at the terminal stage of the disease are tricky to interpret. ...
Dysfunction of the RNA-binding protein (RBP) FUS implicated in RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. Mutations affecting FUS nuclear localization can drive RNA splicing defects and stimulate the formation of non-amyloid inclusions in affected neurons. However, the mechanism by which FUS mutations contribute to the development of ALS remains uncertain. Here we describe a pattern of RNA splicing changes in the dynamics of the continuous proteinopathy induced by mislocalized FUS. We show that the decrease in intron retention of FUS-associated transcripts represents the hallmark of the pathogenesis of ALS and is the earliest molecular event in the course of progression of the disease. As FUS aggregation increases, the pattern of RNA splicing changes, becoming more complex, including a decrease in the inclusion of neuron-specific microexons and induction of cryptic exon splicing due to the sequestration of additional RBPs into FUS aggregates. Crucially, the identified features of the pathological splicing pattern are also observed in ALS patients in both sporadic and familial cases. Our data provide evidence that both a loss of nuclear FUS function due to mislocalization and the subsequent cytoplasmic aggregation of mutant protein lead to the disruption of RNA splicing in a multistep fashion during FUS aggregation.
... and microRNA-mediated gene silencing 10,11 . It also interacts with spliceosomal U snRNAs [35][36][37][38] and we previously reported the interaction of FUS with U7 snRNA 9,16 . Moreover, many microRNAs and other small RNAs, including snoRNAs, were differentially expressed in sporadic ALS patients compared to healthy age-matched controls 39 . ...
... Recent studies suggests the role of another RNA binding protein involved in ALS, TDP-43 15,42 , in regulating site-specific 2'-O-methylation of U1 and U2 snRNAs, by controlling the localization of a subset of C/D box scaRNAs 43 . FUS is involved in major and minor intron splicing by its direct association with U1, U2, U11, and U12 snRNAs as well as in replication-dependent histone gene expression by interaction with U7 snRNA 9,16,36,44 . These interactions are disturbed by ALS-associated mutations in FUS 16,37,45 . ...
FUS is a multifunctional protein involved in many aspects of RNA metabolism, including transcription, splicing, translation, miRNA processing, and replication-dependent histone gene expression. In this work, we show that FUS depletion results in the differential expression of numerous small nucleolar RNAs (snoRNAs) that guide 2’-O methylation (2’-O-Me) and pseudouridylation of specific positions in ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). Using RiboMeth-seq and HydraPsiSeq for the profiling of 2’-O-Me and pseudouridylation status of rRNA species, we demonstrated considerable hypermodification at several sites in HEK293T and SH-SY5Y cells with FUS knockout (FUS KO) compared to wild-type cells. We observed a similar direction of changes in rRNA modification in differentiated SH-SY5Y cells with the FUS mutation (R495X) related to the severe disease phenotype of amyotrophic lateral sclerosis (ALS). Furthermore, the pattern of modification of some rRNA positions was correlated with the abundance of corresponding guide snoRNAs in FUS KO and FUS R495X cells. Our findings reveal a new role for FUS in modulating the modification pattern of rRNA molecules, that in turn might generate ribosome heterogeneity and constitute a fine-tuning mechanism for translation efficiency/fidelity. Therefore, we suggest that increased levels of 2’-O-Me and pseudouridylation at particular positions in rRNAs from cells with the ALS-linked FUS mutation may represent a possible new translation-related mechanism that underlies disease development and progression.
... SMA motor neurons have been reported to show alternations in miR-1 and miR-206 expression (Wang et al, 2014;Luchetti et al, 2015;Wertz et al, 2016). In addition, SMN protein binds to miR processing proteins including fragile X mental retardation protein, KH-type splicing regulatory protein, and fused in sarcoma/translocated in liposarcoma (Piazzon et al, 2008;Tadesse et al, 2008;Yamazaki et al, 2012). Therefore, it is thought that SMN deficiency disturbs miR processing, thus altering miR expression. ...
Spinal muscular atrophy (SMA) is a congenital neuromuscular disease caused by the mutation or deletion of the survival motor neuron 1 (SMN1) gene. Although the primary cause of progressive muscle atrophy in SMA has classically been considered the degeneration of motor neurons, recent studies have indicated a skeletal muscle–specific pathological phenotype such as impaired mitochondrial function and enhanced cell death. Here, we found that the down-regulation of SMN causes mitochondrial dysfunction and subsequent cell death in in vitro models of skeletal myogenesis with both a murine C2C12 cell line and human induced pluripotent stem cells. During myogenesis, SMN binds to the upstream genomic regions of MYOD1 and microRNA (miR)-1 and miR-206. Accordingly, the loss of SMN down-regulates these miRs, whereas supplementation of the miRs recovers the mitochondrial function, cell survival, and myotube formation of SMN-deficient C2C12, indicating the SMN-miR axis is essential for myogenic metabolic maturation. In addition, the introduction of the miRs into ex vivo muscle stem cells derived from Δ7-SMA mice caused myotube formation and muscle contraction. In conclusion, our data revealed novel transcriptional roles of SMN during myogenesis, providing an alternative muscle-oriented therapeutic strategy for SMA patients.
... The majority of ALS-causing mutations harbor the LCD regions of SG-related RBPs, resulting in abnormal LLPS abilities that impair SG homeostasis and lead to irreversible and toxic aggregates [100]. In addition, reduced numbers of GEM have been observed in the spinal cords and fibroblasts of patients with ALS [99,101]. These findings indicate the ability of protein aggregates to sequester RNA and the resultant effects on cells. ...
Amyotrophic lateral sclerosis (ALS) is a devastating progressive neurodegenerative disorder characterized by selective loss of lower and upper motor neurons (MNs) in the brain and spinal cord, resulting in paralysis and eventually death due to respiratory insufficiency. Although the fundamental physiological mechanisms underlying ALS are not completely understood, the key neuropathological hallmarks of ALS pathology are the aggregation and accumulation of ubiquitinated protein inclusions within the cytoplasm of degenerating MNs. Herein, we discuss recent insights into the molecular mechanisms that lead to the accumulation of protein aggregates in ALS. This will contribute to a better understanding of the pathophysiology of the disease and may open novel avenues for the development of therapeutic strategies.
... Despite the observation that the restoration of Mdm2/4 expression improved motor functions to some degree, this restoration of Mdm2/4 did not beneficially affect survival of SMA mice [300]. Beside the components of the classical SMN complex, additional SMN interaction partners have been identified; among them hnRNP R [256], TDP-43 [296], FUS [323] and HuD [82], which are involved in many neuronal functions including transcription regulation, nuclear pre-mRNA processing, nuclear export and subcellular transport of many mRNAs [4, 18,81,82,102,104,110,218,255,325,326]. In particular the axonal translocation of the β-actin mRNA is severely disturbed in Smn- [255], hnRNP R- [102] and TDP-43- [33] deficient neurons. ...
Background
Major efforts have been made in the last decade to develop and improve therapies for proximal spinal muscular atrophy (SMA). The introduction of Nusinersen/Spinraza™ as an antisense oligonucleotide therapy, Onasemnogene abeparvovec/Zolgensma™ as an AAV9-based gene therapy and Risdiplam/Evrysdi™ as a small molecule modifier of pre-mRNA splicing have set new standards for interference with neurodegeneration.
Main body
Therapies for SMA are designed to interfere with the cellular basis of the disease by modifying pre-mRNA splicing and enhancing expression of the Survival Motor Neuron (SMN) protein, which is only expressed at low levels in this disorder. The corresponding strategies also can be applied to other disease mechanisms caused by loss of function or toxic gain of function mutations. The development of therapies for SMA was based on the use of cell culture systems and mouse models, as well as innovative clinical trials that included readouts that had originally been introduced and optimized in preclinical studies. This is summarized in the first part of this review. The second part discusses current developments and perspectives for amyotrophic lateral sclerosis, muscular dystrophies, Parkinson's and Alzheimer's disease, as well as the obstacles that need to be overcome to introduce RNA-based therapies and gene therapies for these disorders.
Conclusion
RNA-based therapies offer chances for therapy development of complex neurodegenerative disorders such as amyotrophic lateral sclerosis, muscular dystrophies, Parkinson’s and Alzheimer’s disease. The experiences made with these new drugs for SMA, and also the experiences in AAV gene therapies could help to broaden the spectrum of current approaches to interfere with pathophysiological mechanisms in neurodegeneration.
... FUS is known to regulate microRNA biogenesis and microRNA-mediated gene silencing (10,11). It also interacts with spliceosomal U snRNAs (35)(36)(37)(38) and we previously reported the interaction of FUS with U7 snRNA (9,16). Moreover, many microRNAs and other small RNAs, including snoRNAs, were differentially expressed in sporadic ALS patients compared to healthy age-matched controls (39). ...
... Recent studies suggests the role of another RNA binding protein involved in ALS, TDP-43 (15,46), in regulating site-specific 2'-O-methylation of U1 and U2 snRNAs, by controlling the localization of a subset of C/D box scaRNAs (47). FUS is involved in major and minor intron splicing by its direct association with U1, U2, U11, and U12 snRNAs as well as in replication-dependent histone gene expression by interaction with U7 snRNA (9,16,36,48). These interactions are disturbed by ALS-associated mutations in FUS (16,37,49). ...
FUS is a multifunctional protein involved in many aspects of RNA metabolism, including transcription, splicing, translation, miRNA processing, and replication-dependent histone gene expression. In this paper, we show that FUS depletion results in differential expression of numerous small nucleolar RNAs (snoRNAs) that guide 2-O methylation (2-O-Me) and pseudouridylation of specific positions in ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). Using RiboMeth-seq and HydraPsiSeq for the profiling of 2-O-Me and pseudouridylation status of rRNA species, we demonstrated considerable hypermodification at several sites in HEK293T and SH-SY5Y cells with FUS knockout (FUS KO) compared to wild-type cells. We observed a similar direction of changes in rRNA modification in differentiated SH-SY5Y cells with the FUS mutation (R495X) related to the severe disease phenotype of amyotrophic lateral sclerosis (ALS). Furthermore, the pattern of modification of some rRNA positions was correlated with the abundance of corresponding guide snoRNAs in FUS KO and FUS R495X cells. Our findings reveal a new role for FUS in modulating the modification pattern of rRNA molecules, that in turn might generate ribosome heterogeneity and constitute a fine-tuning mechanism for translation efficiency/fidelity. Therefore, we suggest that increased levels of 2-O-Me and pseudouridylation at particular positions in rRNAs from cells with the ALS-linked FUS mutation may represent a possible new translation-related mechanism that underlies disease development and/or progression.
