scholarly journals Safety and efficacy of C9ORF72-repeat RNA nuclear export inhibition in amyotrophic lateral sclerosis

2021 ◽  
Author(s):  
Lydia M Castelli ◽  
Luisa Cutillo ◽  
Cleide Dos Santos Souza ◽  
Alvaro Sanchez-Martinez ◽  
Ilaria Granata ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Motor Neurons ◽  
Nuclear Export ◽  
Safety And Efficacy ◽  
Gene Therapy Approach ◽  
Genome Wide ◽  
Disease Modifying ◽  
Lateral Sclerosis ◽  
Partial Depletion

Background: Loss of motor neurons in amyotrophic lateral sclerosis (ALS) leads to progressive paralysis and death. Dysregulation of thousands of RNA molecules with roles in multiple cellular pathways hinders the identification of ALS-causing alterations over downstream changes secondary to the neurodegenerative process. How many and which of these pathological gene expression changes require therapeutic normalisation remains a fundamental question. Methods: Here, we investigated genome-wide RNA changes in C9ORF72-ALS patient-derived neurons and Drosophila, as well as upon neuroprotection taking advantage of our gene therapy approach which specifically inhibits the SRSF1-dependent nuclear export of pathological C9ORF72-repeat transcripts. This is a critical study to evaluate (i) the overall safety and efficacy of the partial depletion of SRSF1, a member of a protein family involved itself in gene expression, and (ii) a unique opportunity to identify neuroprotective RNA changes. Results: Our study demonstrates that manipulation of 362 transcripts out of 2,257 pathological changes in C9ORF72-ALS patient-derived neurons is sufficient to confer neuroprotection upon partial depletion of SRSF1. In particular, expression of 90 disease-altered transcripts is fully reverted upon neuroprotection leading to the characterisation of a human C9ORF72-ALS disease-modifying gene expression signature. These findings were further investigated in vivo in diseased and neuroprotected Drosophila transcriptomes, highlighting a list of 21 neuroprotective changes conserved with 16 human orthologues in patient-derived neurons. We also functionally validated the high therapeutic potential of one of these disease-modifying transcripts, demonstrating that inhibition of ALS-upregulated human KCNN1-3 (Drosophila SK) voltage-gated potassium channel orthologs mitigates degeneration of human motor neurons as well as Drosophila motor deficits. Conclusions: Strikingly, manipulating the expression levels of a small proportion of RNAs is sufficient to induce a therapeutic effect, further indicating that the SRSF1-targeted gene therapy approach is safe in the above preclinical models as it does not disrupt globally gene expression. The efficacy of this intervention is also validated at genome-wide level with therapeutically-induced RNA changes involved in the vast majority of biological processes affected in C9ORF72-ALS. Finally, the identification of a characteristic signature with key RNA changes modified in both the disease state and upon neuroprotection also provides potential new therapeutic targets and biomarkers.

scholarly journals SRSF1-dependent inhibition of C9ORF72-repeat RNA nuclear export: genome-wide mechanisms for neuroprotection in amyotrophic lateral sclerosis

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Lydia M. Castelli ◽  
Luisa Cutillo ◽  
Cleide Dos Santos Souza ◽  
Alvaro Sanchez-Martinez ◽  
Ilaria Granata ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amyotrophic Lateral Sclerosis ◽  
Motor Neurons ◽  
Nuclear Export ◽  
Gene Expression Signature ◽  
Critical Study ◽  
Genome Wide ◽  
Disease Modifying ◽  
Lateral Sclerosis ◽  
Partial Depletion

Abstract Background Loss of motor neurons in amyotrophic lateral sclerosis (ALS) leads to progressive paralysis and death. Dysregulation of thousands of RNA molecules with roles in multiple cellular pathways hinders the identification of ALS-causing alterations over downstream changes secondary to the neurodegenerative process. How many and which of these pathological gene expression changes require therapeutic normalisation remains a fundamental question. Methods Here, we investigated genome-wide RNA changes in C9ORF72-ALS patient-derived neurons and Drosophila, as well as upon neuroprotection taking advantage of our gene therapy approach which specifically inhibits the SRSF1-dependent nuclear export of pathological C9ORF72-repeat transcripts. This is a critical study to evaluate (i) the overall safety and efficacy of the partial depletion of SRSF1, a member of a protein family involved itself in gene expression, and (ii) a unique opportunity to identify neuroprotective RNA changes. Results Our study shows that manipulation of 362 transcripts out of 2257 pathological changes, in addition to inhibiting the nuclear export of repeat transcripts, is sufficient to confer neuroprotection in C9ORF72-ALS patient-derived neurons. In particular, expression of 90 disease-altered transcripts is fully reverted upon neuroprotection leading to the characterisation of a human C9ORF72-ALS disease-modifying gene expression signature. These findings were further investigated in vivo in diseased and neuroprotected Drosophila transcriptomes, highlighting a list of 21 neuroprotective changes conserved with 16 human orthologues in patient-derived neurons. We also functionally validated the high neuroprotective potential of one of these disease-modifying transcripts, demonstrating that inhibition of ALS-upregulated human KCNN1–3 (Drosophila SK) voltage-gated potassium channel orthologs mitigates degeneration of human motor neurons and Drosophila motor deficits. Conclusions Strikingly, the partial depletion of SRSF1 leads to expression changes in only a small proportion of disease-altered transcripts, indicating that not all RNA alterations need normalization and that the gene therapeutic approach is safe in the above preclinical models as it does not disrupt globally gene expression. The efficacy of this intervention is also validated at genome-wide level with transcripts modulated in the vast majority of biological processes affected in C9ORF72-ALS. Finally, the identification of a characteristic signature with key RNA changes modified in both the disease state and upon neuroprotection also provides potential new therapeutic targets and biomarkers.

scholarly journals Xrp1 genetically interacts with the ALS-associated FUS orthologue caz and mediates its toxicity

2018 ◽  
Vol 217 (11) ◽  
pp. 3947-3964 ◽  
Author(s):  
Moushami Mallik ◽  
Marica Catinozzi ◽  
Clemens B. Hug ◽  
Li Zhang ◽  
Marina Wagner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Life Span ◽  
Motor Neurons ◽  
Gene Expression Regulation ◽  
Genetic Interaction ◽  
Motor Deficits ◽  
Chromatin Binding ◽  
Mutant Phenotypes ◽  
Gene Expression Dysregulation ◽  
Lateral Sclerosis

Cabeza (caz) is the single Drosophila melanogaster orthologue of the human FET proteins FUS, TAF15, and EWSR1, which have been implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. In this study, we identified Xrp1, a nuclear chromatin-binding protein, as a key modifier of caz mutant phenotypes. Xrp1 expression was strongly up-regulated in caz mutants, and Xrp1 heterozygosity rescued their motor defects and life span. Interestingly, selective neuronal Xrp1 knockdown was sufficient to rescue, and neuronal Xrp1 overexpression phenocopied caz mutant phenotypes. The caz/Xrp1 genetic interaction depended on the functionality of the AT-hook DNA-binding domain in Xrp1, and the majority of Xrp1-interacting proteins are involved in gene expression regulation. Consistently, caz mutants displayed gene expression dysregulation, which was mitigated by Xrp1 heterozygosity. Finally, Xrp1 knockdown substantially rescued the motor deficits and life span of flies expressing ALS mutant FUS in motor neurons, implicating gene expression dysregulation in ALS-FUS pathogenesis.

scholarly journals Single-dose MGTA-145/plerixafor leads to efficient mobilization and in vivo transduction of HSCs with thalassemia correction in mice

2021 ◽  
Vol 5 (5) ◽  
pp. 1239-1249
Author(s):  
Chang Li ◽  
Kevin A. Goncalves ◽  
Tamás Raskó ◽  
Amit Pande ◽  
Sucheol Gil ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Mononuclear Cells ◽  
Vector System ◽  
Hemopoietic Stem Cell ◽  
Peripheral Blood Mononuclear ◽  
Gene Therapy Approach ◽  
Vector Integration ◽  
Genome Wide ◽  
Gfp Marking

Abstract We have developed an in vivo hemopoietic stem cell (HSC) gene therapy approach without the need for myelosuppressive conditioning and autologous HSC transplantation. It involves HSC mobilization and IV injection of a helper-dependent adenovirus HDAd5/35++ vector system. The current mobilization regimen consists of granulocyte colony-stimulating factor (G-CSF) injections over a 4-day period, followed by the administration of plerixafor/AMD3100. We tested a simpler, 2-hour, G-CSF–free mobilization regimen using truncated GRO-β (MGTA-145; a CXCR2 agonist) and plerixafor in the context of in vivo HSC transduction in mice. The MGTA-145+plerixafor combination resulted in robust mobilization of HSCs. Importantly, compared with G-CSF+plerixafor, MGTA-145+plerixafor led to significantly less leukocytosis and no elevation of serum interleukin-6 levels and was thus likely to be less toxic. With both mobilization regimens, after in vivo selection with O6-benzylguanine (O6BG)/BCNU, stable GFP marking was achieved in >90% of peripheral blood mononuclear cells. Genome-wide analysis showed random, multiclonal vector integration. In vivo HSC transduction after mobilization with MGTA-145+plerixafor in a mouse model for thalassemia resulted in >95% human γ-globin+ erythrocytes at a level of 36% of mouse β-globin. Phenotypic analyses showed a complete correction of thalassemia. The γ-globin marking percentage and level were maintained in secondary recipients, further demonstrating that MGTA145+plerixafor mobilizes long-term repopulating HSCs. Our study indicates that brief exposure to MGTA-145+plerixafor may be advantageous as a mobilization regimen for in vivo HSC gene therapy applications across diseases, including thalassemia and sickle cell disease.

scholarly journals Disease-modifying effects of an SCAF4 structural variant in a predominantly SOD1 ALS cohort

2020 ◽  
Vol 6 (4) ◽  
pp. e470
Author(s):  
Julia Pytte ◽  
Loren L. Flynn ◽  
Ryan S. Anderton ◽  
Frank L. Mastaglia ◽  
Frances Theunissen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Age At Onset ◽  
Structural Variant ◽  
Carboxy Terminal Domain ◽  
Sporadic Als ◽  
Evaluation Algorithm ◽  
Disease Modifying ◽  
Independent Effects ◽  
Carboxy Terminal ◽  
Lateral Sclerosis

ObjectiveTo test the hypothesis that rs573116164 will have disease-modifying effects in patients with superoxide dismutase 1 (SOD1) familial amyotrophic lateral sclerosis (fALS), we characterized rs573116164 within a cohort of 190 patients with fALS and 560 healthy age-matched controls to assess the variant for association with various measures of disease.MethodsUsing a previously described bioinformatics evaluation algorithm, a polymorphic short structural variant associated with SOD1 was identified according to its theoretical effect on gene expression. An 12–18 poly-T repeat (rs573116164) within the 3′ untranslated region of serine and arginine rich proteins-related carboxy terminal domain associated factor 4 (SCAF4), a gene that is adjacent to SOD1, was assessed for disease association and influence on survival and age at onset in an fALS cohort using PCR, Sanger sequencing, and capillary separation techniques for allele detection.ResultsIn a North American cohort of predominantly SOD1 fALS patients (n =190) and age-matched healthy controls (n = 560), we showed that carriage of an 18T SCAF4 allele was associated with disease within this cohort (odds ratio [OR] 6.6; 95% confidence interval [CI] 3.9–11.2; p = 4.0e-11), but also within non-SOD1 cases (n = 27; OR 5.3; 95% CI 1.9–14.5; p = 0.0014). This finding suggests genetically SOD1-independent effects of SCAF4 on fALS susceptibility. Furthermore, carriage of an 18T allele was associated with a 26-month reduction in survival time (95% CI 6.6–40.8; p = 0.014), but did not affect age at onset of disease.ConclusionsThe findings in this fALS cohort suggest that rs573116164 could have SOD1-independent and broader relevance in ALS, warranting further investigation in other fALS and sporadic ALS cohorts, as well as studies of functional effects of the 18T variant on gene expression.

scholarly journals The Closely Related RNA helicases, UAP56 and URH49, Preferentially Form Distinct mRNA Export Machineries and Coordinately Regulate Mitotic Progression

2010 ◽  
Vol 21 (16) ◽  
pp. 2953-2965 ◽  
Author(s):  
Tomohiro Yamazaki ◽  
Naoko Fujiwara ◽  
Hiroko Yukinaga ◽  
Miki Ebisuya ◽  
Takuya Shiki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Export ◽  
Mrna Export ◽  
Mitotic Progression ◽  
Rna Helicases ◽  
Target Mrnas ◽  
Genome Wide ◽  
Chromatid Cohesion ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene

Nuclear export of mRNA is an essential process for eukaryotic gene expression. The TREX complex couples gene expression from transcription and splicing to mRNA export. Sub2, a core component of the TREX complex in yeast, has diversified in humans to two closely related RNA helicases, UAP56 and URH49. Here, we show that URH49 forms a novel URH49–CIP29 complex, termed the AREX (alternative mRNA export) complex, whereas UAP56 forms the human TREX complex. The mRNAs regulated by these helicases are different at the genome-wide level. The two sets of target mRNAs contain distinct subsets of key mitotic regulators. Consistent with their target mRNAs, depletion of UAP56 causes mitotic delay and sister chromatid cohesion defects, whereas depletion of URH49 causes chromosome arm resolution defects and failure of cytokinesis. In addition, depletion of the other human TREX components or CIP29 causes mitotic defects similar to those observed in UAP56- or URH49-depleted cells, respectively. Taken together, the two closely related RNA helicases have evolved to form distinct mRNA export machineries, which regulate mitosis at different steps.

scholarly journals A Complex Network of MicroRNAs Expressed in Brain and Genes Associated with Amyotrophic Lateral Sclerosis

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Santosh Shinde ◽  
Neelima Arora ◽  
Utpal Bhadra
Keyword(s):  
Gene Expression ◽  
Amyotrophic Lateral Sclerosis ◽  
Motor Neurons ◽  
Neurological Disease ◽  
Complex Interplay ◽  
Target Sites ◽  
Sites Of Action ◽  
Lateral Sclerosis ◽  
Similar Search

Amyotrophic Lateral Sclerosis (ALS) is a rare neurological disease affecting mainly motor neurons and often leads to paralysis and death in extreme cases. For exploring the role of microRNAs in genes regulation in ALS disease, miRanda was employed for prediction of target sites of miRNAs expressed in various parts of brain and CNS on 35 genes associated with ALS. Similar search was conducted using TargetScan and PicTar for prediction of target sites in3′UTR only. 1456 target sites were predicted using miRanda and more target sites were found in5′UTR and CDS region as compared to3′UTR. 11 target sites were predicted to be common by all the algorithms and, thus, these represent the most significant sites. Target site hotspots were identified and were recognized as hotspots for multiple miRNAs action, thus, acting as favoured sites of action for the repression of gene expression. The complex interplay of genes and miRNAs brought about by multiplicity and cooperativity was explored. This investigation will aid in elucidating the mechanism of action of miRNAs for the considered genes. The intrinsic network of miRNAs expressed in nervous system and genes associated with ALS may provide rapid and effective outcome for therapeutic applications and diagnosis.

scholarly journals A behavioral screen for mediators of age-dependent TDP-43 neurodegeneration identifies SF2/SRSF1 among a group of potent suppressors in both neurons and glia

PLoS Genetics ◽  
2021 ◽  
Vol 17 (11) ◽  
pp. e1009882
Author(s):  
Jorge Azpurua ◽  
Enas Gad El-Karim ◽  
Marvel Tranquille ◽  
Josh Dubnau
Keyword(s):  
Motor Neurons ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Rna Binding ◽  
Negative Effects ◽  
Number Of Genes ◽  
Age Dependent ◽  
Neurological Phenotype ◽  
Lateral Sclerosis ◽  
Downstream Mediator

Cytoplasmic aggregation of Tar-DNA/RNA binding protein 43 (TDP-43) occurs in 97 percent of amyotrophic lateral sclerosis (ALS), ~40% of frontotemporal dementia (FTD) and in many cases of Alzheimer’s disease (AD). Cytoplasmic TDP-43 inclusions are seen in both sporadic and familial forms of these disorders, including those cases that are caused by repeat expansion mutations in the C9orf72 gene. To identify downstream mediators of TDP-43 toxicity, we expressed human TDP-43 in a subset of Drosophila motor neurons. Such expression causes age-dependent deficits in negative geotaxis behavior. Using this behavioral readout of locomotion, we conducted an shRNA suppressor screen and identified 32 transcripts whose knockdown was sufficient to ameliorate the neurological phenotype. The majority of these suppressors also substantially suppressed the negative effects on lifespan seen with glial TDP-43 expression. In addition to identification of a number of genes whose roles in neurodegeneration were not previously known, our screen also yielded genes involved in chromatin regulation and nuclear/import export- pathways that were previously identified in the context of cell based or neurodevelopmental suppressor screens. A notable example is SF2, a conserved orthologue of mammalian SRSF1, an RNA binding protein with roles in splicing and nuclear export. Our identification SF2/SRSF1 as a potent suppressor of both neuronal and glial TDP-43 toxicity also provides a convergence with C9orf72 expansion repeat mediated neurodegeneration, where this gene also acts as a downstream mediator.

scholarly journals Maximizing the Survival of Amyotrophic Lateral Sclerosis Patients: Current Perspectives

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Osama A. Khairoalsindi ◽  
Ahmad R. Abuzinadah
Keyword(s):  
Gene Therapy ◽  
Amyotrophic Lateral Sclerosis ◽  
Stem Cell ◽  
Cell Therapy ◽  
Motor Neurons ◽  
Stem Cell Therapy ◽  
The United States ◽  
Disease Modifying Drugs ◽  
Familial Form ◽  
Lateral Sclerosis

Amyotrophic lateral sclerosis is a neurodegenerative disease that leads to loss of the upper and lower motor neurons. Almost 90% of all cases occur in the sporadic form, with the rest occurring in the familial form. The disease has a poor prognosis, with only two disease-modifying drugs approved by the United States Food and Drug Administration (FDA). The approved drugs for the disease have very limited survival benefits. Edaravone is a new FDA-approved medication that may slow the disease progression by 33% in a selected subgroup of ALS patients. This paper covers the various interventions that may provide survival benefits, such as early diagnosis, medications, gene therapy, stem cell therapy, diet, nutritional supplements, multidisciplinary clinics, and mechanical invasive and noninvasive ventilation. The recent data on masitinib, the role of enteral feeding, gene therapy, and stem cell therapy is discussed.

Gene expression profile of spinal motor neurons in sporadic amyotrophic lateral sclerosis

2005 ◽  
Vol 57 (2) ◽  
pp. 236-251 ◽  
Author(s):  
Yue-Mei Jiang ◽  
Masahiko Yamamoto ◽  
Yasushi Kobayashi ◽  
Tsuyoshi Yoshihara ◽  
Yideng Liang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amyotrophic Lateral Sclerosis ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Motor Neurons ◽  
Sporadic Amyotrophic Lateral Sclerosis ◽  
Spinal Motor Neurons ◽  
Spinal Motor ◽  
Lateral Sclerosis

scholarly journals A cell-penetrant peptide blocking C9ORF72-repeat RNA nuclear export suppresses neurodegeneration

2021 ◽  
Author(s):  
Lydia M Castelli ◽  
Alvaro Sanchez-Martinez ◽  
Ya-Hui Lin ◽  
Santosh Kumar Upadhyay ◽  
Adrian Higginbottom ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Nuclear Export ◽  
Promising Alternative ◽  
Hexanucleotide Repeat ◽  
Repeat Expansions ◽  
A Cell ◽  
Models Of Disease ◽  
Lateral Sclerosis

Hexanucleotide repeat expansions in C9ORF72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), a spectrum of incurable debilitating neurodegenerative diseases. Here, we report a novel ALS/FTD drug concept with in vivo and in vitro therapeutic activity in preclinical models of C9ORF72-ALS/FTD. Our data demonstrate that supplementation or oral administration of a cell-penetrant peptide, which competes with the SRSF1:NXF1 interaction, confers neuroprotection by inhibiting the nuclear export of pathological C9ORF72-repeat transcripts in various models of disease including primary neurons, patient-derived motor neurons and Drosophila. Our drug-like rationale for disrupting the nuclear export of microsatellite repeat transcripts in neurological disorders provides a promising alternative to conventional small molecule inhibitors often limited by poor blood-brain barrier penetrance.

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