scholarly journals The RNA helicase DHX36/G4R1 modulates C9orf72 GGGGCC repeat-associated translation

2021 ◽  
Author(s):  
Yi-Ju Tseng ◽  
Siara N. Sandwith ◽  
Katelyn M. Green ◽  
Antonio E. Chambers ◽  
Amy Krans ◽  
...  
Keyword(s):  
Dependent Manner ◽  
Hexanucleotide Repeat ◽  
G Quadruplex ◽  
Therapeutic Development ◽  
Repeat Expansions ◽  
Nucleotide Repeats ◽  
The Impact ◽  
Lateral Sclerosis ◽  
Ran Translation

ABSTRACTGGGGCC (G4C2) hexanucleotide repeat expansions (HRE) in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat-associated non-AUG (RAN) translation of this expansion generates toxic proteins that accumulate in patient brains and contribute to disease pathogenesis. The DEAH-Box Helicase 36 (DHX36/G4R1) plays active roles in RNA and DNA G-quadruplex (G4) resolution in cells. As G4C2 repeats form G4 structures in vitro, we sought to determine the impact of manipulating DHX36 expression on repeat transcription and RAN translation. We found that DHX36 depletion suppresses RAN translation from reporter constructs in a repeat length dependent manner while overexpression of DHX36 enhances RAN translation from G4C2 reporter RNAs. Taken together, these results suggest that DHX36 is active in regulating G4C2 repeat translation, providing potential implications for therapeutic development in nucleotide repeats expansion disorders.

scholarly journals Glycine-alanine dipeptide repeats spread rapidly in a repeat length- and age-dependent manner in the fly brain

2019 ◽  
Vol 7 (1) ◽  
Author(s):  
Javier Morón-Oset ◽  
Tessa Supèr ◽  
Jacqueline Esser ◽  
Adrian M. Isaacs ◽  
Sebastian Grönke ◽  
...  
Keyword(s):  
Dependent Manner ◽  
Genetic Screens ◽  
Hexanucleotide Repeat ◽  
Repeat Expansions ◽  
Alanine Dipeptide ◽  
The Brain ◽  
Lateral Sclerosis ◽  
Dipeptide Repeat

AbstractHexanucleotide repeat expansions of variable size in C9orf72 are the most prevalent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Sense and antisense transcripts of the expansions are translated by repeat-associated non-AUG translation into five dipeptide repeat proteins (DPRs). Of these, the polyGR, polyPR and, to a lesser extent, polyGA DPRs are neurotoxic, with polyGA the most abundantly detected DPR in patient tissue. Trans-cellular transmission of protein aggregates has recently emerged as a major driver of toxicity in various neurodegenerative diseases. In vitro evidence suggests that the C9 DPRs can spread. However, whether this phenomenon occurs under more complex in vivo conditions remains unexplored. Here, we used the adult fly brain to investigate whether the C9 DPRs can spread in vivo upon expression in a subset of neurons. We found that only polyGA can progressively spread throughout the brain, which accumulates in the shape of aggregate-like puncta inside recipient cells. Interestingly, GA transmission occurred as early as 3 days after expression induction. By comparing the spread of 36, 100 and 200 polyGA repeats, we found that polyGA spread is enhanced upon expression of longer GA DPRs. Transmission of polyGA is greater in older flies, indicating that age-associated factors exacerbate the spread. These data highlight a unique propensity of polyGA to spread throughout the brain, which could contribute to the greater abundance of polyGA in patient tissue. In addition, we present a model of early GA transmission that is suitable for genetic screens to identify mechanisms of spread and its consequences in vivo.

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.

scholarly journals A C. elegans model of C9orf72-associated ALS/FTD uncovers a conserved role for eIF2D in RAN translation

2020 ◽  
Author(s):  
Yoshifumi Sonobe ◽  
Jihad Aburas ◽  
Priota Islam ◽  
Tania F. Gendron ◽  
André E.X. Brown ◽  
...  
Keyword(s):  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
C Elegans ◽  
Hexanucleotide Repeat ◽  
Eukaryotic Translation Initiation ◽  
Hexanucleotide Repeat Expansion ◽  
Lateral Sclerosis ◽  
Ran Translation

ABSTRACTA hexanucleotide repeat expansion GGGGCC in the noncoding region of C9orf72 is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Potentially toxic dipeptide repeats (DPRs) are synthesized from the GGGGCC sequence via repeat associated non-AUG (RAN) translation. We developed C. elegans models that express, either ubiquitously or exclusively in neurons, a transgene with 75 GGGGCC repeats flanked by intronic C9orf72 sequence. The worms generate DPRs (poly-glycine-alanine [poly-GA], poly-glycine-proline [poly-GP]) and display neurodegeneration, locomotor and lifespan defects. Mutation of a non-canonical translation-initiating codon (CUG) upstream of the repeats blocked poly-GA production and ameliorated disease, suggesting poly-GA is pathogenic. Importantly, eukaryotic translation initiation factor 2D (eif-2D/eIF2D) was necessary for RAN translation. Genetic removal of eif-2D increased lifespan in both C. elegans models. In vitro findings in human cells demonstrated a conserved role for eif-2D/eIF2D in RAN translation that could be exploited for ALS and FTD therapy.

VDAC1 Mediated Anticancer Activity of Gallic Acid in Human Lung Adenocarcinoma A549 Cells

2018 ◽  
Vol 18 (2) ◽  
pp. 255-262 ◽  
Author(s):  
Aikebaier Maimaiti ◽  
Amier Aili ◽  
Hureshitanmu Kuerban ◽  
Xuejun Li
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Gallic Acid ◽  
Molecular Mechanisms ◽  
A549 Cells ◽  
Dependent Manner ◽  
Lung Carcinoma Cells ◽  
Induced Apoptosis ◽  
The Impact

Aims: Gallic acid (GA) is generally distributed in a variety of plants and foods, and possesses cell growth-inhibiting activities in cancer cell lines. In the present study, the impact of GA on cell viability, apoptosis induction and possible molecular mechanisms in cultured A549 lung carcinoma cells was investigated. Methods: In vitro experiments showed that treating A549 cells with various concentrations of GA inhibited cell viability and induced apoptosis in a dose-dependent manner. In order to understand the mechanism by which GA inhibits cell viability, comparative proteomic analysis was applied. The changed proteins were identified by Western blot and siRNA methods. Results: Two-dimensional electrophoresis revealed changes that occurred to the cells when treated with or without GA. Four up-regulated protein spots were clearly identified as malate dehydrogenase (MDH), voltagedependent, anion-selective channel protein 1(VDAC1), calreticulin (CRT) and brain acid soluble protein 1(BASP1). VDAC1 in A549 cells was reconfirmed by western blot. Transfection with VDAC1 siRNA significantly increased cell viability after the treatment of GA. Further investigation showed that GA down regulated PI3K/Akt signaling pathways. These data strongly suggest that up-regulation of VDAC1 by GA may play an important role in GA-induced, inhibitory effects on A549 cell viability.

scholarly journals The impact of indole-3-lactic acid on immature intestinal innate immunity and development: a transcriptomic analysis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Wuyang Huang ◽  
Ky Young Cho ◽  
Di Meng ◽  
W. Allan Walker
Keyword(s):  
Lactic Acid ◽  
Mechanistic Model ◽  
Transcriptomic Analysis ◽  
Dependent Manner ◽  
Protective Effects ◽  
Very Preterm Infants ◽  
Anti Inflammatory ◽  
The Impact

AbstractAn excessive intestinal inflammatory response may have a role in the pathogenesis of necrotizing enterocolitis (NEC) in very preterm infants. Indole-3-lactic acid (ILA) of breastmilk tryptophan was identified as the anti-inflammatory metabolite involved in probiotic conditioned media from Bifidobacteria longum subsp infantis. This study aimed to explore the molecular endocytic pathways involved in the protective ILA effect against inflammation. H4 cells, Caco-2 cells, C57BL/6 pup and adult mice were used to compare the anti-inflammatory mechanisms between immature and mature enterocytes in vitro and in vivo. The results show that ILA has pleiotropic protective effects on immature enterocytes including anti-inflammatory, anti-viral, and developmental regulatory potentials in a region-dependent and an age-dependent manner. Quantitative transcriptomic analysis revealed a new mechanistic model in which STAT1 pathways play an important role in IL-1β-induced inflammation and ILA has a regulatory effect on STAT1 pathways. These studies were validated by real-time RT-qPCR and STAT1 inhibitor experiments. Different protective reactions of ILA between immature and mature enterocytes indicated that ILA’s effects are developmentally regulated. These findings may be helpful in preventing NEC for premature infants.

scholarly journals Concomitant gain and loss of function pathomechanisms in C9ORF72 amyotrophic lateral sclerosis

2021 ◽  
Vol 4 (4) ◽  
pp. e202000764
Author(s):  
Arun Pal ◽  
Benedikt Kretner ◽  
Masin Abo-Rady ◽  
Hannes Glaβ ◽  
Banaja P Dash ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Loss Of Function ◽  
Dna Double Strand Breaks ◽  
Hexanucleotide Repeat ◽  
Rna Foci ◽  
Repeat Expansions ◽  
Organelle Motility ◽  
Trafficking Defects ◽  
Induced Pluripotent ◽  
Lateral Sclerosis

Intronic hexanucleotide repeat expansions (HREs) in C9ORF72 are the most frequent genetic cause of amyotrophic lateral sclerosis, a devastating, incurable motoneuron (MN) disease. The mechanism by which HREs trigger pathogenesis remains elusive. The discovery of repeat-associated non-ATG (RAN) translation of dipeptide repeat proteins (DPRs) from HREs along with reduced exonic C9ORF72 expression suggests gain of toxic functions (GOFs) through DPRs versus loss of C9ORF72 functions (LOFs). Through multiparametric high-content (HC) live profiling in spinal MNs from induced pluripotent stem cells and comparison to mutant FUS and TDP43, we show that HRE C9ORF72 caused a distinct, later spatiotemporal appearance of mainly proximal axonal organelle motility deficits concomitant to augmented DNA double-strand breaks (DSBs), RNA foci, DPRs, and apoptosis. We show that both GOFs and LOFs were necessary to yield the overall C9ORF72 pathology. Increased RNA foci and DPRs concurred with onset of axon trafficking defects, DSBs, and cell death, although DSB induction itself did not phenocopy C9ORF72 mutants. Interestingly, the majority of LOF-specific DEGs were shared with HRE-mediated GOF DEGs. Finally, C9ORF72 LOF was sufficient—albeit to a smaller extent—to induce premature distal axonal trafficking deficits and increased DSBs.

scholarly journals C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility

2019 ◽  
Author(s):  
Laura Fumagalli ◽  
Florence L. Young ◽  
Steven Boeynaems ◽  
Mathias De Decker ◽  
Arpan R. Mehta ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Motor Neurons ◽  
Single Molecule ◽  
Induced Pluripotent Stem Cell ◽  
Cytoplasmic Dynein ◽  
Inhibitory Interaction ◽  
Hexanucleotide Repeat ◽  
Repeat Expansions

ABSTRACTHexanucleotide repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. Here, we use human induced pluripotent stem cell-derived motor neurons to show that C9orf72 repeat expansions impair microtubule-based transport of mitochondria, a process critical for maintenance of neuronal function. Cargo transport defects are recapitulated by treating healthy neurons with the arginine-rich dipeptide repeat proteins (DPRs) that are produced by the hexanucleotide repeat expansions. Single-molecule imaging shows that these DPRs perturb motility of purified kinesin-1 and cytoplasmic dynein-1 motors along microtubules in vitro. Additional in vitro and in vivo data indicate that the DPRs impair transport by interacting with both microtubules and the motor complexes. We also show that kinesin-1 is enriched in DPR inclusions in patient brains and that increasing the level of this motor strongly suppresses the toxic effects of arginine-rich DPR expression in a Drosophila model. Collectively, our study implicates an inhibitory interaction of arginine-rich DPRs with the axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to novel potential therapeutic strategies.

scholarly journals Oligodendrocytes contribute to motor neuron death in ALS via SOD1-dependent mechanism

2016 ◽  
Vol 113 (42) ◽  
pp. E6496-E6505 ◽  
Author(s):  
Laura Ferraiuolo ◽  
Kathrin Meyer ◽  
Thomas W. Sherwood ◽  
Jonathan Vick ◽  
Shibi Likhite ◽  
...  
Keyword(s):  
Motor Neuron ◽  
Lactate Production ◽  
Chromosome 9 ◽  
Neuron Death ◽  
Motor Neuron Death ◽  
Repeat Expansions ◽  
Human Oligodendrocytes ◽  
Lateral Sclerosis ◽  
Dependent Mechanism

Oligodendrocytes have recently been implicated in the pathophysiology of amyotrophic lateral sclerosis (ALS). Here we show that, in vitro, mutant superoxide dismutase 1 (SOD1) mouse oligodendrocytes induce WT motor neuron (MN) hyperexcitability and death. Moreover, we efficiently derived human oligodendrocytes from a large number of controls and patients with sporadic and familial ALS, using two different reprogramming methods. All ALS oligodendrocyte lines induced MN death through conditioned medium (CM) and in coculture. CM-mediated MN death was associated with decreased lactate production and release, whereas toxicity in coculture was lactate-independent, demonstrating that MN survival is mediated not only by soluble factors. Remarkably, human SOD1 shRNA treatment resulted in MN rescue in both mouse and human cultures when knockdown was achieved in progenitor cells, whereas it was ineffective in differentiated oligodendrocytes. In fact, early SOD1 knockdown rescued lactate impairment and cell toxicity in all lines tested, with the exclusion of samples carrying chromosome 9 ORF 72 (C9orf72) repeat expansions. These did not respond to SOD1 knockdown nor did they show lactate release impairment. Our data indicate that SOD1 is directly or indirectly involved in ALS oligodendrocyte pathology and suggest that in this cell type, some damage might be irreversible. In addition, we demonstrate that patients with C9ORF72 represent an independent patient group that might not respond to the same treatment.

scholarly journals Antisense Transcription across Nucleotide Repeat Expansions in Neurodegenerative and Neuromuscular Diseases: Progress and Mysteries

Genes ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1418
Author(s):  
Ana F. Castro ◽  
Joana R. Loureiro ◽  
José Bessa ◽  
Isabel Silveira
Keyword(s):  
Gene Expression ◽  
Neuromuscular Diseases ◽  
Antisense Transcription ◽  
Spinocerebellar Ataxia Type ◽  
Spinocerebellar Ataxia Type 7 ◽  
Repeat Expansions ◽  
Cellular Pathways ◽  
The Impact ◽  
Lateral Sclerosis

Unstable repeat expansions and insertions cause more than 30 neurodegenerative and neuromuscular diseases. Remarkably, bidirectional transcription of repeat expansions has been identified in at least 14 of these diseases. More remarkably, a growing number of studies has been showing that both sense and antisense repeat RNAs are able to dysregulate important cellular pathways, contributing together to the observed clinical phenotype. Notably, antisense repeat RNAs from spinocerebellar ataxia type 7, myotonic dystrophy type 1, Huntington’s disease and frontotemporal dementia/amyotrophic lateral sclerosis associated genes have been implicated in transcriptional regulation of sense gene expression, acting either at a transcriptional or posttranscriptional level. The recent evidence that antisense repeat RNAs could modulate gene expression broadens our understanding of the pathogenic pathways and adds more complexity to the development of therapeutic strategies for these disorders. In this review, we cover the amazing progress made in the understanding of the pathogenic mechanisms associated with repeat expansion neurodegenerative and neuromuscular diseases with a focus on the impact of antisense repeat transcription in the development of efficient therapies.

scholarly journals The Impact of ALS-Associated Genes hnRNPA1, MATR3, VCP and UBQLN2 on the Severity of TDP-43 Aggregation

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1791
Author(s):  
Ana Bajc Česnik ◽  
Helena Motaln ◽  
Boris Rogelj
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Rna Binding ◽  
Neurodegenerative Disorder ◽  
Low Complexity ◽  
Wild Type ◽  
Disease Heterogeneity ◽  
Cytoplasmic Inclusions ◽  
The Impact ◽  
Lateral Sclerosis

Amyotrophic lateral sclerosis is a progressive neurodegenerative disorder, characterized by cytoplasmic inclusions of RNA-binding protein TDP-43. Despite decades of research and identification of more than 50 genes associated with amyotrophic lateral sclerosis (ALS), the cause of TDP-43 translocation from the nucleus and its aggregation in the cytoplasm still remains unknown. Our study addressed the impact of selected ALS-associated genes on TDP-43 aggregation behavior in wild-type and aggregation prone TDP-43 in vitro cell models. These were developed by deleting TDP-43 nuclear localization signal and stepwise shortening its low-complexity region. The SH-SY5Y cells were co-transfected with the constructs of aggregation-prone TDP-43 and wild-type or mutant ALS-associated genes hnRNPA1, MATR3, VCP or UBQLN2. The investigated genes displayed a unique impact on TDP-43 aggregation, generating distinct types of cytoplasmic inclusions, similar to those already described as resembling prion strains, which could represent the basis for neurodegenerative disease heterogeneity.

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