RESUMO
Defects in nucleocytoplasmic transport have been identified as a key pathogenic event in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) mediated by a GGGGCC hexanucleotide repeat expansion in C9ORF72, the most common genetic cause of ALS/FTD. Furthermore, nucleocytoplasmic transport disruption has also been implicated in other neurodegenerative diseases with protein aggregation, suggesting a shared mechanism by which protein stress disrupts nucleocytoplasmic transport. Here, we show that cellular stress disrupts nucleocytoplasmic transport by localizing critical nucleocytoplasmic transport factors into stress granules, RNA/protein complexes that play a crucial role in ALS pathogenesis. Importantly, inhibiting stress granule assembly, such as by knocking down Ataxin-2, suppresses nucleocytoplasmic transport defects as well as neurodegeneration in C9ORF72-mediated ALS/FTD. Our findings identify a link between stress granule assembly and nucleocytoplasmic transport, two fundamental cellular processes implicated in the pathogenesis of C9ORF72-mediated ALS/FTD and other neurodegenerative diseases.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Esclerose Lateral Amiotrófica/patologia , Ataxina-2/metabolismo , Proteína C9orf72/genética , Demência Frontotemporal/patologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Idoso , Esclerose Lateral Amiotrófica/metabolismo , Arsenitos/toxicidade , Ataxina-2/antagonistas & inibidores , Ataxina-2/genética , Proteína C9orf72/metabolismo , Expansão das Repetições de DNA/genética , Feminino , Demência Frontotemporal/metabolismo , Células HEK293 , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Compostos de Sódio/toxicidade , alfa Carioferinas/antagonistas & inibidores , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , beta Carioferinas/antagonistas & inibidores , beta Carioferinas/genética , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/antagonistas & inibidores , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismoRESUMO
An attractive approach to reduce gene expression is via the use of antisense oligonucleotides (ASOs) that harness the RNase H1 mechanism. Here we show that RNase H ASOs targeted to introns or exons robustly reduce the level of spliced RNA associated with chromatin. Surprisingly, intron-targeted ASOs reduce the level of pre-mRNA associated with chromatin to a greater extent than exon-targeted ASOs. This indicates that exon-targeted ASOs achieve full activity after the pre-mRNA has undergone splicing, but before the mRNA is released from chromatin. Even though RNase H ASOs can reduce the level of RNA associated with chromatin, the effect of ASO-directed RNA degradation on transcription has never been documented. Here we show that intron-targeted ASOs and, to a lesser extent, exon-targeted ASOs cause RNA polymerase II (Pol II) transcription termination in cultured cells and mice. Furthermore, ASO-directed transcription termination is mediated by the nuclear exonuclease XRN2.
Assuntos
Cromatina/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Precursores de RNA/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Ribonuclease H/metabolismo , Terminação da Transcrição Genética , Animais , Cromatina/genética , Éxons , Exorribonucleases/genética , Exorribonucleases/metabolismo , Feminino , Células HCT116 , Humanos , Íntrons , Camundongos Endogâmicos C57BL , Modelos Genéticos , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Oligonucleotídeos Antissenso/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Precursores de RNA/genética , RNA Mensageiro/genética , Ribonuclease H/genética , Fatores de TempoRESUMO
Mutations in PLP1, the gene that encodes proteolipid protein (PLP), result in failure of myelination and neurological dysfunction in the X-chromosome-linked leukodystrophy Pelizaeus-Merzbacher disease (PMD)1,2. Most PLP1 mutations, including point mutations and supernumerary copy variants, lead to severe and fatal disease. Patients who lack PLP1 expression, and Plp1-null mice, can display comparatively mild phenotypes, suggesting that PLP1 suppression might provide a general therapeutic strategy for PMD1,3-5. Here we show, using CRISPR-Cas9 to suppress Plp1 expression in the jimpy (Plp1jp) point-mutation mouse model of severe PMD, increased myelination and restored nerve conduction velocity, motor function and lifespan of the mice to wild-type levels. To evaluate the translational potential of this strategy, we identified antisense oligonucleotides that stably decrease the levels of Plp1 mRNA and PLP protein throughout the neuraxis in vivo. Administration of a single dose of Plp1-targeting antisense oligonucleotides in postnatal jimpy mice fully restored oligodendrocyte numbers, increased myelination, improved motor performance, normalized respiratory function and extended lifespan up to an eight-month end point. These results suggest that PLP1 suppression could be developed as a treatment for PMD in humans. More broadly, we demonstrate that oligonucleotide-based therapeutic agents can be delivered to oligodendrocytes in vivo to modulate neurological function and lifespan, establishing a new pharmaceutical modality for myelin disorders.
Assuntos
Modelos Animais de Doenças , Proteína Proteolipídica de Mielina/deficiência , Doença de Pelizaeus-Merzbacher/genética , Doença de Pelizaeus-Merzbacher/terapia , Animais , Sistemas CRISPR-Cas , Feminino , Edição de Genes , Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Mutantes , Atividade Motora/genética , Proteína Proteolipídica de Mielina/genética , Proteína Proteolipídica de Mielina/metabolismo , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , Doença de Pelizaeus-Merzbacher/metabolismo , Mutação Puntual , Testes de Função Respiratória , Análise de SobrevidaRESUMO
The MYC proto-oncogene contributes to the pathogenesis of more than half of human cancers. Malignant transformation by MYC transcriptionally up-regulates the core pre-mRNA splicing machinery and causes misregulation of alternative splicing. However, our understanding of how splicing changes are directed by MYC is limited. We performed a signaling pathway-guided splicing analysis to identify MYC-dependent splicing events. These included an HRAS cassette exon repressed by MYC across multiple tumor types. To molecularly dissect the regulation of this HRAS exon, we used antisense oligonucleotide tiling to identify splicing enhancers and silencers in its flanking introns. RNA-binding motif prediction indicated multiple binding sites for hnRNP H and hnRNP F within these cis-regulatory elements. Using siRNA knockdown and cDNA expression, we found that both hnRNP H and F activate the HRAS cassette exon. Mutagenesis and targeted RNA immunoprecipitation implicate two downstream G-rich elements in this splicing activation. Analyses of ENCODE RNA-seq datasets confirmed hnRNP H regulation of HRAS splicing. Analyses of RNA-seq datasets across multiple cancers showed a negative correlation of HNRNPH gene expression with MYC hallmark enrichment, consistent with the effect of hnRNP H on HRAS splicing. Interestingly, HNRNPF expression showed a positive correlation with MYC hallmarks and thus was not consistent with the observed effects of hnRNP F. Loss of hnRNP H/F altered cell cycle progression and induced apoptosis in the PC3 prostate cancer cell line. Collectively, our results reveal mechanisms for MYC-dependent regulation of splicing and point to possible therapeutic targets in prostate cancers.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H , Neoplasias da Próstata , Masculino , Humanos , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Éxons/genética , Processamento Alternativo/genética , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
TGFß1 is a powerful regulator of fibrosis; secreted in a latent form, it becomes active after release from the latent complex. During tissue fibrosis, the EDA + isoform of cellular fibronectin is overexpressed. In pulmonary fibrosis it has been proposed that the fibronectin splice variant including an EDA domain (FN EDA+) activates latent TGFß. Our work investigates the potential of blocking the 'splicing in' of EDA with antisense oligonucleotides to inhibit TGFß1-induced EDA + fibronectin and to prevent the cascade of events initiated by TGFß1 in human renal proximal tubule cells (PTEC). Human primary PTEC were treated with TGFß1 for 48 h, medium removed and the cells transfected with RNase H-independent antisense oligonucleotides (ASO) designed to block EDA exon inclusion (ASO5). The efficacy of ASO to block EDA exon inclusion was assessed by EDA + fibronectin RNA and protein expression; the expression of TGFß, αSMA (α smooth muscle actin), MMP2 (matrix metalloproteinse-2), MMP9 (matrix metalloproteinse-9), Collagen I, K Cadherin and connexin 43 was analysed. Targeting antisense oligonucleotides designed to block EDA exon inclusion in fibronectin pre mRNA were effective in reducing the amount of TGFß1 -induced cellular EDA + fibronectin RNA and secreted EDA + fibronectin protein (assessed by western immunoblotting and immunocytochemistry) in human proximal tubule cells in an in vitro cell culture model. The effect was selective for EDA + exon with no effect on EDB + fibronectin RNA and total fibronectin mRNA. Exogenous TGFß1 induced endogenous TGFß, αSMA, MMP2, MMP9 and Col I mRNA. TGFß1 treatment for 48h reduced the expression of K-Cadherin and increased the expression of connexin-43. These TGFß1-induced pro-fibrotic changes were attenuated by ASO5 treatment. 48 h after the removal of exogenous TGFß, further increases in αSMA, MMP2, MMP9 was observed; ASO5 significantly inhibited this subsequent increase. ASO5 treatment also significantly inhibited ability of the cell culture medium harvested at the end of the experiment (96h) to stimulate SMAD3 reporter cells. The role of endogenous TGFß1 was confirmed by the use of a TGFß receptor inhibitor. Our results demonstrate a critical role of FN EDA+ in a cycle of TGFß driven pro-fibrotic responses in human PTEC and blocking its production with ASO technology offers a potential therapy to interrupt this vicious circle and hence limit the progression of renal fibrosis.
RESUMO
Brain pH is a critical factor for determining neuronal activity, with alkalosis increasing and acidosis reducing excitability. Acid shifts in brain pH through the breathing of carbogen (5% CO2/95% O2) reduces seizure susceptibility in animal models and patients. The molecular mechanisms underlying this seizure protection remain to be fully elucidated. Here, we demonstrate that male and female mice exposed to carbogen are fully protected from thermogenic-triggered seizures. Whole-cell patch-clamp recordings revealed that acid shifts in extracellular pH (pHo) significantly reduce action potential firing in CA1 pyramidal neurons but did not alter firing in hippocampal inhibitory interneurons. In real-time dynamic clamp experiments, acidification reduced simulated action potential firing generated in hybrid model neurons expressing the excitatory neuron predominant NaV1.2 channel. Conversely, acidification had no effect on action potential firing in hybrid model neurons expressing the interneuron predominant NaV1.1 channel. Furthermore, knockdown of Scn2a mRNA in vivo using antisense oligonucleotides reduced the protective effects of carbogen on seizure susceptibility. Both carbogen-mediated seizure protection and the reduction in CA1 pyramidal neuron action potential firing by low pHo were maintained in an Asic1a knock-out mouse ruling out this acid-sensing channel as the underlying molecular target. These data indicate that the acid-mediated reduction in excitatory neuron firing is mediated, at least in part, through the inhibition of NaV1.2 channels, whereas inhibitory neuron firing is unaffected. This reduction in pyramidal neuron excitability is the likely basis of seizure suppression caused by carbogen-mediated acidification.SIGNIFICANCE STATEMENT Brain pH has long been known to modulate neuronal excitability. Here, we confirm that brain acidification reduces seizure susceptibility in a mouse model of thermogenic seizures. Extracellular acidification reduced excitatory pyramidal neuron firing while having no effect on interneuron firing. Acidification also reduced dynamic clamp firing in cells expressing the NaV1.2 channel but not in cells expressing NaV1.1 channels. In vivo knockdown of Scn2a mRNA reduced seizure protection of acidification. In contrast, acid-mediated seizure protection was maintained in the Asic1a knock-out mouse. These data suggest NaV1.2 channel as an important target for acid-mediated seizure protection. Our results have implications on how natural variations in pH can modulate neuronal excitability and highlight potential antiseizure drug development strategies based on the NaV1.2 channel.
Assuntos
Acidose Respiratória , Segmento Inicial do Axônio , Camundongos , Masculino , Animais , Feminino , Dióxido de Carbono , Convulsões/induzido quimicamente , Convulsões/genética , Células Piramidais , Potenciais de Ação , Camundongos Knockout , RNA MensageiroRESUMO
Heterozygous GRN (progranulin) mutations cause frontotemporal dementia (FTD) due to haploinsufficiency, and increasing progranulin levels is a major therapeutic goal. Several microRNAs, including miR-29b, negatively regulate progranulin protein levels. Antisense oligonucleotides (ASOs) are emerging as a promising therapeutic modality for neurological diseases, but strategies for increasing target protein levels are limited. Here, we tested the efficacy of ASOs as enhancers of progranulin expression by sterically blocking the miR-29b binding site in the 3' UTR of the human GRN mRNA. We found 16 ASOs that increase progranulin protein in a dose-dependent manner in neuroglioma cells. A subset of these ASOs also increased progranulin protein in iPSC-derived neurons and in a humanized GRN mouse model. In FRET-based assays, the ASOs effectively competed for miR-29b from binding to the GRN 3' UTR RNA. The ASOs increased levels of newly synthesized progranulin protein by increasing its translation, as revealed by polysome profiling. Together, our results demonstrate that ASOs can be used to effectively increase target protein levels by partially blocking miR binding sites. This ASO strategy may be therapeutically feasible for progranulin-deficient FTD as well as other conditions of haploinsufficiency.
Assuntos
Demência Frontotemporal , MicroRNAs , Oligonucleotídeos Antissenso , Progranulinas , Animais , Humanos , Camundongos , Regiões 3' não Traduzidas , Sítios de Ligação , Demência Frontotemporal/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , MicroRNAs/genética , Mutação , Oligonucleotídeos Antissenso/genética , Progranulinas/genética , RNA Mensageiro/genéticaRESUMO
Frataxin deficiency in Friedreich's ataxia results from transcriptional downregulation of the FXN gene caused by expansion of the intronic trinucleotide guanine-adenine-adenine (GAA) repeats. We used multiple transcriptomic approaches to determine the molecular mechanism of transcription inhibition caused by long GAAs. We uncovered that transcription of FXN in patient cells is prematurely terminated upstream of the expanded repeats leading to the formation of a novel, truncated and stable RNA. This FXN early terminated transcript (FXN-ett) undergoes alternative, non-productive splicing and does not contribute to the synthesis of functional frataxin. The level the FXN-ett RNA directly correlates with the length of the longer of the two expanded GAA tracts. Targeting GAAs with antisense oligonucleotides or excision of the repeats eliminates the transcription impediment, diminishes expression of the aberrant FXN-ett, while increasing levels of FXN mRNA and frataxin. Non-productive transcription may represent a common phenomenon and attractive therapeutic target in diseases caused by repeat-mediated transcription aberrations.
Assuntos
Ataxia de Friedreich , Adenina , Arsenicais , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Gálio , Guanina , Humanos , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Oligonucleotídeos Antissenso , Poliadenilação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , Expansão das Repetições de Trinucleotídeos/genética , FrataxinaRESUMO
BACKGROUND AND AIMS: Paucity of intrahepatic bile ducts (BDs) is caused by various etiologies and often leads to cholestatic liver disease. For example, in patients with Alagille syndrome (ALGS), which is a genetic disease primarily caused by mutations in jagged 1 ( JAG1) , BD paucity often results in severe cholestasis and liver damage. However, no mechanism-based therapy exists to restore the biliary system in ALGS or other diseases associated with BD paucity. Based on previous genetic observations, we investigated whether postnatal knockdown of the glycosyltransferase gene protein O -glucosyltransferase 1 ( Poglut1) can improve the ALGS liver phenotypes in several mouse models generated by removing one copy of Jag1 in the germline with or without reducing the gene dosage of sex-determining region Y-box 9 in the liver. APPROACH AND RESULTS: Using an ASO established in this study, we show that reducing Poglut1 levels in postnatal livers of ALGS mouse models with moderate to profound biliary abnormalities can significantly improve BD development and biliary tree formation. Importantly, ASO injections prevent liver damage in these models without adverse effects. Furthermore, ASO-mediated Poglut1 knockdown improves biliary tree formation in a different mouse model with no Jag1 mutations. Cell-based signaling assays indicate that reducing POGLUT1 levels or mutating POGLUT1 modification sites on JAG1 increases JAG1 protein level and JAG1-mediated signaling, suggesting a likely mechanism for the observed in vivo rescue. CONCLUSIONS: Our preclinical studies establish ASO-mediated POGLUT1 knockdown as a potential therapeutic strategy for ALGS liver disease and possibly other diseases associated with BD paucity.
Assuntos
Síndrome de Alagille , Glicosiltransferases , Fígado , Oligonucleotídeos Antissenso , Animais , Camundongos , Síndrome de Alagille/genética , Síndrome de Alagille/metabolismo , Síndrome de Alagille/patologia , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Proteínas de Ligação ao Cálcio/genética , Colestase/genética , Colestase/metabolismo , Inativação Gênica , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Fígado/metabolismo , Fígado/patologia , Proteínas de Membrana/genética , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Fenótipo , Proteínas Serrate-Jagged/genética , Proteínas Serrate-Jagged/metabolismoRESUMO
TAR DNA binding protein 43 (TDP-43) pathology is a key feature of over 95% of amyotrophic lateral sclerosis (ALS) and nearly half of frontotemporal dementia (FTD) cases. The pathogenic mechanisms of TDP-43 dysfunction are poorly understood, however, activation of cell stress pathways may contribute to pathogenesis. We, therefore, sought to identify which cell stress components are critical for driving disease onset and neurodegeneration in ALS and FTD. We studied the rNLS8 transgenic mouse model, which expresses human TDP-43 with a genetically-ablated nuclear localisation sequence within neurons of the brain and spinal cord resulting in cytoplasmic TDP-43 pathology and progressive motor dysfunction. Amongst numerous cell stress-related biological pathways profiled using qPCR arrays, several critical integrated stress response (ISR) effectors, including CCAAT/enhancer-binding homologous protein (Chop/Ddit3) and activating transcription factor 4 (Atf4), were upregulated in the cortex of rNLS8 mice prior to disease onset. This was accompanied by early up-regulation of anti-apoptotic gene Bcl2 and diverse pro-apoptotic genes including BH3-interacting domain death agonist (Bid). However, pro-apoptotic signalling predominated after onset of motor phenotypes. Notably, pro-apoptotic cleaved caspase-3 protein was elevated in the cortex of rNLS8 mice at later disease stages, suggesting that downstream activation of apoptosis drives neurodegeneration following failure of early protective responses. Unexpectedly, suppression of Chop in the brain and spinal cord using antisense oligonucleotide-mediated silencing had no effect on overall TDP-43 pathology or disease phenotypes in rNLS8 mice. Cytoplasmic TDP-43 accumulation therefore causes very early activation of ISR and both anti- and pro-apoptotic signalling that switches to predominant pro-apoptotic activation later in disease. These findings suggest that precise temporal modulation of cell stress and death pathways may be beneficial to protect against neurodegeneration in ALS and FTD.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Humanos , Camundongos , Animais , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Camundongos TransgênicosRESUMO
Change History: This Article has been retracted; see accompanying Retraction. Corrected online 20 January: In this Article, author Frank Rigo was incorrectly listed with a middle initial; this has been corrected in the online versions of the paper.
RESUMO
Heterozygous genetic variants within the TREM2 gene show a strong association with increased Alzheimer's disease (AD) risk. Amyloid beta-depositing mouse models haploinsufficient or null for Trem2 have identified important relationships among TREM2, microglia, and AD pathology; however, results are challenging to interpret in the context of varying microglial phenotypes and disease progression. We hypothesized that acute Trem2 reduction may alter amyloid pathology and microglial responses independent of genetic Trem2 deletion in mouse models. We developed antisense oligonucleotides (ASOs) that potently but transiently lower Trem2 messenger RNA throughout the brain and administered them to APP/PS1 mice at varying stages of plaque pathology. Late-stage ASO-mediated Trem2 knockdown significantly reduced plaque deposition and attenuated microglial association around plaque deposits when evaluated 1 mo after ASO injection. Changes in microglial gene signatures 1 wk after ASO administration and phagocytosis measured in ASO-treated cells together indicate that microglia may be activated with short-term Trem2 reduction. These results suggest a time- and/or dose-dependent role for TREM2 in mediating plaque deposition and microglial responses in which loss of TREM2 function may be beneficial for microglial activation and plaque removal in an acute context.
Assuntos
Amiloide/metabolismo , Glicoproteínas de Membrana/metabolismo , Microglia/patologia , Fagocitose , Receptores Imunológicos/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Biomarcadores/metabolismo , Encéfalo/metabolismo , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Fagocitose/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Placa Amiloide/patologia , Presenilina-1/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteínas tau/metabolismoRESUMO
RNA toxicity underlies the pathogenesis of disorders such as myotonic dystrophy type 1 (DM1). Muscular dystrophy is a key element of the pathology of DM1. The means by which RNA toxicity causes muscular dystrophy in DM1 is unclear. Here, we have used the DM200 mouse model of RNA toxicity due to the expression of a mutant DMPK 3'UTR mRNA to model the effects of RNA toxicity on muscle regeneration. Using a BaCl2-induced damage model, we find that RNA toxicity leads to decreased expression of PAX7, and decreased numbers of satellite cells, the stem cells of adult skeletal muscle (also known as MuSCs). This is associated with a delay in regenerative response, a lack of muscle fiber maturation and an inability to maintain a normal number of satellite cells. Repeated muscle damage also elicited key aspects of muscular dystrophy, including fat droplet deposition and increased fibrosis, and the results represent one of the first times to model these classic markers of dystrophic changes in the skeletal muscles of a mouse model of RNA toxicity. Using a ligand-conjugated antisense (LICA) oligonucleotide ASO targeting DMPK sequences for the first time in a mouse model of RNA toxicity in DM1, we find that treatment with IONIS 877864, which targets the DMPK 3'UTR mRNA, is efficacious in correcting the defects in regenerative response and the reductions in satellite cell numbers caused by RNA toxicity. These results demonstrate the possibilities for therapeutic interventions to mitigate the muscular dystrophy associated with RNA toxicity in DM1.
Assuntos
Desenvolvimento Muscular/genética , Distrofia Miotônica/genética , Miotonina Proteína Quinase/genética , Oligonucleotídeos Antissenso/farmacologia , RNA/genética , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Miotônica/patologia , Miotonina Proteína Quinase/antagonistas & inibidores , RNA/toxicidade , RNA Mensageiro/genética , Regeneração/genéticaRESUMO
The G4C2 repeat expansion in the C9orf72 gene is the most common genetic cause of Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Many studies suggest that dipeptide repeat proteins produced from this repeat are toxic, yet, the contribution of repeat RNA toxicity is under investigated and even less is known regarding the pathogenicity of antisense repeat RNA. Recently, two clinical trials targeting G4C2 (sense) repeat RNA via antisense oligonucleotide failed despite a robust decrease in sense-encoded dipeptide repeat proteins demonstrating target engagement. Here, in this brief report, we show that G2C4 antisense, but not G4C2 sense, repeat RNA is sufficient to induce TDP-43 dysfunction in induced pluripotent stem cell (iPSC) derived neurons (iPSNs). Unexpectedly, only G2C4, but not G4C2 sense strand targeting, ASOs mitigate deficits in TDP-43 function in authentic C9orf72 ALS/FTD patient iPSNs. Collectively, our data suggest that the G2C4 antisense repeat RNA may be an important therapeutic target and provide insights into a possible explanation for the recent G4C2 ASO clinical trial failure.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Células-Tronco Pluripotentes Induzidas , Humanos , Oligonucleotídeos Antissenso/farmacologia , Demência Frontotemporal/genética , Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/genética , Proteínas de Ligação a DNA/genética , RNA Antissenso , Dipeptídeos , NeurôniosRESUMO
The global crisis of opioid overdose fatalities has led to an urgent search to discover the neurobiological mechanisms of opioid use disorder (OUD). A driving force for OUD is the dysphoric and emotionally painful state (hyperkatifeia) that is produced during acute and protracted opioid withdrawal. Here, we explored a mechanistic role for extrahypothalamic stress systems in driving opioid addiction. We found that glucocorticoid receptor (GR) antagonism with mifepristone reduced opioid addiction-like behaviors in rats and zebrafish of both sexes and decreased the firing of corticotropin-releasing factor neurons in the rat amygdala (i.e., a marker of brain stress system activation). In support of the hypothesized role of glucocorticoid transcriptional regulation of extrahypothalamic GRs in addiction-like behavior, an intra-amygdala infusion of an antisense oligonucleotide that blocked GR transcriptional activity reduced addiction-like behaviors. Finally, we identified transcriptional adaptations of GR signaling in the amygdala of humans with OUD. Thus, GRs, their coregulators, and downstream systems may represent viable therapeutic targets to treat the "stress side" of OUD.
Assuntos
Transtornos Relacionados ao Uso de Opioides , Síndrome de Abstinência a Substâncias , Corticosteroides , Animais , Hormônio Liberador da Corticotropina , Ratos , Peixe-ZebraRESUMO
Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease that is characterized by motor neuron loss and that leads to paralysis and death 2-5 years after disease onset. Nearly all patients with ALS have aggregates of the RNA-binding protein TDP-43 in their brains and spinal cords, and rare mutations in the gene encoding TDP-43 can cause ALS. There are no effective TDP-43-directed therapies for ALS or related TDP-43 proteinopathies, such as frontotemporal dementia. Antisense oligonucleotides (ASOs) and RNA-interference approaches are emerging as attractive therapeutic strategies in neurological diseases. Indeed, treatment of a rat model of inherited ALS (caused by a mutation in Sod1) with ASOs against Sod1 has been shown to substantially slow disease progression. However, as SOD1 mutations account for only around 2-5% of ALS cases, additional therapeutic strategies are needed. Silencing TDP-43 itself is probably not appropriate, given its critical cellular functions. Here we present a promising alternative therapeutic strategy for ALS that involves targeting ataxin-2. A decrease in ataxin-2 suppresses TDP-43 toxicity in yeast and flies, and intermediate-length polyglutamine expansions in the ataxin-2 gene increase risk of ALS. We used two independent approaches to test whether decreasing ataxin-2 levels could mitigate disease in a mouse model of TDP-43 proteinopathy. First, we crossed ataxin-2 knockout mice with TDP-43 (also known as TARDBP) transgenic mice. The decrease in ataxin-2 reduced aggregation of TDP-43, markedly increased survival and improved motor function. Second, in a more therapeutically applicable approach, we administered ASOs targeting ataxin-2 to the central nervous system of TDP-43 transgenic mice. This single treatment markedly extended survival. Because TDP-43 aggregation is a component of nearly all cases of ALS, targeting ataxin-2 could represent a broadly effective therapeutic strategy.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/terapia , Ataxina-2/deficiência , Proteínas de Ligação a DNA/metabolismo , Longevidade , Oligonucleotídeos Antissenso/uso terapêutico , Agregação Patológica de Proteínas/terapia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Ataxina-2/genética , Sistema Nervoso Central/metabolismo , Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Progressão da Doença , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Destreza Motora/fisiologia , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , Agregação Patológica de Proteínas/genética , Estresse Fisiológico , Análise de SobrevidaRESUMO
There are no disease-modifying treatments for adult human neurodegenerative diseases. Here we test RNA-targeted therapies in two mouse models of spinocerebellar ataxia type 2 (SCA2), an autosomal dominant polyglutamine disease. Both models recreate the progressive adult-onset dysfunction and degeneration of a neuronal network that are seen in patients, including decreased firing frequency of cerebellar Purkinje cells and a decline in motor function. We developed a potential therapy directed at the ATXN2 gene by screening 152 antisense oligonucleotides (ASOs). The most promising oligonucleotide, ASO7, downregulated ATXN2 mRNA and protein, which resulted in delayed onset of the SCA2 phenotype. After delivery by intracerebroventricular injection to ATXN2-Q127 mice, ASO7 localized to Purkinje cells, reduced cerebellar ATXN2 expression below 75% for more than 10 weeks without microglial activation, and reduced the levels of cerebellar ATXN2. Treatment of symptomatic mice with ASO7 improved motor function compared to saline-treated mice. ASO7 had a similar effect in the BAC-Q72 SCA2 mouse model, and in both mouse models it normalized protein levels of several SCA2-related proteins expressed in Purkinje cells, including Rgs8, Pcp2, Pcp4, Homer3, Cep76 and Fam107b. Notably, the firing frequency of Purkinje cells returned to normal even when treatment was initiated more than 12 weeks after the onset of the motor phenotype in BAC-Q72 mice. These findings support ASOs as a promising approach for treating some human neurodegenerative diseases.
Assuntos
Oligonucleotídeos Antissenso/uso terapêutico , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/terapia , Potenciais de Ação , Animais , Ataxina-2/deficiência , Ataxina-2/genética , Ataxina-2/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Movimento , Fenótipo , Células de Purkinje/metabolismo , Células de Purkinje/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Teste de Desempenho do Rota-Rod , Ataxias Espinocerebelares/patologia , Ataxias Espinocerebelares/fisiopatologiaRESUMO
Transcription termination for genes encoding polyadenylated mRNAs requires a functional poly(A) signal (PAS) in the nascent pre-mRNA. Often called PAS-dependent termination, or PADT, it is widely assumed that the PAS requirement reflects an obligatory poly(A) site cleavage requirement for termination. Cleavage is thought to provide entry for a 5'-to-3' exonuclease that targets RNA polymerase II via the nascent transcript-i.e., the torpedo model. To assess the role of cleavage in PADT, we developed a PADT assay using HeLa nuclear extract. Here we examine the basal mechanism of PADT and show that cleavage at the poly(A) site is not required for PADT. Isolated elongation complexes undergo termination in a PAS-dependent manner when incubated in buffer, in the absence of extract, nucleotides, or cleavage at the poly(A) site. Thus, PADT-proficient complexes undergo a conformational change that triggers termination. PADT is inhibited by α-amanitin, which presumably blocks the required conformational change.
Assuntos
Poli A/metabolismo , RNA Mensageiro/química , Terminação da Transcrição Genética , Alfa-Amanitina/farmacologia , Células HeLa , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico/efeitos dos fármacos , RNA Mensageiro/genética , Terminação da Transcrição Genética/efeitos dos fármacosRESUMO
Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing efforts to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal and non-neuronal cells. Therefore, it is important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and target RNA reduction throughout the CNS in neurons, oligodendrocytes, astrocytes and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available.
Assuntos
Sistema Nervoso Central/química , Camundongos/metabolismo , Oligonucleotídeos Antissenso/farmacocinética , Primatas/metabolismo , Ratos/metabolismo , Animais , Sistema Nervoso Central/citologia , Feminino , Hibridização In Situ , Injeções Intraventriculares , Injeções Espinhais , Macaca fascicularis , Masculino , Neuroglia/química , Neurônios/química , Oligonucleotídeos Antissenso/administração & dosagem , Especificidade de Órgãos , RNA Longo não Codificante/análise , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , Ratos Sprague-Dawley , Ribonuclease H , Distribuição TecidualRESUMO
The chaperone protein SmgGDS promotes cell-cycle progression and tumorigenesis in human breast and nonsmall cell lung cancer. Splice variants of SmgGDS, named SmgGDS-607 and SmgGDS-558, facilitate the activation of oncogenic members of the Ras and Rho families of small GTPases through membrane trafficking via regulation of the prenylation pathway. SmgGDS-607 interacts with newly synthesized preprenylated small GTPases, while SmgGDS-558 interacts with prenylated small GTPases. We determined that cancer cells have a high ratio of SmgGDS-607:SmgGDS-558 (607:558 ratio), and this elevated ratio is associated with reduced survival of breast cancer patients. These discoveries suggest that targeting SmgGDS splicing to lower the 607:558 ratio may be an effective strategy to inhibit the malignant phenotype generated by small GTPases. Here we report the development of a splice-switching oligonucleotide, named SSO Ex5, that lowers the 607:558 ratio by altering exon 5 inclusion in SmgGDS pre-mRNA (messenger RNA). Our results indicate that SSO Ex5 suppresses the prenylation of multiple small GTPases in the Ras, Rho, and Rab families and inhibits ERK activity, resulting in endoplasmic reticulum (ER) stress, the unfolded protein response, and ultimately apoptotic cell death in breast and lung cancer cell lines. Furthermore, intraperitoneal (i.p.) delivery of SSO Ex5 in MMTV-PyMT mice redirects SmgGDS splicing in the mammary gland and slows tumorigenesis in this aggressive model of breast cancer. Taken together, our results suggest that the high 607:558 ratio is required for optimal small GTPase prenylation, and validate this innovative approach of targeting SmgGDS splicing to diminish malignancy in breast and lung cancer.