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1.
Methods Mol Biol ; 2587: 153-164, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36401029

RESUMO

Antisense oligonucleotide (ASO)-based exon skipping therapy is thought to be promising for Duchenne muscular dystrophy (DMD). For the screening or assessing patient eligibility before administering ASO to patients, in vitro testing using myoblasts derived from each DMD patient is considered crucial. We previously reported state-of-the-art technology to obtain patient primary myoblasts from MYOD1-induced urine-derived cells (UDCs) as a model of DMD. We hypothesize that the myoblasts may potentially reflect specific pathological phenotypes, leading to a path for precision medicine in DMD patients. Here, we describe a detailed protocol for both acquiring MYOD1-induced myoblasts from UDCs and evaluating the correction of DMD mRNA and protein levels after exon-skipping in the cells.


Assuntos
Distrofia Muscular de Duchenne , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/metabolismo , Distrofina/genética , Distrofina/metabolismo , Éxons , Mioblastos/metabolismo , Fenótipo
2.
Brain Neurosci Adv ; 6: 23982128221086464, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35359460

RESUMO

Synapse loss is associated with cognitive decline in Alzheimer's disease, and owing to their plastic nature, synapses are an ideal target for therapeutic intervention. Oligomeric amyloid beta around amyloid plaques is known to contribute to synapse loss in mouse models and is associated with synapse loss in human Alzheimer's disease brain tissue, but the mechanisms leading from Aß to synapse loss remain unclear. Recent data suggest that the fast-activating and -inactivating voltage-gated potassium channel subtype 3.4 (Kv3.4) may play a role in Aß-mediated neurotoxicity. Here, we tested whether this channel could also be involved in Aß synaptotoxicity. Using adeno-associated virus and clustered regularly interspaced short palindromic repeats technology, we reduced Kv3.4 expression in neurons of the somatosensory cortex of APP/PS1 mice. These mice express human familial Alzheimer's disease-associated mutations in amyloid precursor protein and presenilin-1 and develop amyloid plaques and plaque-associated synapse loss similar to that observed in Alzheimer's disease brain. We observe that reducing Kv3.4 levels ameliorates dendritic spine loss and changes spine morphology compared to control virus. In support of translational relevance, Kv3.4 protein was observed in human Alzheimer's disease and control brain and is associated with synapses in human induced pluripotent stem cell-derived cortical neurons. We also noted morphological changes in induced pluripotent stem cell neurones challenged with human Alzheimer's disease-derived brain homogenate containing Aß but, in this in vitro model, total mRNA levels of Kv3.4 were found to be reduced, perhaps as an early compensatory mechanism for Aß-induced damage. Overall, our results suggest that approaches to reduce Kv3.4 expression and/or function in the Alzheimer's disease brain could be protective against Aß-induced synaptic alterations.

4.
Front Physiol ; 12: 689179, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34721051

RESUMO

Splice-switching antisense oligonucleotide- (SSO-) mediated correction of framedisrupting mutation-containing premessenger RNA (mRNA) transcripts using exon skipping is a highly promising treatment method for muscular diseases such as Duchenne muscular dystrophy (DMD). Phosphorothioate (PS) chemistry, a commonly used oligonucleotide modification, has been shown to increase the stability of and improve the pharmacokinetics of SSOs. However, the effect of PS inclusion in 2'-O-methyl SSOs (2OMe) on cellular uptake and splice switching is less well-understood. At present, we demonstrate that the modification of PS facilitates the uptake of 2OMe in H2k-mdx myoblasts. Furthermore, we found a dependency of SSO nuclear accumulation and high splice-switching activity on PS inclusion in 2OMe (2OMePS), as tested in various reporter cell lines carrying pLuc/705. Increased exon-inclusion activity was observed in muscle, neuronal, liver, and bone cell lineages via both the gymnotic uptake and lipofection of 2OMePS. Using the photoactivatable ribonucleoside-enhanced crosslinking and a subsequent proteomic approach, we identified several 2OMePS-binding proteins, which are likely to play a role in the trafficking of 2OMePS to the nucleus. Ablation of one of them, Ncl by small-interfering RNA (siRNA) enhanced 2OMePS uptake in C2C12 myoblasts and upregulated luciferase RNA splicing in the HeLa Luc/705 reporter cell line. Overall, we demonstrate that PS inclusion increases nuclear delivery and splice switching in muscle, neuronal, liver, and bone cell lineages and that the modulation of 2OMePS-binding partners may improve SSO delivery.

5.
Methods Mol Biol ; 2176: 185-208, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32865792

RESUMO

Several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), have a complex genetic background, in addition to cases where the disease appears to manifest sporadically. The recent discovery of the hexanucleotide repeat expansion in the C9orf72 gene as the causative agent of ALS (C9ALS) gives rise to the opportunity to develop new therapies directed at this mutation , which is responsible for a large proportion of ALS and/or frontotemporal dementia cases. Mammalian models conscientiously replicating the late-onset motor defects and cellular pathologies seen in human patients do not exist. In this context, patient-derived cells give us a platform to test potential antisense oligonucleotide therapies, which could be the key to treat this subtype of motor neuron disease. Recently, we described that locked nucleic acid gapmer oligonucleotide-based treatment targeting C9orf72 repeat expanded transcripts resulted in recovery from the disease-related phenotypes in patient-derived fibroblasts. Our findings highlight the therapeutic potential of C9ALS using this gapmer oligonucleotide-based approach.


Assuntos
Proteína C9orf72/genética , Técnicas de Cultura de Células/métodos , Expansão das Repetições de DNA , Terapia Genética/métodos , Oligonucleotídeos/genética , Transfecção/métodos , Esclerose Lateral Amiotrófica/genética , Células Cultivadas , Vesículas Extracelulares , Fibroblastos , Congelamento , Demência Frontotemporal/genética , Humanos , Immunoblotting , Plasmídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/citologia
6.
Stem Cell Reports ; 14(5): 892-908, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32330447

RESUMO

TDP-43 dysfunction is common to 97% of amyotrophic lateral sclerosis (ALS) cases, including those with mutations in C9orf72. To investigate how C9ORF72 mutations drive cellular pathology in ALS and to identify convergent mechanisms between C9ORF72 and TARDBP mutations, we analyzed motor neurons (MNs) derived from induced pluripotent stem cells (iPSCs) from patients with ALS. C9ORF72 iPSC-MNs have higher Ca2+ release after depolarization, delayed recovery to baseline after glutamate stimulation, and lower levels of calbindin compared with CRISPR/Cas9 genome-edited controls. TARDBP iPS-derived MNs show high glutamate-induced Ca2+ release. We identify here, by RNA sequencing, that both C9ORF72 and TARDBP iPSC-MNs have upregulation of Ca2+-permeable AMPA and NMDA subunits and impairment of mitochondrial Ca2+ buffering due to an imbalance of MICU1 and MICU2 on the mitochondrial Ca2+ uniporter, indicating that impaired mitochondrial Ca2+ uptake contributes to glutamate excitotoxicity and is a shared feature of MNs with C9ORF72 or TARDBP mutations.


Assuntos
Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/genética , Cálcio/metabolismo , Proteínas de Ligação a DNA/genética , Demência Frontotemporal/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios Motores/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Calbindinas/metabolismo , Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular , Demência Frontotemporal/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Neurônios Motores/citologia , Mutação , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo
7.
Brain ; 140(4): 887-897, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28334866

RESUMO

A non-coding hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), however, the precise molecular mechanism by which the C9orf72 hexanucleotide repeat expansion directs C9ALS/FTD pathogenesis remains unclear. Here, we report a novel disease mechanism arising due to the interaction of C9ORF72 with the RAB7L1 GTPase to regulate vesicle trafficking. Endogenous interaction between C9ORF72 and RAB7L1 was confirmed in human SH-SY5Y neuroblastoma cells. The C9orf72 hexanucleotide repeat expansion led to haploinsufficiency resulting in severely defective intracellular and extracellular vesicle trafficking and a dysfunctional trans-Golgi network phenotype in patient-derived fibroblasts and induced pluripotent stem cell-derived motor neurons. Genetic ablation of RAB7L1or C9orf72 in SH-SY5Y cells recapitulated the findings in C9ALS/FTD fibroblasts and induced pluripotent stem cell neurons. When C9ORF72 was overexpressed or antisense oligonucleotides were targeted to the C9orf72 hexanucleotide repeat expansion to upregulate normal variant 1 transcript levels, the defective vesicle trafficking and dysfunctional trans-Golgi network phenotypes were reversed, suggesting that both loss- and gain-of-function mechanisms play a role in disease pathogenesis. In conclusion, we have identified a novel mechanism for C9ALS/FTD pathogenesis highlighting the molecular regulation of intracellular and extracellular vesicle trafficking as an important pathway in C9ALS/FTD pathogenesis.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Demência Frontotemporal/metabolismo , Proteínas/metabolismo , Proteínas rab1 de Ligação ao GTP/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Transporte Biológico , Proteína C9orf72 , Células COS , Linhagem Celular , Chlorocebus aethiops , Expansão das Repetições de DNA , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Humanos , Íntrons , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Oligonucleotídeos Antissenso/farmacologia , Linhagem , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/patologia , Proteínas/genética , Proteínas rab de Ligação ao GTP , Proteínas rab1 de Ligação ao GTP/genética
8.
Mol Pharmacol ; 87(1): 87-95, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25338672

RESUMO

In common with other members of the Cys-loop family of pentameric ligand-gated ion channels, 5-hydroxytryptamine type 3 receptors (5-HT3Rs) are activated by the binding of a neurotransmitter to an extracellular orthosteric site, located at the interface of two adjacent receptor subunits. In addition, a variety of compounds have been identified that modulate agonist-evoked responses of 5-HT3Rs, and other Cys-loop receptors, by binding to distinct allosteric sites. In this study, we examined the pharmacological effects of a group of monoterpene compounds on recombinant 5-HT3Rs expressed in Xenopus oocytes. Two phenolic monoterpenes (carvacrol and thymol) display allosteric agonist activity on human homomeric 5-HT3ARs (64 ± 7% and 80 ± 4% of the maximum response evoked by the endogenous orthosteric agonist 5-HT, respectively). In addition, at lower concentrations, where agonist effects are less apparent, carvacrol and thymol act as potentiators of responses evoked by submaximal concentrations of 5-HT. By contrast, carvacrol and thymol have no agonist or potentiating activity on the closely related mouse 5-HT3ARs. Using subunit chimeras containing regions of the human and mouse 5-HT3A subunits, and by use of site-directed mutagenesis, we have identified transmembrane amino acids that either abolish the agonist activity of carvacrol and thymol on human 5-HT3ARs or are able to confer this property on mouse 5-HT3ARs. By contrast, these mutations have no significant effect on orthosteric activation of 5-HT3ARs by 5-HT. We conclude that 5-HT3ARs can be activated by the binding of ligands to an allosteric transmembrane site, a conclusion that is supported by computer docking studies.


Assuntos
Monoterpenos/farmacologia , Receptores 5-HT3 de Serotonina/metabolismo , Agonistas do Receptor 5-HT3 de Serotonina/farmacologia , Xenopus laevis/embriologia , Aminoácidos , Animais , Sítios de Ligação , Cimenos , Sinergismo Farmacológico , Células HEK293 , Humanos , Camundongos , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Receptores 5-HT3 de Serotonina/genética , Especificidade da Espécie , Timol/farmacologia , Xenopus laevis/genética
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