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1.
Proc Natl Acad Sci U S A ; 120(31): e2207978120, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37487086

RESUMEN

Loss-of-function mutations in the KCNA1(Kv1.1) gene cause episodic ataxia type 1 (EA1), a neurological disease characterized by cerebellar dysfunction, ataxic attacks, persistent myokymia with painful cramps in skeletal muscles, and epilepsy. Precision medicine for EA1 treatment is currently unfeasible, as no drug that can enhance the activity of Kv1.1-containing channels and offset the functional defects caused by KCNA1 mutations has been clinically approved. Here, we uncovered that niflumic acid (NFA), a currently prescribed analgesic and anti-inflammatory drug with an excellent safety profile in the clinic, potentiates the activity of Kv1.1 channels. NFA increased Kv1.1 current amplitudes by enhancing the channel open probability, causing a hyperpolarizing shift in the voltage dependence of both channel opening and gating charge movement, slowing the OFF-gating current decay. NFA exerted similar actions on both homomeric Kv1.2 and heteromeric Kv1.1/Kv1.2 channels, which are formed in most brain structures. We show that through its potentiating action, NFA mitigated the EA1 mutation-induced functional defects in Kv1.1 and restored cerebellar synaptic transmission, Purkinje cell availability, and precision of firing. In addition, NFA ameliorated the motor performance of a knock-in mouse model of EA1 and restored the neuromuscular transmission and climbing ability in Shaker (Kv1.1) mutant Drosophila melanogaster flies (Sh5). By virtue of its multiple actions, NFA has strong potential as an efficacious single-molecule-based therapeutic agent for EA1 and serves as a valuable model for drug discovery.


Asunto(s)
Miocimia , Animales , Ratones , Drosophila melanogaster , Ataxia , Drosophila , Canal de Potasio Kv.1.2
2.
Biochim Biophys Acta Mol Basis Dis ; 1869(8): 166818, 2023 12.
Artículo en Inglés | MEDLINE | ID: mdl-37495086

RESUMEN

SARS-CoV-2, the virus responsible for the coronavirus disease of 2019 (COVID-19), gains cellular entry via interaction with the angiotensin-converting enzyme 2 (ACE2) receptor of host cells. Although SARS-CoV-2 mainly targets the respiratory system, the neuromuscular system also appears to be affected in a large percentage of patients with acute or chronic COVID-19. The cause of the well-described neuromuscular manifestations resulting from SARS-CoV-2 infection remains unresolved. These may result from the neuromuscular-invasive capacity of the virus leading to direct injury. Alternatively, they may be the consequence of ACE2 inactivation either due to viral infection, ACE2 autoantibodies or both. Here, we made use of the Drosophila model to investigate whether ACE2 downregulation is sufficient to induce neuromuscular phenotypes. We show that moderate gene silencing of ACE2 orthologues Ance or Ance3 diminished survival on exposure to thermal stress only upon induction of neuromuscular fatigue driven by increased physical activity. A strong knockdown of Ance or Ance3 directed to muscle reduced or abolished adult viability and caused obvious motoric deficits including reduced locomotion and impaired flight capacity. Selective knockdown of Ance and Ance3 in neurons caused wing defects and an age-dependent decline in motor behaviour, respectively, in adult flies. Interestingly, RNA sequencing allowed us to discover several differentially spliced genes that are required for synaptic function downstream of Ance or Ance3 depletion. Our findings are therefore supportive of the notion that loss of a RAS-independent function for ACE2 contributes to the neuromuscular manifestations associated with SARS-CoV-2 infection.


Asunto(s)
COVID-19 , Animales , COVID-19/complicaciones , COVID-19/genética , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2/genética , Peptidil-Dipeptidasa A/genética , Drosophila
3.
Front Neurosci ; 17: 1164251, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37360176

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neuromuscular disease that has a strong genetic component. Deleterious variants in the DCTN1 gene are known to be a cause of ALS in diverse populations. DCTN1 encodes the p150 subunit of the molecular motor dynactin which is a key player in the bidirectional transport of cargos within cells. Whether DCTN1 mutations lead to the disease through either a gain or loss of function mechanism remains unresolved. Moreover, the contribution of non-neuronal cell types, especially muscle tissue, to ALS phenotypes in DCTN1 carriers is unknown. Here we show that gene silencing of Dctn1, the Drosophila main orthologue of DCTN1, either in neurons or muscles is sufficient to cause climbing and flight defects in adult flies. We also identify Dred, a protein with high homology to Drosophila Dctn1 and human DCTN1, that on loss of function also leads to motoric impairments. A global reduction of Dctn1 induced a significant reduction in the mobility of larvae and neuromuscular junction (NMJ) deficits prior to death at the pupal stage. RNA-seq and transcriptome profiling revealed splicing alterations in genes required for synapse organisation and function, which may explain the observed motor dysfunction and synaptic defects downstream of Dctn1 ablation. Our findings support the possibility that loss of DCTN1 function can lead to ALS and underscore an important requirement for DCTN1 in muscle in addition to neurons.

4.
Neurobiol Aging ; 126: 67-76, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-36944290

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disease mostly resulting from a complex interplay between genetic, environmental and lifestyle factors. Common genetic variants in the Sec1 Family Domain Containing 1 (SCFD1) gene have been associated with increased ALS risk in the most extensive genome-wide association study (GWAS). SCFD1 was also identified as a top-most significant expression Quantitative Trait Locus (eQTL) for ALS. Whether loss of SCFD1 function directly contributes to motor system dysfunction remains unresolved. Here we show that moderate gene silencing of Slh, the Drosophila orthologue of SCFD1, is sufficient to cause climbing and flight defects in adult flies. A more severe knockdown induced a significant reduction in larval mobility and profound neuromuscular junction (NMJ) deficits prior to death before metamorphosis. RNA-seq revealed downregulation of genes encoding chaperones that mediate protein folding downstream of Slh ablation. Our findings support the notion that loss of SCFD1 function is a meaningful contributor to ALS and disease predisposition may result from erosion of the mechanisms protecting against misfolding and protein aggregation.


Asunto(s)
Esclerosis Amiotrófica Lateral , Animales , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Drosophila/genética , Estudio de Asociación del Genoma Completo , Factores de Riesgo
5.
Neurobiol Aging ; 123: 200-207, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36549973

RESUMEN

Genetic risk for amyotrophic lateral sclerosis (ALS) is highly elevated in genetic isolates, like the island population of Malta in the south of Europe, providing a unique opportunity to investigate the genetics of this disease. Here we characterize the clinical phenotype and genetic profile of the largest series of Maltese ALS patients to date identified throughout a 5-year window. Cases and controls underwent neuromuscular assessment and analysis of rare variants in ALS causative or risk genes following whole-genome sequencing. Potentially damaging variants or repeat expansions were identified in more than 45% of all patients. The most commonly affected genes were ALS2, DAO, SETX and SPG11, an infrequent cause of ALS in Europeans. We also confirmed a significant association between ATXN1 intermediate repeats and increased disease risk. Damaging variants in major ALS genes C9orf72, SOD1, TARDBP and FUS were however either absent or rare in Maltese ALS patients. Overall, our study underscores a population that is an outlier within Europe and one that represents a high percentage of genetically explained cases.


Asunto(s)
Esclerosis Amiotrófica Lateral , Predisposición Genética a la Enfermedad , Humanos , Predisposición Genética a la Enfermedad/genética , Estudios de Asociación Genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/epidemiología , Malta/epidemiología , Fenotipo , Proteína C9orf72/genética , Superóxido Dismutasa-1/genética , Mutación/genética , ADN Helicasas/genética , ARN Helicasas/genética , Enzimas Multifuncionales/genética , Proteínas/genética
6.
Neuroscience ; 491: 32-42, 2022 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-35314252

RESUMEN

Increasing evidence points to the involvement of cell types other than motor neurons in both amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), the predominant motor neuron disease in adults and infants, respectively. The contribution of glia to ALS pathophysiology is well documented. Studies have since focused on evaluating the contribution of glia in SMA. Here, we made use of the Drosophila model to ask whether the survival motor neuron (Smn) protein, the causative factor for SMA, is required selectively in glia. We show that the specific loss of Smn function in glia during development reduced survival to adulthood but did not affect motoric performance or neuromuscular junction (NMJ) morphology in flies. In contrast, gain rather than loss of ALS-linked TDP-43, FUS or C9orf72 function in glia induced significant defects in motor behaviour in addition to reduced survival. Furthermore, glia-specific gain of TDP-43 function caused both NMJ defects and muscle atrophy. Smn together with Gemins 2-8 and Unrip, form the Smn complex which is indispensable for the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). We show that glial-selective perturbation of Smn complex components or disruption of key snRNP biogenesis factors pICln and Tgs1, induce deleterious effects on adult fly viability but, similar to Smn reduction, had no negative effect on neuromuscular function. Our findings suggest that the role of Smn in snRNP biogenesis as part of the Smn complex is required in glia for the survival of the organism, underscoring the importance of glial cells in SMA disease formation.


Asunto(s)
Atrofia Muscular Espinal , Envejecimiento , Animales , Proteínas de Unión al ADN/metabolismo , Drosophila/metabolismo , Neuronas Motoras/fisiología , Neuroglía/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Proteínas del Complejo SMN/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo
7.
Eur J Hum Genet ; 30(7): 856-859, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-34616013

RESUMEN

Amyotrophic lateral sclerosis (ALS) is frequently caused by mutations in the SOD1 gene. Here, we report the first SOD1 variant in Malta, an archipelago of three inhabited islands in southern Europe. We describe a patient with a sporadic form of ALS living on the island of Gozo in which the heterozygous SOD1 c.272A>C; p.(Asp91Ala) variant was detected. The patient had a late onset (79 years), sensory impairments and rapid disease progression culminating in respiratory failure. ALS has not yet developed in any of the three additional family members in which the D91A variant was identified. None of the healthy controls from the Maltese population were found to carry this variant. This report underscores the high prevalence of the D91A variant in Europe, despite the presence of a North-South gradient in its frequency, and confirms that this variant can be associated with dominant cases in Mediterranean countries.


Asunto(s)
Esclerosis Amiotrófica Lateral , Esclerosis Amiotrófica Lateral/genética , Europa (Continente) , Heterocigoto , Humanos , Mutación , Superóxido Dismutasa-1/genética
8.
Heliyon ; 7(12): e08555, 2021 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-34901515

RESUMEN

Angiotensin-converting enzyme (ACE) and its homologue ACE2 are key regulators of the renin-angiotensin system and thereby cardiovascular function through their zinc-metallopeptidase activity on vasoactive peptides. ACE2 also serves as the receptor for the cellular entry of various coronaviruses including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the coronavirus disease 2019 (COVID-19). The unprecedented scale of the COVID-19 pandemic has spurred the use of mammalian models to investigate the SARS-ACE2 relationship and knowledge gained from such research has accelerated development of vaccines and therapeutics. Recent studies have just started to underscore the utility of the fruit fly Drosophila melanogaster as a model system to study virus-host interactions and pathogenicity. Notably, the remarkable existence of catalytically functional ACE and ACE2 orthologues in Drosophila, discovered more than two decades ago, provides a unique opportunity for further developing this model organism to better understand COVID-19 in addition to identifying coronavirus preventative and therapeutic interventions targeting ACE2. Here, we review the studies that revealed crucial insights on the biochemistry and physiology of Ance and Acer, two out of the six Drosophila ACE family members with the greatest homology to human ACE and ACE2. We highlight shared in vivo functions outside of the renin-angiotensin system, which is not conserved in flies. Importantly, we identify knowledge gaps that can be filled by further research and outline ways that can raise Drosophila to a powerful model system to combat SARS-CoV-2 and its threatening vaccine-evading variants.

9.
Molecules ; 26(5)2021 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-33799979

RESUMEN

The identification of compounds which protect the double-membrane of mitochondrial organelles from disruption by toxic confomers of amyloid proteins may offer a therapeutic strategy to combat human neurodegenerative diseases. Here, we exploited an extract from the marine brown seaweed Padina pavonica (PPE) as a vital source of natural bioactive compounds to protect mitochondrial membranes against insult by oligomeric aggregates of the amyloidogenic proteins amyloid-ß (Aß), α-synuclein (α-syn) and tau, which are currently considered to be major targets for drug discovery in Alzheimer's disease (AD) and Parkinson's disease (PD). We show that PPE manifested a significant inhibitory effect against swelling of isolated mitochondria exposed to the amyloid oligomers, and attenuated the release of cytochrome c from the mitochondria. Using cardiolipin-enriched synthetic lipid membranes, we also show that dye leakage from fluorophore-loaded vesicles and formation of channel-like pores in planar bilayer membranes are largely prevented by incubating the oligomeric aggregates with PPE. Lastly, we demonstrate that PPE curtails the ability of Aß42 and α-syn monomers to self-assemble into larger ß-aggregate structures, as well as potently disrupts their respective amyloid fibrils. In conclusion, the mito-protective and anti-aggregator biological activities of Padina pavonica extract may be of therapeutic value in neurodegenerative proteinopathies, such as AD and PD.


Asunto(s)
Péptidos beta-Amiloides/toxicidad , Membranas Mitocondriales/efectos de los fármacos , Fragmentos de Péptidos/toxicidad , Phaeophyceae/química , alfa-Sinucleína/toxicidad , Péptidos beta-Amiloides/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Humanos , Membrana Dobles de Lípidos/química , Membranas Mitocondriales/patología , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/metabolismo , Algas Marinas/química , alfa-Sinucleína/metabolismo
10.
Artículo en Inglés | MEDLINE | ID: mdl-33821701

RESUMEN

Objective: Amyotrophic lateral sclerosis (ALS) is a mostly sporadic neurodegenerative disease. The role of environmental factors has been extensively investigated but associations remain controversial. Considering that a substantial proportion of adult life is spent at work, identifying occupations and work-related exposures is considered an effective way to detect factors that increase ALS risk. This process may be further facilitated in population isolates due to environmental and genetic homogeneity. Our study investigated occupations and occupational exposures potentially associated with ALS risk in the isolated island population of Malta, using a case-control study design. Methods: Patients with ALS and randomly identified matched controls (1:1) were recruited throughout a four-year window, from 2017 through 2020. Data on educational level, residence, main occupation, smoking, and alcohol history were collected. Results: We found that compared to controls (44.4%), a higher percentage (73.7%) of ALS patients reported a blue-collar job as their main occupation (OR 2.04, 95% CI 1.2-3.72; p = 0.0072). Through regression analysis, craft and related trades occupations such as carpentry and construction (ISCO-08 major group 7), were found to be positively associated with ALS, with patients in this occupational category found to be more prone to develop bulbar-onset ALS (p = 0.0297). Overall, patients with ALS reported a significantly higher exposure to work-related strenuous physical activity (OR 2.35, 95% CI 1.53-3.59; p = 0.0002). Conclusion: Our findings suggest that manual workers particularly those working in the carpentry and construction industries have an increased ALS risk, possibly due to a history of intense or sustained physical activity.


Asunto(s)
Esclerosis Amiotrófica Lateral , Enfermedades Neurodegenerativas , Exposición Profesional , Adulto , Esclerosis Amiotrófica Lateral/epidemiología , Estudios de Casos y Controles , Humanos , Malta , Exposición Profesional/efectos adversos , Ocupaciones , Factores de Riesgo
11.
Eur J Hum Genet ; 29(4): 604-614, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33414559

RESUMEN

Genetic isolates are compelling tools for mapping genes of inherited disorders. The archipelago of Malta, a sovereign microstate in the south of Europe is home to a geographically and culturally isolated population. Here, we investigate the epidemiology and genetic profile of Maltese patients with amyotrophic lateral sclerosis (ALS), identified throughout a 2-year window. Cases were largely male (66.7%) with a predominant spinal onset of symptoms (70.8%). Disease onset occurred around mid-age (median age: 64 years, men; 59.5 years, female); 12.5% had familial ALS (fALS). Annual incidence rate was 2.48 (95% CI 1.59-3.68) per 100,000 person-years. Male-to-female incidence ratio was 1.93:1. Prevalence was 3.44 (95% CI 2.01-5.52) cases per 100,000 inhabitants on 31st December 2018. Whole-genome sequencing allowed us to determine rare DNA variants that change the protein-coding sequence of ALS-associated genes. Interestingly, the Maltese ALS patient cohort was found to be negative for deleterious variants in C9orf72, SOD1, TARDBP or FUS genes, which are the most commonly mutated ALS genes globally. Nonetheless, ALS-associated repeat expansions were identified in ATXN2 and NIPA1. Variants predicted to be damaging were also detected in ALS2, DAO, DCTN1, ERBB4, SETX, SCFD1 and SPG11. A total of 40% of patients with sporadic ALS had a rare and deleterious variant or repeat expansion in an ALS-associated gene, whilst the genetic cause of two thirds of fALS cases could not be pinpointed to known ALS genes or risk loci. This warrants further studies to elucidate novel genes that cause ALS in this unique population isolate.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Sitios Genéticos , Mutación , Aislamiento Reproductivo , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Esclerosis Amiotrófica Lateral/epidemiología , Femenino , Frecuencia de los Genes , Humanos , Masculino , Malta , Persona de Mediana Edad , Prevalencia , Factores Sexuales
12.
Sci Rep ; 10(1): 17733, 2020 10 20.
Artículo en Inglés | MEDLINE | ID: mdl-33082392

RESUMEN

Studies on the amyloidogenic N-terminal domain of the E. coli HypF protein (HypF-N) have contributed significantly to a detailed understanding of the pathogenic mechanisms in neurodegenerative diseases characterised by the formation of misfolded oligomers, by proteins such as amyloid-ß, α-synuclein and tau. Given that both cell membranes and mitochondria are increasingly recognised as key targets of oligomer toxicity, we investigated the damaging effects of aggregates of HypF-N on mitochondrial membranes. Essentially, we found that HypF-N oligomers characterised by high surface hydrophobicity (type A) were able to trigger a robust permeabilisation of mito-mimetic liposomes possessing cardiolipin-rich membranes and dysfunction of isolated mitochondria, as demonstrated by a combination of mitochondrial shrinking, lowering of mitochondrial membrane potential and cytochrome c release. Furthermore, using single-channel electrophysiology recordings we obtained evidence that the type A aggregates induced currents reflecting formation of ion-conducting pores in mito-mimetic planar phospholipid bilayers, with multi-level conductances ranging in the hundreds of pS at negative membrane voltages. Conversely, HypF-N oligomers with low surface hydrophobicity (type B) could not permeabilise or porate mitochondrial membranes. These results suggest an inherent toxicity of membrane-active aggregates of amyloid-forming proteins to mitochondria, and that targeting of oligomer-mitochondrial membrane interactions might therefore afford protection against such damage.


Asunto(s)
Amiloide/metabolismo , Transferasas de Carboxilo y Carbamoilo/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Membrana Dobles de Lípidos/metabolismo , Mitocondrias/fisiología , Membranas Mitocondriales/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Péptidos beta-Amiloides/metabolismo , Cardiolipinas/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Potencial de la Membrana Mitocondrial , Conformación Proteica , Multimerización de Proteína , Relación Estructura-Actividad , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo
13.
iScience ; 23(1): 100809, 2020 Jan 24.
Artículo en Inglés | MEDLINE | ID: mdl-31927482

RESUMEN

Spinal muscular atrophy (SMA) is a devastating motor neuron disorder caused by mutations in the survival motor neuron (SMN) gene. It remains unclear how SMN deficiency leads to the loss of motor neurons. By screening Schizosaccharomyces pombe, we found that the growth defect of an SMN mutant can be alleviated by deletion of the actin-capping protein subunit gene acp1+. We show that SMN mutated cells have splicing defects in the profilin gene, which thus directly hinder actin cytoskeleton homeostasis including endocytosis and cytokinesis. We conclude that deletion of acp1+ in an SMN mutant background compensates for actin cytoskeleton alterations by restoring redistribution of actin monomers between different types of cellular actin networks. Our data reveal a direct correlation between an impaired function of SMN in snRNP assembly and defects in actin dynamics. They also point to important common features in the pathogenic mechanism of SMA and ALS.

14.
Biochim Biophys Acta Biomembr ; 1862(2): 183064, 2020 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-31521630

RESUMEN

Misfolding and aggregate formation by the tau protein has been closely related with neurotoxicity in a large group of human neurodegenerative disorders, which includes Alzheimer's disease. Here, we investigate the membrane-active properties of tau oligomers on mitochondrial membranes, using minimalist in vitro model systems. Thus, exposure of isolated mitochondria to oligomeric tau evoked a disruption of mitochondrial membrane integrity, as evidenced by a combination of organelle swelling, efflux of cytochrome c and loss of the mitochondrial membrane potential. Tau-induced mitochondrial dysfunction occurred independently of the mitochondrial permeability transition (mPT) pore complex. Notably, mitochondria were rescued by pre-incubation with 10-N-nonyl acridine orange (NAO), a molecule that specifically binds cardiolipin (CL), the signature phospholipid of mitochondrial membranes. Additionally, NAO prevented direct binding of tau oligomers to isolated mitochondria. At the same time, tau proteins exhibited high affinity to CL-enriched membranes, whilst permeabilisation of lipid vesicles also strongly correlated with CL content. Intriguingly, using single-channel electrophysiology, we could demonstrate the formation of non-selective ion-conducting tau nanopores exhibiting multilevel conductances in mito-mimetic bilayers. Taken together, the data presented here advances a scenario in which toxic cytosolic entities of tau protein would target mitochondrial organelles by associating with their CL-rich membrane domains, leading to membrane poration and compromised mitochondrial structural integrity.


Asunto(s)
Cardiolipinas/metabolismo , Membranas Mitocondriales/efectos de los fármacos , Proteínas tau/farmacología , Humanos , Membranas Mitocondriales/metabolismo , Nanoporos , Permeabilidad/efectos de los fármacos , Unión Proteica , Multimerización de Proteína
15.
Sci Rep ; 9(1): 18666, 2019 12 10.
Artículo en Inglés | MEDLINE | ID: mdl-31822699

RESUMEN

The predominant motor neuron disease in infants and adults is spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS), respectively. SMA is caused by insufficient levels of the Survival Motor Neuron (SMN) protein, which operates as part of the multiprotein SMN complex that includes the DEAD-box RNA helicase Gemin3/DDX20/DP103. C9orf72, SOD1, TDP-43 and FUS are ranked as the four major genes causing familial ALS. Accumulating evidence has revealed a surprising molecular overlap between SMA and ALS. Here, we ask the question of whether Drosophila can also be exploited to study shared pathogenic pathways. Focusing on motor behaviour, muscle mass and survival, we show that disruption of either TBPH/TDP-43 or Caz/FUS enhance defects associated with Gemin3 loss-of-function. Gemin3-associated neuromuscular junction overgrowth was however suppressed. Sod1 depletion had a modifying effect in late adulthood. We also show that Gemin3 self-interacts and Gem3ΔN, a helicase domain deletion mutant, retains the ability to interact with its wild-type counterpart. Importantly, mutant:wild-type dimers are favoured more than wild-type:wild-type dimers. In addition to reinforcing the link between SMA and ALS, further exploration of mechanistic overlaps is now possible in a genetically tractable model organism. Notably, Gemin3 can be elevated to a candidate for modifying motor neuron degeneration.


Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Unión al ARN/metabolismo , Superóxido Dismutasa/metabolismo , Factor de Transcripción TFIID/metabolismo , Alelos , Animales , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , ARN Helicasas DEAD-box/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Vuelo Animal , Genotipo , Humanos , Masculino , Fenotipo , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Superóxido Dismutasa/genética , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Factor de Transcripción TFIID/genética
16.
ACS Chem Neurosci ; 10(8): 3815-3829, 2019 08 21.
Artículo en Inglés | MEDLINE | ID: mdl-31356747

RESUMEN

Aggregation of the amyloid-forming α-synuclein (αS) protein is closely associated with the etiology of Parkinson's disease (PD), the most common motor neurodegenerative disorder. Many studies have shown that soluble aggregation intermediates of αS, termed oligomers, permeabilize a variety of phospholipid membranes; thus, membrane disruption may represent a key pathogenic mechanism of αS toxicity. Given the centrality of mitochondrial dysfunction in PD, we therefore probed the formation of ion-permeable pores by αS oligomers in planar lipid bilayers reflecting the complex phospholipid composition of mitochondrial membranes. Using single-channel electrophysiology, we recorded distinct multilevel conductances (100-400 pS) with stepwise current transitions, typical of protein-bound nanopores, in mitochondrial-like membranes. Crucially, we observed that the presence of cardiolipin (CL), the signature phospholipid of mitochondrial membranes, enhanced αS-lipid interaction and the membrane pore-forming activity of αS oligomers. Further, preincubation of isolated mitochondria with a CL-specific dye protected against αS oligomer-induced mitochondrial swelling and release of cytochrome c. Hence, we favor a scenario in which αS oligomers directly porate a local lipid environment rich in CL, for instance outer mitochondrial contact sites or the inner mitochondrial membrane, to induce mitochondrial dysfunction. Pharmacological modulation of αS pore complex formation might thus preserve mitochondrial membrane integrity and alleviate mitochondrial dysfunction in PD.


Asunto(s)
Cardiolipinas/farmacología , Mitocondrias/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , alfa-Sinucleína/metabolismo , Transporte Biológico , Humanos , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Permeabilidad
17.
BMC Res Notes ; 11(1): 378, 2018 Jun 13.
Artículo en Inglés | MEDLINE | ID: mdl-29895323

RESUMEN

OBJECTIVE: Spinal muscular atrophy (SMA) results from insufficient levels of the survival motor neuron (SMN) protein. Drosophila is conducive to large-scale genetic-modifier screens which can reveal novel pathways underpinning the disease mechanism. We tested the ability of a large collection of genomic deletions to enhance SMN-dependent lethality. To test our design, we asked whether our study can identify loci containing genes identified in previous genetic screens. Our objective was to find a common link between genes flagged in independent screens, which would allow us to expose novel functions for SMN in vivo. RESULTS: Out of 128 chromosome deficiency lines, 12 (9.4%) were found to consistently depress adult viability when crossed to SMN loss-of-function heterozygotes. In their majority, the enhancing deletions harboured genes that were previously identified as genetic modifiers, hence, validating the design of the screen. Importantly, gene overlap allowed us to flag genes with a role in post-transcriptional regulation of mRNAs that are crucial for determining the axes of the oocyte and future embryo. We find that SMN is also required for the correct localisation of gurken and oskar mRNAs in oocytes. These findings extend the role of SMN in oogenesis by identifying a key requirement for mRNA trafficking.


Asunto(s)
Proteínas de Drosophila/genética , Genoma de los Insectos/genética , Oocitos/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Animales , Drosophila melanogaster , Factor de Crecimiento Transformador alfa/genética
18.
J Neurosci Methods ; 310: 3-11, 2018 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-29649521

RESUMEN

Motor neuron disease (MND) is characterised by muscle weakness and paralysis downstream of motor neuron degeneration. Genetic factors play a major role in disease pathogenesis and progression. This is best underscored by spinal muscular atrophy (SMA), the most common MND affecting children. Although SMA is caused by homozygous mutations in the survival motor neuron 1 (SMN1) gene, partial compensation by the paralogous SMN2 gene and/or genetic modifiers influence age of onset and disease severity. SMA is also the first MND that is treatable thanks to the recent development of a molecular-based therapy. This key milestone was possible following an intense research campaign in which animal models had a starring role. In this review, we specifically focus on the fruit fly Drosophila melanogaster and highlight its sterling contributions aimed at furthering our understanding of SMA pathogenesis. Methods of gene disruption utilised to generate SMA fly models are discussed and ways through which neuromuscular defects have been characterised are elaborated on. A phenotypic overlap with patients and mammalian models, allowed the use of SMA fly models to identify genetic modifiers, hence spurring investigators to discover pathways that are perturbed in disease. Targeting these can potentially lead to complimentary therapies for SMA. The same output is expected from the use of SMA fly models to identify therapeutic compounds that have an ameliorative effect. We believe that lessons gained from SMA will allow researchers to eagerly exploit Drosophila to confirm novel genes linked to MND, reveal disease mechanisms and ultimately identify therapeutics.


Asunto(s)
Modelos Animales de Enfermedad , Drosophila melanogaster , Enfermedad de la Neurona Motora , Atrofia Muscular Espinal , Animales , Humanos
19.
Biochem Soc Trans ; 46(2): 329-341, 2018 04 17.
Artículo en Inglés | MEDLINE | ID: mdl-29523774

RESUMEN

Gemin3, also known as DDX20 or DP103, is a DEAD-box RNA helicase which is involved in more than one cellular process. Though RNA unwinding has been determined in vitro, it is surprisingly not required for all of its activities in cellular metabolism. Gemin3 is an essential gene, present in Amoeba and Metazoa. The highly conserved N-terminus hosts the helicase core, formed of the helicase- and DEAD-domains, which, based on crystal structure determination, have key roles in RNA binding. The C-terminus of Gemin3 is highly divergent between species and serves as the interaction site for several accessory factors that could recruit Gemin3 to its target substrates and/or modulate its function. This review article focuses on the known roles of Gemin3, first as a core member of the survival motor neuron (SMN) complex, in small nuclear ribonucleoprotein biogenesis. Although mechanistic details are lacking, a critical function for Gemin3 in this pathway is supported by numerous in vitro and in vivo studies. Gene expression activities of Gemin3 are next underscored, mainly messenger ribonucleoprotein trafficking, gene silencing via microRNA processing, and transcriptional regulation. The involvement of Gemin3 in abnormal cell signal transduction pathways involving p53 and NF-κB is also highlighted. Finally, the clinical implications of Gemin3 deregulation are discussed including links to spinal muscular atrophy, poliomyelitis, amyotrophic lateral sclerosis, and cancer. Impressive progress made over the past two decades since the discovery of Gemin3 bodes well for further work that refines the mechanism(s) underpinning its multiple activities.


Asunto(s)
Proteína 20 DEAD-Box/metabolismo , ARN Helicasas DEAD-box/metabolismo , Animales , Carcinogénesis , Proteína 20 DEAD-Box/genética , ARN Helicasas DEAD-box/genética , Expresión Génica , Silenciador del Gen , Humanos , FN-kappa B/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Transducción de Señal , Relación Estructura-Actividad , Proteína p53 Supresora de Tumor/metabolismo
20.
FEBS Lett ; 591(21): 3600-3614, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-28949413

RESUMEN

The Spinal Muscular Atrophy disease protein Survival Motor Neuron (SMN) operates as part of a multiprotein complex whose components also include Gemins 2-8 and Unrip. The fruit fly Drosophila melanogaster is thought to have a slightly smaller SMN complex comprised of SMN, Gemin2/3/5 and, possibly, Unrip. Based upon in vivo interaction methods, we have identified novel interacting partners of the Drosophila SMN complex with homologies to Gemin4/6/7/8. The Gemin4 and Gemin8 orthologues are required for neuromuscular function and survival. The Gemin6/7/Unrip module can be recruited via the SMN-associated Gemin8, hence mirroring the human SMN complex architecture. Our findings lead us to propose that an elaborate SMN complex that is typical in metazoans is also present in Drosophila.


Asunto(s)
Proteínas del Complejo SMN/metabolismo , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Humanos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas del Complejo SMN/genética
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