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1.
J Neurovirol ; 30(1): 39-51, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38172412

RESUMEN

Sarbecoviruses such as SARS and SARS-CoV-2 have been responsible for two major outbreaks in humans, the latter resulting in a global pandemic. While sarbecoviruses primarily cause an acute respiratory infection, they have been shown to infect the nervous system. However, mechanisms of sarbecovirus neuroinvasion and neuropathogenesis remain unclear. In this study, we examined the infectivity and trans-synaptic transmission potential of the sarbecoviruses SARS and SARS-CoV-2 in human stem cell-derived neural model systems. We demonstrated limited ability of sarbecoviruses to infect and replicate in human stem cell-derived neurons. Furthermore, we demonstrated an inability of sarbecoviruses to transmit between synaptically connected human stem cell-derived neurons. Finally, we determined an absence of SARS-CoV-2 infection in olfactory neurons in experimentally infected ferrets. Collectively, this study indicates that sarbecoviruses exhibit low potential to infect human stem cell-derived neurons, lack an ability to infect ferret olfactory neurons, and lack an inbuilt molecular mechanism to utilise retrograde axonal trafficking and trans-synaptic transmission to spread within the human nervous system.


Asunto(s)
Axones , COVID-19 , Hurones , SARS-CoV-2 , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Humanos , SARS-CoV-2/patogenicidad , SARS-CoV-2/fisiología , Animales , COVID-19/virología , COVID-19/transmisión , Axones/virología , Hurones/virología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Neuronas/virología , Replicación Viral , Chlorocebus aethiops , Células-Madre Neurales/virología , Células Vero
2.
Sci Rep ; 13(1): 20467, 2023 11 22.
Artículo en Inglés | MEDLINE | ID: mdl-37993492

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a severely debilitating neurodegenerative condition that is part of the same disease spectrum as frontotemporal dementia (FTD). Mutations in the CCNF gene, encoding cyclin F, are present in both sporadic and familial ALS and FTD. However, the pathophysiological mechanisms underlying neurodegeneration remain unclear. Proper functioning of the endoplasmic reticulum (ER) and Golgi apparatus compartments is essential for normal physiological activities and to maintain cellular viability. Here, we demonstrate that ALS/FTD-associated variant cyclin FS621G inhibits secretory protein transport from the ER to Golgi apparatus, by a mechanism involving dysregulation of COPII vesicles at ER exit sites. Consistent with this finding, cyclin FS621G also induces fragmentation of the Golgi apparatus and activates ER stress, ER-associated degradation, and apoptosis. Induction of Golgi fragmentation and ER stress were confirmed with a second ALS/FTD variant cyclin FS195R, and in cortical primary neurons. Hence, this study provides novel insights into pathogenic mechanisms associated with ALS/FTD-variant cyclin F, involving perturbations to both secretory protein trafficking and ER-Golgi homeostasis.


Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Humanos , Esclerosis Amiotrófica Lateral/metabolismo , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Degradación Asociada con el Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Mutación , Ciclinas/metabolismo
3.
Traffic ; 24(3): 146-157, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36479968

RESUMEN

The nucleolus is a common target of viruses and viral proteins, but for many viruses the functional outcomes and significance of this targeting remains unresolved. Recently, the first intranucleolar function of a protein of a cytoplasmically-replicating negative-sense RNA virus (NSV) was identified, with the finding that the matrix (M) protein of Hendra virus (HeV) (genus Henipavirus, family Paramyxoviridae) interacts with Treacle protein within nucleolar subcompartments and mimics a cellular mechanism of the nucleolar DNA-damage response (DDR) to suppress ribosomal RNA (rRNA) synthesis. Whether other viruses utilise this mechanism has not been examined. We report that sub-nucleolar Treacle targeting and modulation is conserved between M proteins of multiple Henipaviruses, including Nipah virus and other potentially zoonotic viruses. Furthermore, this function is also evident for P3 protein of rabies virus, the prototype virus of a different RNA virus family (Rhabdoviridae), with Treacle depletion in cells also found to impact virus production. These data indicate that unrelated proteins of viruses from different families have independently developed nucleolar/Treacle targeting function, but that modulation of Treacle has distinct effects on infection. Thus, subversion of Treacle may be an important process in infection by diverse NSVs, and so could provide novel targets for antiviral approaches with broad specificity.


Asunto(s)
Virus Hendra , Lyssavirus , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN Ribosómico , Lyssavirus/genética , Lyssavirus/metabolismo , Ribosomas/metabolismo , Virus Hendra/genética , Virus Hendra/metabolismo , Factores de Transcripción
4.
Viruses ; 16(1): 33, 2023 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-38229694

RESUMEN

Australian bat lyssavirus (ABLV) shows similar clinical symptoms as rabies, but there are currently no protein structures available for ABLV proteins. In lyssaviruses, the interaction between nucleoprotein (N) and phosphoprotein (N) in the absence of RNA generates a complex (N0P) that is crucial for viral assembly, and understanding the interface between these two proteins has the potential to provide insight into a key feature: the viral lifecycle. In this study, we used recombinant chimeric protein expression and X-ray crystallography to determine the structure of ABLV nucleoprotein bound to residues 1-40 of its phosphoprotein chaperone. Comparison of our results with the recently generated structure of RABV CVS-11 N0P demonstrated a highly conserved interface in this complex. Because the N0P interface is conserved in the lyssaviruses of phylogroup I, it is an attractive therapeutic target for multiple rabies-causing viral species.


Asunto(s)
Quirópteros , Lyssavirus , Rabia , Infecciones por Rhabdoviridae , Animales , Lyssavirus/genética , Nucleoproteínas/genética , Australia , Fosfoproteínas/genética , Infecciones por Rhabdoviridae/veterinaria
5.
Transbound Emerg Dis ; 69(2): 297-307, 2022 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33400387

RESUMEN

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an emerging virus that has caused significant human morbidity and mortality since its detection in late 2019. With the rapid emergence has come an unprecedented programme of vaccine development with at least 300 candidates under development. Ferrets have proven to be an appropriate animal model for testing safety and efficacy of SARS-CoV-2 vaccines due to quantifiable virus shedding in nasal washes and oral swabs. Here, we outline our efforts early in the SARS-CoV-2 outbreak to propagate and characterize an Australian isolate of the virus in vitro and in an ex vivo model of human airway epithelium, as well as to demonstrate the susceptibility of domestic ferrets (Mustela putorius furo) to SARS-CoV-2 infection following intranasal challenge.


Asunto(s)
COVID-19 , Hurones , Animales , Australia , COVID-19/veterinaria , Vacunas contra la COVID-19 , Humanos , SARS-CoV-2
6.
Front Cell Infect Microbiol ; 11: 783140, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-35004351

RESUMEN

Despite being vaccine preventable, rabies (lyssavirus) still has a significant impact on global mortality, disproportionally affecting children under 15 years of age. This neurotropic virus is deft at avoiding the immune system while travelling through neurons to the brain. Until recently, research efforts into the role of non-coding RNAs in rabies pathogenicity and detection have been hampered by a lack of human in vitro neuronal models. Here, we utilized our previously described human stem cell-derived neural model to investigate the effect of lyssavirus infection on microRNA (miRNA) expression in human neural cells and their secreted exosomes. Conventional differential expression analysis identified 25 cellular and 16 exosomal miRNAs that were significantly altered (FDR adjusted P-value <0.05) in response to different lyssavirus strains. Supervised machine learning algorithms determined 6 cellular miRNAs (miR-99b-5p, miR-346, miR-5701, miR-138-2-3p, miR-651-5p, and miR-7977) were indicative of lyssavirus infection (100% accuracy), with the first four miRNAs having previously established roles in neuronal function, or panic and impulsivity-related behaviors. Another 4-miRNA signatures in exosomes (miR-25-3p, miR-26b-5p, miR-218-5p, miR-598-3p) can independently predict lyssavirus infected cells with >99% accuracy. Identification of these robust lyssavirus miRNA signatures offers further insight into neural lineage responses to infection and provides a foundation for utilizing exosome miRNAs in the development of next-generation molecular diagnostics for rabies.


Asunto(s)
Exosomas , Lyssavirus , MicroARNs , Humanos , Lyssavirus/genética , Aprendizaje Automático , MicroARNs/genética , Neuronas , Células Madre
7.
Viruses ; 12(6)2020 06 24.
Artículo en Inglés | MEDLINE | ID: mdl-32599823

RESUMEN

The respiratory Influenza A Viruses (IAVs) and emerging zoonotic viruses such as Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) pose a significant threat to human health. To accelerate our understanding of the host-pathogen response to respiratory viruses, the use of more complex in vitro systems such as normal human bronchial epithelial (NHBE) cell culture models has gained prominence as an alternative to animal models. NHBE cells were differentiated under air-liquid interface (ALI) conditions to form an in vitro pseudostratified epithelium. The responses of well-differentiated (wd) NHBE cells were examined following infection with the 2009 pandemic Influenza A/H1N1pdm09 strain or following challenge with the dsRNA mimic, poly(I:C). At 30 h postinfection with H1N1pdm09, the integrity of the airway epithelium was severely impaired and apical junction complex damage was exhibited by the disassembly of zona occludens-1 (ZO-1) from the cell cytoskeleton. wdNHBE cells produced an innate immune response to IAV-infection with increased transcription of pro- and anti-inflammatory cytokines and chemokines and the antiviral viperin but reduced expression of the mucin-encoding MUC5B, which may impair mucociliary clearance. Poly(I:C) produced similar responses to IAV, with the exception of MUC5B expression which was more than 3-fold higher than for control cells. This study demonstrates that wdNHBE cells are an appropriate ex-vivo model system to investigate the pathogenesis of respiratory viruses.


Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/fisiología , Gripe Humana/virología , Mucosa Respiratoria/citología , Mucosa Respiratoria/virología , Animales , Bronquios/citología , Bronquios/virología , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Perros , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/epidemiología , Uniones Intercelulares , Células de Riñón Canino Madin Darby , Modelos Biológicos , Mucina 5AC/metabolismo , Pandemias , Cultivo de Virus
8.
Viruses ; 12(4)2020 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-32218146

RESUMEN

Rabies is a zoonotic neurological infection caused by lyssavirus that continues to result in devastating loss of human life. Many aspects of rabies pathogenesis in human neurons are not well understood. Lack of appropriate ex-vivo models for studying rabies infection in human neurons has contributed to this knowledge gap. In this study, we utilize advances in stem cell technology to characterize rabies infection in human stem cell-derived neurons. We show key cellular features of rabies infection in our human neural cultures, including upregulation of inflammatory chemokines, lack of neuronal apoptosis, and axonal transmission of viruses in neuronal networks. In addition, we highlight specific differences in cellular pathogenesis between laboratory-adapted and field strain lyssavirus. This study therefore defines the first stem cell-derived ex-vivo model system to study rabies pathogenesis in human neurons. This new model system demonstrates the potential for enabling an increased understanding of molecular mechanisms in human rabies, which could lead to improved control methods.


Asunto(s)
Lyssavirus/fisiología , Neuronas/virología , Células Madre/citología , Células Madre/metabolismo , Animales , Apoptosis , Axones/metabolismo , Axones/virología , Biomarcadores , Calcio/metabolismo , Supervivencia Celular , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Interacciones Huésped-Patógeno , Humanos , Inmunohistoquímica , Ratones , Imagen Molecular , Virus de la Rabia/fisiología , Infecciones por Rhabdoviridae/virología
9.
PLoS Pathog ; 16(2): e1008343, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32069324

RESUMEN

Neurotropic viral infections continue to pose a serious threat to human and animal wellbeing. Host responses combatting the invading virus in these infections often cause irreversible damage to the nervous system, resulting in poor prognosis. Rabies is the most lethal neurotropic virus, which specifically infects neurons and spreads through the host nervous system by retrograde axonal transport. The key pathogenic mechanisms associated with rabies infection and axonal transmission in neurons remains unclear. Here we studied the pathogenesis of different field isolates of lyssavirus including rabies using ex-vivo model systems generated with mouse primary neurons derived from the peripheral and central nervous systems. In this study, we show that neurons activate selective and compartmentalized degeneration of their axons and dendrites in response to infection with different field strains of lyssavirus. We further show that this axonal degeneration is mediated by the loss of NAD and calpain-mediated digestion of key structural proteins such as MAP2 and neurofilament. We then analysed the role of SARM1 gene in rabies infection, which has been shown to mediate axonal self-destruction during injury. We show that SARM1 is required for the accelerated execution of rabies induced axonal degeneration and the deletion of SARM1 gene significantly delayed axonal degeneration in rabies infected neurons. Using a microfluidic-based ex-vivo neuronal model, we show that SARM1-mediated axonal degeneration impedes the spread of rabies virus among interconnected neurons. However, this neuronal defense mechanism also results in the pathological loss of axons and dendrites. This study therefore identifies a potential host-directed mechanism behind neurological dysfunction in rabies infection. This study also implicates a novel role of SARM1 mediated axonal degeneration in neurotropic viral infection.


Asunto(s)
Proteínas del Dominio Armadillo/metabolismo , Axones/metabolismo , Proteínas del Citoesqueleto/metabolismo , Rabia/fisiopatología , Animales , Proteínas del Dominio Armadillo/genética , Proteínas del Dominio Armadillo/fisiología , Transporte Axonal/fisiología , Axones/fisiología , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/fisiología , Modelos Animales de Enfermedad , Ganglios Espinales/virología , Lyssavirus/patogenicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuritas/metabolismo , Neuritas/virología , Neuronas/metabolismo , Neuronas/virología , Rabia/metabolismo , Virus de la Rabia/metabolismo , Virus de la Rabia/patogenicidad
11.
Cell Mol Life Sci ; 77(19): 3859-3873, 2020 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31802140

RESUMEN

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative diseases that are related genetically and pathologically. Mutations in the UBQLN2 gene, encoding the ubiquitin-like protein ubiquilin2, are associated with familial ALS/FTD, but the pathophysiological mechanisms remain unclear. Here, we demonstrate that ALS/FTD UBQLN2 mutants P497H and P506T inhibit protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in neuronal cells. In addition, we observed that Sec31-positive ER exit sites are clustered in UBQLN2T487I patient spinal cord tissues. Both the ER-Golgi intermediate (ERGIC) compartment and the Golgi become disorganised and fragmented. This activates ER stress and inhibits ER-associated degradation. Hence, this study highlights perturbations in secretory protein trafficking and ER homeostasis as pathogenic mechanisms associated with ALS/FTD-associated forms of UBQLN2.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Proteínas Relacionadas con la Autofagia/genética , Células Cultivadas , Estrés del Retículo Endoplásmico , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Neuronas/citología , Neuronas/metabolismo , Transporte de Proteínas
12.
Pathogens ; 8(3)2019 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-31461898

RESUMEN

Chikungunya virus (CHIKV) is transmitted by Aedes mosquitoes and causes prolonged arthralgia in patients. After crossing the mosquito midgut barrier, the virus disseminates to tissues including the head and salivary glands. To better understand the interaction between Aedes albopictus and CHIKV, we performed RNASeq analysis on pools of mosquito heads and parts of the thorax 8 days post infection, which identified 159 differentially expressed transcripts in infected mosquitos compared to uninfected controls. After validation using RT-qPCR (reverse transcriptase-quantitative polymerase chain reaction), inhibitor of Bruton's tyrosine kinase (BTKi), which has previously been shown to be anti-inflammatory in mammals after viral infection, was further evaluated for its functional significance. Knockdown of BTKi using double-stranded RNA in a mosquito cell line showed no significant difference in viral RNA or infectivity titer. However, BTKi gene knocked-down cells showed increased apoptosis 24 hours post-infection compared with control cells, suggesting involvement of BTKi in the mosquito response to viral infection. Since BTK in mammals promotes an inflammatory response and has been shown to be involved in osteoclastogenesis, a hallmark of CHIKV pathogenesis, our results suggest a possible conserved mechanism at play between mosquitoes and mammals. Taken together, these results will add to our understanding of Aedes Albopictus interactions with CHIKV.

13.
Virol J ; 15(1): 79, 2018 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-29703263

RESUMEN

BACKGROUND: Zika virus infection in new born is linked to congenital syndromes, especially microcephaly. Studies have shown that these neuropathies are the result of significant death of neuronal progenitor cells in the central nervous system of the embryo, targeted by the virus. Although cell death via apoptosis is well acknowledged, little is known about possible pathogenic cellular mechanisms triggering cell death in neurons. METHODS: We used in vitro embryonic mouse primary neuron cultures to study possible upstream cellular mechanisms of cell death. Neuronal networks were grown on microelectrode array and electrical activity was recorded at different times post Zika virus infection. In addition to this method, we used confocal microscopy and Q-PCR techniques to observe morphological and molecular changes after infection. RESULTS: Zika virus infection of mouse primary neurons triggers an early spiking excitation of neuron cultures, followed by dramatic loss of this activity. Using NMDA receptor antagonist, we show that this excitotoxicity mechanism, likely via glutamate, could also contribute to the observed nervous system defects in human embryos and could open new perspective regarding the causes of adult neuropathies. CONCLUSIONS: This model of excitotoxicity, in the context of neurotropic virus infection, highlights the significance of neuronal activity recording with microelectrode array and possibility of more than one lethal mechanism after Zika virus infection in the nervous system.


Asunto(s)
Potenciales de Acción/fisiología , Muerte Celular , Red Nerviosa/virología , Neuronas/virología , Infección por el Virus Zika/virología , Virus Zika/fisiología , Animales , Encéfalo/citología , Encéfalo/virología , Células Cultivadas , Ácido Glutámico/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Neurológicos , Red Nerviosa/patología , Neuronas/metabolismo , Neuronas/patología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Transducción de Señal/genética , Transmisión Sináptica , Replicación Viral , Infección por el Virus Zika/patología
14.
Cell Mol Life Sci ; 75(2): 335-354, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-28852778

RESUMEN

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase (SCFcyclin F) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin-proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin FS621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin FWT. Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin FS621G-expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin FS621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal-lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin FS621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is implicated in ALS pathogenesis.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Autofagia/genética , Ciclinas/genética , Demencia Frontotemporal/genética , Ubiquitinación/genética , Esclerosis Amiotrófica Lateral/complicaciones , Células Cultivadas , Demencia Frontotemporal/complicaciones , Células HEK293 , Humanos , Lisina/metabolismo , Mutación Missense/fisiología
15.
Open Biol ; 7(10)2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-29021214

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that is characterized by progressive weakness, paralysis and muscle loss often resulting in patient death within 3-5 years of diagnosis. Recently, we identified disease-linked mutations in the CCNF gene, which encodes the cyclin F protein, in cohorts of patients with familial and sporadic ALS and frontotemporal dementia (FTD) (Williams KL et al 2016 Nat. Commun.7, 11253. (doi:10.1038/ncomms11253)). Cyclin F is a part of a Skp1-Cul-F-box (SCF) E3 ubiquitin-protein ligase complex and is responsible for ubiquitylating proteins for degradation by the proteasome. In this study, we investigated the phosphorylation status of cyclin F and the effect of the serine to glycine substitution at site 621 (S621G) on E3 ligase activity. This specific mutation (S621G) was found in a multi-generational Australian family with ALS/FTD. We identified seven phosphorylation sites on cyclin F, of which five are newly reported including Ser621. These phosphorylation sites were mostly identified within the PEST (proline, glutamic acid, serine and threonine) sequence located at the C-terminus of cyclin F. Additionally, we determined that casein kinase II (CK2) can phosphorylate Ser621 and thereby regulate the E3 ligase activity of the SCF(cyclin F) complex. Furthermore, the S621G mutation in cyclin F prevents phosphorylation by CK2 and confers elevated Lys48-ubiquitylation activity, a hallmark of ALS/FTD pathology. These findings highlight the importance of phosphorylation in regulating the activity of the SCF(cyclin F) E3 ligase complex that can affect downstream processes and may lead to defective motor neuron development, neuron degeneration and ultimately ALS and FTD.


Asunto(s)
Quinasa de la Caseína II/metabolismo , Ciclinas/metabolismo , Complejos Multiproteicos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular Tumoral , Cromatografía Liquida , Activación Enzimática , Células HEK293 , Humanos , Lisina , Espectrometría de Masas , Modelos Moleculares , Fosfatidilserinas , Fosforilación , Unión Proteica , Ubiquitinación
18.
Artículo en Inglés | MEDLINE | ID: mdl-27534431

RESUMEN

Mutations in the optineurin gene (OPTN) have been identified in a small proportion (<1%) of sporadic and familial ALS cases, and the exact role of optineurin in the pathogenesis of ALS remains unclear. To further examine the role of OPTN in ALS, we sought to identify novel ALS variants in OPTN and examine their potential for pathogenicity in vitro. Whole exome sequence data from 74 familial ALS cases were analysed for the presence of novel OPTN mutations. Pathogenicity was assessed by analysing effects on Golgi fragmentation, endoplasmic reticulum (ER) stress-linked CHOP activation, and cellular localization of optineurin in motor neuron-like NSC-34 cells expressing mutant optineurin. We identified a novel heterozygous missense mutation in OPTN (c.883G > T, p.Val295Phe) in a single familial ALS case. This mutation induced recognized cellular features of ALS pathogenesis including Golgi fragmentation and ER stress in NSC-34 cells. In conclusion, the identification of a novel OPTN mutation in an Australian ALS family, and its capacity to induce ALS-like pathological features in vitro, further strengthens evidence for the role of optineurin in the pathogenesis of ALS.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Estrés del Retículo Endoplásmico/genética , Mutación/genética , Factor de Transcripción TFIIIA/genética , Animales , Australia , Proteínas de Ciclo Celular , Línea Celular Transformada , Estudios de Cohortes , Análisis Mutacional de ADN , Salud de la Familia , Femenino , Aparato de Golgi/genética , Aparato de Golgi/patología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Masculino , Proteínas de Transporte de Membrana , Factor de Transcripción CHOP/genética , Transfección
19.
Nat Commun ; 7: 11253, 2016 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-27080313

RESUMEN

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are overlapping, fatal neurodegenerative disorders in which the molecular and pathogenic basis remains poorly understood. Ubiquitinated protein aggregates, of which TDP-43 is a major component, are a characteristic pathological feature of most ALS and FTD patients. Here we use genome-wide linkage analysis in a large ALS/FTD kindred to identify a novel disease locus on chromosome 16p13.3. Whole-exome sequencing identified a CCNF missense mutation at this locus. Interrogation of international cohorts identified additional novel CCNF variants in familial and sporadic ALS and FTD. Enrichment of rare protein-altering CCNF variants was evident in a large sporadic ALS replication cohort. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase complex (SCF(Cyclin F)). Expression of mutant CCNF in neuronal cells caused abnormal ubiquitination and accumulation of ubiquitinated proteins, including TDP-43 and a SCF(Cyclin F) substrate. This implicates common mechanisms, linked to protein homeostasis, underlying neuronal degeneration.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Ciclinas/genética , Demencia Frontotemporal/genética , Predisposición Genética a la Enfermedad/genética , Mutación Missense , Adulto , Anciano , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Mapeo Cromosómico , Cromosomas Humanos Par 16/genética , Salud de la Familia , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Persona de Mediana Edad , Linaje , Análisis de Secuencia de ADN/métodos , Homología de Secuencia de Aminoácido
20.
Front Neurosci ; 9: 400, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26578862

RESUMEN

Amyotrophic Lateral Sclerosis (ALS) is an invariably fatal neurodegenerative disorder, which specifically targets motor neurons in the brain, brain stem and spinal cord. Whilst the etiology of ALS remains unknown, fragmentation of the Golgi apparatus is detected in ALS patient motor neurons and in animal/cellular disease models. The Golgi is a highly dynamic organelle that acts as a dispatching station for the vesicular transport of secretory/transmembrane proteins. It also mediates autophagy and maintains endoplasmic reticulum (ER) and axonal homeostasis. Both the trigger for Golgi fragmentation and the functional consequences of a fragmented Golgi apparatus in ALS remain unclear. However, recent evidence has highlighted defects in vesicular trafficking as a pathogenic mechanism in ALS. This review summarizes the evidence describing Golgi fragmentation in ALS, with possible links to other disease processes including cellular trafficking, ER stress, defective autophagy, and axonal degeneration.

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