RESUMO
Spinocerebellar ataxia type 3 (SCA3, also known as Machado Joseph disease) is a fatal neurodegenerative disease caused by the expansion of the trinucleotide repeat region within the ATXN3/MJD gene. Mutation of ATXN3 causes formation of ataxin-3 protein aggregates, neurodegeneration, and motor deficits. Here we investigated the therapeutic potential and mechanistic activity of sodium butyrate (SB), the sodium salt of butyric acid, a metabolite naturally produced by gut microbiota, on cultured SH-SY5Y cells and transgenic zebrafish expressing human ataxin-3 containing 84 glutamine (Q) residues to model SCA3. SCA3 SH-SY5Y cells were found to contain high molecular weight ataxin-3 species and detergent-insoluble protein aggregates. Treatment with SB increased the activity of the autophagy protein quality control pathway in the SCA3 cells, decreased the presence of ataxin-3 aggregates and presence of high molecular weight ataxin-3 in an autophagy-dependent manner. Treatment with SB was also beneficial in vivo, improving swimming performance, increasing activity of the autophagy pathway, and decreasing the presence of insoluble ataxin-3 protein species in the transgenic SCA3 zebrafish. Co-treating the SCA3 zebrafish with SB and chloroquine, an autophagy inhibitor, prevented the beneficial effects of SB on zebrafish swimming, indicating that the improved swimming performance was autophagy-dependent. To understand the mechanism by which SB induces autophagy we performed proteomic analysis of protein lysates from the SB-treated and untreated SCA3 SH-SY5Y cells. We found that SB treatment had increased activity of Protein Kinase A and AMPK signaling, with immunoblot analysis confirming that SB treatment had increased levels of AMPK protein and its substrates. Together our findings indicate that treatment with SB can increase activity of the autophagy pathway process and that this has beneficial effects in vitro and in vivo. While our results suggested that this activity may involve activity of a PKA/AMPK-dependent process, this requires further confirmation. We propose that treatment with sodium butyrate warrants further investigation as a potential treatment for neurodegenerative diseases underpinned by mechanisms relating to protein aggregation including SCA3.
Assuntos
Doença de Machado-Joseph , Neuroblastoma , Doenças Neurodegenerativas , Humanos , Animais , Ácido Butírico/farmacologia , Ataxina-3/genética , Doença de Machado-Joseph/tratamento farmacológico , Doença de Machado-Joseph/genética , Peixe-Zebra , Proteínas Quinases Ativadas por AMP , Agregados Proteicos , Proteômica , Autofagia , Animais Geneticamente Modificados , Proteínas Quinases Dependentes de AMP CíclicoRESUMO
Introduction: Teleneurology has become widely adopted during severe acute respiratory syndrome coronavirus 2 pandemic. However, provider impressions about the teleneurology experience are not well described. Methods: A novel questionnaire was developed to collect provider impressions about video teleneurology encounters. All providers in the University of Pennsylvania Health System (UPHS) Neurology Department (N = 162) were asked to complete a questionnaire after each video teleneurology patient encounter between April and August 2020. Individual patient and encounter-level data were extracted from the electronic medical record. Results: One thousand six hundred three surveys were completed by 55 providers (response rate of 10.12%). The history obtained and the ability to connect with the patient were considered the same or better than an in-person visit in almost all encounters. The quality of the physician-patient relationship was good or excellent in 93%, while the overall experience was the same as an in-person visit in 73% of visits and better in 12%. Sixty-eight percent of respondents reported that none of the elements of the neurological examination if performed in person would have changed the assessment and plan. Assessment of the visit as the same or better increased from 83% in April to 89% in July and 95% in August. Headache (91%), multiple sclerosis and neuroimmunology (96%), and movement disorder (89%) providers had the highest proportion of ratings of same or better overall experience and neuromuscular providers the lowest (60%). Conclusions: Provider impressions about the teleneurology history, examination, and provider-patient relationship are favorable in the majority of responses. Important differences emerge between provider specialty and visit characteristics groups.
Assuntos
COVID-19 , Neurologia , Telemedicina , COVID-19/epidemiologia , Humanos , Pandemias , SARS-CoV-2RESUMO
The coronavirus disease-19 (COVID-19) pandemic has compelled health care practices and academic departments to evaluate the suitability of telemedicine for various specialties and attempt rapid implementation to enable continuation of health care that is safe for providers and patients. Many patients with neurological disorders are well suited to evaluations through video. The department of neurology at the University of Pennsylvania (Penn Neurology), with the support of the health system, rapidly expanded telemedicine services to meet the needs of our patient population. This accomplishment required the complex coordination of multiple disciplines and roles within the department and the health care system, including faculty, residents, administrative staff, research and technical staff, information services, and the connected health team. Procedures for provider and patient education were established. Surveys of the provider and patient experience were developed and deployed. The process has demonstrated the vital role telemedicine in neurology (teleneurology) should play in the care of neurological patients beyond the pandemic. We describe our experience as a template for other departments and practices seeking to establish teleneurology programs, as well as an illustration of the challenges and barriers to its implementation.
Assuntos
COVID-19 , Neurologia , Telemedicina , Humanos , Pandemias , SARS-CoV-2RESUMO
Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurodegenerative disease characterised by the death of upper and lower motor neurons. Approximately 10% of cases have a known family history of ALS and disease-linked mutations in multiple genes have been identified. ALS-linked mutations in CCNF were recently reported, however the pathogenic mechanisms associated with these mutations are yet to be established. To investigate possible disease mechanisms, we developed in vitro and in vivo models based on an ALS-linked missense mutation in CCNF. Proteomic analysis of the in vitro models identified the disruption of several cellular pathways in the mutant model, including caspase-3 mediated cell death. Transient overexpression of human CCNF in zebrafish embryos supported this finding, with fish expressing the mutant protein found to have increased levels of cleaved (activated) caspase-3 and increased cell death in the spinal cord. The mutant CCNF fish also developed a motor neuron axonopathy consisting of shortened primary motor axons and increased frequency of aberrant axonal branching. Importantly, we demonstrated a significant correlation between the severity of the CCNF-induced axonopathy and a reduced motor response to a light stimulus (photomotor response). This is the first report of an ALS-linked CCNF mutation in vivo and taken together with the in vitro model identifies the disruption of cell death pathways as a significant consequence of this mutation. Additionally, this study presents a valuable new tool for use in ongoing studies investigating the pathobiology of ALS-linked CCNF mutations.
Assuntos
Esclerose Lateral Amiotrófica/genética , Ciclinas/genética , Demência Frontotemporal/genética , Medula Espinal/patologia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Animais Geneticamente Modificados , Axônios/patologia , Caspase 3/metabolismo , Morte Celular/genética , Ciclinas/biossíntese , Ciclinas/metabolismo , Modelos Animais de Doenças , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Humanos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Mutação de Sentido Incorreto , Medula Espinal/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Peixe-ZebraRESUMO
BACKGROUND: Cryptogenic stroke, now defined as embolic stroke of undetermined source (ESUS), represents about a quarter of all ischemic strokes and the reoccurrence is high. Understanding this stroke subtype better would likely guide treatment recommendations. In this study, we tested the hypothesis that left atrial (LA) shape and function at rest, as well as with exercise, are abnormal compared to matched normal controls. METHODS: The study design was prospective enrollment of ESUS subjects who underwent measurement of LA function at rest and exercise by 2D and 3D echocardiograms. The exercise portion of the study was conducted using a ramped supine bicycle protocol during which LA function was measured. Stroke subjects were matched with normal subjects by age, gender, and body surface area. RESULTS: Over a 1-year enrollment period, 18 ESUS patients met inclusionary criteria and were studied. Their average age was 58 years old and 44% were female. ESUS subjects have larger LA end-diastolic volume at rest (14 versus 11 mL/m2, Pâ¯=â¯.04) and with exercise (11 versus 6 mL/m2, Pâ¯=â¯.001) compared to normal controls. In ESUS, there was a lack of response to maximal exercise of LA function as measured by the LA ejection fraction (61% versus 73% Pâ¯=â¯.001) and the LA function index (.68 versus .82, Pâ¯=â¯.02). The 3D analysis showed spherical remodeling of the LA in ESUS. This remodeling was documented by the sphericity index, which was increased in both diastole (.40 versus .32, Pâ¯=â¯.02) and systole (.63 versus .71 Pâ¯=â¯.03). CONCLUSIONS: In support of our hypothesis, we found that ESUS subjects have LA dysfunction and remodeling at rest and exercise in comparison to healthy, matched controls. Evaluation of the left atrium in this high-risk stroke subtype has potential to inform stroke prevention strategies and to suggest pathways for research.
Assuntos
Função do Átrio Esquerdo , Remodelamento Atrial , Átrios do Coração/fisiopatologia , Cardiopatias/complicações , Embolia Intracraniana/etiologia , Acidente Vascular Cerebral/etiologia , Idoso , Estudos de Casos e Controles , Ecocardiografia sob Estresse/métodos , Ecocardiografia Tridimensional , Teste de Esforço , Feminino , Átrios do Coração/diagnóstico por imagem , Cardiopatias/diagnóstico por imagem , Cardiopatias/fisiopatologia , Humanos , Embolia Intracraniana/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Estudos Prospectivos , Medição de Risco , Fatores de Risco , Acidente Vascular Cerebral/diagnóstico por imagemRESUMO
The neurodegenerative disease Machado-Joseph disease (MJD), also known as spinocerebellar ataxin-3, affects neurons of the brain and spinal cord, disrupting control of the movement of muscles. We have successfully established the first transgenic zebrafish (Danio rerio) model of MJD by expressing human ataxin-3 protein containing either 23 glutamines (23Q, wild-type) or 84Q (MJD-causing) within neurons. Phenotypic characterization of the zebrafish (male and female) revealed that the ataxin-3-84Q zebrafish have decreased survival compared with ataxin-3-23Q and develop ataxin-3 neuropathology, ataxin-3 cleavage fragments and motor impairment. Ataxin-3-84Q zebrafish swim shorter distances than ataxin-3-23Q zebrafish as early as 6 days old, even if expression of the human ataxin-3 protein is limited to motor neurons. This swimming phenotype provides a valuable readout for drug treatment studies. Treating the EGFP-ataxin-3-84Q zebrafish with the calpain inhibitor compound calpeptin decreased levels of ataxin-3 cleavage fragments, but also removed all human ataxin-3 protein (confirmed by ELISA) and prevented the early MJD zebrafish motor phenotype. We identified that this clearance of ataxin-3 protein by calpeptin treatment resulted from an increase in autophagic flux (indicated by decreased p62 levels and increased LC3II). Cotreatment with the autophagy inhibitor chloroquine blocked the decrease in human ataxin-3 levels and the improved movement produced by calpeptin treatment. This study demonstrates that this first transgenic zebrafish model of MJD is a valuable tool for testing potential treatments for MJD. Calpeptin treatment is protective in this model of MJD and removal of human ataxin-3 through macro-autophagy plays an important role in this beneficial effect.SIGNIFICANCE STATEMENT We have established the first transgenic zebrafish model of the neurodegenerative disease MJD, and identified relevant disease phenotypes, including impaired movement from an early age, which can be used in rapid drug testing studies. We have found that treating the MJD zebrafish with the calpain inhibitor compound calpeptin produces complete removal of human ataxin-3 protein, due to induction of the autophagy quality control pathway. This improves the movement of the MJD zebrafish. Artificially blocking the autophagy pathway prevents the removal of human ataxin-3 and improved movement produced by calpeptin treatment. These findings indicate that induction of autophagy, and removal of ataxin-3 protein, plays an important role in the protective effects of calpain inhibition for the treatment of MJD.
Assuntos
Ataxina-3/metabolismo , Autofagia/fisiologia , Calpaína/metabolismo , Modelos Animais de Doenças , Glicoproteínas/farmacologia , Doença de Machado-Joseph/metabolismo , Proteínas Repressoras/metabolismo , Animais , Animais Geneticamente Modificados , Ataxina-3/genética , Autofagia/efeitos dos fármacos , Calpaína/antagonistas & inibidores , Calpaína/genética , Feminino , Glicoproteínas/uso terapêutico , Humanos , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/prevenção & controle , Masculino , Proteínas Repressoras/genética , Peixe-ZebraRESUMO
BACKGROUND: Malignant cerebral edema (CED) complicates at least 20 % of large hemispheric infarcts (LHI) and may result in neurological deterioration or death. Midline shift (MLS) is a standard but crude measure of edema severity. We propose that volumetric analysis of shifts in cerebrospinal fluid (CSF) over time provides a reliable means of quantifying the spectrum of edema severity after LHI. METHODS: We identified 38 patients from 2008 to 2014 with NIHSS ≥8, baseline CT <6 h after stroke onset, at least 1 follow-up (FU) CT, and no parenchymal hematoma. The volumes of CSF (sulci, ventricles, and cisterns) ipsilateral (IL) and contralateral (CL) to infarct on baseline and FU CTs were quantified by manually assisted outlining with MIPAV image analysis software, as was infarct volume and MLS on FU CTs. Percentage change in CSF volumes (∆CSF) from baseline to FU scans was correlated with MLS and compared in those with vs. without malignant edema (defined as hemicraniectomy, osmotic therapy, or death/neurological deterioration with MLS ≥5 mm). RESULTS: 11 of 38 subjects (29 %) developed malignant edema. Neither baseline NIHSS nor CSF volume differed between those with and without edema (median NIHSS 18 vs. 13, p = 0.12, CSF volume 102 vs. 124 ml, p = 0.16). Inter-rater reliability for CSF measurements was excellent (intraclass correlation coefficient 0.97). ∆CSF correlated strongly with MLS at peak edema (r = -0.75), even adjusting for infarct volume (p = 0.009). ∆CSF was also greater in those with malignant edema [-55 % (IQR -49 to -62) vs. -36 % (-27 to -45), p = 0.004]. ∆CSF was the greatest within IL sulci [-97 % (-86 to -99) vs. -71 % (-41 to -79), p = 0.002] but also significantly greater within CL sulci in those with malignant edema [-50 % (-29 to -65) vs. -25 % (0 to -31), p = 0.014]. More than half this CSF volume reduction occurred by the time of first FU CT around 24 h after stroke, while MLS rose later. CONCLUSIONS: Volumetric CSF analysis reliably quantifies CED and distinguishes those with malignant edema and MLS from those with a more benign course after LHI. ∆CSF may provide an earlier and more sensitive indicator of edema severity across a broader dynamic range than MLS.
Assuntos
Edema Encefálico/líquido cefalorraquidiano , Edema Encefálico/diagnóstico por imagem , Infarto Encefálico/líquido cefalorraquidiano , Infarto Encefálico/diagnóstico por imagem , Infarto Encefálico/fisiopatologia , Tomografia Computadorizada por Raios X/normas , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Estudos Retrospectivos , Índice de Gravidade de Doença , Tomografia Computadorizada por Raios X/métodosRESUMO
BACKGROUND: The modified Rankin Scale (mRS) is commonly used to measure disability after stroke, traditionally assessed through telephone or in-person evaluation. Here, we investigated the validity of mRS assessment through an automated text messaging system based on the simplified mRS questionnaire as an alternative method to traditional methods of assessment. METHODS AND RESULTS: A total of 250 patients admitted to 3 hospitals within the University of Pennsylvania Health System with ischemic or hemorrhagic stroke were enrolled. Participants received automated text messages sent 48 hours before their outpatient appointment at about 90 days after stroke. The mRS scores were assigned on the basis of participant responses to 2 to 4 text questions eliciting yes/no responses. The mRS was then evaluated in person or by telephone interview for comparison. Responses were compared with κ. A total of 142 patients (57%) completed the study. The spontaneous response rate to text messages was 46.5% and up to 72% with an additional direct in-person or phone call reminder. Agreement was substantial (quadratic-weighted κ=0.87 [95% CI, 0.83-0.89]) between responses derived from the automated text messaging and traditional interviews. Agreement for distinguishing functional independence (mRS 0-1) from dependence (mRS 2-5) was substantial (unweighted κ=0.79 [95% CI, 0.69-0.90]). CONCLUSIONS: An automated text messaging system is a feasible method for remotely obtaining the mRS after stroke and a potential alternative to traditional in-person or telephone assessment. Further studies are needed to evaluate the generalizability of text message-based approaches to stroke outcome measurement.
Assuntos
Avaliação da Deficiência , Envio de Mensagens de Texto , Humanos , Feminino , Masculino , Idoso , Pessoa de Meia-Idade , Fatores de Tempo , AVC Isquêmico/diagnóstico , Reprodutibilidade dos Testes , Acidente Vascular Cerebral/diagnóstico , Estado Funcional , Idoso de 80 Anos ou mais , Inquéritos e Questionários , Recuperação de Função Fisiológica , Reabilitação do Acidente Vascular Cerebral/métodos , Acidente Vascular Cerebral Hemorrágico/diagnóstico , Pennsylvania , Valor Preditivo dos TestesRESUMO
BACKGROUND: Stroke knowledge is critical to treatment adherence and poststroke outcomes. Here, we aimed to quantify the impact of a personalized video-based educational platform to test the hypothesis that it improves patient satisfaction and stroke knowledge. METHODS AND RESULTS: In a single-center pilot randomized trial, all patients with stroke and caregivers received standard stroke education during the hospitalization, but half were randomized to receive access to MyStroke, a personalized educational platform that provided brief videos about their stroke, risk factors, medications, and poststroke lifestyle. Satisfaction, stroke knowledge, and quality of life were assessed 7, 30, and 90 days after discharge. A total of 120 subjects (96 patients and 24 caregivers) were randomized to standard education (n=59) or MyStroke. At 90 days post-stroke, those who received MyStroke were more likely to be satisfied with the stroke education the received (90% versus 73%, P=0.05) and more likely to correctly identify their stroke cause (67% versus 32%, P=0.003). However, MyStroke was not associated with a difference in self-reported quality of life (EuroQol Visual Analogue Scale: 80 versus 75, P=0.06) or general stroke knowledge (total Stroke Patient Education Retention: 5 versus 5, P=0.47). With respect to secondary end points, MyStroke increased risk factor awareness 7 and 30 days poststroke, but this difference was not significant at 90 days. CONCLUSIONS: The MyStroke personalized video-based education platform improved patient and caregiver satisfaction while improving some aspects of personalized stroke knowledge without improving general stroke knowledge. A multicenter trial is needed to confirm these results, clarify generalizability, and target clinically relevant metrics such as stroke recurrence or adherence. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT05118503.
Assuntos
Conhecimentos, Atitudes e Prática em Saúde , Educação de Pacientes como Assunto , Satisfação do Paciente , Qualidade de Vida , Acidente Vascular Cerebral , Gravação em Vídeo , Humanos , Feminino , Masculino , Educação de Pacientes como Assunto/métodos , Idoso , Pessoa de Meia-Idade , Projetos Piloto , Fatores de Risco , Cuidadores/educação , Fatores de TempoRESUMO
Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3, is a fatal neurodegenerative disease that causes loss of balance and motor co-ordination, eventually leading to paralysis. It is caused by the autosomal dominant inheritance of a long CAG trinucleotide repeat sequence within the ATXN3 gene, encoding for an expanded polyglutamine (polyQ) repeat sequence within the ataxin-3 protein. Ataxin-3 containing an expanded polyQ repeat is known to be highly prone to intraneuronal aggregation, and previous studies have demonstrated that protein quality control pathways, such as autophagy, are impaired in MJD patients and animal models of the disease. In this study, we tested the therapeutic potential of spermidine on zebrafish and rodent models of MJD to determine its capacity to induce autophagy and improve functional output. Spermidine treatment of transgenic MJD zebrafish induced autophagy and resulted in increased distances swum by the MJD zebrafish. Interestingly, treatment of the CMVMJD135 mouse model of MJD with spermidine added to drinking water did not produce any improvement in motor behaviour assays, neurological testing or neuropathology. In fact, wild type mice treated with spermidine were found to have decreased rotarod performance when compared to control animals. Immunoblot analysis of protein lysates extracted from mouse cerebellar tissue found little differences between the groups, except for an increased level of phospho-ULK1 in spermidine treated animals, suggesting that autophagy was indeed induced. As we detected decreased motor performance in wild type mice following treatment with spermidine, we conducted follow up studies into the effects of spermidine treatment in zebrafish. Interestingly, we found that in addition to inducing autophagy, spermidine treatment also induced apoptosis, particularly in wild type zebrafish. These findings suggest that spermidine treatment may not be therapeutically beneficial for the treatment of MJD, and in fact warrants caution due to the potential negative side effects caused by induction of apoptosis.
Assuntos
Doença de Machado-Joseph , Doenças Neurodegenerativas , Humanos , Animais , Camundongos , Espermidina/farmacologia , Espermidina/uso terapêutico , Peixe-Zebra , Apoptose , Autofagia , Modelos Animais de DoençasRESUMO
BACKGROUND: Spinocerebellar ataxia 3 (SCA3, also known as Machado Joseph disease) is a fatal neurodegenerative disease caused by the expansion of the trinucleotide repeat region within the ATXN3/MJD gene. The presence of this genetic expansion results in an ataxin-3 protein containing a polyglutamine repeat region, which renders the ataxin-3 protein aggregation prone. Formation of ataxin-3 protein aggregates is linked with neuronal loss and, therefore, the development of motor deficits. METHODS: Here, we investigated whether the autophagy protein quality control pathway, which is important in the process of protein aggregate removal, is impaired in a cell culture and zebrafish model of SCA3. RESULTS: We found that SH-SY5Y cells expressing human ataxin-3 containing polyglutamine expansion exhibited aberrant levels of autophagy substrates, including increased p62 and decreased LC3II (following bafilomycin treatment), compared to the controls. Similarly, transgenic SCA3 zebrafish showed signs of autophagy impairment at early disease stages (larval), as well as p62 accumulation at advanced age stages (18 months old). We then examined whether treating with compounds known to induce autophagy activity, would aid removal of human ataxin-3 84Q and improve the swimming of the SCA3 zebrafish larvae. We found that treatment with loperamide, trehalose, rapamycin, and MG132 each improved the swimming of the SCA3 zebrafish compared to the vehicle-treated controls. CONCLUSION: We propose that signs of autophagy impairment occur in the SH-SY5Y model of SCA3 and SCA3 zebrafish at larval and advanced age stages. Treatment of the larval SCA3 zebrafish with various compounds with autophagy induction capacity was able to produce the improved swimming of the zebrafish, suggesting the potential benefit of autophagy-inducing compounds for the treatment of SCA3.
Assuntos
Doença de Machado-Joseph , Neuroblastoma , Doenças Neurodegenerativas , Animais , Humanos , Lactente , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/metabolismo , Ataxina-3/genética , Peixe-Zebra/metabolismo , Autofagia/fisiologiaRESUMO
A 19-year-old man with history of Kawasaki disease (KD) at age 12 developed intractable fevers, swelling in hands and feet, arthralgias, and conjunctivitis, followed by strawberry tongue and desquamation of distal extremities. Laboratory studies revealed leukocytosis, thrombocytosis, anemia, elevated erythrocyte sedimentation rate and C-reactive protein levels, and mildly elevated liver enzymes. He was empirically treated with broad-spectrum antibiotics without any improvement. Extensive infectious and rheumatologic workup remained negative. Recurrence of KD was diagnosed based on characteristic mucocutaneous changes and systemic inflammatory response. His symptoms and laboratory values responded rapidly to intravenous immunoglobulin and aspirin therapy. An echocardiogram did not show any coronary abnormality. We report the third case of pediatric KD relapsing in adulthood. Similar to childhood and adult KD, these recurrent episodes respond well to intravenous immunoglobulin therapy. Although recurrent KD is rare, our case highlights the importance of considering it in the differential of febrile illness in the appropriate individual.
Assuntos
Imunoglobulinas Intravenosas/administração & dosagem , Infecções/diagnóstico , Síndrome de Linfonodos Mucocutâneos , Fatores Etários , Sedimentação Sanguínea , Diagnóstico Diferencial , Febre/diagnóstico , Febre/etiologia , Dermatoses da Mão/etiologia , Dermatoses da Mão/patologia , Dermatoses da Mão/fisiopatologia , Humanos , Fatores Imunológicos/administração & dosagem , Dermatoses da Perna/etiologia , Dermatoses da Perna/patologia , Dermatoses da Perna/fisiopatologia , Testes de Função Hepática , Masculino , Síndrome de Linfonodos Mucocutâneos/complicações , Síndrome de Linfonodos Mucocutâneos/patologia , Síndrome de Linfonodos Mucocutâneos/fisiopatologia , Síndrome de Linfonodos Mucocutâneos/terapia , Recidiva , Doenças da Língua/etiologia , Doenças da Língua/patologia , Resultado do Tratamento , Adulto JovemRESUMO
Mesoporous silica-coated hollow manganese oxide (HMnO@mSiO(2)) nanoparticles were developed as a novel T(1) magnetic resonance imaging (MRI) contrast agent. We hypothesized that the mesoporous structure of the nanoparticle shell enables optimal access of water molecules to the magnetic core, and consequently, an effective longitudinal (R(1)) relaxation enhancement of water protons, which value was measured to be 0.99 (mM(-1)s(-1)) at 11.7 T. Adipose-derived mesenchymal stem cells (MSCs) were efficiently labeled using electroporation, with much shorter T(1) values as compared to direct incubation without electroporation, which was also evidenced by signal enhancement on T(1)-weighted MR images in vitro. Intracranial grafting of HMnO@mSiO(2)-labeled MSCs enabled serial MR monitoring of cell transplants over 14 days. These novel nanoparticles may extend the arsenal of currently available nanoparticle MR contrast agents by providing positive contrast on T(1)-weighted images at high magnetic field strengths.
Assuntos
Tecido Adiposo/citologia , Meios de Contraste/análise , Imageamento por Ressonância Magnética/métodos , Compostos de Manganês/análise , Células-Tronco Mesenquimais/citologia , Nanopartículas/química , Óxidos/análise , Dióxido de Silício/análise , Animais , Sobrevivência Celular , Células Cultivadas , Meios de Contraste/química , Compostos de Manganês/química , Transplante de Células-Tronco Mesenquimais , Camundongos , Nanopartículas/ultraestrutura , Óxidos/química , Dióxido de Silício/químicaRESUMO
The mechanisms underlying the exaggerated distal airway inflammation and hyperresponsiveness that characterize acute exacerbations of asthma are largely unknown. Using BALB/c mouse experimental models, we demonstrated a potentially important role for alveolar macrophages (AM) in the development of an allergen-induced exacerbation of asthma. To induce features of airway inflammation and remodeling characteristic of mild chronic asthma, animals were systemically sensitized and exposed to low mass concentrations (≈3 mg/m(3)) of aerosolized ovalbumin for 30 minutes per day, 3 days per week, for 4 weeks. A subsequent single moderate-level challenge (≈30 mg/m(3)) was used to trigger an acute exacerbation. In chronically challenged animals, cytokine expression by AM was not increased, whereas after an acute exacerbation, AM exhibited significantly enhanced expression of proinflammatory cytokines, including interleukin (IL) 1ß, IL-6, CXCL-1, and tumor necrosis factor α. In parallel, there was a marked increase in the expression of several cytokines by CD4(+) T-lymphocytes, notably the Th2 cytokines IL-4 and IL-13. Importantly, AM from an acute exacerbation stimulated the expression of Th2 cytokines when cocultured with CD4(+) cells from chronically challenged animals, and their ability to do so was significantly greater than AM from either chronically challenged or naïve controls. Stimulation was partly dependent on interactions involving CD80/86. We conclude that in an acute exacerbation of asthma, enhanced cytokine expression by AM may play a critical role in triggering increased expression of cytokines by pulmonary CD4(+) T-lymphocytes.
Assuntos
Asma/imunologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/metabolismo , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Células Th2/imunologia , Alérgenos/imunologia , Animais , Asma/patologia , Hiper-Reatividade Brônquica , Líquido da Lavagem Broncoalveolar , Linfócitos T CD4-Positivos/patologia , Doença Crônica , Citocinas/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Volume Expiratório Forçado , Inflamação/imunologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/fisiologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Th2/patologiaRESUMO
Spinocerebellar ataxia type 3 (SCA3) is a hereditary ataxia caused by inheritance of a mutated form of the human ATXN3 gene containing an expanded CAG repeat region, encoding a human ataxin-3 protein with a long polyglutamine (polyQ) repeat region. Previous studies have demonstrated that ataxin-3 containing a long polyQ length is highly aggregation prone. Cleavage of the ataxin-3 protein by calpain proteases has been demonstrated to be enhanced in SCA3 models, leading to an increase in the aggregation propensity of the protein. Here, we tested the therapeutic potential of a novel calpain inhibitor BLD-2736 for the treatment of SCA3 by testing its efficacy on a transgenic zebrafish model of SCA3. We found that treatment with BLD-2736 from 1 to 6 days post-fertilisation (dpf) improves the swimming of SCA3 zebrafish larvae and decreases the presence of insoluble protein aggregates. Furthermore, delaying the commencement of treatment with BLD-2736, until a timepoint when protein aggregates were already known to be present in the zebrafish larvae, was still successful at removing enhanced green fluorescent protein (EGFP) fused-ataxin-3 aggregates and improving the zebrafish swimming. Finally, we demonstrate that treatment with BLD-2736 increased the synthesis of LC3II, increasing the activity of the autophagy protein quality control pathway. Together, these findings suggest that BLD-2736 warrants further investigation as a treatment for SCA3 and related neurodegenerative diseases.
Assuntos
Antineoplásicos/uso terapêutico , Ataxina-3/efeitos dos fármacos , Glicoproteínas/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Modelos Animais de Doenças , Peixe-ZebraRESUMO
Spinocerebellar ataxia 3 (SCA3, also known as Machado-Joseph disease) is a neurodegenerative disease caused by inheritance of a CAG repeat expansion within the ATXN3 gene, resulting in polyglutamine (polyQ) repeat expansion within the ataxin-3 protein. In this study, we have identified protein aggregates in both neuronal-like (SHSY5Y) cells and transgenic zebrafish expressing human ataxin-3 with expanded polyQ. We have adapted a previously reported flow cytometry methodology named flow cytometric analysis of inclusions and trafficking, allowing rapid quantification of detergent insoluble forms of ataxin-3 fused to a GFP in SHSY5Y cells and cells dissociated from the zebrafish larvae. Flow cytometric analysis revealed an increased number of detergent-insoluble ataxin-3 particles per nuclei in cells and in zebrafish expressing polyQ-expanded ataxin-3 compared to those expressing wild-type human ataxin-3. Treatment with compounds known to modulate autophagic activity altered the number of detergent-insoluble ataxin-3 particles in cells and zebrafish expressing mutant human ataxin-3. We conclude that flow cytometry can be harnessed to rapidly count ataxin-3 aggregates, both in vitro and in vivo, and can be used to compare potential therapies targeting protein aggregates. This article has an associated First Person interview with the first author of the paper.
Assuntos
Citometria de Fluxo , Doença de Machado-Joseph/patologia , Agregados Proteicos , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Ataxina-3/metabolismo , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/metabolismo , Humanos , Neurônios/metabolismo , Peptídeos , SolubilidadeRESUMO
Machado-Joseph disease (MJD, also known as spinocerebellar ataxia type 3) is a fatal neurodegenerative disease that impairs control and coordination of movement. Here we tested whether treatment with the histone deacetylase inhibitor sodium valproate (valproate) prevented a movement phenotype that develops in larvae of a transgenic zebrafish model of the disease. We found that treatment with valproate improved the swimming of the MJD zebrafish, affected levels of acetylated histones 3 and 4, but also increased expression of polyglutamine expanded human ataxin-3. Proteomic analysis of protein lysates generated from the treated and untreated MJD zebrafish also predicted that valproate treatment had activated the sirtuin longevity signaling pathway and this was confirmed by findings of increased SIRT1 protein levels and sirtuin activity in valproate treated MJD zebrafish and HEK293 cells expressing ataxin-3 84Q, respectively. Treatment with resveratrol (another compound known to activate the sirtuin pathway), also improved swimming in the MJD zebrafish. Co-treatment with valproate alongside EX527, a SIRT1 activity inhibitor, prevented induction of autophagy by valproate and the beneficial effects of valproate on the movement in the MJD zebrafish, supporting that they were both dependent on sirtuin activity. These findings provide the first evidence of sodium valproate inducing activation of the sirtuin pathway. Further, they indicate that drugs that target the sirtuin pathway, including sodium valproate and resveratrol, warrant further investigation for the treatment of MJD and related neurodegenerative diseases.
Assuntos
Inibidores de Histona Desacetilases/uso terapêutico , Doença de Machado-Joseph/tratamento farmacológico , Sirtuínas/efeitos dos fármacos , Ácido Valproico/uso terapêutico , Acetilação , Animais , Animais Geneticamente Modificados , Ataxina-3/antagonistas & inibidores , Ataxina-3/genética , Ataxina-3/metabolismo , Autofagia/efeitos dos fármacos , Carbazóis/farmacologia , Carbazóis/uso terapêutico , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Genes Reporter , Células HEK293 , Inibidores de Histona Desacetilases/farmacologia , Histonas/metabolismo , Humanos , Peptídeos/genética , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Resveratrol/farmacologia , Resveratrol/uso terapêutico , Transdução de Sinais , Sirtuína 1/fisiologia , Sirtuínas/fisiologia , Natação , Expansão das Repetições de Trinucleotídeos , Ácido Valproico/farmacologia , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Pathological forms of TAR DNA-binding protein 43 (TDP-43) are present in almost all cases of amyotrophic lateral sclerosis (ALS), and 20% of familial ALS cases are due to mutations in superoxide dismutase 1 (SOD1). Redox regulation is critical to maintain cellular homeostasis, although how this relates to ALS is unclear. Here, we demonstrate that the redox function of protein disulfide isomerase (PDI) is protective against protein misfolding, cytoplasmic mislocalization of TDP-43, ER stress, ER-Golgi transport dysfunction, and apoptosis in neuronal cells expressing mutant TDP-43 or SOD1, and motor impairment in zebrafish expressing mutant SOD1. Moreover, previously described PDI mutants present in patients with ALS (D292N, R300H) lack redox activity and were not protective against ALS phenotypes. Hence, these findings implicate the redox activity of PDI centrally in ALS, linking it to multiple cellular processes. They also imply that therapeutics based on PDI's redox activity will be beneficial in ALS.
RESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons. ALS can be modeled in zebrafish (Danio rerio) through the expression of human ALS-causing genes, such as superoxide dismutase 1 (SOD1). Overexpression of mutated human SOD1 protein causes aberrant branching and shortening of spinal motor axons. Despite this, the functional relevance of this axon morphology remains elusive. Our aim was to determine whether this motor axonopathy is correlated with impaired movement in mutant (MT) SOD1-expressing zebrafish. Transgenic zebrafish embryos that express blue fluorescent protein (mTagBFP) in motor neurons were injected with either wild-type (WT) or MT (A4V) human SOD1 messenger ribonucleic acid (mRNA). At 48 hours post-fertilization, larvae movement (distance traveled during behavioral testing) was examined, followed by quantification of motor axon length. Larvae injected with MT SOD1 mRNA had significantly shorter and more aberrantly branched motor axons (p < 0.002) and traveled a significantly shorter distance during behavioral testing (p < 0.001) when compared with WT SOD1 and noninjected larvae. Furthermore, there was a positive correlation between distance traveled and motor axon length (R2 = 0.357, p < 0.001). These data represent the first correlative investigation of motor axonopathies and impaired movement in SOD1-expressing zebrafish, confirming functional relevance and validating movement as a disease phenotype for the testing of disease treatments for ALS.