RESUMO
Abnormalities in the endosomal-autophagic-lysosomal (EAL) system are an early event in Alzheimer's disease (AD) pathogenesis. However, the mechanisms underlying these abnormalities are unclear. The transient receptor potential channel mucolipin 1(TRPML1, also known as MCOLN1), a vital endosomal-lysosomal Ca2+ channel whose loss of function leads to neurodegeneration, has not been investigated with respect to EAL pathogenesis in late-onset AD (LOAD). Here, we identify pathological hallmarks of TRPML1 dysregulation in LOAD neurons, including increased perinuclear clustering and vacuolation of endolysosomes. We reveal that induced pluripotent stem cell (iPSC)-derived human cortical neurons expressing APOE ε4, the strongest genetic risk factor for LOAD, have significantly diminished TRPML1-induced endolysosomal Ca2+ release. Furthermore, we found that blocking TRPML1 function in primary neurons by depleting the TRPML1 agonist PI(3,5)P2 via PIKfyve inhibition, recreated multiple features of EAL neuropathology evident in LOAD. This included increased endolysosomal Ca2+ content, enlargement and perinuclear clustering of endolysosomes, autophagic vesicle accumulation and early endosomal enlargement. Strikingly, these AD-like neuronal EAL defects were rescued by TRPML1 reactivation using its synthetic agonist ML-SA1. These findings implicate defects in TRPML1 in LOAD EAL pathogenesis and present TRPML1 as a potential therapeutic target.
Assuntos
Doença de Alzheimer , Canais de Potencial de Receptor Transitório , Humanos , Cálcio/metabolismo , Doença de Alzheimer/patologia , Canais de Potencial de Receptor Transitório/genética , Lisossomos/metabolismo , AutofagiaRESUMO
Ca2+ signaling is vital for various cellular processes including synaptic vesicle exocytosis, muscle contraction, regulation of secretion, gene transcription, and cellular proliferation. The endoplasmic reticulum (ER) is the largest intracellular Ca2+ store, and dysregulation of ER Ca2+ signaling and homeostasis contributes to the pathogenesis of various complex disorders and Mendelian disease traits. We describe four unrelated individuals with a complex multisystem disorder characterized by woolly hair, liver dysfunction, pruritus, dysmorphic features, hypotonia, and global developmental delay. Through whole-exome sequencing and family-based genomics, we identified bi-allelic variants in CCDC47 that encodes the Ca2+-binding ER transmembrane protein CCDC47. CCDC47, also known as calumin, has been shown to bind Ca2+ with low affinity and high capacity. In mice, loss of Ccdc47 leads to embryonic lethality, suggesting that Ccdc47 is essential for early development. Characterization of cells from individuals with predicted likely damaging alleles showed decreased CCDC47 mRNA expression and protein levels. In vitro cellular experiments showed decreased total ER Ca2+ storage, impaired Ca2+ signaling mediated by the IP3R Ca2+ release channel, and reduced ER Ca2+ refilling via store-operated Ca2+ entry. These results, together with the previously described role of CCDC47 in Ca2+ signaling and development, suggest that bi-allelic loss-of-function variants in CCDC47 underlie the pathogenesis of this multisystem disorder.
RESUMO
Lysosomal storage diseases are the most common cause of neurodegeneration in children. They are characterised at the cellular level by the accumulation of storage material within lysosomes. There are very limited therapeutic options, and the search for novel therapies has been hampered as few good small animal models are available. Here, we describe the use of light sheet microscopy to assess lipid storage in drug and morpholino induced zebrafish models of two diseases of cholesterol homeostasis with lysosomal dysfunction: First, Niemann-Pick type C disease (NPC), caused by mutations in the lysosomal transmembrane protein NPC1, characterised by intralysosomal accumulation of cholesterol and several other lipids. Second, Smith-Lemli-Opitz syndrome (SLOS), caused by mutations in 7-dehydrocholesterol reductase, which catalyses the last step of cholesterol biosynthesis and is characterised by intralysosomal accumulation of dietary cholesterol. This is the first description of a zebrafish SLOS model. We find that zebrafish accurately model lysosomal storage and disease-specific phenotypes in both diseases. Increased cholesterol and ganglioside GM1 were observed in sections taken from NPC model fish, and decreased cholesterol in SLOS model fish, but these are of limited value as resolution is poor, and accurate anatomical comparisons difficult. Using light sheet microscopy, we were able to observe lipid changes in much greater detail and identified an unexpected accumulation of ganglioside GM1 in SLOS model fish. Our data demonstrate, for the first time in zebrafish, the immense potential that light sheet microscopy has in aiding the resolution of studies involving lysosomal and lipid disorders.
Assuntos
Colesterol/análise , Modelos Animais de Doenças , Gangliosídeo G(M1)/análise , Doença de Niemann-Pick Tipo C/diagnóstico , Síndrome de Smith-Lemli-Opitz/diagnóstico , Peixe-Zebra , Animais , Colesterol/metabolismo , Gangliosídeo G(M1)/metabolismo , Lisossomos/metabolismo , Microscopia de Fluorescência , Doença de Niemann-Pick Tipo C/metabolismo , Síndrome de Smith-Lemli-Opitz/metabolismoRESUMO
BACKGROUND: The ongoing pandemic has placed an unprecedented strain on global society, health care, governments, and mass media. Public dissemination of government policies, medical interventions, and misinformation has been remarkably rapid and largely unregulated during the COVID-19 pandemic, resulting in increased misinterpretations, miscommunication, and public panic. Being the first full-scale global pandemic of the digital age, COVID-19 has presented novel challenges pertinent to government advice, the spread of news and misinformation, and the trade-off between the accessibility of science and the premature public use of unproven medical interventions. OBJECTIVE: This study aims to assess the use of internet search terms relating to COVID-19 information and misinformation during the global pandemic, identify which were most used in six affected countries, investigate any temporal trends and the likely propagators of key search terms, and determine any correlation between the per capita cases and deaths with the adoption of these search terms in each of the six countries. METHODS: This study uses relative search volume data extracted from Google Trends for search terms linked to the COVID-19 pandemic alongside per capita case and mortality data extracted from the European Open Data Portal to identify the temporal dynamics of the spread of news and misinformation during the global pandemic in six affected countries (Australia, Germany, Italy, Spain, the United Kingdom, and the United States). A correlation analysis was carried out to ascertain any correlation between the temporal trends of search term use and the rise of per capita mortality and disease cases. RESULTS: Of the selected search terms, most were searched immediately following promotion by governments, public figures, or viral circulation of information, but also in relation to the publication of scientific resources, which were sometimes misinterpreted before further dissemination. Strong correlations were identified between the volume of these COVID-19-related search terms (overall mean Spearman rho 0.753, SD 0.158), and per capita mortality (mean per capita deaths Spearman rho 0.690, SD 0.168) and cases (mean per capita cases Spearman rho 0.800, SD 0.112). CONCLUSIONS: These findings illustrate the increased rate and volume of the public consumption of novel information during a global health care crisis. The positive correlation between mortality and online searching, particularly in countries with lower COVID-19 testing rates, may demonstrate the imperative to safeguard official communications and dispel misinformation in these countries. Online news, government briefings, and social media provide a powerful tool for the dissemination of important information to the public during pandemics, but their misuse and the presentation of misrepresented medical information should be monitored, minimized, and addressed to safeguard public safety. Ultimately, governments, public health authorities, and scientists have a moral imperative to safeguard the truth and maintain an accessible discourse with the public to limit fear.
Assuntos
Comunicação , Infecções por Coronavirus/epidemiologia , Educação em Saúde/estatística & dados numéricos , Internacionalidade , Internet , Meios de Comunicação de Massa/estatística & dados numéricos , Pneumonia Viral/epidemiologia , Betacoronavirus , COVID-19 , Teste para COVID-19 , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Saúde Global , Educação em Saúde/normas , Humanos , Pandemias , Saúde Pública , SARS-CoV-2 , Mídias SociaisRESUMO
TRPML1 is a ubiquitously expressed cation channel found on lysosomes and late endosomes. Mutations in TRPML1 cause mucolipidosis type IV and it has been implicated in Alzheimer's disease and HIV. However, the mechanisms by which TRPML1 activity is regulated are not well understood. This review summarizes the current understanding of TRPML1 activation and regulation.
Assuntos
Doença de Alzheimer/genética , Infecções por HIV/genética , Mucolipidoses/genética , Canais de Potencial de Receptor Transitório/biossíntese , Doença de Alzheimer/patologia , Endossomos/genética , Regulação da Expressão Gênica , Infecções por HIV/patologia , Humanos , Lisossomos/genética , Mucolipidoses/patologia , Mutação , Canais de Potencial de Receptor Transitório/genéticaRESUMO
BACKGROUND: Adhesion G protein-coupled receptors (aGPCR) constitute a structurally and functionally diverse class of seven-transmembrane receptor proteins. Although for some of the members important roles in immunology, neurology, as well as developmental biology have been suggested, most receptors have been poorly characterized. RESULTS: We have studied evolution, expression, and function of an entire receptor group containing four uncharacterized aGPCR: Gpr110, Gpr111, Gpr115, and Gpr116. We show that the genomic loci of these four receptors are clustered tightly together in mouse and human genomes and that this cluster likely derives from a single common ancestor gene. Using transcriptional profiling on wild-type and knockout/LacZ reporter knockin mice strains, we have obtained detailed expression maps that show ubiquitous expression of Gpr116, co-expression of Gpr111 and Gpr115 in developing skin, and expression of Gpr110 in adult kidney. Loss of Gpr110, Gpr111, or Gpr115 function did not result in detectable defects, indicating that genes of this aGPCR group might function redundantly. CONCLUSIONS: The aGPCR cluster Gpr110, Gpr111, Gpr115, and Gpr116 developed from one common ancestor in vertebrates. Expression suggests a role in epithelia, and one can speculate about a possible redundant function of GPR111 and GPR115.
Assuntos
Evolução Molecular , Loci Gênicos/genética , Família Multigênica/genética , Receptores Acoplados a Proteínas G/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Primers do DNA/genética , Epitélio/metabolismo , Galactosídeos , Perfilação da Expressão Gênica , Humanos , Indóis , Rim/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Receptores Acoplados a Proteínas G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Pele/metabolismo , Especificidade da EspécieRESUMO
Bacillus thuringiensis (Bt) proteins are an environmentally safe and effective alternative to chemical pesticides and have been used as biopesticides, with great commercial success, for over 50 years. Global agricultural production is predicted to require a 70% increase until 2050 to provide for an increasing population. In addition to agriculture, Bt proteins are utilized to control human vectors of disease-namely mosquitoes-which account for >700 000 deaths annually. The evolution of resistance to Bt pesticial toxins threatens the progression of sustainable agriculture. Whilst Bt protein toxins are heavily utilized, the exact mechanisms behind receptor binding and toxicity are unknown. It is critical to gain a better understanding of these mechanisms in order to engineer novel toxin variants and to predict, and prevent, future resistance evolution. This review focuses on the role of carbohydrate binding in the toxicity of the most utilized group of Bt pesticidal proteins-three domain Cry (3D-Cry) toxins.
Assuntos
Bacillus thuringiensis , Inseticidas , Animais , Humanos , Inseticidas/metabolismo , Endotoxinas/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/toxicidade , Mosquitos Vetores , Toxinas de Bacillus thuringiensis/metabolismo , Bacillus thuringiensis/genética , GlicoconjugadosRESUMO
Tpp80Aa1 from Bacillus thuringiensis is a Toxin_10 family protein (Tpp) with reported action against Culex mosquitoes. Here, we demonstrate an expanded target range, showing Tpp80Aa1 is also active against the larvae of Anopheles gambiae and Aedes aegypti mosquitoes. We report the first crystal structure of Tpp80Aa1 at a resolution of 1.8 Å, which shows Tpp80Aa1 consists of two domains: an N-terminal ß-trefoil domain resembling a ricin B lectin and a C-terminal putative pore-forming domain sharing structural similarity with the aerolysin family. Similar to other Tpp family members, we observe Tpp80Aa1 binds to the mosquito midgut, specifically the posterior midgut and the gastric caecum. We also identify that Tpp80Aa1 can interact with galactose-containing glycolipids and galactose, and this interaction is critical for exerting full insecticidal action against mosquito target cell lines.
Assuntos
Aedes , Bacillus thuringiensis , Culex , Inseticidas , Animais , Bacillus thuringiensis/metabolismo , Galactose/metabolismo , Aedes/metabolismo , Inseticidas/química , Culex/metabolismo , Proteínas de Bactérias/metabolismo , Larva/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismoRESUMO
Until recently, the mechanisms that regulate endolysosomal calcium homoeostasis were poorly understood. The discovery of the molecular target of NAADP (nicotinic acid-adenine dinucleotide phosphate) as the two-pore channels resident in the endolysosomal system has highlighted this compartment as an important calcium store. The recent findings that dysfunctional NAADP release leads to defective endocytic function which in turn results in secondary lipid accumulation in the lysosomal storage disease Niemann-Pick type C, is the first evidence of a direct connection between a human disease and defective lysosomal calcium release. In the present review, we provide a summary of the current knowledge on mechanisms of calcium homoeostasis within the endolysosomal system and how these mechanisms may be affected in human metabolic disorders.
Assuntos
Cálcio/metabolismo , Endossomos/metabolismo , Doenças por Armazenamento dos Lisossomos/metabolismo , Lisossomos/metabolismo , Canais de Cálcio/metabolismo , Homeostase , Humanos , NADP/análogos & derivados , NADP/metabolismoRESUMO
Calcium (Ca2+) signaling is an essential process in all cells that is maintained by a plethora of channels, pumps, transporters, receptors, and intracellular Ca2+ sequestering stores. Changes in cytosolic Ca2+ concentration govern processes as far reaching as fertilization, cell growth, and motility through to cell death. In recent years, lysosomes have emerged as a major intracellular Ca2+ storage organelle with an increasing involvement in triggering or regulating cellular functions such as endocytosis, autophagy, and Ca2+ release from the endoplasmic reticulum. This review will summarize recent work in the area of lysosomal Ca2+ signaling and homeostasis, including newly identified functions, and the involvement of lysosome-derived Ca2+ signals in human disease. In addition, we explore recent controversies in the techniques used for measurement of lysosomal Ca2+ content.
Assuntos
Cálcio/metabolismo , Lisossomos/metabolismo , Transdução de Sinais , Animais , Autofagia , Sinalização do Cálcio/fisiologia , Citosol/metabolismo , Endocitose , Retículo Endoplasmático/metabolismo , Homeostase , Humanos , Íons , Proteínas de Membrana Transportadoras/metabolismo , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Neurosteroids are steroid hormones synthesised de novo in the brain and peripheral nervous tissues. In contrast to adrenal steroid hormones that act on intracellular nuclear receptors, neurosteroids directly modulate plasma membrane ion channels and regulate intracellular signalling. This review provides an overview of the work that led to the discovery of neurosteroids, our current understanding of their intracellular biosynthetic machinery, and their roles in regulating the development and function of nervous tissue. Neurosteroids mediate signalling in the brain via multiple mechanisms. Here, we describe in detail their effects on GABA (inhibitory) and NMDA (excitatory) receptors, two signalling pathways of opposing function. Furthermore, emerging evidence points to altered neurosteroid function and signalling in neurological disease. This review focuses on neurodegenerative diseases associated with altered neurosteroid metabolism, mainly Niemann-Pick type C, multiple sclerosis and Alzheimer disease. Finally, we summarise the use of natural and synthetic neurosteroids as current and emerging therapeutics alongside their potential use as disease biomarkers.
Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Esclerose Múltipla/metabolismo , Neuroesteroides/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Sistema Nervoso Periférico/metabolismo , Transdução de Sinais , Animais , Humanos , Membranas Mitocondriais/metabolismo , Receptores de GABA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Inositol monophosphatase (IMPase) is inhibited by lithium, which is the most efficacious treatment for bipolar disorder. Several therapies have been approved, or are going through clinical trials, aimed at the replacement of lithium in the treatment of bipolar disorder. One candidate small molecule is ebselen, a selenium-containing antioxidant, which has been demonstrated to produce lithium-like effects both in a murine model and in clinical trials. Here, the crystallization and the first structure of human IMPase covalently complexed with ebselen, a 1.47â Å resolution crystal structure (PDB entry 6zk0), are presented. In the complex with human IMPase, ebselen in a ring-opened conformation is covalently attached to Cys141, a residue located away from the active site. IMPase is a dimeric enzyme and in the crystal structure two adjacent dimers share four ebselen molecules, creating a tetramer with approximate 222 symmetry. In the crystal structure presented in this publication, the active site in the tetramer is still accessible, suggesting that ebselen may function as an allosteric inhibitor or may block the binding of partner proteins.
Assuntos
Antioxidantes/metabolismo , Azóis/metabolismo , Cristalografia por Raios X/métodos , Cisteína/metabolismo , Compostos Organosselênicos/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Cisteína/química , Humanos , Isoindóis , Ligação Proteica , Conformação Proteica , Domínios ProteicosRESUMO
Good's buffers are commonly used for cell culture and, although developed to have minimal to no biological impact, they cause alterations in cellular processes such as autophagy and lysosomal enzyme activity. Using Chinese hamster ovary cells and induced pluripotent stem cell-derived neurons, this study explores the effect of zwitterionic buffers, specifically HEPES, on lysosomal volume and Ca2+ levels. Certain zwitterionic buffers lead to lysosomal expansion and reduced lysosomal Ca2+. Care should be taken when selecting buffers for growth media to avoid detrimental impacts on lysosomal function.
RESUMO
Post-translational protein modifications such as acetylation have significant regulatory roles in metabolic processes, but their relationship to both variation in gene expression and DNA sequence is unclear. We address this question in the Goto-Kakizaki (GK) rat inbred strain, a model of polygenic type 2 diabetes. Expression of the NAD-dependent deacetylase Sirtuin-3 is down-regulated in GK rats compared to normoglycemic Brown Norway (BN) rats. We show first that a promoter SNP causes down-regulation of Sirtuin-3 expression in GK rats. We then use mass-spectrometry to identify proteome-wide differential lysine acetylation of putative Sirtuin-3 protein targets in livers of GK and BN rats. These include many proteins in pathways connected to diabetes and metabolic syndrome. We finally sequence GK and BN liver transcriptomes and find that mRNA expression of these targets does not differ significantly between GK and BN rats, in contrast to other components of the same pathways. We conclude that physiological differences between GK and BN rats are mediated by a combination of differential protein acetylation and gene transcription and that genetic variation can modulate acetylation independently of expression.
Assuntos
Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Acetilação , Acetiltransferases/genética , Aminoácidos/metabolismo , Animais , Ciclo do Ácido Cítrico , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica , Gluconeogênese , Glicólise , Fígado/metabolismo , Masculino , Via de Pentose Fosfato , Polimorfismo Genético , Processamento de Proteína Pós-Traducional , Proteômica , Purinas/metabolismo , Pirimidinas/metabolismo , Ratos , Análise de Sequência de RNA , Sirtuína 3/genética , Especificidade da Espécie , Transcrição GênicaRESUMO
Nutritional factors play important roles in the etiology of obesity, type 2 diabetes mellitus and their complications through genotype x environment interactions. We have characterised molecular adaptation to high fat diet (HFD) feeding in inbred mouse strains widely used in genetic and physiological studies. We carried out physiological tests, plasma lipid assays, obesity measures, liver histology, hepatic lipid measurements and liver genome-wide gene transcription profiling in C57BL/6J and BALB/c mice fed either a control or a high fat diet. The two strains showed marked susceptibility (C57BL/6J) and relative resistance (BALB/c) to HFD-induced insulin resistance and non alcoholic fatty liver disease (NAFLD). Global gene set enrichment analysis (GSEA) of transcriptome data identified consistent patterns of expression of key genes (Srebf1, Stard4, Pnpla2, Ccnd1) and molecular pathways in the two strains, which may underlie homeostatic adaptations to dietary fat. Differential regulation of pathways, including the proteasome, the ubiquitin mediated proteolysis and PPAR signalling in fat fed C57BL/6J and BALB/c suggests that altered expression of underlying diet-responsive genes may be involved in contrasting nutrigenomic predisposition and resistance to insulin resistance and NAFLD in these models. Collectively, these data, which further demonstrate the impact of gene x environment interactions on gene expression regulations, contribute to improved knowledge of natural and pathogenic adaptive genomic regulations and molecular mechanisms associated with genetically determined susceptibility and resistance to metabolic diseases.
Assuntos
Dieta Hiperlipídica , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Nutrigenômica , Obesidade/complicações , Obesidade/etiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Adaptação Fisiológica , Animais , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Glucose/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Hepatopatia Gordurosa não Alcoólica , Reprodutibilidade dos Testes , Transdução de Sinais , TranscriptomaRESUMO
Adhesion-GPCRs provide essential cell-cell and cell-matrix interactions in development, and have been implicated in inherited human diseases like Usher Syndrome and bilateral frontoparietal polymicrogyria. They are the second largest subfamily of seven-transmembrane spanning proteins in vertebrates, but the function of most of these receptors is still not understood. The orphan Adhesion-GPCR GPR126 has recently been shown to play an essential role in the myelination of peripheral nerves in zebrafish. In parallel, whole-genome association studies have implicated variation at the GPR126 locus as a determinant of body height in the human population. The physiological function of GPR126 in mammals is still unknown. We describe a targeted mutation of GPR126 in the mouse, and show that GPR126 is required for embryonic viability and cardiovascular development.
Assuntos
Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Receptores Acoplados a Proteínas G/genética , Animais , Anormalidades Cardiovasculares/embriologia , Anormalidades Cardiovasculares/genética , Anormalidades Cardiovasculares/metabolismo , Embrião de Mamíferos/embriologia , Feminino , Genótipo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , Placenta/metabolismo , Gravidez , Receptores Acoplados a Proteínas G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Understanding the mechanisms that coordinate the orientation of cell division planes during embryogenesis and morphogenesis is a fundamental problem in developmental biology. Here we show that the orphan receptor lat-1, a homolog of vertebrate latrophilins, plays an essential role in the establishment of tissue polarity in the C. elegans embryo. We provide evidence that lat-1 is required for the alignment of cell division planes to the anterior-posterior axis and acts in parallel to known polarity and morphogenesis signals. lat-1 is a member of the Adhesion-GPCR protein family and is structurally related to flamingo/CELSR, an essential component of the planar cell polarity pathway. We dissect the molecular requirements of lat-1 signaling and implicate lat-1 in an anterior-posterior tissue polarity pathway in the premorphogenesis stage of C. elegans development.