RESUMEN
Chorea-acanthocytosis (ChAc) and McLeod syndrome are diseases with shared clinical manifestations caused by mutations in VPS13A and XK, respectively. Key features of these conditions are the degeneration of caudate neurons and the presence of abnormally shaped erythrocytes. XK belongs to a family of plasma membrane (PM) lipid scramblases whose action results in exposure of PtdSer at the cell surface. VPS13A is an endoplasmic reticulum (ER)-anchored lipid transfer protein with a putative role in the transport of lipids at contacts of the ER with other membranes. Recently VPS13A and XK were reported to interact by still unknown mechanisms. So far, however, there is no evidence for a colocalization of the two proteins at contacts of the ER with the PM, where XK resides, as VPS13A was shown to be localized at contacts between the ER and either mitochondria or lipid droplets. Here we show that VPS13A can also localize at ER-PM contacts via the binding of its PH domain to a cytosolic loop of XK, that such interaction is regulated by an intramolecular interaction within XK, and that both VPS13A and XK are highly expressed in the caudate neurons. Binding of the PH domain of VPS13A to XK is competitive with its binding to intracellular membranes that mediate other tethering functions of VPS13A. Our findings support a model according to which VPS13A-dependent lipid transfer between the ER and the PM is coupled to lipid scrambling within the PM. They raise the possibility that defective cell surface exposure of PtdSer may be responsible for neurodegeneration.
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Proteínas Portadoras , Membrana Celular , Lípidos , Proteínas de Transporte Vesicular , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/metabolismo , Humanos , Neuroacantocitosis/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
PURPOSE: The function of FAM177A1 and its relationship to human disease is largely unknown. Recent studies have demonstrated FAM177A1 to be a critical immune-associated gene. One previous case study has linked FAM177A1 to a neurodevelopmental disorder in 4 siblings. METHODS: We identified 5 individuals from 3 unrelated families with biallelic variants in FAM177A1. The physiological function of FAM177A1 was studied in a zebrafish model organism and human cell lines with loss-of-function variants similar to the affected cohort. RESULTS: These individuals share a characteristic phenotype defined by macrocephaly, global developmental delay, intellectual disability, seizures, behavioral abnormalities, hypotonia, and gait disturbance. We show that FAM177A1 localizes to the Golgi complex in mammalian and zebrafish cells. Intersection of the RNA sequencing and metabolomic data sets from FAM177A1-deficient human fibroblasts and whole zebrafish larvae demonstrated dysregulation of pathways associated with apoptosis, inflammation, and negative regulation of cell proliferation. CONCLUSION: Our data shed light on the emerging function of FAM177A1 and defines FAM177A1-related neurodevelopmental disorder as a new clinical entity.
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SWI/SNF-related intellectual disability disorders (SSRIDDs) are rare neurodevelopmental disorders characterized by developmental disability, coarse facial features, and fifth digit/nail hypoplasia that are caused by pathogenic variants in genes that encode for members of the SWI/SNF (or BAF) family of chromatin remodeling complexes. We have identified 12 individuals with rare variants (10 loss-of-function, 2 missense) in the BICRA (BRD4 interacting chromatin remodeling complex-associated protein) gene, also known as GLTSCR1, which encodes a subunit of the non-canonical BAF (ncBAF) complex. These individuals exhibited neurodevelopmental phenotypes that include developmental delay, intellectual disability, autism spectrum disorder, and behavioral abnormalities as well as dysmorphic features. Notably, the majority of individuals lack the fifth digit/nail hypoplasia phenotype, a hallmark of most SSRIDDs. To confirm the role of BICRA in the development of these phenotypes, we performed functional characterization of the zebrafish and Drosophila orthologs of BICRA. In zebrafish, a mutation of bicra that mimics one of the loss-of-function variants leads to craniofacial defects possibly akin to the dysmorphic facial features seen in individuals harboring putatively pathogenic BICRA variants. We further show that Bicra physically binds to other non-canonical ncBAF complex members, including the BRD9/7 ortholog, CG7154, and is the defining member of the ncBAF complex in flies. Like other SWI/SNF complex members, loss of Bicra function in flies acts as a dominant enhancer of position effect variegation but in a more context-specific manner. We conclude that haploinsufficiency of BICRA leads to a unique SSRIDD in humans whose phenotypes overlap with those previously reported.
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Proteínas Cromosómicas no Histona/genética , Discapacidades del Desarrollo/genética , Mutación Missense , Fenotipo , Proteínas Supresoras de Tumor/genética , Adolescente , Animales , Niño , Preescolar , Proteínas de Drosophila/genética , Drosophila melanogaster , Femenino , Genes Dominantes , Variación Genética , Haploinsuficiencia , Humanos , Lactante , Masculino , Microscopía Confocal , Neuroglía/metabolismo , Neuronas/metabolismo , Unión Proteica , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Forward genetic screens using chemical mutagens have been successful in defining the function of thousands of genes in eukaryotic model organisms. The main drawback of this strategy is the time-consuming identification of the molecular lesions causative of the phenotypes of interest. With whole-genome sequencing (WGS), it is now possible to sequence hundreds of strains, but determining which mutations are causative among thousands of polymorphisms remains challenging. We have sequenced 394 mutant strains, generated in a chemical mutagenesis screen, for essential genes on the Drosophila X chromosome and describe strategies to reduce the number of candidate mutations from an average of -3500 to 35 single-nucleotide variants per chromosome. By combining WGS with a rough mapping method based on large duplications, we were able to map 274 (-70%) mutations. We show that these mutations are causative, using small 80-kb duplications that rescue lethality. Hence, our findings demonstrate that combining rough mapping with WGS dramatically expands the toolkit necessary for assigning function to genes.
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Mapeo Cromosómico/métodos , Drosophila melanogaster/genética , Mutagénesis , Animales , Metanosulfonato de Etilo , Femenino , Genes Esenciales , Genes de Insecto , Masculino , Datos de Secuencia Molecular , Mutágenos , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN/métodos , Cromosoma XRESUMEN
Modification by sialylated glycans can affect protein functions, underlying mechanisms that control animal development and physiology. Sialylation relies on a dedicated pathway involving evolutionarily conserved enzymes, including CMP-sialic acid synthetase (CSAS) and sialyltransferase (SiaT) that mediate the activation of sialic acid and its transfer onto glycan termini, respectively. In Drosophila, CSAS and DSiaT genes function in the nervous system, affecting neural transmission and excitability. We found that these genes function in different cells: the function of CSAS is restricted to glia, while DSiaT functions in neurons. This partition of the sialylation pathway allows for regulation of neural functions via a glia-mediated control of neural sialylation. The sialylation genes were shown to be required for tolerance to heat and oxidative stress and for maintenance of the normal level of voltage-gated sodium channels. Our results uncovered a unique bipartite sialylation pathway that mediates glia-neuron coupling and regulates neural excitability and stress tolerance.
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Drosophila , Fenómenos Fisiológicos del Sistema Nervioso , Animales , Drosophila/metabolismo , Transmisión Sináptica/fisiología , Neuronas/metabolismo , Neuroglía/metabolismo , Polisacáridos/metabolismoRESUMEN
Mutations in VPS13B, a member of a protein family implicated in bulk lipid transport between adjacent membranes, cause Cohen syndrome. VPS13B is known to be concentrated in the Golgi complex, but its precise location within this organelle and thus the site(s) where it achieves lipid transport remains unclear. Here we show that VPS13B is localized at the interface between cis and trans Golgi sub-compartments and that Golgi complex re-formation after Brefeldin A (BFA) induced disruption is delayed in VPS13B KO cells. This delay is phenocopied by loss of FAM177A1, a Golgi complex protein of unknown function reported to be a VPS13B interactor and whose mutations also result in a developmental disorder. In zebrafish, the vps13b orthologue, not previously annotated in this organism, genetically interacts with fam177a1. Collectively, these findings raise the possibility that bulk lipid transport by VPS13B may play a role in expanding Golgi membranes and that VPS13B may be assisted in this function by FAM177A1.
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Amyotrophic lateral sclerosis (ALS) is a fatal, complex neurodegenerative disorder that causes selective degeneration of motor neurons. ALS patients exhibit symptoms consistent with altered cellular energetics such as hypermetabolism, weight loss, dyslipidemia, insulin resistance, and altered glucose tolerance. Although evidence supports metabolic changes in ALS patients, metabolic alterations at a cellular level remain poorly understood. Here, we used a Drosophila model of ALS based on TDP-43 expression in motor neurons that recapitulates hallmark features of motor neuron disease including TDP-43 aggregation, locomotor dysfunction, and reduced lifespan. To gain insights into metabolic changes caused by TDP-43, we performed global metabolomic profiling in larvae expressing TDP-43 (WT or ALS associated mutant variant, G298S) and identified significant alterations in several metabolic pathways. Here, we report alterations in multiple metabolic pathways and highlight upregulation of Tricarboxylic acid (TCA) cycle metabolites and defects in neurotransmitter levels. We also show that modulating TCA cycle flux either genetically or by dietary intervention mitigates TDP-43-dependent locomotor defects. In addition, dopamine levels are significantly reduced in the context of TDP-43G298S, and we find that treatment with pramipexole, a dopamine agonist, improves locomotor function in vivo in Drosophila models of TDP-43 proteinopathy.
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Naturally produced peptides (<100 amino acids) are important regulators of physiology, development, and metabolism. Recent studies have predicted that thousands of peptides may be translated from transcripts containing small open-reading frames (smORFs). Here, we describe two peptides in Drosophila encoded by conserved smORFs, Sloth1 and Sloth2. These peptides are translated from the same bicistronic transcript and share sequence similarities, suggesting that they encode paralogs. Yet, Sloth1 and Sloth2 are not functionally redundant, and loss of either peptide causes animal lethality, reduced neuronal function, impaired mitochondrial function, and neurodegeneration. We provide evidence that Sloth1/2 are highly expressed in neurons, imported to mitochondria, and regulate mitochondrial complex III assembly. These results suggest that phenotypic analysis of smORF genes in Drosophila can provide a wealth of information on the biological functions of this poorly characterized class of genes.
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Drosophila , Complejo III de Transporte de Electrones , Animales , Drosophila/genética , Complejo III de Transporte de Electrones/genética , Sistemas de Lectura Abierta , Péptidos/genética , Péptidos/química , NeuronasRESUMEN
The evolutionarily conserved VPS13 family proteins have been implicated in several cellular processes. Mutations in each of the four human VPS13s cause neurodevelopmental or neurodegenerative disorders. Until recently, the molecular function of VPS13 remained elusive. Genetic, functional and structural studies have now revealed that VPS13 acts at contact sites between intracellular organelles to transport lipids by a novel mechanism: direct transfer between bilayers via a hydrophobic channel that spans its entire rod-like N-terminal half. Predicted similarities to the autophagy protein ATG2 suggested a similar role for ATG2 that has now been confirmed by structural and functional studies. Here, after a brief review of this evidence, we discuss what is known of human VPS13 proteins in physiology and disease.
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Proteínas Relacionadas con la Autofagia/metabolismo , Encéfalo/fisiología , Enfermedades Neurodegenerativas/patología , Trastornos del Neurodesarrollo/patología , Proteínas de Transporte Vesicular/metabolismo , Humanos , Enfermedades Neurodegenerativas/metabolismo , Trastornos del Neurodesarrollo/metabolismoRESUMEN
The Alzheimer's disease (AD) susceptibility gene, CD2-associated protein (CD2AP), encodes an actin binding adaptor protein, but its function in the nervous system is largely unknown. Loss of the Drosophila ortholog cindr enhances neurotoxicity of human Tau, which forms neurofibrillary tangle pathology in AD. We show that Cindr is expressed in neurons and present at synaptic terminals. cindr mutants show impairments in synapse maturation and both synaptic vesicle recycling and release. Cindr associates and genetically interacts with 14-3-3ζ, regulates the ubiquitin-proteasome system, and affects turnover of Synapsin and the plasma membrane calcium ATPase (PMCA). Loss of cindr elevates PMCA levels and reduces cytosolic calcium. Studies of Cd2ap null mice support a conserved role in synaptic proteostasis, and CD2AP protein levels are inversely related to Synapsin abundance in human postmortem brains. Our results reveal CD2AP neuronal requirements with relevance to AD susceptibility, including for proteostasis, calcium handling, and synaptic structure and function.
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Proteínas 14-3-3/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Enfermedad de Alzheimer , Proteínas del Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Microfilamentos/metabolismo , Neuronas/metabolismo , Proteostasis , Proteínas 14-3-3/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas del Citoesqueleto/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Humanos , Masculino , Ratones , Proteínas de Microfilamentos/genética , Neuronas/citología , Proteoma/análisis , Proteoma/metabolismo , Transmisión SinápticaRESUMEN
Neurotransmission is a tightly regulated Ca2+-dependent process. Upon Ca2+ influx, Synaptotagmin1 (Syt1) promotes fusion of synaptic vesicles (SVs) with the plasma membrane. This requires regulation at multiple levels, but the role of metabolites in SV release is unclear. Here, we uncover a role for isocitrate dehydrogenase 3a (idh3a), a Krebs cycle enzyme, in neurotransmission. Loss of idh3a leads to a reduction of the metabolite, alpha-ketoglutarate (αKG), causing defects in synaptic transmission similar to the loss of syt1. Supplementing idh3a flies with αKG suppresses these defects through an ATP or neurotransmitter-independent mechanism. Indeed, αKG, but not glutamate, enhances Syt1-dependent fusion in a reconstitution assay. αKG promotes interaction between the C2-domains of Syt1 and phospholipids. The data reveal conserved metabolic regulation of synaptic transmission via αKG. Our studies provide a synaptic role for αKG, a metabolite that has been proposed as a treatment for aging and neurodegenerative disorders.
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Ciclo del Ácido Cítrico , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Drosophila melanogaster/fisiología , Isocitrato Deshidrogenasa/metabolismo , Mitocondrias/metabolismo , Transmisión Sináptica , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Drosophila melanogaster/ultraestructura , Ácidos Cetoglutáricos/metabolismo , Larva/metabolismo , Mitocondrias/ultraestructura , Unión Neuromuscular/metabolismo , Unión Neuromuscular/ultraestructura , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Unión Proteica , Dominios Proteicos , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestructura , Sinaptotagminas/química , Sinaptotagminas/metabolismoRESUMEN
Many of the internal organ systems of Drosophila melanogaster are functionally analogous to those in vertebrates, including humans. Although humans and flies differ greatly in terms of their gross morphological and cellular features, many of the molecular mechanisms that govern development and drive cellular and physiological processes are conserved between both organisms. The morphological differences are deceiving and have led researchers to undervalue the study of invertebrate organs in unraveling pathogenic mechanisms of diseases. In this review and accompanying poster, we highlight the physiological and molecular parallels between fly and human organs that validate the use of Drosophila to study the molecular pathogenesis underlying human diseases. We discuss assays that have been developed in flies to study the function of specific genes in the central nervous system, heart, liver and kidney, and provide examples of the use of these assays to address questions related to human diseases. These assays provide us with simple yet powerful tools to study the pathogenic mechanisms associated with human disease-causing genes.