RESUMO
There is considerable inter-individual variability in susceptibility to weight gain despite an equally obesogenic environment in large parts of the world. Whereas many studies have focused on identifying the genetic susceptibility to obesity, we performed a GWAS on metabolically healthy thin individuals (lowest 6th percentile of the population-wide BMI spectrum) in a uniquely phenotyped Estonian cohort. We discovered anaplastic lymphoma kinase (ALK) as a candidate thinness gene. In Drosophila, RNAi mediated knockdown of Alk led to decreased triglyceride levels. In mice, genetic deletion of Alk resulted in thin animals with marked resistance to diet- and leptin-mutation-induced obesity. Mechanistically, we found that ALK expression in hypothalamic neurons controls energy expenditure via sympathetic control of adipose tissue lipolysis. Our genetic and mechanistic experiments identify ALK as a thinness gene, which is involved in the resistance to weight gain.
Assuntos
Quinase do Linfoma Anaplásico/genética , Magreza/genética , Tecido Adiposo/metabolismo , Adulto , Animais , Linhagem Celular , Estudos de Coortes , Drosophila/genética , Estônia , Feminino , Humanos , Leptina/genética , Lipólise/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/genética , Interferência de RNA/fisiologia , Adulto JovemRESUMO
Although ACE2 is the primary receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here, we use whole-genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe Coronavirus Disease 2019 (COVID-19) infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans. Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , COVID-19/genética , Antivirais/farmacologia , Enzima de Conversão de Angiotensina 2/metabolismo , Fibroblastos/metabolismo , Ligação Proteica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismoRESUMO
Over 1 billion people are estimated to be overweight, placing them at risk for diabetes, cardiovascular disease, and cancer. We performed a systems-level genetic dissection of adiposity regulation using genome-wide RNAi screening in adult Drosophila. As a follow-up, the resulting approximately 500 candidate obesity genes were functionally classified using muscle-, oenocyte-, fat-body-, and neuronal-specific knockdown in vivo and revealed hedgehog signaling as the top-scoring fat-body-specific pathway. To extrapolate these findings into mammals, we generated fat-specific hedgehog-activation mutant mice. Intriguingly, these mice displayed near total loss of white, but not brown, fat compartments. Mechanistically, activation of hedgehog signaling irreversibly blocked differentiation of white adipocytes through direct, coordinate modulation of early adipogenic factors. These findings identify a role for hedgehog signaling in white/brown adipocyte determination and link in vivo RNAi-based scanning of the Drosophila genome to regulation of adipocyte cell fate in mammals.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/metabolismo , Obesidade/genética , Adipócitos Marrons/metabolismo , Adipócitos Brancos/metabolismo , Adipogenia , Animais , AMP Cíclico/metabolismo , Glucocorticoides/metabolismo , Humanos , Camundongos , Camundongos Knockout , Células Musculares/metabolismo , Proteínas Repressoras/genéticaRESUMO
Worldwide, acute, and chronic pain affects 20% of the adult population and represents an enormous financial and emotional burden. Using genome-wide neuronal-specific RNAi knockdown in Drosophila, we report a global screen for an innate behavior and identify hundreds of genes implicated in heat nociception, including the α2δ family calcium channel subunit straightjacket (stj). Mice mutant for the stj ortholog CACNA2D3 (α2δ3) also exhibit impaired behavioral heat pain sensitivity. In addition, in humans, α2δ3 SNP variants associate with reduced sensitivity to acute noxious heat and chronic back pain. Functional imaging in α2δ3 mutant mice revealed impaired transmission of thermal pain-evoked signals from the thalamus to higher-order pain centers. Intriguingly, in α2δ3 mutant mice, thermal pain and tactile stimulation triggered strong cross-activation, or synesthesia, of brain regions involved in vision, olfaction, and hearing.
Assuntos
Canais de Cálcio/genética , Proteínas de Drosophila/genética , Drosophila/genética , Dor/genética , Adulto , Animais , Dor nas Costas/genética , Canais de Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Técnicas de Silenciamento de Genes , Estudo de Associação Genômica Ampla , Temperatura Alta , Humanos , Camundongos , Polimorfismo de Nucleotídeo Único , Interferência de RNARESUMO
Heart diseases are the most common causes of morbidity and death in humans. Using cardiac-specific RNAi-silencing in Drosophila, we knocked down 7061 evolutionarily conserved genes under conditions of stress. We present a first global roadmap of pathways potentially playing conserved roles in the cardiovascular system. One critical pathway identified was the CCR4-Not complex implicated in transcriptional and posttranscriptional regulatory mechanisms. Silencing of CCR4-Not components in adult Drosophila resulted in myofibrillar disarray and dilated cardiomyopathy. Heterozygous not3 knockout mice showed spontaneous impairment of cardiac contractility and increased susceptibility to heart failure. These heart defects were reversed via inhibition of HDACs, suggesting a mechanistic link to epigenetic chromatin remodeling. In humans, we show that a common NOT3 SNP correlates with altered cardiac QT intervals, a known cause of potentially lethal ventricular tachyarrhythmias. Thus, our functional genome-wide screen in Drosophila can identify candidates that directly translate into conserved mammalian genes involved in heart function.
Assuntos
Drosophila melanogaster/fisiologia , Modelos Animais , Animais , Cardiomiopatias/genética , Cardiomiopatias/fisiopatologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Feminino , Estudo de Associação Genômica Ampla , Coração/embriologia , Coração/fisiologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , Interferência de RNARESUMO
BACKGROUND: Widescale evidence points to the involvement of glia and immune pathways in the progression of Alzheimer's disease (AD). AD-associated iPSC-derived glial cells show a diverse range of AD-related phenotypic states encompassing cytokine/chemokine release, phagocytosis and morphological profiles, but to date studies are limited to cells derived from PSEN1, APOE and APP mutations or sporadic patients. The aim of the current study was to successfully differentiate iPSC-derived microglia and astrocytes from patients harbouring an AD-causative PSEN2 (N141I) mutation and characterise the inflammatory and morphological profile of these cells. METHODS: iPSCs from three healthy control individuals and three familial AD patients harbouring a heterozygous PSEN2 (N141I) mutation were used to derive astrocytes and microglia-like cells and cell identity and morphology were characterised through immunofluorescent microscopy. Cellular characterisation involved the stimulation of these cells by LPS and Aß42 and analysis of cytokine/chemokine release was conducted through ELISAs and multi-cytokine arrays. The phagocytic capacity of these cells was then indexed by the uptake of fluorescently-labelled fibrillar Aß42. RESULTS: AD-derived astrocytes and microglia-like cells exhibited an atrophied and less complex morphological appearance than healthy controls. AD-derived astrocytes showed increased basal expression of GFAP, S100ß and increased secretion and phagocytosis of Aß42 while AD-derived microglia-like cells showed decreased IL-8 secretion compared to healthy controls. Upon immunological challenge AD-derived astrocytes and microglia-like cells showed exaggerated secretion of the pro-inflammatory IL-6, CXCL1, ICAM-1 and IL-8 from astrocytes and IL-18 and MIF from microglia. CONCLUSION: Our study showed, for the first time, the differentiation and characterisation of iPSC-derived astrocytes and microglia-like cells harbouring a PSEN2 (N141I) mutation. PSEN2 (N141I)-mutant astrocytes and microglia-like cells presented with a 'primed' phenotype characterised by reduced morphological complexity, exaggerated pro-inflammatory cytokine secretion and altered Aß42 production and phagocytosis.
Assuntos
Doença de Alzheimer , Células-Tronco Pluripotentes Induzidas , Humanos , Astrócitos/metabolismo , Microglia/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Interleucina-8/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Citocinas/metabolismo , Fenótipo , Peptídeos beta-Amiloides/metabolismo , Presenilina-2/genética , Presenilina-2/metabolismoRESUMO
TM2 domain containing (TM2D) proteins are conserved in metazoans and encoded by three separate genes in each model organism species that has been sequenced. Rare variants in TM2D3 are associated with Alzheimer's disease (AD) and its fly ortholog almondex is required for embryonic Notch signaling. However, the functions of this gene family remain elusive. We knocked-out all three TM2D genes (almondex, CG11103/amaretto, CG10795/biscotti) in Drosophila and found that they share the same maternal-effect neurogenic defect. Triple null animals are not phenotypically worse than single nulls, suggesting these genes function together. Overexpression of the most conserved region of the TM2D proteins acts as a potent inhibitor of Notch signaling at the γ-secretase cleavage step. Lastly, Almondex is detected in the brain and its loss causes shortened lifespan accompanied by progressive motor and electrophysiological defects. The functional links between all three TM2D genes are likely to be evolutionarily conserved, suggesting that this entire gene family may be involved in AD.
Assuntos
Proteínas de Drosophila , Proteínas de Membrana , Neurogênese , Receptores Notch , Animais , Drosophila melanogaster/genética , Proteínas de Drosophila/genética , Técnicas de Inativação de Genes , Proteínas de Membrana/genética , Mutação/genética , Neurogênese/genética , Neurônios/metabolismo , Receptores Notch/genética , Transdução de Sinais/genéticaRESUMO
Obesity has become a serious global public health problem, affecting over 988 million people worldwide. Nevertheless, current pharmacotherapies have proven inadequate. Natural compounds have garnered significant attention due to their potential antiobesity effects. Over the past three decades, ca. 50 natural compounds have been evaluated for the preventive and/or therapeutic effects on obesity in animals and humans. However, variations in the antiobesity efficacies among these natural compounds have been substantial, owing to differences in experimental designs, including variations in animal models, dosages, treatment durations, and administration methods. The feasibility of employing these natural compounds as pharmacotherapies for obesity remained uncertain. In this review, we systematically summarized the antiobesity efficacy and mechanisms of action of each natural compound in animal models. This comprehensive review furnishes valuable insights for the development of antiobesity medications based on natural compounds.
Assuntos
Fármacos Antiobesidade , Obesidade , Humanos , Animais , Obesidade/tratamento farmacológico , Fármacos Antiobesidade/farmacologia , Fármacos Antiobesidade/uso terapêuticoRESUMO
BACKGROUND: Eusociality is widely considered to evolve through kin selection, where the reproductive success of an individual's close relative is favored at the expense of its own. High genetic relatedness is thus considered a prerequisite for eusociality. While ants are textbook examples of eusocial animals, not all ants form colonies of closely related individuals. One such example is the ectatommine ant Rhytidoponera metallica, which predominantly forms queen-less colonies that have such a low intra-colony relatedness that they have been proposed to represent a transient, unstable form of eusociality. However, R. metallica is among the most abundant and widespread ants on the Australian continent. This apparent contradiction provides an example of how inclusive fitness may not by itself explain the maintenance of eusociality and raises the question of what other selective advantages maintain the eusocial lifestyle of this species. RESULTS: We provide a comprehensive portrait of the venom of R. metallica and show that the colony-wide venom consists of an exceptionally high diversity of functionally distinct toxins for an ant. These toxins have evolved under strong positive selection, which is normally expected to reduce genetic variance. Yet, R. metallica exhibits remarkable intra-colony variation, with workers sharing only a relatively small proportion of toxins in their venoms. This variation is not due to the presence of chemical castes, but has a genetic foundation that is at least in part explained by toxin allelic diversity. CONCLUSIONS: Taken together, our results suggest that the toxin diversity contained in R. metallica colonies may be maintained by a form of group selection that selects for colonies that can exploit more resources and defend against a wider range of predators. We propose that increased intra-colony genetic variance resulting from low kinship may itself provide a selective advantage in the form of an expanded pharmacological venom repertoire. These findings provide an example of how group selection on adaptive phenotypes may contribute to maintaining eusociality where a prerequisite for kin selection is diminished.
Assuntos
Formigas , Animais , Formigas/genética , Peçonhas , Austrália , Reprodução , Comportamento SocialRESUMO
The mechanistic (or mammalian) target of rapamycin complex 1 (mTORC1) controls cell growth, proliferation, and metabolism in response to diverse stimuli. Two major parallel pathways are implicated in mTORC1 regulation including a growth factor-responsive pathway mediated via TSC2/Rheb and an amino acid-responsive pathway mediated via the Rag GTPases. Here, we identify and characterize three highly conserved growth factor-responsive phosphorylation sites on RagC, a component of the Rag heterodimer, implicating cross talk between amino acid and growth factor-mediated regulation of mTORC1. We find that RagC phosphorylation is associated with destabilization of mTORC1 and is essential for both growth factor and amino acid-induced mTORC1 activation. Functionally, RagC phosphorylation suppresses starvation-induced autophagy, and genetic studies in Drosophila reveal that RagC phosphorylation plays an essential role in regulation of cell growth. Finally, we identify mTORC1 as the upstream kinase of RagC on S21. Our data highlight the importance of RagC phosphorylation in its function and identify a previously unappreciated auto-regulatory mechanism of mTORC1 activity.
Assuntos
Aminoácidos/metabolismo , Drosophila melanogaster/metabolismo , Homeostase , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos/metabolismo , Sequência de Aminoácidos , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Células HEK293 , Células HeLa , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Complexos Multiproteicos/genética , Fosforilação , Homologia de Sequência , Transdução de SinaisRESUMO
COVID-19 patients display a wide range of disease severity, ranging from asymptomatic to critical symptoms with high mortality risk. Our ability to understand the interaction of SARS-CoV-2 infected cells within the lung, and of protective or dysfunctional immune responses to the virus, is critical to effectively treat these patients. Currently, our understanding of cell-cell interactions across different disease states, and how such interactions may drive pathogenic outcomes, is incomplete. Here, we developed a generalizable and scalable workflow for identifying cells that are differentially interacting across COVID-19 patients with distinct disease outcomes and use this to examine eight public single-cell RNA-seq datasets (six from peripheral blood mononuclear cells, one from bronchoalveolar lavage and one from nasopharyngeal), with a total of 211 individual samples. By characterizing the cell-cell interaction patterns across epithelial and immune cells in lung tissues for patients with varying disease severity, we illustrate diverse communication patterns across individuals, and discover heterogeneous communication patterns among moderate and severe patients. We further illustrate patterns derived from cell-cell interactions are potential signatures for discriminating between moderate and severe patients. Overall, this workflow can be generalized and scaled to combine multiple scRNA-seq datasets to uncover cell-cell interactions.
Assuntos
COVID-19 , Comunicação Celular , Humanos , Leucócitos Mononucleares , SARS-CoV-2 , Fluxo de TrabalhoRESUMO
Autosomal dominant mutations in the gene encoding α-synuclein (SNCA) were the first to be linked with hereditary Parkinson's disease (PD). Duplication and triplication of SNCA has been observed in PD patients, together with mutations at the N-terminal of the protein, among which A30P and A53T influence the formation of fibrils. By overexpressing human α-synuclein in the neuronal system of Drosophila, we functionally validated the ability of IP3K2, an ortholog of the GWAS identified risk gene, Inositol-trisphosphate 3-kinase B (ITPKB), to modulate α-synuclein toxicity in vivo. ITPKB mRNA and protein levels were also increased in SK-N-SH cells overexpressing wild-type α-synuclein, A53T or A30P mutants. Kinase overexpression was detected in the cytoplasmatic and in the nuclear compartments in all α-synuclein cell types. By quantifying mRNAs in the cortex of PD patients, we observed higher levels of ITPKB mRNA when SNCA was expressed more (p < 0.05), compared to controls. A positive correlation was also observed between SNCA and ITPKB expression in the cortex of patients, which was not seen in the controls. We replicated this observation in a public dataset. Our data, generated in SK-N-SH cells and in cortex from PD patients, show that the expression of α-synuclein and ITPKB is correlated in pathological situations.
Assuntos
Doença de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Mutação , Neurônios/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismoRESUMO
Within mammalian brain circuits, activity-dependent synaptic adaptations, such as synaptic scaling, stabilize neuronal activity in the face of perturbations. Stability afforded through synaptic scaling involves uniform scaling of quantal amplitudes across all synaptic inputs formed on neurons, as well as on the postsynaptic side. It remains unclear whether activity-dependent uniform scaling also operates within peripheral circuits. We tested for such scaling in a Drosophila larval neuromuscular circuit, where the muscle receives synaptic inputs from different motoneurons. We used motoneuron-specific genetic manipulations to increase the activity of only one motoneuron and recordings of postsynaptic currents from inputs formed by the different motoneurons. We discovered an adaptation which caused uniform downscaling of evoked neurotransmitter release across all inputs through decreases in release probabilities. This "presynaptic downscaling" maintained the relative differences in neurotransmitter release across all inputs around a homeostatic set point, caused a compensatory decrease in synaptic drive to the muscle affording robust and stable muscle activity, and was induced within hours. Presynaptic downscaling was associated with an activity-dependent increase in Drosophila vesicular glutamate transporter expression. Activity-dependent uniform scaling can therefore manifest also on the presynaptic side to produce robust and stable circuit outputs. Within brain circuits, uniform downscaling on the postsynaptic side is implicated in sleep- and memory-related processes. Our results suggest that evaluation of such processes might be broadened to include uniform downscaling on the presynaptic side.SIGNIFICANCE STATEMENT To date, compensatory adaptations which stabilise target cell activity through activity-dependent global scaling have been observed only within central circuits, and on the postsynaptic side. Considering that maintenance of stable activity is imperative for the robust function of the nervous system as a whole, we tested whether activity-dependent global scaling could also manifest within peripheral circuits. We uncovered a compensatory adaptation which causes global scaling within a peripheral circuit and on the presynaptic side through uniform downscaling of evoked neurotransmitter release. Unlike in central circuits, uniform scaling maintains functionality over a wide, rather than a narrow, operational range, affording robust and stable activity. Activity-dependent global scaling therefore operates on both the presynaptic and postsynaptic sides to maintain target cell activity.
Assuntos
Drosophila/fisiologia , Ácido Glutâmico/fisiologia , Neurotransmissores/metabolismo , Animais , Potenciais Evocados/fisiologia , Homeostase , Imuno-Histoquímica , Locomoção/fisiologia , Neurônios Motores/fisiologia , Músculos/inervação , Músculos/fisiologia , Junção Neuromuscular/fisiologia , Técnicas de Patch-Clamp , Sinapses/fisiologia , Potenciais Sinápticos/fisiologia , Proteínas Vesiculares de Transporte de Glutamato/metabolismoRESUMO
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) together with CRISPR-associated (Cas) proteins have catalysed a revolution in genetic engineering. Native CRISPR-Cas systems exist in many bacteria and archaea where they provide an adaptive immune response through sequence-specific degradation of an invading pathogen's genome. This system has been reconfigured for use in genome editing, drug development, gene expression regulation, diagnostics, the prevention and treatment of cancers, and the treatment of genetic and infectious diseases. In recent years, CRISPR-Cas systems have been used in the diagnosis and control of viral diseases, for example, CRISPR-Cas12/13 coupled with new amplification techniques to improve the specificity of sequence-specific fluorescent probe detection. Importantly, CRISPR applications are both sensitive and specific and usually only require commonly available lab equipment. Unlike the canonical Cas9 which is guided to double-stranded DNA sites of interest, Cas13 systems target RNA sequences and thus can be employed in strategies directed against RNA viruses or for transcriptional silencing. Many challenges remain for these approach, including issues with specificity and the requirement for better mammalian delivery systems. In this review, we summarize the applications of CRISPR-Cas systems in controlling mammalian viral infections. Following necessary improvements, it is expected that CRISPR-Cas systems will be used effectively for such applications in the future.
Assuntos
Sistemas CRISPR-Cas/genética , Engenharia Genética , Genoma/genética , Viroses/genética , Animais , Edição de Genes , Humanos , Mamíferos , Viroses/terapia , Viroses/virologia , Vírus/genética , Vírus/patogenicidadeRESUMO
Oncogenic mutations in the small GTPase Ras contribute to ~30% of human cancers. However, Ras mutations alone are insufficient for tumorigenesis, therefore it is paramount to identify cooperating cancer-relevant signaling pathways. We devised an in vivo near genome-wide, functional screen in Drosophila and discovered multiple novel, evolutionarily-conserved pathways controlling Ras-driven epithelial tumorigenesis. Human gene orthologs of the fly hits were significantly downregulated in thousands of primary tumors, revealing novel prognostic markers for human epithelial tumors. Of the top 100 candidate tumor suppressor genes, 80 were validated in secondary Drosophila assays, identifying many known cancer genes and multiple novel candidate genes that cooperate with Ras-driven tumorigenesis. Low expression of the confirmed hits significantly correlated with the KRASG12 mutation status and poor prognosis in pancreatic cancer. Among the novel top 80 candidate cancer genes, we mechanistically characterized the function of the top hit, the Tetraspanin family member Tsp29Fb, revealing that Tsp29Fb regulates EGFR signaling, epithelial architecture and restrains tumor growth and invasion. Our functional Drosophila screen uncovers multiple novel and evolutionarily conserved epithelial cancer genes, and experimentally confirmed Tsp29Fb as a key regulator of EGFR/Ras induced epithelial tumor growth and invasion.
Assuntos
Proteínas de Drosophila/genética , IMP Desidrogenase/genética , Neoplasias/genética , Tetraspanina 29/genética , Animais , Animais Geneticamente Modificados , Carcinogênese/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Genes ras , Testes Genéticos/métodos , Humanos , IMP Desidrogenase/metabolismo , Masculino , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Oncogenes , Transdução de Sinais , Tetraspanina 29/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
OBJECTIVE: Heightened somatic symptoms are reported by a wide range of patients with chronic pain and have been associated with emotional distress and physical dysfunction. Despite their clinical significance, molecular mechanisms leading to their manifestation are not understood. METHODS: We used an association study design based on a curated list of 3,295 single nucleotide polymorphisms mapped to 358 genes to test somatic symptoms reporting using the Pennebaker Inventory of Limbic Languidness questionnaire from a case-control cohort of orofacial pain (n = 1,607). A replication meta-analysis of 3 independent cohorts (n = 3,189) was followed by functional validation, including in silico molecular dynamics, in vitro enzyme assays, and measures of serotonin (5-HT) plasma concentration. RESULTS: An association with the T allele of rs11575542 coding for an arginine to glutamine substitution in the L-aromatic amino acid decarboxylase (AADC) enzyme was replicated in a meta-analysis of 3 independent cohorts. In a combined meta-analysis of all cohorts, this association reached p = 6.43 × 10-8 . In silico studies demonstrated that this substitution dramatically reduces the conformational dynamics of AADC, potentially lowering its binding capacity to a cofactor. in vitro enzymatic assays showed that this substitution reduces the maximum kinetic velocity of AADC, hence lowering 5-HT levels. Finally, plasma samples from 90 subjects showed correlation between low 5-HT levels and heightened somatic symptoms. INTERPRETATION: Using functional genomics approaches, we identified a polymorphism in the AADC enzyme that contributes to somatic symptoms through reduced levels of 5-HT. Our findings suggest a molecular mechanism underlying the pathophysiology of somatic symptoms and opens up new treatment options targeting the serotonergic system. ANN NEUROL 2019;86:168-180.
Assuntos
Substituição de Aminoácidos/genética , Descarboxilases de Aminoácido-L-Aromático/genética , Dor Facial/genética , Estudos de Associação Genética/métodos , Sintomas Inexplicáveis , Serotonina/genética , Adolescente , Adulto , Estudos de Casos e Controles , Dor Facial/diagnóstico , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Estrutura Secundária de Proteína , Transdução de Sinais/genética , Adulto JovemRESUMO
Rhodopsins (Rhs) are light sensors, and Rh1 is the major Rh in the Drosophila photoreceptor rhabdomere membrane. Upon photoactivation, a fraction of Rh1 is internalized and degraded, but it remains unclear how the rhabdomeric Rh1 pool is replenished and what molecular players are involved. Here, we show that Crag, a DENN protein, is a guanine nucleotide exchange factor for Rab11 that is required for the homeostasis of Rh1 upon light exposure. The absence of Crag causes a light-induced accumulation of cytoplasmic Rh1, and loss of Crag or Rab11 leads to a similar photoreceptor degeneration in adult flies. Furthermore, the defects associated with loss of Crag can be partially rescued with a constitutive active form of Rab11. We propose that upon light stimulation, Crag is required for trafficking of Rh from the trans-Golgi network to rhabdomere membranes via a Rab11-dependent vesicular transport.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Rodopsina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Envelhecimento/metabolismo , Animais , Citoplasma/metabolismo , Citoplasma/efeitos da radiação , Drosophila melanogaster/genética , Drosophila melanogaster/efeitos da radiação , Eletrorretinografia , Feminino , Técnicas de Silenciamento de Genes , Genes de Insetos/genética , Luz , Masculino , Mutação/genética , Células Fotorreceptoras de Invertebrados/patologia , Células Fotorreceptoras de Invertebrados/efeitos da radiação , Células Fotorreceptoras de Invertebrados/ultraestrutura , Ligação Proteica/efeitos da radiação , Transporte Proteico/efeitos da radiação , Degeneração Retiniana/patologia , Degeneração Retiniana/fisiopatologiaRESUMO
The ability to perceive noxious stimuli is critical for an animal's survival in the face of environmental danger, and thus pain perception is likely to be under stringent evolutionary pressure. Using a neuronal-specific RNAi knock-down strategy in adult Drosophila, we recently completed a genome-wide functional annotation of heat nociception that allowed us to identify α2δ3 as a novel pain gene. Here we report construction of an evolutionary-conserved, system-level, global molecular pain network map. Our systems map is markedly enriched for multiple genes associated with human pain and predicts a plethora of novel candidate pain pathways. One central node of this pain network is phospholipid signaling, which has been implicated before in pain processing. To further investigate the role of phospholipid signaling in mammalian heat pain perception, we analysed the phenotype of PIP5Kα and PI3Kγ mutant mice. Intriguingly, both of these mice exhibit pronounced hypersensitivity to noxious heat and capsaicin-induced pain, which directly mapped through PI3Kγ kinase-dead knock-in mice to PI3Kγ lipid kinase activity. Using single primary sensory neuron recording, PI3Kγ function was mechanistically linked to a negative regulation of TRPV1 channel transduction. Our data provide a systems map for heat nociception and reinforces the extraordinary conservation of molecular mechanisms of nociception across different species.
Assuntos
Drosophila , Redes Reguladoras de Genes , Dor Nociceptiva , Fosfolipídeos , Transdução de Sinais , Animais , Capsaicina/toxicidade , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Classe Ib de Fosfatidilinositol 3-Quinase/fisiologia , Drosophila/genética , Drosophila/fisiologia , Temperatura Alta , Humanos , Hipersensibilidade/genética , Camundongos , Neurônios Aferentes/metabolismo , Dor Nociceptiva/induzido quimicamente , Dor Nociceptiva/genética , Dor Nociceptiva/fisiopatologia , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Fosfolipídeos/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/fisiologiaRESUMO
Chronic pain has an enormous impact on the quality of life of billions of patients, families, and caregivers worldwide. Current therapies do not adequately address pain for most patients. A basic understanding of the conserved genetic framework controlling pain may help us develop better, non-addictive pain therapies. Here we identify new conserved and druggable analgesic targets using tissue-specific functional genomic screening of candidate "pain" genes in the fly. From these efforts we describe 23 new pain genes for further consideration. This included Acsl, a fatty acid-metabolizing enzyme and mammalian orthologs are involved in arachidonic acid metabolism. Acsl knockdown and mutant larvae showed delayed nocifensive responses to localized and global noxious heat. Mechanistically, knockdown of Acsl reduced dendritic branching of nociceptive neurons. Surprisingly, the pain phenotype in these animals could be rescued through dietary intervention with vitamin B5, highlighting the interplay between genetics, metabolism and nutrient environment to establish sensory perception thresholds. Together, our functional genomic screening within the sensory nociceptor has identified new nociception genes that provide a better understanding of pain biology and can help guide the development of new painkillers.
RESUMO
Clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) has become an integral part of the molecular biology toolkit. CRISPRa genetic screens are an exciting high-throughput means of identifying genes the upregulation of which is sufficient to elicit a given phenotype. Activation machinery is continually under development to achieve greater, more robust, and more consistent activation. In this review, we offer a succinct technological overview of available CRISPRa architectures and a comprehensive summary of pooled CRISPRa screens. Furthermore, we discuss contemporary applications of CRISPRa across broad fields of research, with the aim of presenting a view of exciting emerging applications for CRISPRa screening.