RESUMO
Chronic stress has become a predominant factor associated with a variety of psychiatric disorders, such as depression and anxiety, in both human and animal models. Although multiple studies have looked at transcriptional changes after social defeat stress, these studies primarily focus on bulk tissues, which might dilute important molecular signatures of social interaction in activated cells. In this study, we employed the Arc-GFP mouse model in conjunction with chronic social defeat (CSD) to selectively isolate activated nuclei (AN) populations in the ventral hippocampus (vHIP) and prefrontal cortex (PFC) of resilient and susceptible animals. Nuclear RNA-seq of susceptible vs. resilient populations revealed distinct transcriptional profiles linked predominantly with neuronal and synaptic regulation mechanisms. In the vHIP, susceptible AN exhibited increased expression of genes related to the cytoskeleton and synaptic organization. At the same time, resilient AN showed upregulation of cell adhesion genes and differential expression of major glutamatergic subunits. In the PFC, susceptible mice exhibited upregulation of synaptotagmins and immediate early genes (IEGs), suggesting a potentially over-amplified neuronal activity state. Our findings provide a novel view of stress-exposed neuronal activation and the molecular response mechanisms in stress-susceptible vs. resilient animals, which may have important implications for understanding mental resilience.
RESUMO
Common variation in the gene encoding the neuron-specific RNA splicing factor RNA Binding Fox-1 Homolog 1 (RBFOX1) has been identified as a risk factor for several psychiatric conditions, and rare genetic variants have been found causal for autism spectrum disorder (ASD). Here, we explored the genetic landscape of RBFOX1 more deeply, integrating evidence from existing and new human studies as well as studies in Rbfox1 knockout mice. Mining existing data from large-scale studies of human common genetic variants, we confirmed gene-based and genome-wide association of RBFOX1 with risk tolerance, major depressive disorder and schizophrenia. Data on six mental disorders revealed copy number losses and gains to be more frequent in ASD cases than in controls. Consistently, RBFOX1 expression appeared decreased in post-mortem frontal and temporal cortices of individuals with ASD and prefrontal cortex of individuals with schizophrenia. Brain-functional MRI studies demonstrated that carriers of a common RBFOX1 variant, rs6500744, displayed increased neural reactivity to emotional stimuli, reduced prefrontal processing during cognitive control, and enhanced fear expression after fear conditioning, going along with increased avoidance behaviour. Investigating Rbfox1 neuron-specific knockout mice allowed us to further specify the role of this gene in behaviour. The model was characterised by pronounced hyperactivity, stereotyped behaviour, impairments in fear acquisition and extinction, reduced social interest, and lack of aggression; it provides excellent construct and face validity as an animal model of ASD. In conclusion, convergent translational evidence shows that common variants in RBFOX1 are associated with a broad spectrum of psychiatric traits and disorders, while rare genetic variation seems to expose to early-onset neurodevelopmental psychiatric disorders with and without developmental delay like ASD, in particular. Studying the pleiotropic nature of RBFOX1 can profoundly enhance our understanding of mental disorder vulnerability.
Assuntos
Transtorno do Espectro Autista , Transtorno Depressivo Maior , Transtornos Mentais , Animais , Camundongos , Humanos , Transtorno do Espectro Autista/genética , Transtorno Depressivo Maior/genética , Estudo de Associação Genômica Ampla , Transtornos Mentais/genética , Camundongos Knockout , Fatores de Processamento de RNA/genéticaRESUMO
BACKGROUND: Next Generation Sequencing (NGS) is the fundament of various studies, providing insights into questions from biology and medicine. Nevertheless, integrating data from different experimental backgrounds can introduce strong biases. In order to methodically investigate the magnitude of systematic errors in single nucleotide variant calls, we performed a cross-sectional observational study on a genomic cohort of 99 subjects each sequenced via (i) Illumina HiSeq X, (ii) Illumina HiSeq, and (iii) Complete Genomics and processed with the respective bioinformatic pipeline. We also repeated variant calling for the Illumina cohorts with GATK, which allowed us to investigate the effect of the bioinformatics analysis strategy separately from the sequencing platform's impact. RESULTS: The number of detected variants/variant classes per individual was highly dependent on the experimental setup. We observed a statistically significant overrepresentation of variants uniquely called by a single setup, indicating potential systematic biases. Insertion/deletion polymorphisms (indels) were associated with decreased concordance compared to single nucleotide polymorphisms (SNPs). The discrepancies in indel absolute numbers were particularly prominent in introns, Alu elements, simple repeats, and regions with medium GC content. Notably, reprocessing sequencing data following the best practice recommendations of GATK considerably improved concordance between the respective setups. CONCLUSION: We provide empirical evidence of systematic heterogeneity in variant calls between alternative experimental and data analysis setups. Furthermore, our results demonstrate the benefit of reprocessing genomic data with harmonized pipelines when integrating data from different studies.
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Biologia Computacional , Sequenciamento de Nucleotídeos em Larga Escala , Estudos Transversais , Genômica , Humanos , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos TestesRESUMO
Dysregulated mammalian target of rapamycin (mTOR) activity is associated with various neurodevelopmental disorders ranging from idiopathic autism spectrum disorders (ASD) to syndromes caused by single gene defects. This suggests that maintaining mTOR activity levels in a physiological range is essential for brain development and functioning. Upon activation, mTOR regulates a variety of cellular processes such as cell growth, autophagy, and metabolism. On a molecular level, however, the consequences of mTOR activation in the brain are not well understood. Low levels of cholesterol are associated with a wide variety of neurodevelopmental disorders. We here describe numerous genes of the sterol/cholesterol biosynthesis pathway to be transcriptionally regulated by mTOR complex 1 (mTORC1) signaling in vitro in primary neurons and in vivo in the developing cerebral cortex of the mouse. We find that these genes are shared targets of the transcription factors SREBP, SP1, and NF-Y. Prenatal as well as postnatal mTORC1 inhibition downregulated expression of these genes which directly translated into reduced cholesterol levels, pointing towards a substantial metabolic function of the mTORC1 signaling cascade. Altogether, our results indicate that mTORC1 is an essential transcriptional regulator of the expression of sterol/cholesterol biosynthesis genes in the developing brain. Altered expression of these genes may be an important factor contributing to the pathogenesis of neurodevelopmental disorders associated with dysregulated mTOR signaling.
Assuntos
Colesterol/genética , Neurônios/metabolismo , Proteínas Quinases/genética , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Serina-Treonina Quinases TOR/genética , Animais , Autofagia/genética , Fator de Ligação a CCAAT/genética , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Colesterol/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Neurogênese/genética , Cultura Primária de Células , Transdução de Sinais/genética , Transcrição Gênica/genéticaRESUMO
The S-Phase Cyclin A Associated Protein In The ER (SCAPER) gene is a ubiquitously expressed gene with unknown function in the brain. Recently, biallelic SCAPER variants were described in four patients from three families with retinitis pigmentosa (RP) and intellectual disability (ID). Here, we expand the spectrum of pathogenic variants in SCAPER and report on 10 further patients from four families with ID, RP, and additional dysmorphic features carrying homozygous variants in SCAPER. The variants found comprise frameshift, nonsense, and missense variants as well as an intragenic homozygous deletion, which spans SCAPER exons 15 and 16 and introduces a frameshift and a premature stop codon. Analyses of SCAPER expression in human and mouse brain revealed an upregulation of SCAPER expression during cortical development and a higher expression of SCAPER in neurons compared to neural progenitors. In the adult brain SCAPER is expressed in several regions including the cerebral cortex where it shows a layer-specific expression with an expression peak in lower layer glutamatergic neurons. Our study supports the role of SCAPER variants in the pathogenesis of ID and RP, expands the variant spectrum and highlights the need for functional studies concerning the role of SCAPER during brain development and function.
Assuntos
Proteínas de Transporte/genética , Homozigoto , Deficiência Intelectual/genética , Mutação , Retinose Pigmentar/genética , Adolescente , Adulto , Animais , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Criança , Consanguinidade , Família , Feminino , Expressão Gênica , Humanos , Deficiência Intelectual/complicações , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Masculino , Camundongos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neurônios/metabolismo , Neurônios/patologia , Linhagem , Retinose Pigmentar/complicações , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , SíndromeRESUMO
BACKGROUND: Huntington's disease (HD) is a rare, genetic neurodegenerative disorder often presenting with emotional, cognitive and behavioral abnormalities before manifestation of disease defining motor symptoms. Cognitive impairment is a frequent clinical feature caused by different dementia subtypes. Imaging cortical and subcortical glucose metabolism via 18F-FDG PET/CT can help to discriminate the underlying disease. CASE PRESENTATION: The patient is a 54-year old man presenting with progressive cognitive impairment and mild orofacial dyskinesia. 18F-FDG PET/CT of the brain revealed a severe bilateral hypometabolism in the striatum. Following imaging Huntington's disease was suspected and a molecular genetic testing confirmed the diagnosis. CONCLUSIONS: Huntington's disease is a rare but important differential diagnosis of cognitive impairment, especially before motor symptoms are manifest. 18F-FDG PET is capable to show early striatal dysfunction in HD even when structural imaging is normal. We conclude that, in cases with negative family history the HD characteristic metabolic pattern can lead to the diagnosis when no other dementia-suspected changes are present.
Assuntos
Encéfalo/diagnóstico por imagem , Doença de Huntington/diagnóstico por imagem , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Fluordesoxiglucose F18/metabolismo , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
SHH (Sonic Hedgehog)-GLI signaling plays an important role during embryogenesis and in tumorigenesis. The survival and growth of several types of cancer depend on autonomously activated SHH-GLI signaling. A protein complex containing the ubiquitin ligase MID1 and protein phosphatase 2A regulates the nuclear localization and transcriptional activity of GLI3, a transcriptional effector molecule of SHH, in cancer cell lines with autonomously activated SHH signaling. However, the exact molecular mechanisms that mediate the interaction between MID1 and GLI3 remained unknown. Here, we show that MID1 catalyzes the ubiquitination and proteasomal cleavage of the GLI3 regulator Fu. Our data suggest that Fu ubiquitination and cleavage is one of the key elements connecting the MID1-PP2A protein complex with GLI3 activity control.
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Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas dos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/química , Catálise , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Primers do DNA , Regulação Neoplásica da Expressão Gênica , Células HeLa , Proteínas Hedgehog/metabolismo , Humanos , Lisina/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais , Ubiquitina/química , Ubiquitinação , Proteína Gli3 com Dedos de ZincoRESUMO
BACKGROUND: High androgen receptor (AR) level in primary tumour predicts increased prostate cancer (PCa)-specific mortality. Furthermore, activations of the AR, PI3K, mTOR, NFκB and Hedgehog (Hh) signaling pathways are involved in the fatal development of castration-resistant prostate cancer during androgen ablation therapy. MID1, a negative regulator of the tumor-suppressor PP2A, is known to promote PI3K, mTOR, NFκB and Hh signaling. Here we investigate the interaction of MID1 and AR. METHODS: AR and MID1 mRNA and protein levels were measured by qPCR, Western blot and immunohistochemistry. Co-immunoprecipitation followed by PCR and RNA-pull-down followed by Western blot was used to investigate protein-mRNA interaction, chromatin-immunoprecipitation followed by next-generation sequencing for identification of AR chromatin binding sites. AR transcriptional activity and activity of promoter binding sites for AR were analyzed by reporter gene assays. For knockdown or overexpression of proteins of interest prostate cancer cells were transfected with siRNA or expression plasmids, respectively. RESULTS: The microtubule-associated MID1 protein complex associates with AR mRNA via purine-rich trinucleotide repeats, expansions of which are known to correlate with ataxia and cancer. The level of MID1 directly correlates with the AR protein level in PCa cells. Overexpression of MID1 results in a several fold increase in AR protein and activity without major changes in mRNA-levels, whereas siRNA-triggered knockdown of MID1 mRNA reduces AR-protein levels significantly. Upregulation of AR protein by MID1 occurs via increased translation as no major changes in AR protein stability could be observed. AR on the other hand, regulates MID1 via several functional AR binding sites in the MID1 gene, and, in the presence of androgens, exerts a negative feedback loop on MID1 transcription. Thus, androgen withdrawal increases MID1 and concomitantly AR-protein levels. In line with this, MID1 is significantly over-expressed in PCa in a stage-dependent manner. CONCLUSION: Promotion of AR, in addition to enhancement of the Akt-, NFκB-, and Hh-pathways by sustained MID1-upregulation during androgen deprivation therapy provides a powerful proliferative scenario for PCa progression into castration resistance. Thus MID1 represents a novel, multi-faceted player in PCa and a promising target to treat castration resistant prostate cancer.
Assuntos
Proteínas dos Microtúbulos/genética , Neoplasias Hormônio-Dependentes/genética , Proteínas Nucleares/genética , Neoplasias de Próstata Resistentes à Castração/genética , Receptores Androgênicos/metabolismo , Fatores de Transcrição/genética , Androgênios/metabolismo , Linhagem Celular Tumoral , Retroalimentação Fisiológica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Proteínas dos Microtúbulos/biossíntese , Neoplasias Hormônio-Dependentes/patologia , Proteínas Nucleares/biossíntese , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Neoplasias de Próstata Resistentes à Castração/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fatores de Transcrição/biossíntese , Ubiquitina-Proteína LigasesRESUMO
BACKGROUND: Metformin is an approved drug prescribed for diabetes. Its role as an anti-cancer agent has drawn significant attention because of its minimal side effects and low cost. However, its mechanism of anti-tumour action has not yet been fully clarified. METHODS: The effect on cell growth was assessed by cell counting. Western blot was used for analysis of protein levels, Boyden chamber assays for analyses of cell migration and co-immunoprecipitation (CoIP) followed by western blot, PCR or qPCR for analysis of protein-protein and protein-mRNA interactions. RESULTS: Metformin showed an anti-proliferative effect on a wide range of prostate cancer cells. It disrupted the AR translational MID1 regulator complex leading to release of the associated AR mRNA and subsequently to downregulation of AR protein in AR positive cell lines. Inhibition of AR positive and negative prostate cancer cells by metformin suggests involvement of additional targets. The inhibitory effect of metformin was mimicked by disruption of the MID1-α4/PP2A protein complex by siRNA knockdown of MID1 or α4 whereas AMPK activation was not required. CONCLUSIONS: Findings reported herein uncover a mechanism for the anti-tumor activity of metformin in prostate cancer, which is independent of its anti-diabetic effects. These data provide a rationale for the use of metformin in the treatment of hormone naïve and castration-resistant prostate cancer and suggest AR is an important indirect target of metformin.
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Antineoplásicos/farmacologia , Regulação para Baixo/fisiologia , Metformina/farmacologia , Proteínas dos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Fatores de Transcrição/metabolismo , Antineoplásicos/uso terapêutico , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Regulação para Baixo/efeitos dos fármacos , Humanos , Masculino , Metformina/uso terapêutico , Proteínas dos Microtúbulos/antagonistas & inibidores , Proteínas dos Microtúbulos/genética , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Receptores Androgênicos/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Resultado do Tratamento , Ubiquitina-Proteína LigasesRESUMO
NDST1 was recently proposed as a candidate gene for autosomal recessive intellectual disability in two families. It encodes a bifunctional GlcNAc N-deacetylase/N-sulfotransferase with important functions in heparan sulfate biosynthesis. In mice, Ndst1 is crucial for embryonic development and homozygous null mutations are perinatally lethal. We now report on two additional unrelated families with homozygous missense NDST1 mutations. All mutations described to date predict the substitution of conserved amino acids in the sulfotransferase domain, and mutation modeling predicts drastic alterations in the local protein conformation. Comparing the four families, we noticed significant overlap in the clinical features, including both demonstrated and apparent intellectual disability, muscular hypotonia, epilepsy, and postnatal growth deficiency. Furthermore, in Drosophila, knockdown of sulfateless, the NDST ortholog, impairs long-term memory, highlighting its function in cognition. Our data confirm NDST1 mutations as a cause of autosomal recessive intellectual disability with a distinctive phenotype, and support an important function of NDST1 in human development.
Assuntos
Genes Recessivos , Deficiência Intelectual/genética , Mutação de Sentido Incorreto , Sulfotransferases/genética , Adolescente , Adulto , Animais , Animais Geneticamente Modificados , Comportamento Animal , Criança , Pré-Escolar , Biologia Computacional , Consanguinidade , Análise Mutacional de DNA , Drosophila/genética , Fácies , Feminino , Técnicas de Silenciamento de Genes , Estudo de Associação Genômica Ampla , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Deficiência Intelectual/diagnóstico , Masculino , Modelos Moleculares , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único , Conformação Proteica , Sulfotransferases/química , Adulto JovemRESUMO
Mutations in the MID1 gene are causally linked to X-linked Opitz BBB/G syndrome (OS), a congenital disorder that primarily affects the formation of diverse ventral midline structures. The MID1 protein has been shown to function as an E3 ligase targeting the catalytic subunit of protein phosphatase 2A (PP2A-C) for ubiquitin-mediated degradation. However, the molecular pathways downstream of the MID1/PP2A axis that are dysregulated in OS and that translate dysfunctional MID1 and elevated levels of PP2A-C into the OS phenotype are poorly understood. Here, we show that perturbations in MID1/PP2A affect mTORC1 signaling. Increased PP2A levels, resulting from proteasome inhibition or depletion of MID1, lead to disruption of the mTOR/Raptor complex and down-regulated mTORC1 signaling. Congruously, cells derived from OS patients that carry MID1 mutations exhibit decreased mTORC1 formation, S6K1 phosphorylation, cell size, and cap-dependent translation, all of which is rescued by expression of wild-type MID1 or an activated mTOR allele. Our findings define mTORC1 signaling as a downstream pathway regulated by the MID1/PP2A axis, suggesting that mTORC1 plays a key role in OS pathogenesis.
Assuntos
Hipertelorismo/etiologia , Hipospadia/etiologia , Proteínas dos Microtúbulos/fisiologia , Proteínas Nucleares/fisiologia , Proteínas/metabolismo , Transdução de Sinais , Fatores de Transcrição/fisiologia , Tamanho Celular , Células Cultivadas , Esôfago/anormalidades , Esôfago/patologia , Humanos , Hipertelorismo/patologia , Hipospadia/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos , Fosforilação , Proteína Fosfatase 2/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR , Ubiquitina-Proteína LigasesRESUMO
Neuronal networks possess the ability to regulate their activity states in response to disruptions. How and when neuronal networks turn from physiological into pathological states, leading to the manifestation of neuropsychiatric disorders, remains largely unknown. Here, we propose that neuronal networks intrinsically maintain network stability even at the cost of neuronal loss. Despite the new stable state being potentially maladaptive, neural networks may not reverse back to states associated with better long-term outcomes. These maladaptive states are often associated with hyperactive neurons, marking the starting point for activity-dependent neurodegeneration. Transitions between network states may occur rapidly, and in discrete steps rather than continuously, particularly in neurodegenerative disorders. The self-stabilizing, metastable, and noncontinuous characteristics of these network states can be mathematically described as attractors. Maladaptive attractors may represent a distinct pathophysiological entity that could serve as a target for new therapies and for fostering resilience.
Assuntos
Encéfalo , Neurônios , Humanos , Neurônios/fisiologia , Redes Neurais de ComputaçãoRESUMO
Adaptive nanopore sequencing as a diagnostic method for imprinting disorders and episignature analysis revealed an intragenic duplication of Exon 6 and 7 in UBE3A (NM_000462.5) in a patient with relatively mild Angelman-like syndrome. In an all-in-one nanopore sequencing analysis DNA hypomethylation of the SNURF:TSS-DMR, known contributing deletions on the maternal allele and point mutations in UBE3A could be ruled out as disease drivers. In contrast, breakpoints and orientation of the tandem duplication could clearly be defined. Segregation analysis in the family showed that the duplication derived de novo in the maternal grandfather. Our study shows the benefits of an all-in-one nanopore sequencing approach for the diagnostics of Angelman syndrome and other imprinting disorders.
Assuntos
Síndrome de Angelman , Metilação de DNA , Duplicação Gênica , Sequenciamento por Nanoporos , Ubiquitina-Proteína Ligases , Humanos , Síndrome de Angelman/genética , Síndrome de Angelman/diagnóstico , Ubiquitina-Proteína Ligases/genética , Sequenciamento por Nanoporos/métodos , Metilação de DNA/genética , Feminino , Duplicação Gênica/genética , Masculino , Éxons/genética , Linhagem , Impressão Genômica/genéticaRESUMO
The X-linked form of Opitz BBB/G syndrome (OS) is a monogenic disorder in which symptoms are established early during embryonic development. OS is caused by pathogenic variants in the X-linked gene MID1 Disease-associated variants are distributed across the entire gene locus, except for the N-terminal really interesting new gene (RING) domain that encompasses the E3 ubiquitin ligase activity. By using genome-edited human induced pluripotent stem cell lines, we here show that absence of isoforms containing the RING domain of MID1 causes severe patterning defects in human brain organoids. We observed a prominent neurogenic deficit with a reduction in neural tissue and a concomitant increase in choroid plexus-like structures. Transcriptome analyses revealed a deregulation of patterning pathways very early on, even preceding neural induction. Notably, the observed phenotypes starkly contrast with those observed in MID1 full-knockout organoids, indicating the presence of a distinct mechanism that underlies the patterning defects. The severity and early onset of these phenotypes could potentially account for the absence of patients carrying pathogenic variants in exon 1 of the MID1 gene coding for the N-terminal RING domain.
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Esôfago , Hipertelorismo , Hipospadia , Células-Tronco Pluripotentes Induzidas , Proteínas Nucleares , Humanos , Encéfalo/metabolismo , Esôfago/anormalidades , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas dos Microtúbulos/química , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Multiple neurodegenerative disorders are linked to aberrant phosphorylation of microtubule-associated proteins (MAPs). Protein phosphatase 2A (PP2A) is the major MAP phosphatase; however, little is known about its regulation at microtubules. α4 binds the PP2A catalytic subunit (PP2Ac) and the microtubule-associated E3 ubiquitin ligase MID1, and through unknown mechanisms can both reduce and enhance PP2Ac stability. We show MID1-dependent monoubiquitination of α4 triggers calpain-mediated cleavage and switches α4's activity from protective to destructive, resulting in increased Tau phosphorylation. This regulatory mechanism appears important in MAP-dependent pathologies as levels of cleaved α4 are decreased in Opitz syndrome and increased in Alzheimer disease, disorders characterized by MAP hypophosphorylation and hyperphosphorylation, respectively. These findings indicate that regulated inter-domain cleavage controls the dual functions of α4, and dysregulation of α4 cleavage may contribute to Opitz syndrome and Alzheimer disease.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Fosfatase 2/metabolismo , Ubiquitinação/fisiologia , Western Blotting , Linhagem Celular , Humanos , Imunoprecipitação , Espectrometria de Massas , Proteínas Associadas aos Microtúbulos/genética , Fosforilação/genética , Fosforilação/fisiologia , Proteína Fosfatase 2/genética , Estabilidade Proteica , Ubiquitinação/genéticaRESUMO
Hyperphosphorylated tau plays an important role in the formation of neurofibrillary tangles in brains of patients with Alzheimer's disease (AD) and related tauopathies and is a crucial factor in the pathogenesis of these disorders. Though diverse kinases have been implicated in tau phosphorylation, protein phosphatase 2A (PP2A) seems to be the major tau phosphatase. Using murine primary neurons from wild-type and human tau transgenic mice, we show that the antidiabetic drug metformin induces PP2A activity and reduces tau phosphorylation at PP2A-dependent epitopes in vitro and in vivo. This tau dephosphorylating potency can be blocked entirely by the PP2A inhibitors okadaic acid and fostriecin, confirming that PP2A is an important mediator of the observed effects. Surprisingly, metformin effects on PP2A activity and tau phosphorylation seem to be independent of AMPK activation, because in our experiments (i) metformin induces PP2A activity before and at lower levels than AMPK activity and (ii) the AMPK activator AICAR does not influence the phosphorylation of tau at the sites analyzed. Affinity chromatography and immunoprecipitation experiments together with PP2A activity assays indicate that metformin interferes with the association of the catalytic subunit of PP2A (PP2Ac) to the so-called MID1-α4 protein complex, which regulates the degradation of PP2Ac and thereby influences PP2A activity. In summary, our data suggest a potential beneficial role of biguanides such as metformin in the prophylaxis and/or therapy of AD.
Assuntos
Metformina/farmacologia , Emaranhados Neurofibrilares/metabolismo , Proteína Fosfatase 2/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas tau/metabolismo , Adenilato Quinase/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Animais , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Epitopos , Células HeLa , Humanos , Hipoglicemiantes/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Transgênicos , Complexos Multiproteicos , Emaranhados Neurofibrilares/patologia , Neurônios/citologia , Neurônios/metabolismo , Ácido Okadáico/farmacologia , Fosforilação , Proteína Fosfatase 2/genética , Proteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética , Proteínas tau/genéticaRESUMO
We found mutations in the gene PQBP1 in 5 of 29 families with nonsyndromic (MRX) and syndromic (MRXS) forms of X-linked mental retardation (XLMR). Clinical features in affected males include mental retardation, microcephaly, short stature, spastic paraplegia and midline defects. PQBP1 has previously been implicated in the pathogenesis of polyglutamine expansion diseases. Our findings link this gene to XLMR and shed more light on the pathogenesis of this common disorder.
Assuntos
Deficiência Intelectual Ligada ao Cromossomo X/genética , Mutação/genética , Oligopeptídeos/genética , Proteínas de Transporte/genética , Proteínas de Ligação a DNA , Feminino , Ligação Genética , Humanos , Masculino , Deficiência Intelectual Ligada ao Cromossomo X/classificação , Deficiência Intelectual Ligada ao Cromossomo X/etiologia , Dados de Sequência Molecular , Proteínas Nucleares/genética , Linhagem , SíndromeRESUMO
In mammals, X-chromosomal genes are expressed from a single copy since males (XY) possess a single X chromosome, while females (XX) undergo X inactivation. To compensate for this reduction in dosage compared with two active copies of autosomes, it has been proposed that genes from the active X chromosome exhibit dosage compensation. However, the existence and mechanisms of X-to-autosome dosage compensation are still under debate. Here we show that X-chromosomal transcripts have fewer m6A modifications and are more stable than their autosomal counterparts. Acute depletion of m6A selectively stabilizes autosomal transcripts, resulting in perturbed dosage compensation in mouse embryonic stem cells. We propose that higher stability of X-chromosomal transcripts is directed by lower levels of m6A, indicating that mammalian dosage compensation is partly regulated by epitranscriptomic RNA modifications.
Assuntos
Mecanismo Genético de Compensação de Dose , Cromossomo X , Masculino , Feminino , Animais , Camundongos , Metilação , Cromossomo X/genética , Mamíferos/genética , Estabilidade de RNARESUMO
Background: People with complex symptomatology but unclear diagnosis presenting to a centre for rare diseases (CRD) may present with mental (co-)morbidity. We hypothesised that combining an expert in somatic medicine with a mental health specialist working in tandem will improve the diagnostic outcome. Methods: Patients aged 12 years and older who presented to one of the 11 participating German CRDs with an unknown diagnosis were recruited into this prospective cohort trial with a two-phase cohort design. From October 1, 2018 to September 30, 2019, participants were allocated to standard care (SC, N = 684), and from October 1, 2019 to January 31, 2021 to innovative care (IC, N = 695). The cohorts consisted mainly of adult participants with only a minority of children included (N = 67). IC included the involvement of a mental health specialist in all aspects of care (e.g., assessing medical records, clinic visits, telehealth care, and case conferences). Clinicaltrials.gov identifier: NCT03563677. Findings: The proportion of patients with diagnoses established within 12 months after the first visit to the CRD explaining the entire symptomatology (primary outcome) was 19% (N = 131 of 672) in the SC and 42% (N = 286 of 686) in the IC cohort (OR adjusted for centre effects 3.45 [95% CrI: 1.99-5.65]). The difference was mainly due to a higher prevalence of mental disorders and non-rare somatic diseases in the IC cohort. The median time to explaining diagnoses was one month shorter with IC (95% CrI: 1-2), and significantly more patients could be referred to local regular care in the IC (27.5%; N = 181 of 659) compared to the SC (12.3%; N = 81 of 658) cohort (OR adjusted for centre effects 2.70 [95% CrI: 2.02-3.60]). At 12-month follow-up, patient satisfaction with care was significantly higher in the IC compared to the SC cohort, while quality of life was not different between cohorts. Interpretation: Our findings suggested that including a mental health specialist in the entire evaluation process of CRDs for undiagnosed adolescents and adults should become an integral part of the assessment of individuals with a suspected rare disease. Funding: The study was funded by the Global Innovation Fund from the Joint Federal Committee in Germany (Innovationsfonds des Gemeinsamen Bundesausschusses), grant number 01NVF17031.
RESUMO
We have shown previously that the ubiquitin ligase MID1, mutations of which cause the midline malformation Opitz BBB/G syndrome (OS), serves as scaffold for a microtubule-associated protein complex that regulates protein phosphatase 2A (PP2A) activity in a ubiquitin-dependent manner. Here, we show that the MID1 protein complex associates with mRNAs via a purine-rich sequence motif called MIDAS (MID1 association sequence) and thereby increases stability and translational efficiency of these mRNAs. Strikingly, inclusion of multiple copies of the MIDAS motif into mammalian mRNAs increases production of the encoded proteins up to 20-fold. Mutated MID1, as found in OS patients, loses its influence on MIDAS-containing mRNAs, suggesting that the malformations in OS patients could be caused by failures in the regulation of cytoskeleton-bound protein translation. This is supported by the observation that the majority of mRNAs that carry MIDAS motifs is involved in developmental processes and/or energy homeostasis. Further analysis of one of the proteins encoded by a MIDAS-containing mRNA, namely PDPK-1 (3-phosphoinositide dependent protein kinase-1), which is an important regulator of mammalian target of rapamycin/PP2A signaling, showed that PDPK-1 protein synthesis is significantly reduced in cells from an OS patient compared with an age-matched control and can be rescued by functional MID1. Together, our data uncover a novel messenger ribonucleoprotein complex that regulates microtubule-associated protein translation. They suggest a novel mechanism underlying OS and point at an enormous potential of the MIDAS motif to increase the efficiency of biotechnological protein production in mammalian cells.