RESUMEN
The Vps10p domain receptor SorCS2 is crucial for the development and function of the nervous system and essential for brain-derived neurotrophic factor (BDNF)-induced changes in neuronal morphology and plasticity. SorCS2 regulates the subcellular trafficking of the BDNF signaling receptor TrkB as well as selected neurotransmitter receptors in a manner that is dependent on the SorCS2 intracellular domain (ICD). However, the cellular machinery and adaptor protein (AP) interactions that regulate receptor trafficking via the SorCS2 ICD are unknown. We here identify four splice variants of human SorCS2 differing in the insertion of an acidic cluster motif and/or a serine residue within the ICD. We show that each variant undergoes posttranslational proteolytic processing into a one- or two-chain receptor, giving rise to eight protein isoforms, the expression of which differs between neuronal and nonneuronal tissues and is affected by cellular stressors. We found that the only variants without the serine were able to rescue BDNF-induced branching of SorCS2 knockout hippocampal neurons, while variants without the acidic cluster showed increased interactions with clathrin-associated APs AP-1, AP-2, and AP-3. Using yeast two-hybrid screens, we further discovered that all variants bound dynein light chain Tctex-type 3; however, only variants with an acidic cluster motif bound kinesin light chain 1. Accordingly, splice variants showed markedly different trafficking properties and localized to different subcellular compartments. Taken together, our findings demonstrate the existence of eight functional SorCS2 isoforms with differential capacity for interactions with cytosolic ligands dynein light chain Tctex-type 3 and kinesin light chain 1, which potentially allows cell-type specific SorCS2 trafficking and BDNF signaling.
Asunto(s)
Empalme Alternativo , Sistema Nervioso Central , Receptores de Superficie Celular , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Empalme Alternativo/fisiología , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Dineínas/metabolismo , Cinesinas/metabolismo , Unión Proteica , Isoformas de Proteínas/metabolismo , Receptor trkB/metabolismo , Receptores de Superficie Celular/metabolismo , Sistema Nervioso Central/crecimiento & desarrollo , Procesamiento Proteico-Postraduccional , Transporte de Proteínas/genéticaRESUMEN
Proteinuria is a prominent feature of chronic kidney disease. Interventions that reduce proteinuria slow the progression of chronic kidney disease and the associated risk of cardiovascular disease. Here, we propose a mechanistic coupling between proteinuria and proprotein convertase subtilisin/kexin type 9 (PCSK9), a regulator of cholesterol and a therapeutic target in cardiovascular disease. PCSK9 undergoes glomerular filtration and is captured by megalin, the receptor responsible for driving protein reabsorption in the proximal tubule. Accordingly, megalin-deficient mice and patients carrying megalin pathogenic variants (Donnai Barrow syndrome) were characterized by elevated urinary PCSK9 excretion. Interestingly, PCSK9 knockout mice displayed increased kidney megalin while PCSK9 overexpression resulted in its reduction. Furthermore, PCSK9 promoted trafficking of megalin to lysosomes in cultured proximal tubule cells, suggesting that PCSK9 is a negative regulator of megalin. This effect can be accelerated under disease conditions since either genetic destruction of the glomerular filtration barrier in podocin knockout mice or minimal change disease (a common cause of nephrotic syndrome) in patients resulted in enhanced tubular PCSK9 uptake and urinary PCSK9 excretion. Pharmacological PCSK9 inhibition increased kidney megalin while reducing urinary albumin excretion in nephrotic mice. Thus, glomerular damage increases filtration of PCSK9 and concomitantly megalin degradation, resulting in escalated proteinuria.
Asunto(s)
Enfermedades Cardiovasculares , Síndrome Nefrótico , Insuficiencia Renal Crónica , Humanos , Ratones , Animales , Síndrome Nefrótico/patología , Proproteína Convertasa 9/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad , Enfermedades Cardiovasculares/metabolismo , Proteinuria/genética , Túbulos Renales Proximales/patología , Insuficiencia Renal Crónica/patología , Ratones Noqueados , Subtilisinas/metabolismoRESUMEN
SORCS2 is one of five proteins that constitute the Vps10p-domain receptor family. Members of this family play important roles in cellular processes linked to neuronal survival, differentiation and function. Genetic and functional studies implicate SORCS2 in cognitive function, as well as in neurodegenerative and psychiatric disorders. DNA damage and DNA repair deficits are linked to ageing and neurodegeneration, and transient neuronal DNA double-strand breaks (DSBs) also occur as a result of neuronal activity. Here, we report a novel role for SORCS2 in DSB formation. We show that SorCS2 loss is associated with elevated DSB levels in the mouse dentate gyrus and that knocking out SORCS2 in a human neuronal cell line increased Topoisomerase IIß-dependent DSB formation and reduced neuronal viability. Neuronal stimulation had no impact on levels of DNA breaks in vitro, suggesting that the observed differences may not be the result of aberrant neuronal activity in these cells. Our findings are consistent with studies linking the VPS10 receptors and DNA damage to neurodegenerative conditions.
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Roturas del ADN de Doble Cadena , Reparación del ADN , Humanos , Animales , Ratones , Neuronas/metabolismo , Daño del ADN , Línea Celular , Receptores de Superficie Celular/genética , Proteínas del Tejido Nervioso/metabolismoRESUMEN
The 22q11.2 hemizygous deletion confers high risk for multiple neurodevelopmental disorders. Inhibitory signaling, largely regulated through GABAA receptors, is suggested to serve a multitude of brain functions that are disrupted in the 22q11.2 deletion syndrome. We investigated the putative deficit of GABAA receptors and the potential substrates contributing to the inhibitory and excitatory dysregulations in hippocampal networks of the Df(h22q11)/+ mouse model of the 22q11.2 hemizygous deletion. The Df(h22q11)/+ mice exhibited impairments in several hippocampus-related functional domains, represented by impaired spatial memory and sensory gating functions. Autoradiography using the [3H]muscimol tracer revealed a significant reduction in GABAA receptor binding in the CA1 and CA3 subregions, together with a loss of GAD67+ interneurons in CA1 of Df(h22q11)/+ mice. Furthermore, electrophysiology recordings exhibited significantly higher neuronal activity in CA3, in response to the GABAA receptor antagonist, bicuculline, as compared with wild type mice. Density and volume of dendritic spines in pyramidal neurons were reduced and Sholl analysis also showed a reduction in the complexity of basal dendritic tree in CA1 and CA3 subregions of Df(h22q11)/+ mice. Overall, our findings demonstrate that hemizygous deletion in the 22q11.2 locus leads to dysregulations in the inhibitory circuits, involving reduced binding levels of GABAA receptors, in addition to functional and structural modulations of the excitatory networks of hippocampus.
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Hipocampo , Receptores de GABA-A , Animales , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Ratones , Muscimol/metabolismo , Muscimol/farmacología , Células Piramidales/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The 15q13.3 deletion is associated with multiple neurodevelopmental disorders including epilepsy, schizophrenia, and autism. The Df(h15q13)/+ mouse model was recently generated that recapitulates several phenotypic features of the human 15q13.3 deletion syndrome (DS). However, the biological substrates underlying these phenotypes in Df(h15q13)/+ mice have not yet been fully characterized. RNA sequencing followed by real-time quantitative PCR, western blotting, liquid chromatography-mass spectrometry, and stereological analysis were employed to dissect the molecular, structural, and neurochemical phenotypes of the medial prefrontal cortex (mPFC) circuits in Df(h15q13)/+ mouse model. Transcriptomic profiling revealed enrichment for astrocyte-specific genes among differentially expressed genes, translated by a decrease in the number of glial fibrillary acidic protein positive cells in mPFC of Df(h15q13)/+ mice compared with wild-type mice. mPFC in Df(h15q13)/+ mice also showed a deficit of the inhibitory presynaptic marker GAD65, in addition to a reduction in dendritic arborization and spine density of pyramidal neurons from layers II/III. mPFC levels of GABA and glutamate neurotransmitters were not different between genotypes. Our results suggest that the 15q13.3 deletion modulates nonneuronal circuits in mPFC and confers molecular and morphometric alterations in the inhibitory and excitatory neurocircuits, respectively. These alterations potentially contribute to the phenotypes accompanied with the 15q13.3DS.
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Astrocitos/patología , Trastornos de los Cromosomas/patología , Trastornos de los Cromosomas/fisiopatología , Discapacidad Intelectual/patología , Discapacidad Intelectual/fisiopatología , Corteza Prefrontal/patología , Corteza Prefrontal/fisiopatología , Convulsiones/patología , Convulsiones/fisiopatología , Sinapsis/patología , Animales , Deleción Cromosómica , Cromosomas Humanos Par 15 , Modelos Animales de Enfermedad , Masculino , RatonesRESUMEN
BACKGROUND: Chronic kidney disease is a risk factor for premature development of coronary atherosclerosis and mortality. A high level of proprotein convertase subtilisin/kexin type 9 (PCSK9) is a recently recognized cardiovascular risk factor and has become the target of effective inhibitory treatment. In 167 kidney transplantation candidates, we aimed to: (i) compare levels of PCSK9 with those of healthy controls, (ii) examine the association between levels of PCSK9 and low-density lipoprotein cholesterol (LDL-c) and the degree of coronary artery disease (CAD) and (iii) evaluate if levels of PCSK9 predict major adverse cardiac events (MACE) and mortality. METHODS: Kidney transplant candidates (n = 167) underwent coronary computed tomography angiography (CCTA) and invasive coronary angiography (ICA) before transplantation. MACE and mortality data were extracted from the Western Denmark Heart Registry, a review of patient records and patient interviews. A group of 79 healthy subjects were used as controls. RESULTS: Mean PCSK9 levels did not differ between healthy controls and kidney transplant candidates. In patients not receiving lipid-lowering therapy, PCSK9 correlated positively with LDL-c (rho = 0.24, P < 0.05). Mean PCSK9 was similar in patients with and without obstructive CAD at both CCTA and ICA. In a multiple regression analysis, PCSK9 was associated with neither LDL-c (ß=-6.45, P = 0.44) nor coronary artery calcium score (ß=2.17, P = 0.84). During a follow-up of 3.7 years, PCSK9 levels were not associated with either MACE or mortality. CONCLUSIONS: The ability of PCSK9 levels to predict cardiovascular disease and prognosis does not seem to apply to a cohort of kidney transplant candidates.
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Biomarcadores/sangre , Enfermedades Cardiovasculares/diagnóstico , LDL-Colesterol/sangre , Proproteína Convertasa 9/sangre , Insuficiencia Renal Crónica/complicaciones , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/etiología , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Prospectivos , Insuficiencia Renal Crónica/sangre , Insuficiencia Renal Crónica/patología , Factores de RiesgoRESUMEN
BACKGROUND: Efforts to promote the cessation of harmful alcohol use are hindered by the affective and physiological components of alcohol withdrawal (AW), which can include life-threatening seizures. Although previous studies of AW and relapse have highlighted the detrimental role of stress, little is known about genetic risk factors. METHODS: We conducted a genome-wide association study of AW symptom count in uniformly assessed subjects with histories of serious AW, followed by additional genotyping in independent AW subjects. RESULTS: The top association signal for AW severity was in sortilin family neurotrophin receptor gene SORCS2 on chromosome 4 (European American meta-analysis n = 1,478, p = 4.3 × 10-9 ). There were no genome-wide significant findings in African Americans (n = 1,231). Bioinformatic analyses were conducted using publicly available high-throughput transcriptomic and epigenomic data sets, showing that in humans SORCS2 is most highly expressed in the nervous system. The identified SORCS2 risk haplotype is predicted to disrupt a stress hormone-modulated regulatory element that has tissue-specific activity in human hippocampus. We used human neural lineage cells to demonstrate in vitro a causal relationship between stress hormone levels and SORCS2 expression, and show that SORCS2 levels in culture are increased upon ethanol exposure and withdrawal. CONCLUSIONS: Taken together, these findings indicate that the pathophysiology of withdrawal may involve the effects of stress hormones on neurotrophic factor signaling. Further investigation of these pathways could produce new approaches to managing the aversive consequences of abrupt alcohol cessation.
Asunto(s)
Convulsiones por Abstinencia de Alcohol/genética , Receptores de Superficie Celular/genética , Adulto , Población Negra , Línea Celular , Biología Computacional , Dexametasona/farmacología , Femenino , Estudio de Asociación del Genoma Completo , Hipocampo/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Factores de Riesgo , Población BlancaRESUMEN
BACKGROUND: The identification of biomarkers for depression is of great clinical relevance as the diagnosis is currently subjective. Recent research points towards sortilin as a potential biomarker for depression, and the aim of the current study was to investigate the serum sortilin level in response to antidepressant treatment. METHODS: The study included 56 depressed individuals of which 41 responded to treatment. Depression scores and serum levels of sortilin were measured at baseline and after 12 weeks of antidepressant treatment. Statistical analyses were performed using Stata 13. RESULTS: The depression score and response to treatment were not predicted by the sortilin level. Likewise, we observed no significant change in serum sortilin levels following 12 weeks of antidepressant treatment. Furthermore, no association between the serum sortilin level and depression score was observed. CONCLUSION: The results do not point towards sortilin as a state-dependent biomarker.
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Proteínas Adaptadoras del Transporte Vesicular/sangre , Antidepresivos/uso terapéutico , Trastorno Depresivo Mayor/diagnóstico , Trastorno Depresivo Mayor/tratamiento farmacológico , Adulto , Biomarcadores/sangre , Trastorno Depresivo Mayor/sangre , Femenino , Humanos , Masculino , Escalas de Valoración Psiquiátrica , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Adulto JovenRESUMEN
Ischemic heart disease is the main cause of death worldwide and is accelerated by increased levels of low-density lipoprotein cholesterol (LDL-C). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a potent circulating regulator of LDL-C through its ability to induce degradation of the LDL receptor (LDLR) in the lysosome of hepatocytes. Only in the last few years, a number of breakthroughs in the understanding of PCSK9 biology have been reported illustrating how PCSK9 activity is tightly regulated at several levels by factors influencing its transcription, secretion, or by extracellular inactivation and clearance. Two humanized antibodies directed against the LDLR-binding site in PCSK9 received approval by the European and US authorities and additional PCSK9 directed therapeutics are climbing up the phases of clinical trials. The first outcome data of the PCSK9 inhibitor evolocumab reported a significant reduction in the composite endpoint (cardiovascular death, myocardial infarction, or stroke) and further outcome data are awaited. Meanwhile, it became evident that PCSK9 has (patho)physiological roles in several cardiovascular cells. In this review, we summarize and discuss the recent biological and clinical data on PCSK9, the regulation of PCSK9, its extra-hepatic activities focusing on cardiovascular cells, molecular concepts to target PCSK9, and finally briefly summarize the data of recent clinical studies.
Asunto(s)
Enfermedades Cardiovasculares/metabolismo , Proproteína Convertasa 9/metabolismo , Animales , Humanos , Receptores de LDL/metabolismoRESUMEN
BACKGROUND: Sortilin and SorCS2 are part of the Vps10p receptor family. They have both been studied in nervous tissue with several important functions revealed, while their expression and possible functions in developing peripheral tissue remain poorly understood. Here we deliver a thorough characterization of the prenatal localization of sortilin and SorCS2 in mouse peripheral tissue. RESULTS: Sortilin is highly expressed in epithelial tissues of the developing lung, nasal cavity, kidney, pancreas, salivary gland and developing intrahepatic bile ducts. Furthermore tissues such as the thyroid gland, developing cartilage and ossifying bone also show high expression of sortilin together with cell types such as megakaryocytes in the liver. SorCS2 is primarily expressed in mesodermally derived tissues such as striated muscle, adipose tissue, ossifying bone and general connective tissue throughout the body, as well as in lung epithelia. Furthermore, the adrenal gland and liver show high expression of SorCS2 in embryos 13.5 days old. CONCLUSIONS: The possible functions relating to the expression patterns of Sortilin and SorCS2 in development are numerous and hopefully this paper will help to generate new hypotheses to further our understanding of the Vps10p receptor family.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Ratones/embriología , Ratones/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Organogénesis , Receptores de Superficie Celular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo , Epitelio/embriología , Epitelio/metabolismo , Femenino , Riñón/embriología , Riñón/metabolismo , Pulmón/embriología , Pulmón/metabolismo , Masculino , Ratones/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Páncreas/embriología , Páncreas/metabolismo , Transporte de Proteínas , Receptores de Superficie Celular/genética , Glándulas Salivales/embriología , Glándulas Salivales/metabolismoRESUMEN
The development and progression of Alzheimer's disease is linked to excessive production of toxic amyloid-ß peptide, initiated by ß-secretase cleavage of the amyloid precursor protein (APP). In contrast, soluble APPα (sAPPα) generated by the α-secretase is known to stimulate dendritic branching and enhance synaptic function. Regulation of APP processing, and the shift from neurotrophic to neurotoxic APP metabolism remains poorly understood, but the cellular localization of APP and its interaction with various receptors is considered important. We here identify sortilin as a novel APP interaction partner. Like the related APP receptor SorLA, sortilin is highly expressed in the CNS, but whereas SorLA mainly colocalizes with APP in the soma, sortilin interacts with APP in neurites. The presence of sortilin promotes α-secretase cleavage of APP, unlike SorLA, which inhibits the generation of all soluble products. Also, sortilin and SorLA both bind and mediate internalization of sAPP but to different cellular compartments. The interaction involves the 6A domain of APP, present in both neuronal and non-neuronal APP isoforms. This is important as sAPP receptors described so far only bind the non-neuronal isoforms, leaving SorLA and sortilin as the only receptors for sAPP generated by neurons. Together, our findings establish sortilin, as a novel APP interaction partner that influences both production and cellular uptake of sAPP.
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Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas Relacionadas con Receptor de LDL/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Neuronas/metabolismo , Animales , Células CHO , Cricetinae , Células HEK293 , Humanos , Neuritas/metabolismo , Transporte de Proteínas/fisiologíaRESUMEN
The plasma concentration of the placentally derived proMBP (proform of eosinophil major basic protein) increases in pregnancy, and three different complexes containing proMBP have been isolated from pregnancy plasma and serum: a 2:2 complex with the metalloproteinase, PAPP-A (pregnancy-associated plasma protein-A), a 2:2 complex with AGT (angiotensinogen) and a 2:2:2 complex with AGT and complement C3dg. In the present study we show that during human pregnancy, all of the circulating proMBP exists in covalent complexes, bound to either PAPP-A or AGT. We also show that the proMBP-AGT complex constitutes the major fraction of circulating HMW (high-molecular weight) AGT in late pregnancy, and that this complex is able to further associate with complement C3 derivatives post-sampling. Clearance experiments in mice suggest that complement C3-based complexes are removed faster from the circulation compared to monomeric AGT and the proMBP-AGT complex. Furthermore, we have used recombinant proteins to analyse the formation of the proMBP-PAPP-A and the proMBP-AGT complexes, and we demonstrate that they are competing reactions, depending on the same cysteine residue of proMBP, but differentially on the redox potential, potentially important for the relative amounts of the complexes in vivo. These findings may be important physiologically, since the biochemical properties of the proteins change as a consequence of complex formation.
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Angiotensinógeno/química , Angiotensinógeno/metabolismo , Proteína Mayor Básica del Eosinófilo/química , Proteína Mayor Básica del Eosinófilo/metabolismo , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Proteoglicanos/química , Proteoglicanos/metabolismo , Animales , Biomarcadores/química , Biomarcadores/metabolismo , Femenino , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Peso Molecular , Oxidación-Reducción , Embarazo , Proteínas Gestacionales/química , Proteínas Gestacionales/metabolismoRESUMEN
BDNF-induced signaling is essential for the development of the central nervous system and critical for plasticity in adults. Mature BDNF signals through TrkB, while its precursor proBDNF employs p75(NTR), resulting in activation of signaling cascades with opposite effects on neuronal survival, growth cone decisions, and synaptic plasticity. Accordingly, variations in the genes encoding BDNF and its receptors sometimes have opposing influences in psychiatric disorders, and despite the vast literature, consensus is lacking about the behavioral consequences of disrupting the activity of the BDNF system in mice. To dissect the behavioral traits affected by dysfunctional BDNF/TrkB vs. proBDNF/p75(NTR) activity, we studied Bdnf(+/-), Ntrk2(+/-), and Ngfr(-/-) mice in parallel with respect to exploratory behavior, anxiety, startle, and fear acquisition. Our data reveal that the effect of proBDNF/BDNF and its receptors on behavior is more complex than expected. Strikingly, receptor-deficient mice displayed increased risk-taking behavior in the open field and elevated plus maze, whereas lack of proBDNF/BDNF had the opposite effect on mouse behavior. On the other hand, although TrkB signaling is instrumental for acquisition of fear memory in an inhibitory avoidance experiment, lack of p75(NTR) or proBDNF/BDNF conferred increased memory in this task. Importantly, none of the genotypes displayed any deficits in startle reflex, indicating unimpaired response to shock. The combined data illustrate an apparent paradox in the role of the BDNF system in controlling complex behavior and suggest that the individual components may also engage independently in separate signaling pathways.
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Ansiedad/fisiopatología , Factor Neurotrófico Derivado del Encéfalo/fisiología , Condicionamiento Psicológico/fisiología , Miedo/fisiología , Receptor trkB/fisiología , Receptores de Factor de Crecimiento Nervioso/fisiología , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Ratones , Ratones Noqueados , Modelos Animales , Receptor trkB/genética , Receptores de Factor de Crecimiento Nervioso/genéticaRESUMEN
Alzheimer's disease accounts for 60-70% of dementia cases. Current treatments are inadequate and there is a need to develop new approaches to drug discovery. Recently, in cancer, morphological profiling has been used in combination with high-throughput screening of small-molecule libraries in human cells in vitro. To test feasibility of this approach for Alzheimer's disease, we developed a cell morphology-based drug screen centred on the risk gene, SORL1 (which encodes the protein SORLA). Increased Alzheimer's disease risk has been repeatedly linked to variants in SORL1, particularly those conferring loss or decreased expression of SORLA, and lower SORL1 levels are observed in post-mortem brain samples from individuals with Alzheimer's disease. Consistent with its role in the endolysosomal pathway, SORL1 deletion is associated with enlarged endosomes in neural progenitor cells and neurons. We, therefore, hypothesized that multi-parametric, image-based cell phenotyping would identify features characteristic of SORL1 deletion. An automated morphological profiling method (Cell Painting) was adapted to neural progenitor cells and used to determine the phenotypic response of SORL1-/- neural progenitor cells to treatment with compounds from a small internationally approved drug library (TargetMol, 330 compounds). We detected distinct phenotypic signatures for SORL1-/- neural progenitor cells compared to isogenic wild-type controls. Furthermore, we identified 16 compounds (representing 14 drugs) that reversed the mutant morphological signatures in neural progenitor cells derived from three SORL1-/- induced pluripotent stem cell sub-clones. Network pharmacology analysis revealed the 16 compounds belonged to five mechanistic groups: 20S proteasome, aldehyde dehydrogenase, topoisomerase I and II, and DNA synthesis inhibitors. Enrichment analysis identified DNA synthesis/damage/repair, proteases/proteasome and metabolism as key pathways/biological processes. Prediction of novel targets revealed enrichment in pathways associated with neural cell function and Alzheimer's disease. Overall, this work suggests that (i) a quantitative phenotypic metric can distinguish induced pluripotent stem cell-derived SORL1-/- neural progenitor cells from isogenic wild-type controls and (ii) phenotypic screening combined with multi-parametric high-content image analysis is a viable option for drug repurposing and discovery in this human neural cell model of Alzheimer's disease.
RESUMEN
Neurotrophins comprise a group of neuronal growth factors that are essential for the development and maintenance of the nervous system. However, the immature pro-neurotrophins promote apoptosis by engaging in a complex with sortilin and the p75 neurotrophin receptor (p75(NTR)). To identify the interaction site between sortilin and p75(NTR), we analyzed binding between chimeric receptor constructs and truncated p75(NTR) variants by co-immunoprecipitation experiments, surface plasmon resonance analysis, and FRET. We found that complex formation between sortilin and p75(NTR) relies on contact points in the extracellular domains of the receptors. We also determined that the interaction critically depends on an extracellular juxtamembrane 23-amino acid sequence of p75(NTR). Functional studies further revealed an important regulatory function of the sortilin intracellular domain in p75(NTR)-regulated intramembrane proteolysis and apoptosis. Thus, although the intracellular domain of sortilin does not contribute to p75(NTR) binding, it does regulate the rates of p75(NTR) cleavage, which is required to mediate pro-neurotrophin-stimulated cell death.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Apoptosis/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteolisis , Receptores de Factor de Crecimiento Nervioso/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Línea Celular Tumoral , Células HEK293 , Humanos , Proteínas del Tejido Nervioso/genética , Mapeo Peptídico , Estructura Terciaria de Proteína , Ratas , Receptores de Factor de Crecimiento Nervioso/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Many different tissues and cell types exhibit regulated secretion of lipoprotein lipase (LPL). However, the sorting of LPL in the trans Golgi network has not, hitherto, been understood in detail. Here, we characterize the role of SorLA (officially known as SorLA-1 or sortilin-related receptor) in the intracellular trafficking of LPL. We found that LPL bound to SorLA under neutral and acidic conditions, and in cells this binding mainly occurred in vesicular structures. SorLA expression changed the subcellular distribution of LPL so it became more concentrated in endosomes. From the endosomes, LPL was further routed to the lysosomes, which resulted in a degradation of newly synthesized LPL. Consequently, an 80% reduction of LPL activity was observed in cells that expressed SorLA. By analogy, SorLA regulated the vesicle-like localization of LPL in primary neuronal cells. Thus, LPL binds to SorLA in the biosynthetic pathway and is subsequently transported to endosomes. As a result of this SorLA mediated-transport, newly synthesized LPL can be routed into specialized vesicles and eventually sent to degradation, and its activity thereby regulated.
Asunto(s)
Espacio Intracelular/metabolismo , Proteínas Relacionadas con Receptor de LDL/metabolismo , Lipoproteína Lipasa/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Animales , Bovinos , Línea Celular , Cricetinae , Humanos , Espacio Intracelular/química , Espacio Intracelular/enzimología , Espacio Intracelular/genética , Proteínas Relacionadas con Receptor de LDL/genética , Lipoproteína Lipasa/química , Lipoproteína Lipasa/genética , Proteínas de Transporte de Membrana/genética , Unión Proteica , Estructura Terciaria de Proteína , Transporte de ProteínasRESUMEN
Lowering blood cholesterol levels efficiently reduces the risk of developing atherosclerotic cardiovascular disease (ASCVD), including coronary artery disease (CAD), which is the main cause of death worldwide. CAD is caused by plaque formation, comprising cholesterol deposits in the coronary arteries. Proprotein convertase subtilisin kexin/type 9 (PCSK9) was discovered in the early 2000s and later identified as a key regulator of cholesterol metabolism. PCSK9 induces lysosomal degradation of the low-density lipoprotein (LDL) receptor in the liver, which is responsible for clearing LDL-cholesterol (LDL-C) from the circulation. Accordingly, gain-of-function PCSK9 mutations are causative of familial hypercholesterolemia, a severe condition with extremely high plasma cholesterol levels and increased ASCVD risk, whereas loss-of-function PCSK9 mutations are associated with very low LDL-C levels and protection against CAD. Since the discovery of PCSK9, extensive investigations in developing PCSK9 targeting therapies have been performed. The combined delineation of clear biology, genetic risk variants, and PCSK9 crystal structures have been major drivers in developing antagonistic molecules. Today, two antibody-based PCSK9 inhibitors have successfully progressed to clinical application and shown to be effective in reducing cholesterol levels and mitigating the risk of ASCVD events, including myocardial infarction, stroke, and death, without any major adverse effects. A third siRNA-based inhibitor has been FDA-approved but awaits cardiovascular outcome data. In this review, we outline the PCSK9 biology, focusing on the structure and nonsynonymous mutations reported in the PCSK9 gene and elaborate on PCSK9-lowering strategies under development. Finally, we discuss future perspectives with PCSK9 inhibition in other severe disorders beyond cardiovascular disease.
Asunto(s)
Anticolesterolemiantes , Aterosclerosis , Enfermedades Cardiovasculares , Enfermedad de la Arteria Coronaria , Hipercolesterolemia , Humanos , Proproteína Convertasa 9/genética , Proproteína Convertasa 9/metabolismo , LDL-Colesterol , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/prevención & control , Enfermedades Cardiovasculares/tratamiento farmacológico , Hipercolesterolemia/tratamiento farmacológico , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Aterosclerosis/tratamiento farmacológico , Anticolesterolemiantes/uso terapéuticoRESUMEN
INTRODUCTION: The use of abbreviations in patient records in Danish hospitals is extensive. The purpose of this study was to investigate the use of abbreviations, how they affect the comprehension, and how much time was saved writing abbreviations. METHODS: The study consisted of four sub-studies. First, a prospective collection of abbreviations from patient records. From these, three sentences were constructed and used in the other three sub-studies: a questionnaire for doctors concerning the use of abbreviations, an evaluation of time used to understand abbreviated versus non-abbreviated sentences, and a theoretical analysis of time saved by reducing the number of written characters. RESULTS: We found several abbreviations with multiple meanings. Writing a sentence with abbreviations saved 20 seconds. Comprehension of an abbreviated sentence took an extra 12-85 seconds. There was no difference in comprehension of abbreviations based on medical experience. Finally, data showed that neurologists' self-rated comprehension of complicated abbreviated sentences was very good. DISCUSSION: Numerous abbreviations were used in Danish patient records, many which could not be looked up. The use of abbreviations in patient records might not live up to the Danish record-keeping order, and we proposed four solutions to overcome the problem: more bureaucracy and administration; embrace and expand use of abbreviations; introduction of artificial intelligence to interpret abbreviations; or usage of speech recognition software in all Danish hospitals. FUNDING: none. TRIAL REGISTRATION: none.
Asunto(s)
Inteligencia Artificial , Médicos , Humanos , Estudios Prospectivos , Registros Médicos , Encuestas y CuestionariosRESUMEN
PCSK9 induces lysosomal degradation of the low-density lipoprotein (LDL) receptor (LDLR) in the liver, hereby preventing removal of LDL cholesterol from the circulation. Accordingly, PCSK9 inhibitory antibodies and siRNA potently reduce LDL cholesterol to unprecedented low levels and are approved for treatment of hypercholesterolemia. In addition, PCSK9 inactivation alters the levels of several other circulating lipid classes and species. Brain function is critically influenced by cholesterol and lipid composition. However, it remains unclear how the brain is affected long-term by the reduction in circulating lipids as achieved with potent lipid lowering therapeutics such as PCSK9 inhibitors. Furthermore, it is unknown if locally expressed PCSK9 affects neuronal circuits through regulation of receptor levels. We have studied the effect of lifelong low peripheral cholesterol levels on brain lipid composition and behavior in adult PCSK9 KO mice. In addition, we studied the effect of PCSK9 on neurons in culture and in vivo in the developing cerebral cortex. We found that PCSK9 reduced LDLR and neurite complexity in cultured neurons, but neither PCSK9 KO nor overexpression affected cortical development in vivo. Interestingly, PCSK9 deficiency resulted in changes of several lipid classes in the adult cortex and cerebellum. Despite the observed changes, PCSK9 KO mice had unchanged behavior compared to WT controls. In conclusion, our findings demonstrate that altered PCSK9 levels do not compromise brain development or function in mice, and are in line with clinical trials showing that PCSK9 inhibitors have no adverse effects on cognitive function.
RESUMEN
Peripheral nerve regeneration relies on the ability of Schwann cells to support the regrowth of damaged axons. Schwann cells re-differentiate when reestablishing contact with the sprouting axons, with large fibers becoming remyelinated and small nociceptive fibers ensheathed and collected into Remak bundles. We have previously described how the receptor sortilin facilitates neurotrophin signaling in peripheral neurons via regulated trafficking of Trk receptors. This study aims to characterize the effects of sortilin deletion on nerve regeneration following sciatic crush injury. We found that Sort1 - / - mice displayed functional motor recovery like that of WT mice, with no detectable differences in relation to nerve conduction velocities and morphological aspects of myelinated fibers. In contrast, we found abnormal ensheathment of regenerated C-fibers in injured Sort1 - / - mice, demonstrating a role of sortilin for Remak bundle formation following injury. Further studies on Schwann cell signaling pathways showed a significant reduction of MAPK/ERK, RSK, and CREB phosphorylation in Sort1 - / - Schwann cells after stimulation with neurotrophin-3 (NT-3), while Schwann cell migration and myelination remained unaffected. In conclusion, our results demonstrate that loss of sortilin blunts NT-3 signaling in Schwann cells which might contribute to the impaired Remak bundle regeneration after sciatic nerve injury.