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1.
Mol Psychiatry ; 2023 Jul 04.
Artículo en Inglés | MEDLINE | ID: mdl-37402854

RESUMEN

While most of the efforts to uncover mechanisms contributing to bipolar disorder (BD) focused on phenotypes at the mature neuron stage, little research has considered events that may occur during earlier timepoints of neurodevelopment. Further, although aberrant calcium (Ca2+) signaling has been implicated in the etiology of this condition, the possible contribution of store-operated Ca2+ entry (SOCE) is not well understood. Here, we report Ca2+ and developmental dysregulations related to SOCE in BD patient induced pluripotent stem cell (iPSC)-derived neural progenitor cells (BD-NPCs) and cortical-like glutamatergic neurons. First, using a Ca2+ re-addition assay we found that BD-NPCs and neurons had attenuated SOCE. Intrigued by this finding, we then performed RNA-sequencing and uncovered a unique transcriptome profile in BD-NPCs suggesting accelerated neurodifferentiation. Consistent with these results, we measured a slower rate of proliferation, increased neurite outgrowth, and decreased size in neurosphere formations with BD-NPCs. Also, we observed decreased subventricular areas in developing BD cerebral organoids. Finally, BD NPCs demonstrated high expression of the let-7 family while BD neurons had increased miR-34a, both being microRNAs previously implicated in neurodevelopmental deviations and BD etiology. In summary, we present evidence supporting an accelerated transition towards the neuronal stage in BD-NPCs that may be indicative of early pathophysiological features of the disorder.

2.
J Am Soc Nephrol ; 33(8): 1546-1567, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35906089

RESUMEN

BACKGROUND: Maintenance of the kidney filtration barrier requires coordinated interactions between podocytes and the underlying glomerular basement membrane (GBM). GBM ligands bind podocyte integrins, which triggers actin-based signaling events critical for adhesion. Nck1/2 adaptors have emerged as essential regulators of podocyte cytoskeletal dynamics. However, the precise signaling mechanisms mediated by Nck1/2 adaptors in podocytes remain to be fully elucidated. METHODS: We generated podocytes deficient in Nck1 and Nck2 and used transcriptomic approaches to profile expression differences. Proteomic techniques identified specific binding partners for Nck1 and Nck2 in podocytes. We used cultured podocytes and mice deficient in Nck1 and/or Nck2, along with podocyte injury models, to comprehensively verify our findings. RESULTS: Compound loss of Nck1/2 altered expression of genes involved in actin binding, cell adhesion, and extracellular matrix composition. Accordingly, Nck1/2-deficient podocytes showed defects in actin organization and cell adhesion in vitro, with podocyte detachment and altered GBM morphology present in vivo. We identified distinct interactomes for Nck1 and Nck2 and uncovered a mechanism by which Nck1 and Nck2 cooperate to regulate actin bundling at focal adhesions via α actinin-4. Furthermore, loss of Nck1 or Nck2 resulted in increased matrix deposition in vivo, with more prominent defects in Nck2-deficient mice, consistent with enhanced susceptibility to podocyte injury. CONCLUSION: These findings reveal distinct, yet complementary, roles for Nck proteins in regulating podocyte adhesion, controlling GBM composition, and sustaining filtration barrier integrity.


Asunto(s)
Podocitos , Actinina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Membrana Basal Glomerular/metabolismo , Ratones , Proteínas Oncogénicas/metabolismo , Podocitos/metabolismo , Proteómica
3.
Int J Mol Sci ; 22(4)2021 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-33673213

RESUMEN

Glioblastoma multiforme (GBM) is amongst the deadliest of human cancers, with a median survival rate of just over one year following diagnosis. Characterized by rapid proliferation and diffuse infiltration into the brain, GBM is notoriously difficult to treat, with tumor cells showing limited response to existing therapies and eventually developing resistance to these interventions. As such, there is intense interest in better understanding the molecular alterations in GBM to guide the development of more efficient targeted therapies. GBM tumors can be classified into several molecular subtypes which have distinct genetic signatures, and they show aberrant activation of numerous signal transduction pathways, particularly those connected to receptor tyrosine kinases (RTKs) which control glioma cell growth, survival, migration, invasion, and angiogenesis. There are also non-canonical modes of RTK signaling found in GBM, which involve G-protein-coupled receptors and calcium channels. This review uses The Cancer Genome Atlas (TCGA) GBM dataset in combination with a data-mining approach to summarize disease characteristics, with a focus on select molecular pathways that drive GBM pathogenesis. We also present a unique genomic survey of RTKs that are frequently altered in GBM subtypes, as well as catalog the GBM disease association scores for all RTKs. Lastly, we discuss current RTK targeted therapies and highlight emerging directions in GBM research.


Asunto(s)
Neoplasias Encefálicas/enzimología , Proliferación Celular , Glioblastoma/enzimología , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Glioblastoma/genética , Glioblastoma/patología , Humanos , Proteínas de Neoplasias/genética , Fosforilación/genética , Proteínas Tirosina Quinasas Receptoras/genética
4.
J Am Soc Nephrol ; 29(1): 92-103, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29018139

RESUMEN

Nephrin is a key structural component of the podocyte slit diaphragm, and proper expression of nephrin on the cell surface is critical to ensure integrity of the blood filtration barrier. Maintenance of nephrin within this unique cell junction has been proposed to require dynamic phosphorylation events and endocytic recycling, although the molecular mechanisms that control this interplay are poorly understood. Here, we investigated the possibility that the phosphotyrosine adaptor protein ShcA regulates nephrin turnover. Western blotting and immunostaining analysis confirmed that ShcA is expressed in podocytes. In immunoprecipitation and pulldown assays, ShcA, via its SH2 domain, was associated with several phosphorylated tyrosine residues on nephrin. Overexpression of ShcA promoted nephrin tyrosine phosphorylation and reduced nephrin signaling and cell surface expression in vitro In a rat model of reversible podocyte injury and proteinuria, phosphorylated nephrin temporally colocalized with endocytic structures coincident with upregulation of ShcA expression. In vivo biotinylation assays confirmed that nephrin expression decreased at the cell surface and correspondingly increased in the cytosol during the injury time course. Finally, immunostaining in kidney biopsy specimens demonstrated overexpression of ShcA in several human proteinuric kidney diseases compared with normal conditions. Our results suggest that increases in ShcA perturb nephrin phosphosignaling dynamics, leading to aberrant nephrin turnover and slit diaphragm disassembly.


Asunto(s)
Endocitosis , Enfermedades Renales/metabolismo , Proteínas de la Membrana/metabolismo , Podocitos/metabolismo , Proteinuria/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/metabolismo , Animales , Biotinilación , Membrana Celular/metabolismo , Citosol/metabolismo , Células HEK293 , Humanos , Enfermedades Renales/patología , Masculino , Nefrosis/inducido químicamente , Fosforilación , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/genética , Tirosina/metabolismo , Regulación hacia Arriba
5.
J Biol Chem ; 292(14): 5748-5759, 2017 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-28213521

RESUMEN

Proteins of the Src homology and collagen (Shc) family are typically involved in signal transduction events involving Ras/MAPK and PI3K/Akt pathways. In the nervous system, they function proximal to the neurotrophic factors that regulate cell survival, differentiation, and neuron-specific characteristics. The least characterized homolog, ShcD, is robustly expressed in the developing and mature nervous system, but its contributions to neural cell circuitry are largely uncharted. We now report that ShcD binds to active Ret, TrkA, and TrkB neurotrophic factor receptors predominantly via its phosphotyrosine-binding (PTB) domain. However, in contrast to the conventional Shc adaptors, ShcD suppresses distal phosphorylation of the Erk MAPK. Accordingly, genetic knock-out of mouse ShcD enhances Erk phosphorylation in the brain. In cultured cells, this capacity is tightly aligned to phosphorylation of ShcD CH1 region tyrosine motifs, which serve as docking platforms for signal transducers, such as Grb2. Erk suppression is relieved through independent mutagenesis of the PTB domain and the CH1 tyrosine residues, and successive substitution of these tyrosines breaks the interaction between ShcD and Grb2, thereby promoting TrkB-Grb2 association. Erk phosphorylation can also be restored in the presence of wild type ShcD through Grb2 overexpression. Conversely, mutation of the ShcD SH2 domain results in enhanced repression of Erk. Although the SH2 domain is a less common binding interface in Shc proteins, we demonstrate that it associates with the Ptpn11 (Shp2) phosphatase, which in turn regulates ShcD tyrosine phosphorylation. We therefore propose a model whereby ShcD competes with neurotrophic receptors for Grb2 binding and opposes activation of the MAPK cascade.


Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Glicoproteínas de Membrana/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-ret/metabolismo , Receptor trkA/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Secuencias de Aminoácidos , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/genética , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Fosforilación/fisiología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-ret/genética , Receptor trkA/genética , Receptor trkB , Proteínas Adaptadoras de la Señalización Shc/genética
6.
PLoS One ; 18(11): e0291382, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37939060

RESUMEN

Lyme disease, caused by vector-borne Borrelia bacteria, can present with diverse multi-system symptoms that resemble other conditions. The objective of this study was to evaluate disease presentations and Borrelia seroreactivity in individuals experiencing a spectrum of chronic and complex illnesses. We recruited 157 participants from Eastern Canada who reported one or more diagnoses of Lyme disease, neurological, rheumatic, autoimmune, inflammatory, gastrointestinal, or cardiovascular illnesses, or were asymptomatic and presumed healthy. Intake categories were used to classify participants based on their perceived proximity to Lyme disease, distinguishing between those with a disclosed history of Borrelia infection, those with lookalike conditions (e.g. fibromyalgia syndrome), and those with unrelated ailments (e.g. intestinal polyps). Participants completed three questionnaires, the SF-36 v1, SIQR, and HMQ, to capture symptoms and functional burden, and provided blood serum for analysis at an accredited diagnostic lab. Two-tiered IgG and IgM serological assessments (whole cell ELISA and Western blot) were performed in a blinded fashion on all samples. The pattern of symptoms and functional burden were similarly profound in the presumptive Lyme and Lyme-like disease categories. Borrelia seroprevalence across the study cohort was 10% for each of IgG and IgM, and occurred within and beyond the Lyme disease intake category. Western blot positivity in the absence of reactive ELISA was also substantial. Fibromyalgia was the most common individual diagnostic tag disclosed by two-tier IgG-positive participants who did not report a history of Lyme disease. Within the IgG seropositive cohort, the presence of antibodies against the 31 kDa Outer Surface Protein A (OspA) was associated with significantly better health outcomes. Previously, this marker has been linked to treatment-refractory Lyme arthritis. Overall, our findings support prior observations of phenotypic overlap between Lyme and other diseases. Seropositivity associated with non-specific symptoms and functional impairment warrants further mechanistic investigation and therapeutic optimization.


Asunto(s)
Borrelia burgdorferi , Borrelia , Fibromialgia , Enfermedad de Lyme , Humanos , Fibromialgia/epidemiología , Estudios Seroepidemiológicos , Canadá/epidemiología , Enfermedad de Lyme/diagnóstico , Enfermedad de Lyme/epidemiología , Enfermedad Crónica , Anticuerpos Antibacterianos , Inmunoglobulina G , Inmunoglobulina M
7.
Mol Cancer Res ; 19(5): 757-770, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33495401

RESUMEN

Gliomas are characterized by diffuse infiltration of tumor cells into surrounding brain tissue, and this highly invasive nature contributes to disease recurrence and poor patient outcomes. The molecular mechanisms underlying glioma cell invasion remain incompletely understood, limiting development of new targeted therapies. Here, we have identified phosphotyrosine adaptor protein ShcD as upregulated in malignant glioma and shown that it associates with receptor tyrosine kinase Tie2 to facilitate invasion. In human glioma cells, we find that expression of ShcD and Tie2 increases invasion, and this significant synergistic effect is disrupted with a ShcD mutant that cannot bind Tie2 or hyperphosphorylate the receptor. Expression of ShcD and/or Tie2 further increases invadopodia formation and matrix degradation in U87 glioma cells. In a coculture model, we show that U87-derived tumor spheroids expressing both ShcD and Tie2 display enhanced infiltration into cerebral organoids. Mechanistically, we identify changes in focal adhesion kinase phosphorylation in the presence of ShcD and/or Tie2 in U87 cells upon Tie2 activation. Finally, we identify a strong correlation between transcript levels of ShcD and Tie2 signaling components as well as N-cadherin in advanced gliomas and those with classical or mesenchymal subtypes, and we show that elevated expression of ShcD correlates with a significant reduction in patient survival in higher grade gliomas with mesenchymal signature. Altogether, our data highlight a novel Tie2-ShcD signaling axis in glioma cell invasion, which may be of clinical significance. IMPLICATIONS: ShcD cooperates with Tie2 to promote glioma cell invasion and its elevated expression correlates with poor patient outcome in advanced gliomas.


Asunto(s)
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Receptor TIE-2/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Secuencia de Aminoácidos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Glioma/genética , Glioma/patología , Células HEK293 , Humanos , Invasividad Neoplásica , Transfección
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