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1.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-33810326

RESUMEN

Musashi-1 (MSI1) is an RNA-binding protein that regulates progenitor cells in adult and developing organisms to maintain self-renewal capacities. The role of musashi-1 in the bone healing environment and its relation with other osteogenic factors is unknown. In the current study, we analyze the expression of MSI1 in an experimental model of rat femoral bone fractures. We also analyze the relation between MSI1 expression and the expression of two osteogenic markers: periostin (POSTN) and runt-related transcription factor 2 (RUNX2). We use histological, immunohistochemical, and qPCR techniques to evaluate bone healing and the expression of MSI1, POSTN, and RUNX2 over time (4, 7, and 14 days). We compare our findings with non-fractured controls. We find that in bone calluses, the number of cells expressing MSI1 and RUNX2 increase over time and the intensity of POSTN expression decreases over time. Within bone calluses, we find the presence of MSI1 expression in mesenchymal stromal cells, osteoblasts, and osteocytes but not in hypertrophic chondrocytes. After 14 days, the expression of MSI1, POSTN, and RUNX2 was significantly correlated. Thus, we conclude that musashi-1 potentially serves in the osteogenic differentiation of mesenchymal stromal cells and bone healing. Therefore, further studies are needed to determine the possibility of musashi-1's role as a clinical biomarker of bone healing and therapeutic agent for bone regeneration.


Asunto(s)
Curación de Fractura , Proteínas del Tejido Nervioso/metabolismo , Osteogénesis , Proteínas de Unión al ARN/metabolismo , Animales , Callo Óseo/citología , Callo Óseo/metabolismo , Callo Óseo/fisiología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Condrocitos/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Masculino , Células Madre Mesenquimatosas/metabolismo , Proteínas del Tejido Nervioso/genética , Osteoblastos/metabolismo , Osteocitos/metabolismo , Proteínas de Unión al ARN/genética , Ratas , Ratas Wistar
2.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-33802633

RESUMEN

The current study was designed to investigate the protective role of diosmin against cyclophosphamide-induced premature ovarian insufficiency (POI). Female Swiss albino rats received a single intraperitoneal dose of cyclophosphamide (200 mg/kg) followed by 8 mg/kg/day for the next 15 consecutive days either alone or in combination with oral diosmin at 50 or 100 mg/kg. Histopathological examination of ovarian tissues, hormonal assays for follicle stimulating hormone (FSH), estradiol (E2), and anti-Mullerian hormone (AMH), assessment of the oxidative stress status, as well as measurement of the relative expression of miRNA-145 and its target genes [vascular endothelial growth factor B (VEGF-B) and regulator of cell cycle (RGC32)] were performed. Diosmin treatment ameliorated the levels of E2, AMH, and oxidative stress markers. Additionally, both low and high diosmin doses significantly reduced the histopathological alterations and nearly preserved the normal ovarian reserve. MiRNA-145 expression was upregulated after treatment with diosmin high dose. miRNA-145 target genes were over-expressed after both low and high diosmin administration. Based on our findings, diosmin has a dose-dependent protective effect against cyclophosphamide-induced ovarian toxicity in rats.


Asunto(s)
Diosmina/uso terapéutico , Insuficiencia Ovárica Primaria/inducido químicamente , Insuficiencia Ovárica Primaria/tratamiento farmacológico , Animales , Biomarcadores/metabolismo , Peso Corporal/efectos de los fármacos , Caspasa 3/metabolismo , Catalasa/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Colágeno/metabolismo , Ciclofosfamida , Diosmina/farmacología , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hormonas/sangre , Malondialdehído/sangre , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Tamaño de los Órganos/efectos de los fármacos , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/crecimiento & desarrollo , Folículo Ovárico/patología , Estrés Oxidativo/efectos de los fármacos , Insuficiencia Ovárica Primaria/sangre , Insuficiencia Ovárica Primaria/patología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo
3.
Nat Commun ; 12(1): 2182, 2021 04 12.
Artículo en Inglés | MEDLINE | ID: mdl-33846329

RESUMEN

Autosomal genetic analyses of blood lipids have yielded key insights for coronary heart disease (CHD). However, X chromosome genetic variation is understudied for blood lipids in large sample sizes. We now analyze genetic and blood lipid data in a high-coverage whole X chromosome sequencing study of 65,322 multi-ancestry participants and perform replication among 456,893 European participants. Common alleles on chromosome Xq23 are strongly associated with reduced total cholesterol, LDL cholesterol, and triglycerides (min P = 8.5 × 10-72), with similar effects for males and females. Chromosome Xq23 lipid-lowering alleles are associated with reduced odds for CHD among 42,545 cases and 591,247 controls (P = 1.7 × 10-4), and reduced odds for diabetes mellitus type 2 among 54,095 cases and 573,885 controls (P = 1.4 × 10-5). Although we observe an association with increased BMI, waist-to-hip ratio adjusted for BMI is reduced, bioimpedance analyses indicate increased gluteofemoral fat, and abdominal MRI analyses indicate reduced visceral adiposity. Co-localization analyses strongly correlate increased CHRDL1 gene expression, particularly in adipose tissue, with reduced concentrations of blood lipids.


Asunto(s)
Cromosomas Humanos X/genética , Lípidos/sangre , Proteínas del Ojo/metabolismo , Femenino , Regulación de la Expresión Génica , Estudios de Asociación Genética , Sitios Genéticos , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Proteínas del Tejido Nervioso/metabolismo , Fenómica , Polimorfismo de Nucleótido Simple/genética , Tejido Subcutáneo/metabolismo , Secuenciación Completa del Genoma
4.
Int J Mol Sci ; 22(6)2021 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-33807010

RESUMEN

Glycine N-methyltransferase (GNMT) regulates S-adenosylmethionine (SAMe), a methyl donor in methylation. Over-expressed SAMe may cause neurogenic capacity reduction and memory impairment. GNMT knockout mice (GNMT-KO) was applied as an experimental model to evaluate its effect on neurons. In this study, proteins from brain tissues were studied using proteomic approaches, Haemotoxylin and Eosin staining, immunohistochemistry, Western blotting, and ingenuity pathway analysis. The expression of Receptor-interacting protein 1(RIPK1) and Caspase 3 were up-regulated and activity-dependent neuroprotective protein (ADNP) was down-regulated in GNMT-KO mice regardless of the age. Besides, proteins related to neuropathology, such as excitatory amino acid transporter 2, calcium/calmodulin-dependent protein kinase type II subunit alpha, and Cu-Zn superoxide dismutase were found only in the group of aged wild-type mice; 4-aminobutyrate amino transferase, limbic system-associated membrane protein, sodium- and chloride-dependent GABA transporter 3 and ProSAAS were found only in the group of young GNMT-KO mice and are related to function of neurons; serum albumin and Rho GDP dissociation inhibitor 1 were found only in the group of aged GNMT-KO mice and are connected to neurodegenerative disorders. With proteomic analyses, a pathway involving Gonadotropin-releasing hormone (GnRH) signal was found to be associated with aging. The GnRH pathway could provide additional information on the mechanism of aging and non-aging related neurodegeneration, and these protein markers may be served in developing future therapeutic treatments to ameliorate aging and prevent diseases.


Asunto(s)
Envejecimiento/metabolismo , Biomarcadores , Enfermedades Neurodegenerativas/metabolismo , Animales , Biomarcadores/metabolismo , Encéfalo , Senescencia Celular , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Inmunohistoquímica , Ratones , Proteínas del Tejido Nervioso/metabolismo , Enfermedades Neurodegenerativas/diagnóstico , Enfermedades Neurodegenerativas/etiología , Neuronas/metabolismo , Pronóstico , Proteoma , Proteómica/métodos , Transducción de Señal/efectos de los fármacos
5.
Int J Mol Sci ; 22(6)2021 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-33809984

RESUMEN

The Perlecan-Semaphorin 3A-Plexin A1-Neuropilin-1 (PSPN) Complex at the cell surface of prostate cancer (PCa) cells influences cell-cell cohesion and dyscohesion. We investigated matrix metalloproteinase-7/matrilysin (MMP-7)'s ability to digest components of the PSPN Complex in bone metastatic PCa cells using in silico analyses and in vitro experiments. Results demonstrated that in addition to the heparan sulfate proteoglycan, perlecan, all components of the PSPN Complex were degraded by MMP-7. To investigate the functional consequences of PSPN Complex cleavage, we developed a preformed microtumor model to examine initiation of cell dispersion after MMP-7 digestion. We found that while perlecan fully decorated with glycosaminoglycan limited dispersion of PCa microtumors, MMP-7 initiated rapid dyscohesion and migration even with perlecan present. Additionally, we found that a bioactive peptide (PLN4) found in perlecan domain IV in a region subject to digestion by MMP-7 further enhanced cell dispersion along with MMP-7. We found that digestion of the PSPN Complex with MMP-7 destabilized cell-cell junctions in microtumors evidenced by loss of co-registration of E-cadherin and F-actin. We conclude that MMP-7 plays a key functional role in PCa cell transition from a cohesive, indolent phenotype to a dyscohesive, migratory phenotype favoring production of circulating tumor cells and metastasis to bone.


Asunto(s)
Metaloproteinasa 7 de la Matriz/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neoplasias de la Próstata/metabolismo , Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Movimiento Celular , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Modelos Biológicos , Neuropilina-1/metabolismo , Neoplasias de la Próstata/etiología , Unión Proteica , Proteolisis
6.
Nat Commun ; 12(1): 1465, 2021 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-33674582

RESUMEN

Atoh7 has been believed to be essential for establishing the retinal ganglion cell (RGC) lineage, and Pou4f2 and Isl1 are known to regulate RGC specification and differentiation. Here we report our further study of the roles of these transcription factors. Using bulk RNA-seq, we identify genes regulated by the three transcription factors, which expand our understanding of the scope of downstream events. Using scRNA-seq on wild-type and mutant retinal cells, we reveal a transitional cell state of retinal progenitor cells (RPCs) co-marked by Atoh7 and other genes for different lineages and shared by all early retinal lineages. We further discover the unexpected emergence of the RGC lineage in the absence of Atoh7. We conclude that competence of RPCs for different retinal fates is defined by lineage-specific genes co-expressed in the transitional state and that Atoh7 defines the RGC competence and collaborates with other factors to shepherd transitional RPCs to the RGC lineage.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Retina/metabolismo , Células Ganglionares de la Retina/metabolismo , Transcriptoma , Animales , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Mutación con Pérdida de Función , Ratones , ARN Citoplasmático Pequeño , Análisis de Secuencia , Células Madre , Factor de Transcripción Brn-3B/genética , Factor de Transcripción Brn-3B/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
7.
Int J Mol Sci ; 22(4)2021 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-33672695

RESUMEN

Circadian desynchrony induced by a long period of irregular feeding leads to metabolic diseases, such as obesity and diabetes mellitus. The recently identified neurosecretory protein GL (NPGL) and neurosecretory protein GM (NPGM) are hypothalamic small proteins that stimulate food intake and fat accumulation in several animals. To clarify the mechanisms that evoke feeding behavior and induce energy metabolism at the appropriate times in accordance with a circadian rhythm, diurnal fluctuations in Npgl and Npgm mRNA expression were investigated in mice. Quantitative RT-PCR analysis revealed that the mRNAs of these two genes were highly expressed in the mediobasal hypothalamus during the active dark phase under ad libitum feeding. In mice restricted to 3 h of feeding during the inactive light phase, the Npgl mRNA level was augmented in the moment prior to the feeding period and the midnight peak of Npgm mRNA was attenuated. Moreover, the mRNA expression levels of clock genes, feeding regulatory neuropeptides, and lipid metabolic enzymes in the central and peripheral tissues were comparable to those of central Npgl and Npgm. These data suggest that Npgl and Npgm transcription fluctuates daily and likely mediates feeding behavior and/or energy metabolism at an appropriate time according to the meal timing.


Asunto(s)
Conducta Alimentaria/fisiología , Regulación de la Expresión Génica , Hipotálamo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Análisis de Varianza , Animales , Anorexia/sangre , Anorexia/genética , Glucemia/metabolismo , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Perfilación de la Expresión Génica , Insulina/sangre , Metabolismo de los Lípidos/genética , Masculino , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/genética , Orexinas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Tiempo
8.
Arch Insect Biochem Physiol ; 106(4): e21779, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33660341

RESUMEN

Shrub (CG8055) encodes the vps32/snf7 protein, a filament-forming subunit of the ESCRT (endosomal sorting complexes required for transport)-III complex involved in inward membrane budding. It was reported that shrub was required for abscission in female germline stem cells. In this study, we showed that the expression level of shrub in the testis was significantly higher than that in the ovary of 1-day-old Drosophila melanogaster, suggesting a role in male reproduction. Then we used nosGal4 driver to knockdown shrub specifically in the fly testis and found that this resulted in a significantly lower paternal effect egg hatch rate relative to the control group. Immunofluorescence staining showed that shrub knockdown in fly testes caused an accumulation of early-stage germ cells and lack of spectrin caps. In the late stages (spermiogenesis), the control testis contained multiple compacted spermatid bundles and individualization complexes (ICs) consisting of actin cones, whereas there were scattered spermatid nuclei and only a few ICs with disorganized actin cones in the shrub knockdown testis. Finally, the control seminal vesicle was full of mature sperms with needle-like heads, but in shrub knockdown testis 75% of seminal vesicles had no mature sperms. We also found that knockdown of shrub in fly testes led to upregulated expression of several cytoskeleton-associated genes, and an accumulation of ubiquitylated proteins. These results suggest that knockdown of shrub in fly testes might damage spermatogenesis by affecting transportability.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas del Tejido Nervioso/metabolismo , Espermatogénesis/fisiología , Animales , Drosophila melanogaster/metabolismo , Drosophila melanogaster/fisiología , Femenino , Masculino , Ovario/metabolismo , Testículo/metabolismo
9.
Nat Commun ; 12(1): 1848, 2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33758193

RESUMEN

Neuroligin 3 (NLGN3) and neurexins (NRXNs) constitute a canonical transsynaptic cell-adhesion pair, which has been implicated in autism. In autism spectrum disorder (ASD) development of sociality can be impaired. However, the molecular mechanism underlying NLGN3-mediated social development is unclear. Here, we identify non-canonical interactions between NLGN3 and protein tyrosine phosphatase δ (PTPδ) splice variants, competing with NRXN binding. NLGN3-PTPδ complex structure revealed a splicing-dependent interaction mode and competition mechanism between PTPδ and NRXNs. Mice carrying a NLGN3 mutation that selectively impairs NLGN3-NRXN interaction show increased sociability, whereas mice where the NLGN3-PTPδ interaction is impaired exhibit impaired social behavior and enhanced motor learning, with imbalance in excitatory/inhibitory synaptic protein expressions, as reported in the Nlgn3 R451C autism model. At neuronal level, the autism-related Nlgn3 R451C mutation causes selective impairment in the non-canonical pathway. Our findings suggest that canonical and non-canonical NLGN3 pathways compete and regulate the development of sociality.


Asunto(s)
Trastorno del Espectro Autista/genética , Proteínas de Unión al Calcio/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/metabolismo , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Secuencia de Aminoácidos , Animales , Trastorno del Espectro Autista/metabolismo , Escala de Evaluación de la Conducta , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Moléculas de Adhesión Celular Neuronal/química , Moléculas de Adhesión Celular Neuronal/genética , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Humanos , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Moléculas de Adhesión de Célula Nerviosa/química , Moléculas de Adhesión de Célula Nerviosa/genética , Dominios Proteicos , Empalme de Proteína , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genética , Proteínas Recombinantes , Transducción de Señal/genética , Transducción de Señal/fisiología , Conducta Social , Sinapsis/genética
10.
Nat Commun ; 12(1): 1832, 2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33758201

RESUMEN

Synthetic glucocorticoids (GCs), one of the most effective treatments for chronic inflammatory and autoimmune conditions in children, have adverse effects on the growing skeleton. GCs inhibit angiogenesis in growing bone, but the underlying mechanisms remain unclear. Here, we show that GC treatment in young mice induces vascular endothelial cell senescence in metaphysis of long bone, and that inhibition of endothelial cell senescence improves GC-impaired bone angiogenesis with coupled osteogenesis. We identify angiogenin (ANG), a ribonuclease with pro-angiogenic activity, secreted by osteoclasts as a key factor for protecting the neighboring vascular cells against senescence. ANG maintains the proliferative activity of endothelial cells through plexin-B2 (PLXNB2)-mediated transcription of ribosomal RNA (rRNA). GC treatment inhibits ANG production by suppressing osteoclast formation in metaphysis, resulting in impaired endothelial cell rRNA transcription and subsequent cellular senescence. These findings reveal the role of metaphyseal blood vessel senescence in mediating the action of GCs on growing skeleton and establish the ANG/PLXNB2 axis as a molecular basis for the osteoclast-vascular interplay in skeletal angiogenesis.


Asunto(s)
Senescencia Celular/efectos de los fármacos , Células Endoteliales/metabolismo , Glucocorticoides/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Osteoclastos/metabolismo , Ribonucleasa Pancreática/metabolismo , Animales , Apoptosis/efectos de los fármacos , Desarrollo Óseo/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Senescencia Celular/genética , Células Endoteliales/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Metilprednisolona/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neovascularización Patológica , Proteínas del Tejido Nervioso/genética , Osteoclastos/efectos de los fármacos , Osteoclastos/enzimología , Osteogénesis/efectos de los fármacos , ARN Ribosómico/biosíntesis , ARN Interferente Pequeño , Proteínas Recombinantes , Ribonucleasa Pancreática/genética , Ribonucleasa Pancreática/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Tomógrafos Computarizados por Rayos X
11.
Nat Commun ; 12(1): 1932, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33771998

RESUMEN

The physical distance between presynaptic Ca2+ channels and the Ca2+ sensors triggering the release of neurotransmitter-containing vesicles regulates short-term plasticity (STP). While STP is highly diversified across synapse types, the computational and behavioral relevance of this diversity remains unclear. In the Drosophila brain, at nanoscale level, we can distinguish distinct coupling distances between Ca2+ channels and the (m)unc13 family priming factors, Unc13A and Unc13B. Importantly, coupling distance defines release components with distinct STP characteristics. Here, we show that while Unc13A and Unc13B both contribute to synaptic signalling, they play distinct roles in neural decoding of olfactory information at excitatory projection neuron (ePN) output synapses. Unc13A clusters closer to Ca2+ channels than Unc13B, specifically promoting fast phasic signal transfer. Reduction of Unc13A in ePNs attenuates responses to both aversive and appetitive stimuli, while reduction of Unc13B provokes a general shift towards appetitive values. Collectively, we provide direct genetic evidence that release components of distinct nanoscopic coupling distances differentially control STP to play distinct roles in neural decoding of sensory information.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Plasticidad Neuronal/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Animales , Animales Modificados Genéticamente , Conducta Apetitiva/fisiología , Calcio/metabolismo , Canales de Calcio/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Femenino , Interneuronas/metabolismo , Interneuronas/fisiología , Proteínas de la Membrana/genética , Microscopía Confocal , Proteínas del Tejido Nervioso/genética , Plasticidad Neuronal/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN , Sinapsis/metabolismo , Transmisión Sináptica/genética , Vesículas Sinápticas/metabolismo
12.
Nat Commun ; 12(1): 1920, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33772001

RESUMEN

Adipogenesis associated Mth938 domain containing (AAMDC) represents an uncharacterized oncogene amplified in aggressive estrogen receptor-positive breast cancers. We uncover that AAMDC regulates the expression of several metabolic enzymes involved in the one-carbon folate and methionine cycles, and lipid metabolism. We show that AAMDC controls PI3K-AKT-mTOR signaling, regulating the translation of ATF4 and MYC and modulating the transcriptional activity of AAMDC-dependent promoters. High AAMDC expression is associated with sensitization to dactolisib and everolimus, and these PI3K-mTOR inhibitors exhibit synergistic interactions with anti-estrogens in IntClust2 models. Ectopic AAMDC expression is sufficient to activate AKT signaling, resulting in estrogen-independent tumor growth. Thus, AAMDC-overexpressing tumors may be sensitive to PI3K-mTORC1 blockers in combination with anti-estrogens. Lastly, we provide evidence that AAMDC can interact with the RabGTPase-activating protein RabGAP1L, and that AAMDC, RabGAP1L, and Rab7a colocalize in endolysosomes. The discovery of the RabGAP1L-AAMDC assembly platform provides insights for the design of selective blockers to target malignancies having the AAMDC amplification.


Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Antineoplásicos/farmacología , Neoplasias de la Mama/genética , Proteínas de Ciclo Celular/genética , Everolimus/farmacología , Femenino , Proteínas Activadoras de GTPasa/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Imidazoles/farmacología , Proteínas del Tejido Nervioso/metabolismo , Oncogenes/genética , Unión Proteica , Quinolinas/farmacología , Receptores Estrogénicos/metabolismo , Transducción de Señal/efectos de los fármacos
13.
J Vis Exp ; (169)2021 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-33749674

RESUMEN

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining and imaging of thick brain samples. However, this advanced technology has not yet been used for whole-mount retinas. Although the retina is partially transparent, its thickness of approximately 200 µm (in mice) still limits the penetration of antibodies into the deep tissue as well as reducing light penetration for high-resolution imaging. Here, we adapted the CLARITY method for whole-mount mouse retinas by polymerizing them with an acrylamide monomer to form a nanoporous hydrogel and then clearing them in sodium dodecyl sulfate to minimize protein loss and avoid tissue damage. CLARITY-processed retinas were immunostained with antibodies for retinal neurons, glial cells, and synaptic proteins, mounted in a refractive index matching solution, and imaged. Our data demonstrate that CLARITY can improve the quality of standard immunohistochemical staining and imaging for retinal neurons and glial cells in whole-mount preparation. For instance, 3D resolution of fine axon-like and dendritic structures of dopaminergic amacrine cells were much improved by CLARITY. Compared to non-processed whole-mount retinas, CLARITY can reveal immunostaining for synaptic proteins such as postsynaptic density protein 95. Our results show that CLARITY renders the retina more optically transparent after the removal of lipids and preserves fine structures of retinal neurons and their proteins, which can be routinely used for obtaining high-resolution imaging of retinal neurons and their subcellular structures in whole-mount preparation.


Asunto(s)
Retina/metabolismo , Coloración y Etiquetado/métodos , Células Amacrinas/fisiología , Animales , Dendritas/fisiología , Neuronas Dopaminérgicas/fisiología , Procesamiento de Imagen Asistido por Computador , Ratones , Microscopía Confocal/métodos , Proteínas del Tejido Nervioso/metabolismo , Receptores AMPA/metabolismo , Refractometría
15.
Int J Mol Sci ; 22(5)2021 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-33669069

RESUMEN

Osteoclasts, bone-specified multinucleated cells produced by monocyte/macrophage, are involved in numerous bone destructive diseases such as arthritis, osteoporosis, and inflammation-induced bone loss. The osteoclast differentiation mechanism suggests a possible strategy to treat bone diseases. In this regard, we recently examined the in vivo impact of kalkitoxin (KT), a marine product obtained from the marine cyanobacterium Moorena producens (previously Lyngbya majuscula), on the macrophage colony-stimulating factor (M-CSF) and on the receptor activator of nuclear factor κB ligand (RANKL)-stimulated in vitro osteoclastogenesis and inflammation-mediated bone loss. We have now examined the molecular mechanism of KT in greater detail. KT decreased RANKL-induced bone marrow-derived macrophages (BMMs) tartrate-resistant acid phosphatase (TRAP)-multinucleated cells at a late stage. Likewise, KT suppressed RANKL-induced pit area and actin ring formation in BMM cells. Additionally, KT inhibited several RANKL-induced genes such as cathepsin K, matrix metalloproteinase (MMP-9), TRAP, and dendritic cell-specific transmembrane protein (DC-STAMP). In line with these results, RANKL stimulated both genes and protein expression of c-Fos and nuclear factor of activated T cells (NFATc1), and this was also suppressed by KT. Moreover, KT markedly decreased RANKL-induced p-ERK1/2 and p-JNK pathways at different time points. As a result, KT prevented inflammatory bone loss in mice, such as bone mineral density (BMD) and osteoclast differentiation markers. These experiments demonstrated that KT markedly inhibited osteoclast formation and inflammatory bone loss through NFATc1 and mitogen-activated protein kinase (MAPK) signaling pathways. Therefore, KT may have potential as a treatment for destructive bone diseases.


Asunto(s)
Resorción Ósea/tratamiento farmacológico , Lípidos/uso terapéutico , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Factores de Transcripción NFATC/metabolismo , Osteoclastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Tiazoles/uso terapéutico , Actinas/genética , Actinas/metabolismo , Animales , Densidad Ósea/efectos de los fármacos , Resorción Ósea/metabolismo , Catepsina K/genética , Catepsina K/metabolismo , Supervivencia Celular , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Quinasas Janus/metabolismo , Lípidos/farmacología , Lipopolisacáridos/toxicidad , Sistema de Señalización de MAP Quinasas/genética , Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Factor Estimulante de Colonias de Macrófagos/metabolismo , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos ICR , Factores de Transcripción NFATC/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Osteoclastos/metabolismo , Osteogénesis/genética , Fosforilación , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ligando RANK/antagonistas & inhibidores , Ligando RANK/metabolismo , Ligando RANK/farmacología , Fosfatasa Ácida Tartratorresistente/genética , Fosfatasa Ácida Tartratorresistente/metabolismo , Tiazoles/farmacología
16.
Nucleic Acids Res ; 49(5): 2946-2958, 2021 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-33577684

RESUMEN

RBM45 is an RNA-binding protein involved in neural development, whose aggregation is associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). However, the mechanisms of RNA-binding and aggregation of RBM45 remain unelucidated. Here, we report the crystal structure of the N-terminal tandem RRM domains of human RBM45 in complex with single-stranded DNA (ssDNA). Our structural and biochemical results revealed that both the RRM1 and RRM2 of RBM45 recognized the GAC sequence of RNA/ssDNA. Two aromatic residues and an arginine residue in each RRM were critical for RNA-binding, and the interdomain linker was also involved in RNA-binding. Two RRMs formed a pair of antiparallel RNA-binding sites, indicating that the N-terminal tandem RRM domains of RBM45 bound separate GAC motifs in one RNA strand or GAC motifs in different RNA strands. Our findings will be helpful in the identification of physiologic targets of RBM45 and provide evidence for understanding the physiologic and pathologic functions of RBM45.


Asunto(s)
Proteínas del Tejido Nervioso/química , Proteínas de Unión al ARN/química , ARN/química , Cristalografía por Rayos X , ADN de Cadena Simple/química , Humanos , Modelos Moleculares , Proteínas del Tejido Nervioso/metabolismo , Motivos de Nucleótidos , Unión Proteica , ARN/metabolismo , Motivo de Reconocimiento de ARN , Proteínas de Unión al ARN/metabolismo
17.
Nat Commun ; 12(1): 841, 2021 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-33547291

RESUMEN

A new life begins with the unification of the maternal and paternal chromosomes upon fertilization. The parental chromosomes first become enclosed in two separate pronuclei near the surface of the fertilized egg. The mechanisms that then move the pronuclei inwards for their unification are only poorly understood in mammals. Here, we report two mechanisms that act in concert to unite the parental genomes in fertilized mouse eggs. The male pronucleus assembles within the fertilization cone and is rapidly moved inwards by the flattening cone. Rab11a recruits the actin nucleation factors Spire and Formin-2 into the fertilization cone, where they locally nucleate actin and further accelerate the pronucleus inwards. In parallel, a dynamic network of microtubules assembles that slowly moves the male and female pronuclei towards the cell centre in a dynein-dependent manner. Both mechanisms are partially redundant and act in concert to unite the parental pronuclei in the zygote's centre.


Asunto(s)
Núcleo Celular/metabolismo , Fertilización/genética , Forminas/genética , Proteínas de Microfilamentos/genética , Proteínas del Tejido Nervioso/genética , Cigoto/metabolismo , Proteínas de Unión al GTP rab/genética , Actinas/genética , Actinas/metabolismo , Animales , Núcleo Celular/ultraestructura , Femenino , Forminas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Proteínas de Microfilamentos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Movimiento , Proteínas del Tejido Nervioso/metabolismo , Oocitos/metabolismo , Oocitos/ultraestructura , Espermatozoides/metabolismo , Espermatozoides/ultraestructura , Cigoto/ultraestructura , Proteínas de Unión al GTP rab/metabolismo
18.
Nat Commun ; 12(1): 1295, 2021 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-33637754

RESUMEN

Deep brain stimulation (DBS) relieves motor dysfunction in Parkinson's disease, and other movement disorders. Here, we demonstrate the potential benefits of DBS in a model of ataxia by targeting the cerebellum, a major motor center in the brain. We use the Car8 mouse model of hereditary ataxia to test the potential of using cerebellar nuclei DBS plus physical activity to restore movement. While low-frequency cerebellar DBS alone improves Car8 mobility and muscle function, adding skilled exercise to the treatment regimen additionally rescues limb coordination and stepping. Importantly, the gains persist in the absence of further stimulation. Because DBS promotes the most dramatic improvements in mice with early-stage ataxia, we postulated that cerebellar circuit function affects stimulation efficacy. Indeed, genetically eliminating Purkinje cell neurotransmission blocked the ability of DBS to reduce ataxia. These findings may be valuable in devising future DBS strategies.


Asunto(s)
Ataxia Cerebelosa/metabolismo , Cerebelo/fisiología , Movimiento/fisiología , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Ataxia Cerebelosa/genética , Núcleos Cerebelosos/fisiología , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Enfermedad de Parkinson , Células de Purkinje/fisiología , Transmisión Sináptica
19.
Toxicol Lett ; 341: 68-79, 2021 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-33548343

RESUMEN

BACKGROUND: General anesthetics such as sevoflurane interfere with dendritic development and synaptogenesis, resulting in cognitive impairment. The collapsin response mediator protein2 (CRMP2) plays important roles in dendritic development and synaptic plasticity and its phosphorylation is regulated by cycline dependent kinase-5 (Cdk5) and glycogen synthase kinase-3ß (GSK-3ß). Here we investigated whether Cdk5/CRMP2 or GSK-3ß/CRMP2 pathway is involved in sevoflurane-induced developmental neurotoxicity. METHODS: Rats at postnatal day 7 (PND7) were i.p. injected with Cdk5 inhibitor roscovitine, GSK-3ß inhibitor SB415286 or saline 20 min. before exposure to 2.8% sevoflurane for 4 h. Western-blotting was applied to measure the expression of Cdk5/CRMP2 and GSK-3ß/CRMP2 pathway proteins in the hippocampus 6 h after the sevoflurane exposure. When rats grew to adolescence (from PND25), they were tested for open-field and contextual fear conditioning, and then long term potentiation (LTP) from hippocampal slices was recorded, and morphology of pyramidal neuron was examined by Golgi staining and synaptic plasticity-related proteins expression in hippocampus were measured by western-blotting. In another batch of experiment, siRNA-CRMP2 or vehicle control was injected into hippocampus on PND5. RESULTS: Sevoflurane activated Cdk5/CRMP2 and GSK-3ß/CRMP2 pathways in the hippocampus of neonatal rats, reduced dendritic length, branches and the density of dendritic spine in pyramidal neurons. It also reduced the expressions of PSD-95, drebrin and synaptophysin in hippocampus, impaired memory ability of rats and inhibited LTP in hippocampal slices. All the impairment effects by sevoflurane were attenuated by pretreatment with inhibitor of Cdk5 or GSK-3ß. Furthermore, rat transfected with siRNA-CRMP2 eliminated the neuroprotective effects of Cdk5 or GSK-3ß blocker in neurobehavioral and LTP tests. CONCLUSION: Cdk5/CRMP2 and GSK-3ß/CRMP2 pathways participate in sevoflurane-induced dendritic development abnormalities and cognitive dysfunction in developing rats.


Asunto(s)
Disfunción Cognitiva/inducido químicamente , Quinasa 5 Dependiente de la Ciclina/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Sevoflurano/toxicidad , Aminofenoles/farmacología , Animales , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 5 Dependiente de la Ciclina/genética , Dendritas/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta/genética , Hipocampo/citología , Hipocampo/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/genética , Maleimidas/farmacología , Proteínas del Tejido Nervioso/genética , Inhibidores de Proteínas Quinasas/farmacología , Células Piramidales/efectos de los fármacos , Ratas , Roscovitina/farmacología
20.
Science ; 371(6530)2021 02 12.
Artículo en Inglés | MEDLINE | ID: mdl-33574182

RESUMEN

The evolutionarily conserved splicing regulator neuro-oncological ventral antigen 1 (NOVA1) plays a key role in neural development and function. NOVA1 also includes a protein-coding difference between the modern human genome and Neanderthal and Denisovan genomes. To investigate the functional importance of an amino acid change in humans, we reintroduced the archaic allele into human induced pluripotent cells using genome editing and then followed their neural development through cortical organoids. This modification promoted slower development and higher surface complexity in cortical organoids with the archaic version of NOVA1 Moreover, levels of synaptic markers and synaptic protein coassociations correlated with altered electrophysiological properties in organoids expressing the archaic variant. Our results suggest that the human-specific substitution in NOVA1, which is exclusive to modern humans since divergence from Neanderthals, may have had functional consequences for our species' evolution.


Asunto(s)
Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/fisiología , Hombre de Neandertal/genética , Neuronas/fisiología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Alelos , Empalme Alternativo , Sustitución de Aminoácidos , Animales , Sitios de Unión , Evolución Biológica , Sistemas CRISPR-Cas , Proliferación Celular , Corteza Cerebral/citología , Regulación del Desarrollo de la Expresión Génica , Variación Genética , Genoma , Genoma Humano , Haplotipos , Hominidae/genética , Humanos , Células Madre Pluripotentes Inducidas , Red Nerviosa/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Organoides , Sinapsis/fisiología
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