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1.
Stem Cell Reports ; 19(9): 1336-1350, 2024 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-39151428

RESUMEN

Variability between human pluripotent stem cell (hPSC) lines remains a challenge and opportunity in biomedicine. In this study, hPSC lines from multiple donors were differentiated toward neuroectoderm and mesendoderm lineages. We revealed dynamic transcriptomic patterns that delineate the emergence of these lineages, which were conserved across lines, along with individual line-specific transcriptional signatures that were invariant throughout differentiation. These transcriptomic signatures predicted an antagonism between SOX21-driven forebrain fates and retinoic acid-induced hindbrain fates. Replicate lines and paired adult tissue demonstrated the stability of these line-specific transcriptomic traits. We show that this transcriptomic variation in lineage bias had both genetic and epigenetic origins, aligned with the anterior-to-posterior structure of early mammalian development, and was present across a large collection of hPSC lines. These findings contribute to developing systematic analyses of PSCs to define the origin and consequences of variation in the early events orchestrating individual human development.


Asunto(s)
Diferenciación Celular , Linaje de la Célula , Células Madre Pluripotentes , Transcriptoma , Humanos , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Diferenciación Celular/genética , Linaje de la Célula/genética , Línea Celular , Tretinoina/farmacología , Tretinoina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Epigénesis Genética
2.
iScience ; 27(4): 109512, 2024 Apr 19.
Artículo en Inglés | MEDLINE | ID: mdl-38715938

RESUMEN

LMTK3 is a brain-specific transmembrane serine/threonine protein kinase that acts as a scaffold for protein phosphatase-1 (PP1). Although LMKT3 has been identified as a risk factor for autism and epilepsy, its physiological significance is unknown. Here, we demonstrate that LMTK3 copurifies and binds to KCC2, a neuron-specific K+/Cl- transporter. KCC2 activity is essential for Cl--mediated hyperpolarizing GABAAR receptor currents, the unitary events that underpin fast synaptic inhibition. LMTK3 acts to promote the association of KCC2 with PP1 to promote the dephosphorylation of S940 within its C-terminal cytoplasmic domain, a process the diminishes KCC2 activity. Accordingly, acute inhibition of LMTK3 increases KCC2 activity dependent upon S940 and increases neuronal Cl- extrusion. Consistent with this, LMTK3 inhibition reduced intrinsic neuronal excitability and the severity of seizure-like events in vitro. Thus, LMTK3 may have profound effects on neuronal excitability as an endogenous modulator of KCC2 activity.

3.
ACS Appl Mater Interfaces ; 16(25): 32587-32598, 2024 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-38771585

RESUMEN

Passive heat management is crucial in space, especially for extended missions involving protection from sunlight. Thermal coatings with desirable optical properties can drastically reduce the power consumed by active cooling systems, thereby reserving more resources for other critical systems onboard. Specifically, materials with wavelength-dependent reflectance and emittance are desirable for managing incident sunlight and self-cooling by thermal emission. This study demonstrates the use of polymer nanofibers, specifically poly(tetrafluoroethylene) (PTFE), for passive temperature control in space applications. This study describes the electrospinning fabrication process to create nanofibers and how process parameters can be varied to control the fiber geometry. We combine poly(tetrafluoroethylene) (PTFE) and poly(ethylene oxide) (PEO) polymers to fabricate highly reflective thermal control materials by electrospinning. To understand the role of material and fiber geometry, we measure spectral reflectance, absorptance, and transmittance using spectrophotometers interfaced with integrating spheres. We control the materials' fiber geometry and solar reflectance by modifying the solution properties, flow rate, rotating collector speed, and fabrication time. With 220-1560 µm thick electrospun nanofiber materials, we demonstrate an average solar reflectance of 94.73-99.75%, with values approaching 99.9% for thicker samples, which is among the highest for space applications. Meanwhile, a thermal emittance of 81.4% was observed at 300 K for a 3360 µm thick sample. The durability of these samples was also tested under ultraviolet light and atomic oxygen. Compared to the state-of-the-art materials, the electrospun PTFE-PEO fibers present a new paradigm for passive thermal management in space applications.

4.
Int J Behav Nutr Phys Act ; 20(1): 144, 2023 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-38062460

RESUMEN

BACKGROUND: The growth of urban dwelling populations globally has led to rapid increases of research and policy initiatives addressing associations between the built environment and physical activity (PA). Given this rapid proliferation, it is important to identify priority areas and research questions for moving the field forward. The objective of this study was to identify and compare research priorities on the built environment and PA among researchers and knowledge users (e.g., policy makers, practitioners). METHODS: Between September 2022 and April 2023, a three-round, modified Delphi survey was conducted among two independent panels of international researchers (n = 38) and knowledge users (n = 23) to identify similarities and differences in perceived research priorities on the built environment and PA and generate twin 'top 10' lists of the most important research needs. RESULTS: From a broad range of self-identified issues, both panels ranked in common the most pressing research priorities including stronger study designs such as natural experiments, research that examines inequalities and inequities, establishing the cost effectiveness of interventions, safety and injuries related to engagement in active transportation (AT), and considerations for climate change and climate adaptation. Additional priorities identified by researchers included: implementation science, research that incorporates Indigenous perspectives, land-use policies, built environments that support active aging, and participatory research. Additional priorities identified by knowledge users included: built environments and PA among people living with disabilities and a need for national data on trip chaining, multi-modal travel, and non-work or school-related AT. CONCLUSIONS: Five common research priorities between the two groups emerged, including (1) to better understand causality, (2) interactions with the natural environment, (3) economic evaluations, (4) social disparities, and (5) preventable AT-related injuries. The findings may help set directions for future research, interdisciplinary and intersectoral collaborations, and funding opportunities.


Asunto(s)
Ambiente , Ejercicio Físico , Humanos , Técnica Delphi , Entorno Construido , Proyectos de Investigación
5.
BMC Public Health ; 23(1): 1443, 2023 07 28.
Artículo en Inglés | MEDLINE | ID: mdl-37507665

RESUMEN

BACKGROUND: This report describes two L. monocytogenes outbreak investigations that occurred in March and September of 2018 and that linked illness to a food premises located in an Ontario cancer centre. The cancer centre serves patients from across the province. METHODS: In Ontario, local public health agencies follow up with all reported laboratory-confirmed cases of listeriosis to identify possible sources of disease acquisition and to carry out investigations, including at suspected food premises. The Canadian Food Inspection Agency (CFIA) is notified of any Listeria-positive food product collected in relation to a case. The CFIA traces Listeria-positive product through the food distribution system to identify the contamination source and ensure the implicated manufacturing facility implements corrective measures. RESULTS: Outbreaks one and two each involved three outbreak-confirmed listeriosis cases. All six cases were considered genetically related by whole genome sequencing (WGS). In both outbreaks, outbreak-confirmed cases reported consuming meals at a food premises located in a cancer centre (food premises A) before illness onset. Various open deli meat samples and, in outbreak two, environmental swabs (primarily from the meat slicer) collected from food premises A were genetically related to the outbreak-confirmed cases. Food premises A closed as a result of the investigations. CONCLUSIONS: When procuring on-site food premises, healthcare facilities and institutions serving individuals with immuno-compromising conditions should consider the potential health risk of foods available to their patient population.


Asunto(s)
Enfermedades Transmitidas por los Alimentos , Listeria monocytogenes , Listeriosis , Neoplasias , Humanos , Listeria monocytogenes/genética , Enfermedades Transmitidas por los Alimentos/epidemiología , Microbiología de Alimentos , Neoplasias/epidemiología , Listeriosis/epidemiología , Brotes de Enfermedades , Ontario/epidemiología
6.
Cell Rep Med ; 4(3): 100957, 2023 03 21.
Artículo en Inglés | MEDLINE | ID: mdl-36889319

RESUMEN

Hyperpolarizing GABAAR currents, the unitary events that underlie synaptic inhibition, are dependent upon efficient Cl- extrusion, a process that is facilitated by the neuronal specific K+/Cl- co-transporter KCC2. Its activity is also a determinant of the anticonvulsant efficacy of the canonical GABAAR-positive allosteric: benzodiazepines (BDZs). Compromised KCC2 activity is implicated in the pathophysiology of status epilepticus (SE), a medical emergency that rapidly becomes refractory to BDZ (BDZ-RSE). Here, we have identified small molecules that directly bind to and activate KCC2, which leads to reduced neuronal Cl- accumulation and excitability. KCC2 activation does not induce any overt effects on behavior but prevents the development of and terminates ongoing BDZ-RSE. In addition, KCC2 activation reduces neuronal cell death following BDZ-RSE. Collectively, these findings demonstrate that KCC2 activation is a promising strategy to terminate BDZ-resistant seizures and limit the associated neuronal injury.


Asunto(s)
Estado Epiléptico , Simportadores , Ratones , Animales , Benzodiazepinas/farmacología , Benzodiazepinas/uso terapéutico , Estado Epiléptico/tratamiento farmacológico , Convulsiones/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Simportadores/metabolismo
7.
Mol Psychiatry ; 27(3): 1805-1815, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-35165396

RESUMEN

Sensorimotor information processing underlies normal cognitive and behavioral traits and has classically been evaluated through prepulse inhibition (PPI) of a startle reflex. PPI is a behavioral dimension deregulated in several neurological and psychiatric disorders, yet the mechanisms underlying the cross-diagnostic nature of PPI deficits across these conditions remain to be understood. To identify circuitry mechanisms for PPI, we performed circuitry recording over the prefrontal cortex and striatum, two brain regions previously implicated in PPI, using wild-type (WT) mice compared to Disc1-locus-impairment (LI) mice, a model representing neuropsychiatric conditions. We demonstrated that the corticostriatal projection regulates neurophysiological responses during the PPI testing in WT, whereas these circuitry responses were disrupted in Disc1-LI mice. Because our biochemical analyses revealed attenuated brain-derived neurotrophic factor (Bdnf) transport along the corticostriatal circuit in Disc1-LI mice, we investigated the potential role of Bdnf in this circuitry for regulation of PPI. Virus-mediated delivery of Bdnf into the striatum rescued PPI deficits in Disc1-LI mice. Pharmacologically augmenting Bdnf transport by chronic lithium administration, partly via phosphorylation of Huntingtin (Htt) serine-421 and its integration into the motor machinery, restored striatal Bdnf levels and rescued PPI deficits in Disc1-LI mice. Furthermore, reducing the cortical Bdnf expression negated this rescuing effect of lithium, confirming the key role of Bdnf in lithium-mediated PPI rescuing. Collectively, the data suggest that striatal Bdnf supply, collaboratively regulated by Htt and Disc1 along the corticostriatal circuit, is involved in sensorimotor gating, highlighting the utility of dimensional approach in investigating pathophysiological mechanisms across neuropsychiatric disorders.


Asunto(s)
Factor Neurotrófico Derivado del Encéfalo , Cuerpo Estriado , Proteínas del Tejido Nervioso , Corteza Prefrontal , Inhibición Prepulso , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Cuerpo Estriado/metabolismo , Humanos , Ratones , Proteínas del Tejido Nervioso/metabolismo , Corteza Prefrontal/metabolismo , Inhibición Prepulso/fisiología , Reflejo de Sobresalto/fisiología , Filtrado Sensorial/fisiología
8.
Brain ; 145(3): 950-963, 2022 04 29.
Artículo en Inglés | MEDLINE | ID: mdl-34528073

RESUMEN

First-in-line benzodiazepine treatment fails to terminate seizures in about 30% of epilepsy patients, highlighting a need for novel anti-seizure strategies. It is emerging that impaired K+/Cl- cotransporter 2 (KCC2) activity leads to deficits in GABAergic inhibition and increased seizure vulnerability in patients. In neurons, the with-no-lysine (WNK) kinase-STE20/SPS1-related proline/alanine-rich (SPAK) kinase signalling pathway inhibits KCC2 activity via T1007 phosphorylation. Here, we exploit the selective WNK kinase inhibitor WNK463 to test the effects of pharmacological WNK inhibition on KCC2 function, GABAergic inhibition, and epileptiform activity. Immunoprecipitation and western blotting analysis revealed that WNK463 reduces KCC2-T1007 phosphorylation in vitro and in vivo. Using patch-clamp recordings in primary rat neurons, we further observed that WNK463 hyperpolarized the Cl- reversal potential, and enhanced KCC2-mediated Cl- extrusion. In the 4-aminopyridine slice model of acute seizures, WNK463 administration reduced the frequency and number of seizure-like events. In vivo, C57BL/6 mice that received intrahippocampal WNK463 experienced delayed onset of kainic acid-induced status epilepticus, less epileptiform EEG activity, and did not develop pharmaco-resistance to diazepam. Our findings demonstrate that acute WNK463 treatment potentiates KCC2 activity in neurons and limits seizure burden in two well-established models of seizures and epilepsy. In summary, our work suggests that agents which act to increase KCC2 activity may be useful adjunct therapeutics to alleviate diazepam-resistant status epilepticus.


Asunto(s)
Epilepsia , Estado Epiléptico , Simportadores , Animales , Diazepam/metabolismo , Diazepam/farmacología , Hipocampo/metabolismo , Humanos , Lisina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratas , Estado Epiléptico/inducido químicamente , Estado Epiléptico/tratamiento farmacológico , Estado Epiléptico/metabolismo , Simportadores/metabolismo
9.
EMBO J ; 40(14): e100715, 2021 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-34152608

RESUMEN

Clearance of mitochondria following damage is critical for neuronal homeostasis. Here, we investigate the role of Miro proteins in mitochondrial turnover by the PINK1/Parkin mitochondrial quality control system in vitro and in vivo. We find that upon mitochondrial damage, Miro is promiscuously ubiquitinated on multiple lysine residues. Genetic deletion of Miro or block of Miro1 ubiquitination and subsequent degradation lead to delayed translocation of the E3 ubiquitin ligase Parkin onto damaged mitochondria and reduced mitochondrial clearance in both fibroblasts and cultured neurons. Disrupted mitophagy in vivo, upon post-natal knockout of Miro1 in hippocampus and cortex, leads to a dramatic increase in mitofusin levels, the appearance of enlarged and hyperfused mitochondria and hyperactivation of the integrated stress response (ISR). Altogether, our results provide new insights into the central role of Miro1 in the regulation of mitochondrial homeostasis and further implicate Miro1 dysfunction in the pathogenesis of human neurodegenerative disease.


Asunto(s)
Mitocondrias/metabolismo , Mitofagia/fisiología , Neuronas/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Línea Celular Tumoral , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Mitocondriales/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/fisiología
10.
J Neurochem ; 158(5): 1058-1073, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34077555

RESUMEN

Activity-regulated cytoskeleton-associated protein (Arc) is an immediate early gene product that support neuroplastic changes important for cognitive function and memory formation. As a protein with homology to the retroviral Gag protein, a particular characteristic of Arc is its capacity to self-assemble into virus-like capsids that can package mRNAs and transfer those transcripts to other cells. Although a lot has been uncovered about the contributions of Arc to neuron biology and behavior, very little is known about how different functions of Arc are coordinately regulated both temporally and spatially in neurons. The answer to this question we hypothesized must involve the occurrence of different protein post-translational modifications acting to confer specificity. In this study, we used mass spectrometry and sequence prediction strategies to map novel Arc phosphorylation sites. Our approach led us to recognize serine 67 (S67) and threonine 278 (T278) as residues that can be modified by TNIK, which is a kinase abundantly expressed in neurons that shares many functional overlaps with Arc and has, along with its interacting proteins such as the NMDA receptor, and been implicated as a risk factor for psychiatric disorders. Furthermore, characterization of each residue using site-directed mutagenesis to create S67 and T278 mutant variants revealed that TNIK action at those amino acids can strongly influence Arc's subcellular distribution and self-assembly as capsids. Together, our findings reveal an unsuspected connection between Arc and TNIK. Better understanding of the interplay between these two proteins in neuronal cells could lead to new insights about apparition and progression of psychiatric disorders. Cover Image for this issue: https://doi.org/10.1111/jnc.15077.


Asunto(s)
Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Ratones , Neuronas/metabolismo , Fosforilación/fisiología
11.
J Biol Chem ; 296: 100364, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33539918

RESUMEN

The K+/Cl- cotransporter KCC2 (SLC12A5) allows mature neurons in the CNS to maintain low intracellular Cl- levels that are critical in mediating fast hyperpolarizing synaptic inhibition via type A γ-aminobutyric acid receptors (GABAARs). In accordance with this, compromised KCC2 activity results in seizures, but whether such deficits directly contribute to the subsequent changes in neuronal structure and viability that lead to epileptogenesis remains to be assessed. Canonical hyperpolarizing GABAAR currents develop postnatally, which reflect a progressive increase in KCC2 expression levels and activity. To investigate the role that KCC2 plays in regulating neuronal viability and architecture, we have conditionally ablated KCC2 expression in developing and mature neurons. Decreasing KCC2 expression in mature neurons resulted in the rapid activation of the extrinsic apoptotic pathway. Intriguingly, direct pharmacological inhibition of KCC2 in mature neurons was sufficient to rapidly induce apoptosis, an effect that was not abrogated via blockade of neuronal depolarization using tetrodotoxin (TTX). In contrast, ablating KCC2 expression in immature neurons had no discernable effects on their subsequent development, arborization, or dendritic structure. However, removing KCC2 in immature neurons was sufficient to ablate the subsequent postnatal development of hyperpolarizing GABAAR currents. Collectively, our results demonstrate that KCC2 plays a critical role in neuronal survival by limiting apoptosis, and mature neurons are highly sensitive to the loss of KCC2 function. In contrast, KCC2 appears to play a minimal role in mediating neuronal development or architecture.


Asunto(s)
Neuronas/metabolismo , Simportadores/metabolismo , Animales , Apoptosis , Cloruros/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neurogénesis/efectos de los fármacos , Neuronas/fisiología , Potasio/metabolismo , Cultivo Primario de Células , Receptores de GABA/metabolismo , Convulsiones , Simportadores/fisiología , Ácido gamma-Aminobutírico/metabolismo , Cotransportadores de K Cl
12.
Front Mol Neurosci ; 13: 563091, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33192291

RESUMEN

Kcc2 plays a critical role in determining the efficacy of synaptic inhibition, however, the cellular mechanisms neurons use to regulate its membrane trafficking, stability and activity are ill-defined. To address these issues, we used affinity purification to isolate stable multi-protein complexes of K-Cl Co-transporter 2 (Kcc2) from the plasma membrane of murine forebrain. We resolved these using blue-native polyacrylamide gel electrophoresis (BN-PAGE) coupled to LC-MS/MS and label-free quantification. Data are available via ProteomeXchange with identifier PXD021368. Purified Kcc2 migrated as distinct molecular species of 300, 600, and 800 kDa following BN-PAGE. In excess of 90% coverage of the soluble N- and C-termini of Kcc2 was obtained. In total we identified 246 proteins significantly associated with Kcc2. The 300 kDa species largely contained Kcc2, which is consistent with a dimeric quaternary structure for this transporter. The 600 and 800 kDa species represented stable multi-protein complexes of Kcc2. We identified a set of novel structural, ion transporting, immune related and signaling protein interactors, that are present at both excitatory and inhibitory synapses, consistent with the proposed localization of Kcc2. These included spectrins, C1qa/b/c and the IP3 receptor. We also identified interactors more directly associated with phosphorylation; Akap5, Akap13, and Lmtk3. Finally, we used LC-MS/MS on the same purified endogenous plasma membrane Kcc2 to detect phosphorylation sites. We detected 11 sites with high confidence, including known and novel sites. Collectively our experiments demonstrate that Kcc2 is associated with components of the neuronal cytoskeleton and signaling molecules that may act to regulate transporter membrane trafficking, stability, and activity.

13.
BMC Infect Dis ; 20(1): 523, 2020 Jul 18.
Artículo en Inglés | MEDLINE | ID: mdl-32682398

RESUMEN

BACKGROUND: Assessing the burden of rickettsial infections in Ontario, Canada, is challenging since rickettsial infections are not reportable to public health. In the absence of reportable disease data, we assessed the burden of rickettsial infections by examining patient serological data and clinical information. METHODS: Our retrospective, cross-sectional study included patients who had Rickettsia serological testing ordered by their physician, in Ontario, from 2013 to 2018. We tested sera from 2755 non-travel patients for antibodies against spotted fever group rickettsiae (SFGR) and typhus group rickettsiae (TGR) using an indirect immunofluorescence assay (IFA) (positive IgG titers ≥1:64). We classified cases using a sensitive surveillance case definition: confirmed (4-fold increase in IgG titers between acute and convalescent sera with clinical evidence of infection), possible (single positive sera with clinical evidence) and previous rickettsial infection (single positive sera without clinical evidence). We classified cases seropositive for both SFGR and TGR as unspecified Rickettsia infections (URIs). RESULTS: Less than 5% of all patients had paired acute and convalescent sera tested, and of these, we found a single, laboratory-confirmed SFGR case, with a 4-fold increase in IgG titers and evidence of fever, maculopapular rash and headache. There were 45 possible (19 SFGR, 7 TGR, 19 URI) and 580 previous rickettsial infection (183 SFGR, 89 TGR, 308 URI) cases. The rate of positive tests for SFGR, TGR and URI combined (all case classifications) were 4.4 per 100,000 population. For confirmed and possible cases, the most common signs and symptoms were fever, headache, gastrointestinal complaints and maculopapular rash. The odds of having seropositive patients increased annually by 30% (odds ratio = 1.3, 95% confidence interval: 1.23-1.39). CONCLUSIONS: The rates of rickettsial infections in Ontario are difficult to determine. Based on confirmed and possible cases, rates are low, but inclusion of previous rickettsial infection cases would indicate higher rates. We highlight the need for education regarding the importance of testing acute and convalescent sera and consistent completion of the laboratory requisition in confirming rickettsial disease. We suggest further research in Ontario to investigate rickettsial agents in potential vectors and clinical studies employing PCR testing of clinical samples.


Asunto(s)
Rickettsia typhi/inmunología , Rickettsiosis Exantemáticas/diagnóstico , Rickettsiosis Exantemáticas/epidemiología , Tifus Endémico Transmitido por Pulgas/diagnóstico , Tifus Endémico Transmitido por Pulgas/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antibacterianos/sangre , Niño , Preescolar , Estudios Transversales , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Inmunoglobulina G/sangre , Lactante , Masculino , Persona de Mediana Edad , Ontario/epidemiología , Estudios Retrospectivos , Rickettsiosis Exantemáticas/sangre , Rickettsiosis Exantemáticas/microbiología , Tifus Endémico Transmitido por Pulgas/sangre , Tifus Endémico Transmitido por Pulgas/microbiología , Adulto Joven
14.
Nat Commun ; 11(1): 3258, 2020 06 26.
Artículo en Inglés | MEDLINE | ID: mdl-32591533

RESUMEN

Tauopathies are neurodegenerative diseases associated with accumulation of abnormal tau protein in the brain. Patient iPSC-derived neuronal cell models replicate disease-relevant phenotypes ex vivo that can be pharmacologically targeted for drug discovery. Here, we explored autophagy as a mechanism to reduce tau burden in human neurons and, from a small-molecule screen, identify the mTOR inhibitors OSI-027, AZD2014 and AZD8055. These compounds are more potent than rapamycin, and robustly downregulate phosphorylated and insoluble tau, consequently reducing tau-mediated neuronal stress vulnerability. MTORC1 inhibition and autophagy activity are directly linked to tau clearance. Notably, single-dose treatment followed by washout leads to a prolonged reduction of tau levels and toxicity for 12 days, which is mirrored by a sustained effect on mTORC1 inhibition and autophagy. This new insight into the pharmacodynamics of mTOR inhibitors in regulation of neuronal autophagy may contribute to development of therapies for tauopathies.


Asunto(s)
Autofagia , Neuronas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Estrés Fisiológico , Tauopatías/metabolismo , Proteínas tau/metabolismo , Animales , Autofagia/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Persona de Mediana Edad , Modelos Biológicos , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Neuronas/efectos de los fármacos , Fagosomas/efectos de los fármacos , Fagosomas/metabolismo , Fenotipo , Ratas Wistar , Estrés Fisiológico/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Tauopatías/patología , Factores de Tiempo
15.
Mol Brain ; 13(1): 66, 2020 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-32366277

RESUMEN

Alzheimer's disease (AD), the leading cause of dementia, is a chronic neurodegenerative disease. Apolipoprotein E (apoE), which carries lipids in the brain in the form of lipoproteins, plays an undisputed role in AD pathophysiology. A high-throughput phenotypic screen was conducted using a CCF-STTG1 human astrocytoma cell line to identify small molecules that could upregulate apoE secretion. AZ7235, a previously discovered Axl kinase inhibitor, was identified to have robust apoE activity in brain microglia, astrocytes and pericytes. AZ7235 also increased expression of ATP-binding cassette protein A1 (ABCA1), which is involved in the lipidation and secretion of apoE. Moreover, AZ7235 did not exhibit Liver-X-Receptor (LXR) activity and stimulated apoE and ABCA1 expression in the absence of LXR. Target validation studies using AXL-/- CCF-STTG1 cells showed that Axl is required to mediate AZ7235 upregulation of apoE and ABCA1. Intriguingly, apoE expression and secretion was significantly attenuated in AXL-deficient CCF-STTG1 cells and reconstitution of Axl or kinase-dead Axl significantly restored apoE baseline levels, demonstrating that Axl also plays a role in maintaining apoE homeostasis in astrocytes independent of its kinase activity. Lastly, these effects may require human apoE regulatory sequences, as AZ7235 exhibited little stimulatory activity toward apoE and ABCA1 in primary murine glia derived from neonatal human APOE3 targeted-replacement mice. Collectively, we identified a small molecule that exhibits robust apoE and ABCA1 activity independent of the LXR pathway in human cells and elucidated a novel relationship between Axl and apoE homeostasis in human astrocytes.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Apolipoproteínas E/metabolismo , Astrocitos/efectos de los fármacos , Astrocitoma/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transportador 1 de Casete de Unión a ATP/metabolismo , Animales , Apolipoproteínas E/genética , Astrocitoma/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Técnicas de Silenciamiento del Gen , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Microglía/efectos de los fármacos , Microglía/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Tirosina Quinasa del Receptor Axl
16.
Cell Rep ; 31(5): 107599, 2020 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-32375049

RESUMEN

Better understanding of the progression of neural stem cells (NSCs) in the developing cerebral cortex is important for modeling neurogenesis and defining the pathogenesis of neuropsychiatric disorders. Here, we use RNA sequencing, cell imaging, and lineage tracing of mouse and human in vitro NSCs and monkey brain sections to model the generation of cortical neuronal fates. We show that conserved signaling mechanisms regulate the acute transition from proliferative NSCs to committed glutamatergic excitatory neurons. As human telencephalic NSCs develop from pluripotency in vitro, they transition through organizer states that spatially pattern the cortex before generating glutamatergic precursor fates. NSCs derived from multiple human pluripotent lines vary in these early patterning states, leading differentially to dorsal or ventral telencephalic fates. This work furthers systematic analyses of the earliest patterning events that generate the major neuronal trajectories of the human telencephalon.


Asunto(s)
Células Madre Embrionarias/metabolismo , Células-Madre Neurales/citología , Neurogénesis/fisiología , Neuronas/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Transducción de Señal/fisiología
17.
Med Res Rev ; 40(4): 1352-1384, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32043626

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease marked by progressive loss of muscle function. It is the most common adult-onset form of motor neuron disease, affecting about 16 000 people in the United States alone. The average survival is about 3 years. Only two interventional drugs, the antiglutamatergic small-molecule riluzole and the more recent antioxidant edaravone, have been approved for the treatment of ALS to date. Therapeutic strategies under investigation in clinical trials cover a range of different modalities and targets, and more than 70 different drugs have been tested in the clinic to date. Here, we summarize and classify interventional therapeutic strategies based on their molecular targets and phenotypic effects. We also discuss possible reasons for the failure of clinical trials in ALS and highlight emerging preclinical strategies that could provide a breakthrough in the battle against this relentless disease.


Asunto(s)
Esclerosis Amiotrófica Lateral/terapia , Ensayos Clínicos como Asunto , Antiinflamatorios/química , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Autofagia/efectos de los fármacos , Aprobación de Drogas , Humanos , Factores Inmunológicos/química , Factores Inmunológicos/farmacología , Factores Inmunológicos/uso terapéutico
18.
Transl Psychiatry ; 10(1): 16, 2020 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-32066698

RESUMEN

Loss of glutamatergic synapses is thought to be a key cellular pathology associated with neuropsychiatric disorders including schizophrenia (SCZ) and major depressive disorder (MDD). Genetic and cellular studies of SCZ and MDD using in vivo and in vitro systems have supported a key role for dysfunction of excitatory synapses in the pathophysiology of these disorders. Recent clinical studies have demonstrated that the estrogen, 17ß-estradiol can ameliorate many of the symptoms experienced by patients. Yet, to date, our understanding of how 17ß-estradiol exerted these beneficial effects is limited. In this study, we have tested the hypothesis that 17ß-estradiol can restore dendritic spine number in a cellular model that recapitulates the loss of synapses associated with SCZ and MDD. Ectopic expression of wildtype, mutant or shRNA-mediated knockdown of Disrupted in Schizophrenia 1 (DISC1) reduced dendritic spine density in primary cortical neurons. Acute or chronic treatment with 17ß-estradiol increased spine density to control levels in neurons with altered DISC1 levels. In addition, 17ß-estradiol reduced the extent to which ectopic wildtype and mutant DISC1 aggregated. Furthermore, 17ß-estradiol also caused the enrichment of synaptic proteins at synapses and increased the number of dendritic spines containing PSD-95 or that overlapped with the pre-synaptic marker bassoon. Taken together, our data indicates that estrogens can restore lost excitatory synapses caused by altered DISC1 expression, potentially through the trafficking of DISC1 and its interacting partners. These data highlight the possibility that estrogens exert their beneficial effects in SCZ and MDD in part by modulating dendritic spine number.


Asunto(s)
Trastorno Depresivo Mayor , Estradiol , Espinas Dendríticas , Estradiol/farmacología , Estrógenos , Humanos , Sinapsis
19.
Nat Commun ; 11(1): 462, 2020 01 23.
Artículo en Inglés | MEDLINE | ID: mdl-31974374

RESUMEN

Human induced pluripotent stem cells (hiPSCs) are a powerful model of neural differentiation and maturation. We present a hiPSC transcriptomics resource on corticogenesis from 5 iPSC donor and 13 subclonal lines across 9 time points over 5 broad conditions: self-renewal, early neuronal differentiation, neural precursor cells (NPCs), assembled rosettes, and differentiated neuronal cells. We identify widespread changes in the expression of both individual features and global patterns of transcription. We next demonstrate that co-culturing human NPCs with rodent astrocytes results in mutually synergistic maturation, and that cell type-specific expression data can be extracted using only sequencing read alignments without cell sorting. We lastly adapt a previously generated RNA deconvolution approach to single-cell expression data to estimate the relative neuronal maturity of iPSC-derived neuronal cultures and human brain tissue. Using many public datasets, we demonstrate neuronal cultures are maturationally heterogeneous but contain subsets of neurons more mature than previously observed.


Asunto(s)
Diferenciación Celular/genética , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/fisiología , Células-Madre Neurales/fisiología , Transcriptoma , Algoritmos , Animales , Astrocitos/citología , Células Cultivadas , Corteza Cerebral/citología , Técnicas de Cocultivo , Bases de Datos Genéticas , Regulación de la Expresión Génica , Humanos , Modelos Neurológicos , Células-Madre Neurales/citología , Neuronas/citología , Neuronas/fisiología , Ratas
20.
Proc Natl Acad Sci U S A ; 117(1): 677-688, 2020 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-31871190

RESUMEN

A robust body of evidence supports the concept that phosphodiesterase 10A (PDE10A) activity in the basal ganglia orchestrates the control of coordinated movement in human subjects. Although human mutations in the PDE10A gene manifest in hyperkinetic movement disorders that phenocopy many features of early Huntington's disease, characterization of the maladapted molecular mechanisms and aberrant signaling processes that underpin these conditions remains scarce. Recessive mutations in the GAF-A domain have been shown to impair PDE10A function due to the loss of striatal PDE10A protein levels, but here we show that this paucity is caused by irregular intracellular trafficking and increased PDE10A degradation in the cytosolic compartment. In contrast to GAF-A mutants, dominant mutations in the GAF-B domain of PDE10A induce PDE10A misfolding, a common pathological phenotype in many neurodegenerative diseases. These data demonstrate that the function of striatal PDE10A is compromised in disorders where disease-associated mutations trigger a reduction in the fidelity of PDE compartmentalization.


Asunto(s)
Membrana Celular/metabolismo , Enfermedad de Huntington/genética , Neuronas/enzimología , Hidrolasas Diéster Fosfóricas/genética , Dominios Proteicos/genética , Animales , Autofagia/genética , Cuerpo Estriado/citología , Cuerpo Estriado/patología , AMP Cíclico/metabolismo , Embrión de Mamíferos , Células HEK293 , Humanos , Enfermedad de Huntington/patología , Hidrólisis , Isoenzimas/genética , Isoenzimas/metabolismo , Mutación , Neuronas/citología , Técnicas de Placa-Clamp , Hidrolasas Diéster Fosfóricas/metabolismo , Cultivo Primario de Células , Proteolisis , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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