RESUMEN
Frontotemporal dementia (FTD) because of MAPT mutation causes pathological accumulation of tau and glutamatergic cortical neuronal death by unknown mechanisms. We used human induced pluripotent stem cell (iPSC)-derived cerebral organoids expressing tau-V337M and isogenic corrected controls to discover early alterations because of the mutation that precede neurodegeneration. At 2 months, mutant organoids show upregulated expression of MAPT, glutamatergic signaling pathways, and regulators, including the RNA-binding protein ELAVL4, and increased stress granules. Over the following 4 months, mutant organoids accumulate splicing changes, disruption of autophagy function, and build-up of tau and P-tau-S396. By 6 months, tau-V337M organoids show specific loss of glutamatergic neurons as seen in individuals with FTD. Mutant neurons are susceptible to glutamate toxicity, which can be rescued pharmacologically by the PIKFYVE kinase inhibitor apilimod. Our results demonstrate a sequence of events that precede neurodegeneration, revealing molecular pathways associated with glutamate signaling as potential targets for therapeutic intervention in FTD.
Asunto(s)
Cerebro/patología , Proteína 4 Similar a ELAV/genética , Ácido Glutámico/metabolismo , Mutación/genética , Neuronas/patología , Organoides/metabolismo , Empalme del ARN/genética , Proteínas tau/genética , Autofagia/efectos de los fármacos , Autofagia/genética , Biomarcadores/metabolismo , Tipificación del Cuerpo/efectos de los fármacos , Tipificación del Cuerpo/genética , Muerte Celular/efectos de los fármacos , Línea Celular , Humanos , Hidrazonas/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Morfolinas/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Organoides/efectos de los fármacos , Organoides/ultraestructura , Fosforilación/efectos de los fármacos , Pirimidinas/farmacología , Empalme del ARN/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Gránulos de Estrés/efectos de los fármacos , Gránulos de Estrés/metabolismo , Sinapsis/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND: Immune cells play crucial roles after spinal cord injury (SCI). However, incomplete knowledge of immune contributions to injury and repair hinders development of SCI therapies. We leveraged single-cell observations to describe key populations of immune cells present in the spinal cord and changes in their transcriptional profiles from uninjured to subacute and chronic stages of SCI. METHODS: Deep-read single-cell sequencing was performed on CD45+ cells from spinal cords of uninjured and injured Swiss-webster mice. After T9 thoracic contusion, cells were collected 3-, 7-, and 60-day post-injury (dpi). Subpopulations of CD45+ immune cells were identified informatically, and their transcriptional responses characterized with time. We compared gene expression in spinal cord microglia and B cell subpopulations with those in published models of disease and injury. Microglia were compared with Disease Associated Microglia (DAM) and Injury Responsive Microglia (IRM). B cells were compared to developmental lineage states and to an Amyotrophic Lateral Sclerosis (ALS) model. RESULTS: In uninjured and 7 dpi spinal cord, most CD45+ cells isolated were microglia while chronically B cells predominated. B cells accumulating in the spinal cord following injury included immature B to mature stages and were predominantly found in the injury zone. We defined diverse subtypes of microglia and B cells with altered gene expression with time after SCI. Spinal cord microglia gene expression indicates differences from brain microglia at rest and in inflammatory states. Expression analysis of signaling ligand-receptor partners identified microglia-B cell interactions at acute and chronic stages that may be involved in B cell recruitment, retention, and formation of ectopic lymphoid follicles. CONCLUSIONS: Immune cell responses to SCI have region-specific aspects and evolve with time. Developmentally diverse populations of B cells accumulate in the spinal cord following injury. Microglia at subacute stages express B cell recruitment factors, while chronically, they express factors predicted to reduce B cell inflammatory state. In the injured spinal cord, B cells create ectopic lymphoid structures, and express secreted factors potentially acting on microglia. Our study predicts previously unidentified crosstalk between microglia and B cells post-injury at acute and chronic stages, revealing new potential targets of inflammatory responses for SCI repair warranting future functional analyses.
Asunto(s)
Microglía , Traumatismos de la Médula Espinal , Ratones , Animales , Microglía/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Médula Espinal/metabolismo , Linfocitos B/metabolismoRESUMEN
Tau protein aggregation is a hallmark of several neurodegenerative diseases, including Alzheimer's disease, frontotemporal dementia (FTD) and progressive supranuclear palsy (PSP), spurring development of tau-lowering therapeutic strategies. Here, we report fully human bifunctional anti-tau-PEST intrabodies that bind the mid-domain of tau to block aggregation and degrade tau via the proteasome using the ornithine decarboxylase (ODC) PEST degron. They effectively reduced tau protein in human iPSC-derived cortical neurons in 2D cultures and 3D organoids, including those with the disease-associated tau mutations R5L, N279K, R406W, and V337M. Anti-tau-hPEST intrabodies facilitated efficient ubiquitin-independent proteolysis, in contrast to tau-lowering approaches that rely on the cell's ubiquitination system. Importantly, they counteracted the proteasome impairment observed in V337M patient-derived cortical neurons and significantly improved neuronal survival. By serial mutagenesis, we created variants of the PEST degron that achieved graded levels of tau reduction. Moderate reduction was as effective as high reduction against tau V337M-induced neural cell death.
RESUMEN
During embryonic development, endothelial cells (ECs) undergo vasculogenesis to form a primitive plexus and assemble into networks comprised of mural cell-stabilized vessels with molecularly distinct artery and vein signatures. This organized vasculature is established prior to the initiation of blood flow and depends on a sequence of complex signaling events elucidated primarily in animal models, but less studied and understood in humans. Here, we have developed a simple vascular differentiation protocol for human pluripotent stem cells that generates ECs, pericytes, and smooth muscle cells simultaneously. When this protocol is applied in a 3D hydrogel, we demonstrate that it recapitulates the dynamic processes of early human vessel formation, including acquisition of distinct arterial and venous fates, resulting in a vasculogenesis angiogenesis model plexus (VAMP). The VAMP captures the major stages of vasculogenesis, angiogenesis, and vascular network formation and is a simple, rapid, scalable model system for studying early human vascular development in vitro.
RESUMEN
Cerebral cortical-enriched organoids derived from human pluripotent stem cells (hPSCs) are valuable models for studying neurodevelopment, disease mechanisms, and therapeutic development. However, recognized limitations include the high variability of organoids across hPSC donor lines and experimental replicates. We report a 96-slitwell method for efficient, scalable, reproducible cortical organoid production. When hPSCs were cultured with controlled-release FGF2 and an SB431542 concentration appropriate for their TGFBR1 / ALK5 expression level, organoid cortical patterning and reproducibility were significantly improved. Well-patterned organoids included 16 neuronal and glial subtypes by single cell RNA sequencing (scRNA-seq), frequent neural progenitor rosettes and robust BCL11B+ and TBR1+ deep layer cortical neurons at 2 months by immunohistochemistry. In contrast, poorly-patterned organoids contain mesendoderm-related cells, identifiable by negative QC markers including COL1A2 . Using this improved protocol, we demonstrate increased sensitivity to study the impact of different MAPT mutations from patients with frontotemporal dementia (FTD), revealing early changes in key metabolic pathways.
RESUMEN
Mutations in the MAPT gene that encodes tau lead to frontotemporal dementia (FTD) with pathology evident in both cerebral neurons and glia. Human cerebral organoids (hCOs) from individuals harboring pathogenic tau mutations can reveal the earliest downstream effects on molecular pathways within a developmental context, generating interacting neurons and glia. We found that in hCOs carrying the V337M and R406W tau mutations, the cholesterol biosynthesis pathway in astrocytes was the top upregulated gene set compared with isogenic controls by single-cell RNA sequencing (scRNA-seq). The 15 upregulated genes included HMGCR, ACAT2, STARD4, LDLR, and SREBF2. This result was confirmed in a homozygous R406W mutant cell line by immunostaining and sterol measurements. Cholesterol abundance in the brain is tightly regulated by efflux and cholesterol biosynthetic enzyme levels in astrocytes, and dysregulation can cause aberrant phosphorylation of tau. Our findings suggest that cholesterol dyshomeostasis is an early event in the etiology of neurodegeneration caused by tau mutations.
Asunto(s)
Demencia Frontotemporal , Proteínas tau , Colesterol , Demencia Frontotemporal/genética , Humanos , Mutación/genética , Organoides/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Genetic and genomic studies of brain disease increasingly demonstrate disease-associated interactions between the cell types of the brain. Increasingly complex and more physiologically relevant human-induced pluripotent stem cell (hiPSC)-based models better explore the molecular mechanisms underlying disease but also challenge our ability to resolve cell type-specific perturbations. Here, we report an extension of the RiboTag system, first developed to achieve cell type-restricted expression of epitope-tagged ribosomal protein (RPL22) in mouse tissue, to a variety of in vitro applications, including immortalized cell lines, primary mouse astrocytes, and hiPSC-derived neurons. RiboTag expression enables depletion of up to 87 percent of off-target RNA in mixed species co-cultures. Nonetheless, depletion efficiency varies across independent experimental replicates, particularly for hiPSC-derived motor neurons. The challenges and potential of implementing RiboTags in complex in vitro cultures are discussed.
Asunto(s)
Perfilación de la Expresión Génica , Modelos Biológicos , Células-Madre Neurales/metabolismo , Células 3T3 , Animales , Técnicas de Cocultivo , Epítopos/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Células-Madre Neurales/citología , Neuronas/citología , Neuronas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Ribosómicas/metabolismo , Especificidad de la Especie , Transcriptoma/genéticaRESUMEN
The ability of isolated neural stem cells (NSCs) to proliferate as neurospheres is indicative of their competence as stem cells, and depends critically on the polycomb group (PcG) member Bmi1: knockdown of Bmi1 results in defective proliferation and self-renewal of isolated NSCs, whereas overexpression of Bmi1 enhances these properties. Here we report genome-wide changes in gene expression in embryonic and adult NSCs (eNSCs and aNSCs) caused by overexpression of Bmi1. We find that genes whose expression is altered by perturbations in Bmi1 levels in NSCs are mostly distinct from those affected in other multipotent stem/progenitor cells, such as those from liver and lung, aside from a small core of common targets that is enriched for genes associated with cell migration and mobility. We also show that genes differing in expression between prospectively isolated quiescent and activated NSCs are not affected by Bmi1 overexpression. In contrast, a comparison of genes showing altered expression upon Bmi1 overexpression in eNSCs and in aNSCs reveals considerable overlap, in spite of their different provenances in the brain and their differing developmental programs.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Células Madre Embrionarias de Ratones/metabolismo , Células-Madre Neurales/metabolismo , Complejo Represivo Polycomb 1/genética , Proteínas Proto-Oncogénicas/genética , Regulación hacia Arriba , Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Animales , Células Cultivadas , Masculino , Ratones , Células Madre Embrionarias de Ratones/citología , Células-Madre Neurales/citologíaRESUMEN
Francisella tularensis is a category A biothreat agent, and as a result, it has recently generated much research interest. F. tularensis live vaccine strain (LVS) is an attenuated form of the virulent F. tularensis organism and has previously been used as a vaccine. However, because of safety concerns, it is no longer approved for this purpose. Thus, the use of inactivated organisms is preferable for vaccine purposes. Although many studies have been performed that examine human peripheral blood mononuclear cells (PBMC), and in particular CD4 T cells, responses to inactivated F. tularensis, there has been no study identifying the individual human cell populations within a mixed PBMC population that respond to this organism. We sought to address this deficit. Our results indicate that natural killer and CD8 T cells comprise the majority of cells responding to F. tularensis LVS. In addition, data suggest CD8 T cell responses are maximal when antibiotic-treated organisms are used and are minimal when formaldehyde-fixed organisms are used. Given the belief that CD8 T cells can play an important role in protection against F. tularensis infection, these studies have direct relevance to the development of F. tularensis vaccines that use inactivated organisms. In addition, important new knowledge is added to our understanding of the human immune response to F. tularensis LVS.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Vacunas Bacterianas/inmunología , Linfocitos T CD8-positivos/inmunología , Francisella tularensis/inmunología , Células Asesinas Naturales/inmunología , Leucocitos Mononucleares/inmunología , Subgrupos de Linfocitos T/inmunología , Antibacterianos/farmacología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/microbiología , Proliferación Celular , Células Cultivadas , Formaldehído , Francisella tularensis/efectos de los fármacos , Francisella tularensis/efectos de la radiación , Humanos , Separación Inmunomagnética , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/microbiología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/microbiología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/microbiología , Factor de Necrosis Tumoral alfa/metabolismo , Rayos Ultravioleta , Vacunas Atenuadas/inmunologíaRESUMEN
Neural induction is the first fundamental step in nervous system formation. During development, a tightly regulated niche modulates transient extracellular signals to influence neural lineage commitment. To date, however, the cascade of molecular events that sustain these signals in humans is not well understood. Here we show that NPTX1, a secreted protein, is rapidly upregulated during neural induction from human pluripotent stem cells (hPSCs). By manipulating its expression, we were able to reduce or initiate neural lineage commitment. A time-course transcriptome analysis and functional assays show that NPTX1 acts in part by binding the Nodal receptor cofactor TDGF1, reducing both Nodal and BMP signaling. Our findings identify one of the earliest genes expressed upon neural induction and provide insight into human neural lineage specification.
Asunto(s)
Proteína C-Reactiva/metabolismo , Linaje de la Célula , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Proteína C-Reactiva/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/genética , Células-Madre Neurales/citología , Neurogénesis , Unión Proteica , Transcriptoma , Regulación hacia ArribaRESUMEN
An essential aspect of stem cell culture is the successful maintenance of the undifferentiated state. Many types of stem cells are FGF2 dependent, and pluripotent stem cells are maintained by replacing FGF2-containing media daily, while tissue-specific stem cells are typically fed every 3rd day. Frequent feeding, however, results in significant variation in growth factor levels due to FGF2 instability, which limits effective maintenance due to spontaneous differentiation. We report that stabilization of FGF2 levels using controlled release PLGA microspheres improves expression of stem cell markers, increases stem cell numbers and decreases spontaneous differentiation. The controlled release FGF2 additive reduces the frequency of media changes needed to maintain stem cell cultures, so that human embryonic stem cells and induced pluripotent stem cells can be maintained successfully with biweekly feedings.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Células Madre/citología , Animales , Células Cultivadas , Células Inmovilizadas/citología , Células Inmovilizadas/efectos de los fármacos , Medios de Cultivo/farmacología , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/enzimología , Activación Enzimática/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/enzimología , Ácido Láctico , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Microesferas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/enzimología , Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Células Madre/efectos de los fármacos , Células Madre/enzimologíaRESUMEN
The ability of exogenous interleukin-12 (IL-12) to elicit protective innate immune responses against the extracellular pathogen Streptococcus pneumoniae was tested by infecting BALB/c mice intranasally (i.n.) with S. pneumoniae after i.n. administration of IL-12. It was found that administration of IL-12 resulted in lower bacterial burdens in the infected mice and significantly improved survival rates. All IL-12-treated mice contained higher levels of pulmonary gamma interferon (IFN-gamma) after infection and significantly more neutrophils than infected mice not treated with IL-12. IFN-gamma was found to be essential for IL-12-induced resistance and for neutrophil influx into the lungs, and the observed changes correlated with increased levels of the IL-8 homologue keratinocyte-derived chemokine (KC). In addition, in vitro tumor necrosis factor alpha (TNF-alpha) production by alveolar macrophages stimulated with heat-killed pneumococci was enhanced by IFN-gamma, and TNF-alpha in turn could enhance production of KC by lung cells. Finally, IL-12-induced protection was dependent upon the presence of neutrophils and the KC receptor CXCR2. Taken together, the results indicate that exogenous IL-12 can improve innate defense in the lung against S. pneumoniae by inducing IFN-gamma production, which in turn enhances chemokine expression, and promotes pulmonary neutrophil recruitment into the infected lung. The findings show that IL-12 and IFN-gamma can mediate a protective effect against respiratory infection caused by extracellular bacterial pathogens.
Asunto(s)
Interferón gamma/fisiología , Interleucina-12/fisiología , Pulmón/metabolismo , Infiltración Neutrófila/inmunología , Neutrófilos/inmunología , Neutrófilos/patología , Neumonía Neumocócica/metabolismo , Streptococcus pneumoniae/inmunología , Animales , Interferón gamma/deficiencia , Interferón gamma/genética , Pulmón/inmunología , Pulmón/microbiología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neutrófilos/microbiología , Neumonía Neumocócica/inmunología , Neumonía Neumocócica/prevención & controlRESUMEN
Toll-like receptor 2 (TLR2) deficiency enhances murine susceptibility to infection by Francisella tularensis as indicated by accelerated mortality, higher bacterial burden, and greater histopathology. Analysis of pulmonary cytokine levels revealed that TLR2 deficiency results in significantly lower levels of tumor necrosis factor alpha and interleukin-6 but increased amounts of gamma interferon and monocyte chemoattractant protein 1. This pattern of cytokine production may contribute to the exaggerated pathogenesis seen in TLR2-/- mice. Collectively, these findings suggest that TLR2 plays an important role in tempering the host response to pneumonic tularemia.