RESUMO
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.
Assuntos
Cérebro/patologia , Proteína Semelhante a ELAV 4/genética , Ácido Glutâmico/metabolismo , Mutação/genética , Neurônios/patologia , Organoides/metabolismo , Splicing de RNA/genética , Proteínas tau/genética , Autofagia/efeitos dos fármacos , Autofagia/genética , Biomarcadores/metabolismo , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Morte Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Hidrazonas/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Morfolinas/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Organoides/efeitos dos fármacos , Organoides/ultraestrutura , Fosforilação/efeitos dos fármacos , Pirimidinas/farmacologia , Splicing de RNA/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Grânulos de Estresse/efeitos dos fármacos , Grânulos de Estresse/metabolismo , Sinapses/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Self-organizing three-dimensional cellular models derived from human pluripotent stem cells or primary tissue have great potential to provide insights into how the human nervous system develops, what makes it unique and how disorders of the nervous system arise, progress and could be treated. Here, to facilitate progress and improve communication with the scientific community and the public, we clarify and provide a basic framework for the nomenclature of human multicellular models of nervous system development and disease, including organoids, assembloids and transplants.
Assuntos
Consenso , Sistema Nervoso , Organoides , Terminologia como Assunto , Humanos , Modelos Biológicos , Sistema Nervoso/citologia , Sistema Nervoso/patologia , Organoides/citologia , Organoides/patologia , Células-Tronco Pluripotentes/citologiaRESUMO
BACKGROUND: Tau post-translational modifications (PTMs) result in the gradual build-up of abnormal tau and neuronal degeneration in tauopathies, encompassing variants of frontotemporal lobar degeneration (FTLD) and Alzheimer's disease (AD). Tau proteolytically cleaved by active caspases, including caspase-6, may be neurotoxic and prone to self-aggregation. Also, our recent findings show that caspase-6 truncated tau represents a frequent and understudied aspect of tau pathology in AD in addition to phospho-tau pathology. In AD and Pick's disease, a large percentage of caspase-6 associated cleaved-tau positive neurons lack phospho-tau, suggesting that many vulnerable neurons to tau pathology go undetected when using conventional phospho-tau antibodies and possibly will not respond to phospho-tau based therapies. Therefore, therapeutic strategies against caspase cleaved-tau pathology could be necessary to modulate the extent of tau abnormalities in AD and other tauopathies. METHODS: To understand the timing and progression of caspase activation, tau cleavage, and neuronal death, we created two mAbs targeting caspase-6 tau cleavage sites and probed postmortem brain tissue from an individual with FTLD due to the V337M MAPT mutation. We then assessed tau cleavage and apoptotic stress response in cortical neurons derived from induced pluripotent stem cells (iPSCs) carrying the FTD-related V337M MAPT mutation. Finally, we evaluated the neuroprotective effects of caspase inhibitors in these iPSC-derived neurons. RESULTS: FTLD V337M MAPT postmortem brain showed positivity for both cleaved tau mAbs and active caspase-6. Relative to isogenic wild-type MAPT controls, V337M MAPT neurons cultured for 3 months post-differentiation showed a time-dependent increase in pathogenic tau in the form of caspase-cleaved tau, phospho-tau, and higher levels of tau oligomers. Accumulation of toxic tau species in V337M MAPT neurons was correlated with increased vulnerability to pro-apoptotic stress. Notably, this mutation-associated cell death was pharmacologically rescued by the inhibition of effector caspases. CONCLUSIONS: Our results suggest an upstream, time-dependent accumulation of caspase-6 cleaved tau in V337M MAPT neurons promoting neurotoxicity. These processes can be reversed by caspase inhibition. These results underscore the potential of developing caspase-6 inhibitors as therapeutic agents for FTLD and other tauopathies. Additionally, they highlight the promise of using caspase-cleaved tau as biomarkers for these conditions.
RESUMO
STAU2 is a double-stranded RNA-binding protein enriched in the nervous system. During asymmetric divisions in the developing mouse cortex, STAU2 preferentially distributes into the intermediate progenitor cell (IPC), delivering RNA molecules that can impact IPC behavior. Corticogenesis occurs on a precise time schedule, raising the hypothesis that the cargo STAU2 delivers into IPCs changes over time. To test this, we combine RNA-immunoprecipitation with sequencing (RIP-seq) over four stages of mouse cortical development, generating a comprehensive cargo profile for STAU2. A subset of the cargo was 'stable', present at all stages, and involved in chromosome organization, macromolecule localization, translation and DNA repair. Another subset was 'dynamic', changing with cortical stage, and involved in neurogenesis, cell projection organization, neurite outgrowth, and included cortical layer markers. Notably, the dynamic STAU2 cargo included determinants of IPC versus neuronal fates and genes contributing to abnormal corticogenesis. Knockdown of one STAU2 target, Taf13, previously linked to microcephaly and impaired myelination, reduced oligodendrogenesis in vitro. We conclude that STAU2 contributes to the timing of corticogenesis by binding and delivering complex and temporally regulated RNA cargo into IPCs.
Assuntos
Córtex Cerebral/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Células-Tronco/metabolismo , Animais , Células Cultivadas , Reparo do DNA/fisiologia , Feminino , Imunoprecipitação/métodos , Masculino , Camundongos , Neurogênese/fisiologia , Neurônios/metabolismo , GravidezRESUMO
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.
Assuntos
Microglia , Traumatismos da Medula Espinal , Camundongos , Animais , Microglia/metabolismo , Traumatismos da Medula Espinal/metabolismo , Medula Espinal/metabolismo , Linfócitos B/metabolismoRESUMO
Transcriptional profiling is a powerful approach for understanding development and disease. Current cell type-specific RNA purification methods have limitations, including cell dissociation trauma or inability to identify all RNA species. Here, we describe "mouse thiouracil (TU) tagging," a genetic and chemical intersectional method for covalent labeling and purification of cell type-specific RNA in vivo. Cre-induced expression of uracil phosphoribosyltransferase (UPRT) provides spatial specificity; injection of 4-thiouracil (4TU) provides temporal specificity. Only UPRT(+) cells exposed to 4TU produce thio-RNA, which is then purified for RNA sequencing (RNA-seq). This method can purify transcripts from spatially complex and rare (<5%) cells, such as Tie2:Cre(+) brain endothelia/microglia (76% validated by expression pattern), or temporally dynamic transcripts, such as those acutely induced by lipopolysaccharide (LPS) injection. Moreover, generating chimeric mice via UPRT(+) bone marrow transplants identifies immune versus niche spleen RNA. TU tagging provides a novel method for identifying actively transcribed genes in specific cells at specific times within intact mice.
Assuntos
Biologia Molecular/métodos , RNA/isolamento & purificação , Coloração e Rotulagem/métodos , Tiouracila/metabolismo , Animais , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Encéfalo/embriologia , Encéfalo/metabolismo , Quimera , Perfilação da Expressão Gênica , Camundongos , Transgenes/genéticaRESUMO
Retinal degenerative diseases are the leading causes of blindness worldwide. Replacing lost retinal cells via stem cell-based therapies is an exciting, rapidly advancing area of translational research that has already entered the clinic. Here, we review the status of these clinical efforts for several significant retinal diseases, describe the challenges involved and discuss how basic developmental studies have contributed to and are needed to advance clinical goals.
Assuntos
Degeneração Retiniana/terapia , Transplante de Células-Tronco , Animais , Ensaios Clínicos como Assunto , Humanos , Modelos Biológicos , Retina/embriologia , Retina/patologia , Degeneração Retiniana/imunologiaRESUMO
Staufen2 (Stau2) is a double-stranded RNA-binding protein (RBP) involved in posttranscriptional gene expression control in neurons. In flies, staufen contributes to learning and long-term memory formation. To study the impact of mammalian Stau2 on behavior, we generated a novel gene-trap mouse model that yields significant constitutive downregulation of Stau2 (Stau2GT). In order to investigate the effect of Stau2 downregulation on hippocampus-dependent behavior, we performed a battery of behavioral assays, i.e. open field, novel object recognition/location (NOR/L) and Barnes maze. Stau2GT mice displayed reduced locomotor activity in the open field and altered novelty preference in the NOR and NOL paradigms. Adult Stau2GT male mice failed to discriminate between familiar and newly introduced objects but showed enhanced spatial novelty detection. Additionally, we observed deficits in discriminating different spatial contexts in a Barnes maze assay. Together, our data suggest that Stau2 contributes to novelty preference and explorative behavior that is a driver for proper spatial learning in mice.
Assuntos
Comportamento Exploratório/fisiologia , Hipocampo/metabolismo , Aprendizagem/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas de Ligação a RNA/metabolismo , Reconhecimento Psicológico/fisiologia , Animais , Comportamento de Escolha/fisiologia , Camundongos , Camundongos Knockout , Atividade Motora/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas de Ligação a RNA/genéticaRESUMO
Exploration of the epitranscriptome requires the development of highly sensitive and accurate technologies in order to elucidate the contributions of the more than 100 RNA modifications to cell processes. A highly sensitive and accurate ultra-high performance liquid chromatography-tandem mass spectrometry method was developed to simultaneously detect and quantify 28 modified and four major nucleosides in less than 20 min. Absolute concentrations were calculated using extinction coefficients of each of the RNA modifications studied. A comprehensive RNA modifications database of UV profiles and extinction coefficient is reported within a 2.3-5.2 % relative standard deviation. Excellent linearity was observed 0.99227-0.99999 and limit of detection values ranged from 63.75 attomoles to 1.21 femtomoles. The analytical performance was evaluated by analyzing RNA modifications from 100 ng of RNA from human pluripotent stem cell-derived neural cells. Modifications were detected at concentrations four orders of magnitude lower than the corresponding parental nucleosides, and as low as 23.01 femtograms, 64.09 attomoles. Direct and global quantitative analysis of RNA modifications are among the advantages of this new approach.
Assuntos
Perfilação da Expressão Gênica , Células-Tronco Neurais/metabolismo , RNA/genética , Transcriptoma , Células Cultivadas , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Limite de Detecção , Processamento Pós-Transcricional do RNA , Espectrometria de Massas em Tandem/métodosRESUMO
Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule-only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Epitélio Pigmentado da Retina/citologia , Ensaios de Triagem em Larga Escala , Humanos , Células-Tronco Pluripotentes/citologia , Reação em Cadeia da PolimeraseRESUMO
Neural stem cells (NSCs) have great potential for self-renewal, which must be tightly regulated to generate appropriate cell numbers during development and to prevent tumor formation. The Ras-MAPK-ERK pathway affects mitogen-stimulated proliferation, and negative regulators are likely to be important for keeping self-renewal in check. Sprouty-related protein with an EVH1 domain (Spred1) is a recently discovered negative Ras-MAPK-ERK regulator linked to a neurofibromatosis 1 (NF-1)-like human syndrome; however, its role in CNS development has not been explored. We show that Spred1 is highly enriched in CNS germinal zones during neurogenesis. Spred1 knockdown increases NSC self-renewal and progenitor proliferation cell-autonomously, and overexpression causes premature differentiation. Surprisingly, Spred1 knockdown in vivo in the embryonic mouse forebrain frequently resulted in periventricular heterotopia, developmental abnormalities often associated with mutations in genes in the vesicular trafficking pathway that cause disruption of germinal zones and impair cell migration. In cortical progenitor cells, Spred1 localizes within distinct vesicles, indicating a potential role in transport. Spred1 knockdown gradually leads to disruption of the apical ventricular zone and loss of radial glia alignment. This impairs late neuronal migration, resulting in the formation of periventricular masses. Thus, Spred1 is critical for normal cortical development, as it modulates progenitor self-renewal/proliferation and helps maintain the integrity and organization of germinal zones.
Assuntos
Córtex Cerebral/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Genes ras/genética , Quinases de Proteína Quinase Ativadas por Mitógeno , Neurônios/citologia , Proteínas Repressoras/metabolismo , Células-Tronco/citologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Diferenciação Celular , Proliferação de Células , Feminino , Técnicas de Silenciamento de Genes , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Heterotopia Nodular Periventricular/genética , Gravidez , Proteínas Repressoras/genéticaRESUMO
The analysis of time-lapse images showing cells dividing to produce clones of related cells is an important application in biological microscopy. Imaging at the temporal resolution required to establish accurate tracking for vertebrate stem or cancer cells often requires the use of transmitted light or phase-contrast microscopy. Processing these images requires automated segmentation, tracking and lineaging algorithms. There is also a need for any errors in the automated processing to be easily identified and quickly corrected. We have developed LEVER, an open source software tool that combines the automated image analysis for phase-contrast microscopy movies with an easy-to-use interface for validating the results and correcting any errors. AVAILABILITY AND IMPLEMENTATION: LEVER is available free and open source, licensed under the GNU GPLv3. Details on obtaining and using LEVER are available at http://n2t.net/ark:/87918/d9rp4t CONTACT: acohen@coe.drexel.edu.
Assuntos
Linhagem da Célula , Proliferação de Células , Software , Algoritmos , Animais , Humanos , Processamento de Imagem Assistida por Computador , Microscopia , Microscopia de Contraste de FaseRESUMO
Neural stem cells (NSCs) persist throughout life in two forebrain areas: the subventricular zone (SVZ) and the hippocampus. Why forebrain NSCs self-renew more extensively than those from other regions remains unclear. Prior studies have shown that the polycomb factor Bmi-1 is necessary for NSC self-renewal and that it represses the cell cycle inhibitors p16, p19, and p21. Here we show that overexpression of Bmi-1 enhances self-renewal of forebrain NSCs significantly more than those derived from spinal cord, demonstrating a regional difference in responsiveness. We show that forebrain NSCs require the forebrain-specific transcription factor Foxg1 for Bmi-1-dependent self-renewal, and that repression of p21 is a focus of this interaction. Bmi-1 enhancement of NSC self-renewal is significantly greater with increasing age and passage. Importantly, when Bmi-1 is overexpressed in cultured adult forebrain NSCs, they expand dramatically and continue to make neurons even after multiple passages, when control NSCs have become restricted to glial differentiation. Together these findings demonstrate the importance of Bmi-1 and Foxg1 cooperation to maintenance of NSC multipotency and self-renewal, and establish a useful method for generating abundant forebrain neurons ex vivo, outside the neurogenic niche.
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Fatores de Transcrição Forkhead/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Prosencéfalo/citologia , Prosencéfalo/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Células-Tronco/citologia , Animais , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Feminino , Expressão Gênica , Camundongos , Complexo Repressor Polycomb 1 , Gravidez , Prosencéfalo/embriologia , Células-Tronco/metabolismoRESUMO
The retina, like other central nervous system tissues, has poor regenerative properties in humans. Therefore, diseases that cause retinal cell loss, such as Age-related macular degeneration (AMD), retinitis pigmentosa (RP), Leber congenital amaurosis, Usher syndrome, glaucoma, and diabetic retinopathy, typically result in permanent visual impairment. Stem cell technologies have revolutionized our ability to produce neural cells in abundant supply. Much stem cell research effort is focused on producing the required cell types for cell replacement, or to generate disease-in-a-dish models to elucidate novel disease mechanisms for therapeutic development. Here we review the recent advances in stem cell studies relevant to producing RPE and retinal cells, and highlight future directions.
Assuntos
Oftalmopatias/terapia , Medicina Regenerativa/métodos , Transplante de Células-Tronco/métodos , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células/tendências , Técnicas de Reprogramação Celular/métodos , Técnicas de Reprogramação Celular/tendências , Células-Tronco Embrionárias/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Medicina Regenerativa/tendências , Epitélio Pigmentado da Retina/citologia , Células-Tronco/citologiaRESUMO
The retinal pigment epithelium (RPE) is a pigmented cellular monolayer that supports photoreceptor cells located in the overlying neural retina. The RPE is critical for vision and its dysfunction results in numerous pathologies, several with limited available disease-altering strategies. Regeneration of the retina from RPE is robust in lower vertebrates, but is not normally exhibited in mammals. We recently found that a subpopulation of human RPE cells can be stimulated in culture to generate multipotent self-renewing cells-the RPE stem cell (RPESC). RPESC can be expanded to generate RPE progeny that are a potential source for cell replacement therapy. Alternatively, RPESC can produce mesenchymal progeny which serve as a disease model of epiretinal membrane formation. Yet another potential application of RPESCs is activation within the eye to awaken dormant endogenous repair.
Assuntos
Diferenciação Celular , Proliferação de Células , Epitélio Pigmentado da Retina/citologia , Células-Tronco/citologia , Animais , Técnicas de Cultura de Células , Linhagem da Célula , Células Cultivadas , Humanos , Células-Tronco Multipotentes/citologia , Transplante de Células-Tronco/métodosRESUMO
BACKGROUND: Neural stem cells are motile and proliferative cells that undergo mitosis, dividing to produce daughter cells and ultimately generating differentiated neurons and glia. Understanding the mechanisms controlling neural stem cell proliferation and differentiation will play a key role in the emerging fields of regenerative medicine and cancer therapeutics. Stem cell studies in vitro from 2-D image data are well established. Visualizing and analyzing large three dimensional images of intact tissue is a challenging task. It becomes more difficult as the dimensionality of the image data increases to include time and additional fluorescence channels. There is a pressing need for 5-D image analysis and visualization tools to study cellular dynamics in the intact niche and to quantify the role that environmental factors play in determining cell fate. RESULTS: We present an application that integrates visualization and quantitative analysis of 5-D (x,y,z,t,channel) and large montage confocal fluorescence microscopy images. The image sequences show stem cells together with blood vessels, enabling quantification of the dynamic behaviors of stem cells in relation to their vascular niche, with applications in developmental and cancer biology. Our application automatically segments, tracks, and lineages the image sequence data and then allows the user to view and edit the results of automated algorithms in a stereoscopic 3-D window while simultaneously viewing the stem cell lineage tree in a 2-D window. Using the GPU to store and render the image sequence data enables a hybrid computational approach. An inference-based approach utilizing user-provided edits to automatically correct related mistakes executes interactively on the system CPU while the GPU handles 3-D visualization tasks. CONCLUSIONS: By exploiting commodity computer gaming hardware, we have developed an application that can be run in the laboratory to facilitate rapid iteration through biological experiments. We combine unsupervised image analysis algorithms with an interactive visualization of the results. Our validation interface allows for each data set to be corrected to 100% accuracy, ensuring that downstream data analysis is accurate and verifiable. Our tool is the first to combine all of these aspects, leveraging the synergies obtained by utilizing validation information from stereo visualization to improve the low level image processing tasks.
Assuntos
Algoritmos , Linhagem da Célula , Gráficos por Computador , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Células-Tronco Neurais/citologia , Automação , Microscopia Confocal , Microscopia de Fluorescência , SoftwareRESUMO
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.
RESUMO
Neuronal dysfunction has been extensively studied as a central feature of neurodegenerative tauopathies. However, across neurodegenerative diseases, there is strong evidence for active involvement of immune cells like microglia in driving disease pathophysiology. Here, we demonstrate that tau mRNA and protein are expressed in microglia in human brains and in human induced pluripotent stem cell (iPSC)-derived microglia like cells (iMGLs). Using iMGLs harboring the MAPT IVS10+16 mutation and isogenic controls, we demonstrate that a tau mutation is sufficient to alter microglial transcriptional states. We discovered that MAPT IVS10+16 microglia exhibit cytoskeletal abnormalities, stalled phagocytosis, disrupted TREM2/TYROBP networks, and altered metabolism. Additionally, we found that secretory factors from MAPT IVS10+16 iMGLs impact neuronal health, reducing synaptic density in neurons. Key features observed in vitro were recapitulated in human brain tissue and cerebrospinal fluid from MAPT mutations carriers. Together, our findings that MAPT IVS10+16 drives cell-intrinsic dysfunction in microglia that impacts neuronal health has major implications for development of therapeutic strategies.
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Cell-based therapies are being developed for various neurodegenerative diseases that affect the central nervous system (CNS). Concomitantly, the roles of individual cell types in neurodegenerative pathology are being uncovered by genetic and single-cell studies. With a greater understanding of cellular contributions to health and disease and with the arrival of promising approaches to modulate them, effective therapeutic cell products are now emerging. This review examines how the ability to generate diverse CNS cell types from stem cells, along with a deeper understanding of cell-type-specific functions and pathology, is advancing preclinical development of cell products for the treatment of neurodegenerative diseases.