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Stress granules are the RNA/protein condensates assembled in the cells under stress. They play a critical role in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, how stress granule assembly is regulated and related to ALS/FTD pathomechanism is incompletely understood. Mutation in the C9orf72 gene is the most common cause of familial ALS and FTD. C9orf72 mutation causes the formation of toxic dipeptide repeats. Here we show that the two most toxic dipeptide repeats [i.e., poly(GR) and poly(PR)] activate c-Jun N-terminal kinase (JNK) via the ER-stress response protein IRE1 using fly and cellular models. Further, we show that activated JNK promotes stress granule assembly in cells by promoting the transcription of one of the key stress granule proteins (i.e., G3BP1) by inducing histone 3 phosphorylation. Consistent with these findings, JNK or IRE1 inhibition reduced stress granule formation, histone 3 phosphorylation, G3BP1 mRNA and protein levels, and neurotoxicity in cells overexpressing poly(GR) and poly(PR) or neurons derived from male and female C9ALS/FTD patient-induced pluripotent stem cells. Our findings connect ER stress, JNK activation, and stress granule assembly in a unified pathway contributing to C9ALS/FTD neurodegeneration.SIGNIFICANCE STATEMENT c-Jun N-terminal kinase (JNK) is a part of the mitogen-activated protein kinase pathway, which is the central node for the integration of multiple stress signals. Cells are under constant stress in neurodegenerative diseases, and how these cells respond to stress signals is a critical factor in determining their survival or death. Previous studies have shown JNK as a major contributor to cellular apoptosis. Here, we show the role of JNK in stress granule assembly. We identify that toxic dipeptide repeats produced in ALS/FTD conditions activate JNK. The activated JNK in the nucleus can induce histone modifications which increase G3BP1 expression, thus promoting stress granule assembly and neurodegeneration.
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Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Femenino , Humanos , Masculino , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Proteína C9orf72/genética , Dipéptidos/genética , Dipéptidos/metabolismo , ADN Helicasas/metabolismo , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Histonas , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN , Gránulos de Estrés , Drosophila melanogaster , AnimalesRESUMEN
OBJECTIVES: To express a TAT-PBX1 fusion protein using a prokaryotic expression system and to explore potential effects of TAT-PBX1 in the proliferation and senescence of human hair follicle-derived mesenchymal stem cells. RESULTS: The TAT-PBX1 fusion was produced in inclusion bodies and heterogenously expressed in Rosetta (DE3) cells. Immunofluorescence staining showed that TAT-PBX1 fusion proteins were internalized by human hair follicle-derived mesenchymal stem cells. The growth rate of cells was increased after treatment with more than 5.0 µg/mL of TAT-PBX1. The rate of senescence-associated ß-galactosidase positive cells was reduced in the 10.0 µg/mL TAT-PBX1 group (28%) than the 0 µg/mL control group (60%). Cells treated with the TAT-PBX1 fusion protein showed higher expression of p-AKT (1.22-fold that of the control), which indicates that TAT-PBX1 activated AKT pathway after cellular uptake. CONCLUSIONS: The TAT-PBX1 fusion protein increased the proliferation of hair follicle mesenchymal stem cells and delayed their senescence by activating the AKT pathway following internalization by cells.
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Folículo Piloso/citología , Células Madre Mesenquimatosas , Factor de Transcripción 1 de la Leucemia de Células Pre-B/genética , Proteínas Recombinantes de Fusión , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
The use of human mesenchymal stem cells (hMSCs) in cell therapies has increased the demand for strategies that allow efficient cell scale-up. Preliminary data on the three-dimensional (3D) spinner culture describing the potential use of microcarriers for hMSCs culture scale-up have been reported. We exploited a rich source of autologous stem cells (human hair follicle) and demonstrated the robust in vitro long-term expansion of human hair follicle-derived mesenchymal stem cells (hHF-MSCs) by using CultiSpher(®)-G microcarriers. We analyzed the feasibility of 3D culture by using hHF-MSCs/CultiSpher(®)-G microcarrier constructs for its potential applicability in regenerative medicine by comparatively analyzing the performance of hHF-MSCs adhered to the CultiSpher(®)-G microspheres in 3D spinner culture and those grown on the gelatin-coated plastic dishes (2D culture), using various assays. We showed that the hHF-MSCs seeded at various densities quickly adhered to and proliferated well on the microspheres, thus generating at least hundreds of millions of hHF-MSCs on 1 g of CultiSpher(®)-G within 12 days. This resulted in a cumulative cell expansion of greater than 26-fold. Notably, the maximum and average proliferation rates in 3D culture were significantly greater than that of the 2D culture. However, the hHF-MSCs from both the cultures retained surface marker and nestin expression, proliferation capacity and differentiation potentials toward adipocytes, osteoblasts and smooth muscle cells and showed no significant differences as evidenced by Edu incorporation, cell cycle, colony formation, apoptosis, biochemical quantification and qPCR assays.
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Folículo Piloso/metabolismo , Células Madre Mesenquimatosas/metabolismo , Adulto , Diferenciación Celular , Proliferación Celular , Femenino , Folículo Piloso/citología , Humanos , Masculino , Persona de Mediana Edad , Medicina RegenerativaRESUMEN
Tauopathies represent a diverse group of neurodegenerative disorders characterized by the abnormal aggregation of the microtubule-associated protein tau. Despite extensive research, the precise mechanisms underlying the complexity of different types of tau pathology remain incompletely understood. Here we describe an approach for proteomic profiling of aggregate-associated proteomes on slides with formalin-fixed, paraffin-embedded (FFPE) tissue that utilizes proximity labelling upon high preservation of aggregate morphology, which permits the profiling of pathological aggregates regardless of their size. To comprehensively investigate the common and unique protein interactors associated with the variety of tau lesions present across different human tauopathies, Alzheimer's disease (AD), corticobasal degeneration (CBD), Pick's disease (PiD), and progressive supranuclear palsy (PSP), were selected to represent the major tauopathy diseases. Implementation of our widely applicable Probe-dependent Proximity Profiling (ProPPr) strategy, using the AT8 antibody, permitted identification and quantification of proteins associated with phospho-tau lesions in well-characterized human post-mortem tissue. The analysis revealed both common and disease-specific proteins associated with phospho-tau aggregates, highlighting potential targets for therapeutic intervention and biomarker development. Candidate validation through high-resolution co-immunofluorescence of distinct aggregates across disease and control cases, confirmed the association of retromer complex protein VPS35 with phospho-tau lesions across the studied tauopathies. Furthermore, we discovered disease-specific associations of proteins including ferritin light chain (FTL) and the neuropeptide precursor VGF within distinct pathological lesions. Notably, examination of FTL-positive microglia in CBD astrocytic plaques indicate a potential role for microglial involvement in the pathogenesis of these tau lesions. Our findings provide valuable insights into the proteomic landscape of tauopathies, shedding light on the molecular mechanisms underlying tau pathology. This first comprehensive characterization of tau-associated proteomes across different tauopathies enhances our understanding of disease heterogeneity and provides a resource for future functional investigation, as well as development of targeted therapies and diagnostic biomarkers.
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In this issue of Neuron, Liu et al.1 identify DAXX as a C9orf72 hexanucleotide repeat expansion DNA-binding protein that initiates epigenetic modifications and chromatin remodeling, contributing to C9orf72 haploinsufficiency by inhibiting its stress-inducible expression and mediating both loss- and toxic gain-of-function pathology.
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Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Humanos , Expansión de las Repeticiones de ADN/genética , Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Demencia Frontotemporal/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/metabolismoRESUMEN
Stem cell senescence and depletion are major causes of aging and aging-related diseases. The NAD (Nicotinamide adenine dinucleotide) - SIRT1 (Silent Information Regulator 1) - PARP1 (Poly (ADP-ribose) polymerase-1) axis has gained interest owing to its significant role in regulating stem cell senescence and organismal aging. A recent study from our lab showed that pre-B-cell leukemia transcription factor1 (PBX1) overexpression attenuates hair follicle-derived mesenchymal stem cells (HF-MSCs) senescence and apoptosis by regulating ROS-mediated DNA damage via PARP1 downregulation; thus, suggesting that PARP1 downregulation is a common manifestation of the roles of both PBX1 and SIRT1 in HF-MSCs senescence attenuation, and implying a potential link between PBX1 and SIRT1. To this end, HF-MSCs overexpressing PBX1, overexpressing both PBX1 and PARP1, downregulating SIRT1, and overexpressing PBX1 as well as downregulating SIRT1 were generated, and senescence, apoptosis, DNA damage, and repair biomarkers were analyzed. Our results showed that (1) PBX1 overexpression alleviated HF-MSCs senescence and apoptosis accompanied by SIRT1 upregulation, PARP1 downregulation, and increased intracellular NAD and ATP levels. (2) SIRT1 knockdown enhanced cellular senescence and apoptosis, accompanied by increased ROS accumulation, DNA damage aggravation, and decreased intracellular NAD and ATP levels. (3) PBX1 overexpression rescued HF-MSCs senescence and apoptosis induced by SIRT1 knockdown. (4) PBX1 rescued PARP1 overexpression-mediated ATP and NAD depletion, accompanied by increased SIRT1 expression. Collectively, our results revealed that a positive interaction feedback loop exists between PBX1 and SIRT1. To the best of our knowledge we are the first to report that there is a PBX1-SIRT1-PARP1 axis that plays a critical role in alleviating HF-MSCs senescence and apoptosis. We provide a new perspective on the mechanisms underlying stem cell senescence as well as age-related disease prevention and treatment.
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Transducción de Señal , Sirtuina 1 , Especies Reactivas de Oxígeno , Sirtuina 1/genética , Sirtuina 1/metabolismo , NAD/metabolismo , Retroalimentación , Apoptosis/genética , Adenosina TrifosfatoRESUMEN
Hair follicles (HFs) maintain homeostasis through the hair cycles; therefore, disrupting the hair cycle may lead to hair loss. Our previous study showed that apoptosis-inducing factor (AIF) nuclear translocation and poly [ADP-ribose] polymerase 1 (PARP1) upregulation induced apoptosis in mouse hair follicles during the hair cycle transition from anagen to catagen. However, the mechanism underlying this phenomenon remains unclear. In this study, we found that intrinsic ROS levels increased during the hair follicle cycle transition from anagen to catagen, followed by abrupt DNA breaks and activation of homologous recombinant and nonhomologous end joining DNA repair, along with the enhancement of apoptosis. Mice in different stages of the hair cycle were sacrificed, and the dorsal skins were collected. The results of western blot and histological staining indicated that AIF-PARP1 plays a key role in HF apoptosis, but their role in the regulation of the HF cycle is not clear. Mice were treated with inhibitors from anagen to catagen: treatment with BMN 673, a PARP1 inhibitor, increased DNA breaks and activated the cytochrome c/caspase-3-mediated apoptotic pathway, accelerating HF regression. Ac-DEVD-CHO (Ac), a caspase-3 inhibitor, attenuated HF degeneration by upregulating PARP1 expression, suggesting a seesaw relationship between cytochrome c-caspase-3- and AIF-PARP1-mediated apoptosis, wherein PARP1 may be the fulcrum. In addition, macrophages were involved in regulating the hair cycle, and the rate of M1 macrophages around HFs increased during catagen, while more M2 macrophages were found during anagen and telogen. Our results indicate that intrinsic ROS drive HF cycle progression through DNA damage and repair, followed by apoptosis. Intrinsic ROS drive hair follicle cycle progression by modulating DNA damage and repair, and consecutively, hair follicle apoptosis and macrophage polarization work together to promote the hair follicle cycle.
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Citocromos c , Folículo Piloso , Animales , Apoptosis/fisiología , Factor Inductor de la Apoptosis/metabolismo , Caspasa 3/metabolismo , Caspasas/metabolismo , Citocromos c/metabolismo , Daño del ADN , Macrófagos/metabolismo , Ratones , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The most common inherited cause of two genetically and clinico-pathologically overlapping neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), is the presence of expanded GGGGCC intronic hexanucleotide repeats in the C9orf72 gene. Aside from haploinsufficiency and toxic RNA foci, another non-exclusive disease mechanism is the non-canonical translation of the repeat RNA into five different dipeptide repeat proteins (DPRs), which form neuronal inclusions in affected patient brains. While evidence from cellular and animal models supports a toxic gain-of-function of pathologic poly-GA, poly-GR, and poly-PR aggregates in promoting deposition of TDP-43 pathology and neurodegeneration in affected brain areas, the relative contribution of DPRs to the disease process in c9FTD/ALS patients remains unclear. Here we have used the proximity-dependent biotin identification (BioID) proximity proteomics approach to investigate the formation and collective composition of DPR aggregates using cellular models. While interactomes of arginine rich poly-GR and poly-PR aggregates overlapped and were enriched for nucleolar and ribosomal proteins, poly-GA aggregates demonstrated a distinct association with proteasomal components, molecular chaperones (HSPA1A/HSP70, HSPA8/HSC70, VCP/p97), co-chaperones (BAG3, DNAJA1A) and other factors that regulate protein folding and degradation (SQSTM1/p62, CALR, CHIP/STUB1). Experiments in cellular models of poly-GA pathology show that molecular chaperones and co-chaperones are sequestered to the periphery of dense cytoplasmic aggregates, causing depletion from their typical cellular localization. Their involvement in the pathologic process is confirmed in autopsy brain tissue, where HSPA8, BAG3, VCP, and its adapter protein UBXN6 show a close association with poly-GA aggregates in the frontal cortex, temporal cortex, and hippocampus of c9FTLD and c9ALS cases. The association of heat shock proteins and co-chaperones with poly-GA led us to investigate their potential role in reducing its aggregation. We identified HSP40 co-chaperones of the DNAJB family as potent modifiers that increased the solubility of poly-GA, highlighting a possible novel therapeutic avenue and a central role of molecular chaperones in the pathogenesis of human C9orf72-linked diseases.
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Esclerosis Amiotrófica Lateral , Proteína C9orf72 , Demencia Frontotemporal , Agregación Patológica de Proteínas , Secuencias Repetitivas de Ácidos Nucleicos , Dipéptidos , Células HEK293 , Humanos , Chaperonas Moleculares , Proteómica , ARNRESUMEN
BACKGROUND: Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) disease spectrum, causing both nuclear loss-of-function and cytoplasmic toxic gain-of-function phenotypes. While TDP-43 proteinopathy has been associated with defects in nucleocytoplasmic transport, this process is still poorly understood. Here we study the role of karyopherin-ß1 (KPNB1) and other nuclear import receptors in regulating TDP-43 pathology. METHODS: We used immunostaining, immunoprecipitation, biochemical and toxicity assays in cell lines, primary neuron and organotypic mouse brain slice cultures, to determine the impact of KPNB1 on the solubility, localization, and toxicity of pathological TDP-43 constructs. Postmortem patient brain and spinal cord tissue was stained to assess KPNB1 colocalization with TDP-43 inclusions. Turbidity assays were employed to study the dissolution and prevention of aggregation of recombinant TDP-43 fibrils in vitro. Fly models of TDP-43 proteinopathy were used to determine the effect of KPNB1 on their neurodegenerative phenotype in vivo. RESULTS: We discovered that several members of the nuclear import receptor protein family can reduce the formation of pathological TDP-43 aggregates. Using KPNB1 as a model, we found that its activity depends on the prion-like C-terminal region of TDP-43, which mediates the co-aggregation with phenylalanine and glycine-rich nucleoporins (FG-Nups) such as Nup62. KPNB1 is recruited into these co-aggregates where it acts as a molecular chaperone that reverses aberrant phase transition of Nup62 and TDP-43. These findings are supported by the discovery that Nup62 and KPNB1 are also sequestered into pathological TDP-43 aggregates in ALS/FTD postmortem CNS tissue, and by the identification of the fly ortholog of KPNB1 as a strong protective modifier in Drosophila models of TDP-43 proteinopathy. Our results show that KPNB1 can rescue all hallmarks of TDP-43 pathology, by restoring its solubility and nuclear localization, and reducing neurodegeneration in cellular and animal models of ALS/FTD. CONCLUSION: Our findings suggest a novel NLS-independent mechanism where, analogous to its canonical role in dissolving the diffusion barrier formed by FG-Nups in the nuclear pore, KPNB1 is recruited into TDP-43/FG-Nup co-aggregates present in TDP-43 proteinopathies and therapeutically reverses their deleterious phase transition and mislocalization, mitigating neurodegeneration.
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Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Animales , Ratones , Transporte Activo de Núcleo Celular , Autopsia , Proteínas de Unión al ADN , Proteínas de Complejo Poro Nuclear , Humanos , DrosophilaRESUMEN
Tissues and organs undergo structural deterioration and functional decline during aging. DNA damage is considered a major cause of stem cell senescence. Although stem cells develop sophisticated DNA repair systems, when the intrinsic and extrinsic insults exceed the DNA repair capacity, cellular senescence, and age-related diseases inevitably occur. Therefore, the prevention and alleviation of DNA damage is an alternative to DNA repair in attenuating stem cell senescence and preventing age-related diseases. Pre-B-cell leukaemia homeobox 1 (PBX1) participates in maintaining the pluripotency of human embryonic and haematopoietic stem cells. Our recent studies showed that PBX1 promotes hair follicle-derived mesenchymal stem cell (HF-MSC) proliferation, decreases cellular senescence and apoptosis, and enhances induced pluripotent stem cell generation. Whether PBX1 attenuates HF-MSC senescence and apoptosis by alleviating DNA damage or by enhancing DNA repair remains unknown. In this study, we aimed to determine the effects of PBX1 on the intrinsic ROS or extrinsic H2O2-induced cellular senescence of HF-MSCs. To this end, we generated HF-MSCs overexpressing either PBX1, or poly (ADP-ribose) polymerase 1, or both. Our results showed that PBX1 overexpression attenuates HF-MSC senescence and apoptosis by alleviating reactive oxygen species (ROS)-mediated DNA damage instead of enhancing DNA repair. This is the first study to report that PBX1 attenuates stem cell senescence and apoptosis by alleviating DNA damage. It provides new insight into the mechanism of stem cell senescence and lays the foundation for the development of strategies for age-related disease prevention and treatment, and in particular, hair follicle repair and regeneration.
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BACKGROUND: Skin wounding is very common and may be slow to heal. Increasing evidence shows that exosomes derived from mesenchymal stem cells (MSCs) dramatically enhance skin wound healing in a paracrine manner. However, the mechanism underlying this phenomenon has not yet been elucidated. Thus, the objective of the present study was to identify the signaling pathways and paracrine factors by which MSC-derived exosomes promote de novo skin tissue regeneration in response to wound healing. METHODS: In vitro and in vivo skin wound healing models were created by treating immortalized human keratinocytes (HaCaT) with hydrogen peroxide (H2O2) and excising full-thickness mouse skin, respectively. Exosomes were extracted from human umbilical cord Wharton's jelly MSCs (hucMSC-Ex) by ultracentrifugation of cell culture supernatant. RESULTS: The hucMSC-Ex treatment significantly increased HaCaT cell proliferation and migration in a time- and dose-dependent manner, suppressed HaCaT apoptosis induced with H2O2 by inhibiting nuclear translocation of apoptosis-inducing factor (AIF) and upregulating poly ADP ribose polymerase 1 (PARP-1) and poly (ADP-ribose) (PAR). The animal experiments showed that relative to hucMSCs, hucMSC-Ex attenuated full-thickness skin wounding by enhancing epidermal re-epithelialization and dermal angiogenesis. CONCLUSIONS: These findings indicated that direct administration of hucMSC-Ex may effectively treat cutaneous wounding and could be of great value in clinical settings.
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Exosomas , Animales , Apoptosis , Factor Inductor de la Apoptosis/genética , Proliferación Celular , Peróxido de Hidrógeno/farmacología , Cicatrización de HeridasRESUMEN
Melanocytes are the major cells responsible for skin and fair pigmentation in vertebrates. They localize to hair follicles(HFs) and the epidermis during embryonic development. A reduced number or dysfunction of melanocytes results in pigmentation disorders.Thus, methods for isolation, culture, and identification of melanocytes in mouse hair follicles provide an experimental basis for thestudy of of pigmentation disorders. In the current work, we harvested the melanocytes from the anagen phase dorsal skin of C57BL/6 mice.After its separation from the skin, the dermis was digested, and the HFs were released. HFs were then also digested, and the cells released from HFs were cultured in melanocyte growth medium. Immunofluorescence and immunohistochemistry staining were used to localize the distribution of melanocytes in HFs . Reverse transcription polymerase chain reaction was performed to detect the expression of specific melanocyte marker genes. Immunofluorescence, immunohistochemistry, flow cytometry, and western blot were carried out to detect the expression of marker proteins in cells. 3,4-Dihydroxy-L-phenylalanine (L-DOPA) staining was used to detect the pigmentation functionality of melaonocytes. Based on our results, we conclude that mature and functional melanocytes can be successfully obtained from theHFs, providing a cell model to study pigmentation disorders. The current findings provide novel insights for the treatment of pigmentation disorders by autologous cell transplantation. Further, we believe that issues related to skin damage, insufficient numbers of autologous cells, and autoimmune problems can be resolved in future though the use of functional melanocytes.
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Folículo Piloso/patología , Melanocitos/patología , Trastornos de la Pigmentación/patología , Animales , Diferenciación Celular/fisiología , Ratones , Modelos Animales , Pigmentación/fisiologíaRESUMEN
BACKGROUND: PBX homeobox 1 (PBX1) is involved in the maintenance of the pluripotency of human embryonic and hematopoietic stem cells; however, the effects of PBX1 in the self-renewal and reprogramming of hair follicle mesenchymal stem cells (HF-MSCs) are unclear. The AKT/glycogen synthase kinase (GSK) 3ß pathway regulates cell metabolism, proliferation, apoptosis, and reprogramming, and p16 and p21, which act downstream of this pathway, regulate cell proliferation, cell cycle, and apoptosis induced by reprogramming. Here, we aimed to elucidate the roles of PBX1 in regulating the proliferation and reprogramming of HF-MSCs. METHODS: A lentiviral vector designed to carry the PBX1 sequence or PBX1 short hairpin RNA sequence was used to overexpress or knock down PBX1. The roles of PBX1 in proliferation and apoptosis were investigated by flow cytometry. Real-time polymerase chain reaction was performed to evaluate pluripotent gene expression. Dual-luciferase reporter assays were performed to examine the transcriptional activity of the NANOG promoter. Western blotting was performed to identify the molecules downstream of PBX1 involved in proliferation and reprogramming. Caspase3 activity was detected to assess HF-MSC reprogramming. The phosphatidylinositol 3-kinase/AKT inhibitor LY294002 was used to inhibit the phosphorylation and activity of AKT. RESULTS: Overexpression of PBX1 in HF-MSCs increased the phosphorylation of AKT and nuclear translocation of ß-catenin, resulting in the progression of the cell cycle from G0/G1 to S phase. Moreover, transfection with a combination of five transcription factors (SOMKP) in HF-MSCs enhanced the formation of alkaline phosphatase-stained colonies compared with that in HF-MSCs transfected with a combination of four transcription factors (SOMK). PBX1 upregulated Nanog transcription by activating the promoter and promoted the expression of endogenous SOX2 and OCT4. Furthermore, PBX1 expression activated the AKT/glycogen synthase kinase (GSK) 3ß pathway and reduced apoptosis during the early stages of reprogramming. Inhibition of phospho-AKT or knockdown of PBX1 promoted mitochondrion-mediated apoptosis and reduced reprogramming efficiency. CONCLUSIONS: PBX1 enhanced HF-MSC proliferation, and HF-MSCs induced pluripotent stem cells (iPSC) generation by activating the AKT/GSK3ß signaling pathway. During the reprogramming of HF-MSCs into HF-iPSCs, PBX1 activated the NANOG promoter, upregulated NANOG, and inhibited mitochondrion-mediated apoptosis via the AKT/GSK3ß pathway during the early stages of reprogramming.
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Apoptosis , Proliferación Celular , Reprogramación Celular , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Folículo Piloso/citología , Células Madre Mesenquimatosas/citología , Factor de Transcripción 1 de la Leucemia de Células Pre-B/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células Cultivadas , Regulación de la Expresión Génica , Glucógeno Sintasa Quinasa 3 beta/genética , Folículo Piloso/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Factor de Transcripción 1 de la Leucemia de Células Pre-B/genética , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de SeñalRESUMEN
Stem cells derived from elderly donors or harvested by repeated subculture exhibit a marked decrease in proliferative capacity and multipotency, which not only compromises their therapeutic potential but also raises safety concerns for regenerative medicine. NANOG-a well-known core transcription factor-plays an important role in maintaining the self-renewal and pluripotency of stem cells. Unfortunately, the mechanism that NANOG delays mesenchymal stem cell (MSC) senescence is not well-known until now. In our study, we showed that both ectopic NANOG expression and PBX1 overexpression (i) significantly upregulated phosphorylated AKT (p-AKT) and PARP1; (ii) promoted cell proliferation, cell cycle progression, and osteogenesis; (iii) reduced the number of senescence-associated-ß-galactosidase- (SA-ß-gal-) positive cells; and (iv) downregulated the expression of p16, p53, and p21. Western blotting and dual-luciferase activity assays showed that ectopic NANOG expression significantly upregulated PBX1 expression and increased PBX1 promoter activity. In contrast, PBX1 knockdown by RNA interference in hair follicle- (HF-) derived MSCs that were ectopically expressing NANOG resulted in the significant downregulation of p-AKT and the upregulation of p16 and p21. Moreover, blocking AKT with the PI3K/AKT inhibitor LY294002 or knocking down AKT via RNA interference significantly decreased PBX1 expression, while increasing p16 and p21 expression and the number of SA-ß-gal-positive cells. In conclusion, our findings show that NANOG delays HF-MSC senescence by upregulating PBX1 and activating AKT signaling and that a feedback loop likely exists between PBX1 and AKT signaling.
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Folículo Piloso/metabolismo , Células Madre Mesenquimatosas/metabolismo , Proteína Homeótica Nanog/metabolismo , Factor de Transcripción 1 de la Leucemia de Células Pre-B/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Apoptosis/fisiología , Ciclo Celular/fisiología , Proliferación Celular/fisiología , Células Cultivadas , Senescencia Celular/fisiología , Cromonas/farmacología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Activación Enzimática , Células HEK293 , Folículo Piloso/citología , Humanos , Células Madre Mesenquimatosas/citología , Morfolinas/farmacología , Proteína Homeótica Nanog/biosíntesis , Proteína Homeótica Nanog/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Factor de Transcripción 1 de la Leucemia de Células Pre-B/biosíntesis , Factor de Transcripción 1 de la Leucemia de Células Pre-B/genética , Regiones Promotoras Genéticas , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/biosíntesis , Regulación hacia ArribaRESUMEN
The development, maturation, and maintenance of the mammalian nervous system rely on complex spatiotemporal patterns of gene expression. In neurons, this is achieved by the expression of differentially localized isoforms and specific sets of mRNA-binding proteins (mRBPs) that regulate RNA processing, mRNA trafficking, and local protein synthesis at remote sites within dendrites and axons. There is growing evidence that axons contain a specialized transcriptome and are endowed with the machinery that allows them to rapidly alter their local proteome via local translation and protein degradation. This enables axons to quickly respond to changes in their environment during development, and to facilitate axon regeneration and maintenance in adult organisms. Aside from providing autonomy to neuronal processes, local translation allows axons to send retrograde injury signals to the cell soma. In this review, we discuss evidence that disturbances in mRNP transport, granule assembly, axonal localization, and local translation contribute to pathology in various neurodegenerative diseases, including spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease (AD).
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Transporte Axonal , Axones/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Ribonucleoproteínas/metabolismo , Animales , Axones/patología , Gránulos Citoplasmáticos/metabolismo , Dendritas/metabolismo , Dendritas/patología , Humanos , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Neuronas/metabolismo , Neuronas/patología , Biosíntesis de Proteínas , Transporte de ARN/fisiología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/genética , TranscriptomaRESUMEN
The cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a common histopathological hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia disease spectrum (ALS/FTD). However, the composition of aggregates and their contribution to the disease process remain unknown. Here we used proximity-dependent biotin identification (BioID) to interrogate the interactome of detergent-insoluble TDP-43 aggregates and found them enriched for components of the nuclear pore complex and nucleocytoplasmic transport machinery. Aggregated and disease-linked mutant TDP-43 triggered the sequestration and/or mislocalization of nucleoporins and transport factors, and interfered with nuclear protein import and RNA export in mouse primary cortical neurons, human fibroblasts and induced pluripotent stem cell-derived neurons. Nuclear pore pathology is present in brain tissue in cases of sporadic ALS and those involving genetic mutations in TARDBP and C9orf72. Our data strongly implicate TDP-43-mediated nucleocytoplasmic transport defects as a common disease mechanism in ALS/FTD.
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Transporte Activo de Núcleo Celular/fisiología , Esclerosis Amiotrófica Lateral , Corteza Cerebral/citología , Proteínas de Unión al ADN/metabolismo , Demencia Frontotemporal , Poro Nuclear/metabolismo , Transporte Activo de Núcleo Celular/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Animales Modificados Genéticamente , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Proteína C9orf72/ultraestructura , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/ultraestructura , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrión no Mamífero , Femenino , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Humanos , Larva , Masculino , Ratones , Ratones Endogámicos C57BL , Neuroblastoma/patología , Membrana Nuclear/patología , Membrana Nuclear/ultraestructura , Poro Nuclear/genética , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patologíaRESUMEN
The maintenance of highly proliferative capacity and full differentiation potential is a necessary step in the initiation of stem cell-based regenerative medicine. Our recent study showed that epidermal growth factor (EGF) significantly enhanced hair follicle-derived mesenchymal stem cell (HF-MSC) proliferation while maintaining the multilineage differentiation potentials. However, the underlying mechanism remains unclear. Herein, we investigated the role of EGF in HF-MSC proliferation. HF-MSCs were isolated and cultured with or without EGF. Immunofluorescence staining, flow cytometry, cytochemistry, and western blotting were used to assess proliferation, cell signaling pathways related to the EGF receptor (EGFR), and cell cycle progression. HF-MSCs exhibited surface markers of mesenchymal stem cells and displayed trilineage differentiation potentials toward adipocytes, chondrocytes, and osteoblasts. EGF significantly increased HF-MSC proliferation as well as EGFR, ERK1/2, and AKT phosphorylation (p-EGFR, p-ERK1/2, and p-AKT) in a time- and dose-dependent manner, but not STAT3 phosphorylation. EGFR inhibitor (AG1478), PI3K-AKT inhibitor (LY294002), ERK inhibitor (U0126), and STAT3 inhibitor (STA-21) significantly blocked EGF-induced HF-MSC proliferation. Moreover, AG1478, LY294002, and U0126 significantly decreased p-EGFR, p-AKT, and p-ERK1/2 expression. EGF shifted HF-MSCs at the G1 phase to the S and G2 phase. Concomitantly, cyclinD1, phosphorylated Rb, and E2F1expression increased, while that of p16 decreased. In conclusion, EGF induces HF-MSC proliferation through the EGFR/ERK and AKT pathways, but not through STAT-3. The G1/S transition was stimulated by upregulation of cyclinD1 and inhibition of p16 expression.
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Ciclina D1/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Folículo Piloso/citología , Células Madre Mesenquimatosas/citología , Transducción de Señal/efectos de los fármacos , Adulto , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Separación Celular , Regulación hacia Abajo/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/enzimología , Persona de Mediana Edad , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Genetically engineered stem cells that overexpress genes encoding therapeutic products can be exploited to correct metabolic disorders by repairing and regenerating diseased organs or restoring their function. Hair follicles are readily accessible and serve as a rich source of autologous stem cells for cell-based gene therapy. Here we isolated mesenchymal stem cells from human hair follicles (HF-MSCs) and engineered them to overexpress the human insulin gene and release human insulin in a time- and dose-dependent manner in response to rapamycin. The engineered HF-MSCs retained their characteristic cell surface markers and retained their potential to differentiate into adipocytes and osteoblasts. When mice with streptozotocin-induced type 1 diabetes were engrafted with these engineered HF-MSCs, these cells expressed and released a dose of human insulin, dramatically reversed hyperglycemia, and significantly reduced death rate. Moreover, the engineered HF-MSCs did not form detectable tumors throughout the 120-day animal tests in our experiment. Our results show that HF-MSCs can be used to safely and efficiently express therapeutic transgenes and therefore show promise for cell-based gene therapy of human disease.
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Ingeniería Celular , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Folículo Piloso/metabolismo , Hiperglucemia/terapia , Insulina/metabolismo , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Adulto , Animales , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Xenoinjertos , Humanos , Hiperglucemia/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana EdadRESUMEN
INTRODUCTION: Successful stem cell therapy relies on large-scale generation of stem cells and their maintenance in a proliferative multipotent state. This study aimed to establish a three-dimension culture system for large-scale generation of hWJ-MSC and investigated the self-renewal activity, genomic stability and multi-lineage differentiation potential of such hWJ-MSC in enhancing skin wound healing. METHODS: hWJ-MSC were seeded on gelatin microbeads and cultured in spinning bottles (3D). Cell proliferation, karyotype analysis, surface marker expression, multipotent differentiation (adipogenic, chondrogenic, and osteogenic potentials), and expression of core transcription factors (OCT4, SOX2, NANOG, and C-MYC), as well as their efficacy in accelerating skin wound healing, were investigated and compared with those of hWJ-MSC derived from plate cultres (2D), using in vivo and in vitro experiments. RESULTS: hWJ-MSC attached to and proliferated on gelatin microbeads in 3D cultures reaching a maximum of 1.1-1.30×10(7) cells on 0.5 g of microbeads by days 8-14; in contrast, hWJ-MSC derived from 2D cultures reached a maximum of 6.5 -11.5×10(5) cells per well in a 24-well plate by days 6-10. hWJ-MSC derived by 3D culture incorporated significantly more EdU (P<0.05) and had a significantly higher proliferation index (P<0.05) than those derived from 2D culture. Immunofluorescence staining, real-time PCR, flow cytometry analysis, and multipotency assays showed that hWJ-MSC derived from 3D culture retained MSC surface markers and multipotency potential similar to 2D culture-derived cells. 3D culture-derived hWJ-MSC also retained the expression of core transcription factors at levels comparable to their 2D culture counterparts. Direct injection of hWJ-MSC derived from 3D or 2D cultures into animals exhibited similar efficacy in enhancing skin wound healing. CONCLUSIONS: Thus, hWJ-MSC can be expanded markedly in gelatin microbeads, while retaining MSC surface marker expression, multipotent differential potential, and expression of core transcription factors. These cells also efficiently enhanced skin wound healing in vivo, in a manner comparable to that of hWJ-MSC obtained from 2D culture.