RESUMO

Progerin causes Hutchinson-Gilford progeria syndrome (HGPS), but how progerin accelerates aging is still an interesting question. Here, we provide evidence linking nuclear envelope (NE) budding and accelerated aging. Mechanistically, progerin disrupts nuclear lamina to induce NE budding in concert with lamin A/C, resulting in transport of chromatin into the cytoplasm where it is removed via autophagy, whereas emerin antagonizes this process. Primary cells from both HGPS patients and mouse models express progerin and display NE budding and chromatin loss, and ectopically expressing progerin in cells can mimic this process. More excitingly, we screen a NE budding inhibitor chaetocin by high-throughput screening, which can dramatically sequester progerin from the NE and prevent this NE budding through sustaining ERK1/2 activation. Chaetocin alleviates NE budding-induced chromatin loss and ameliorates HGPS defects in cells and mice and significantly extends lifespan of HGPS mice. Collectively, we propose that progerin-induced NE budding participates in the induction of progeria, highlight the roles of chaetocin and sustained ERK1/2 activation in anti-aging, and provide a distinct avenue for treating HGPS.

Assuntos

Lamina Tipo A , Membrana Nuclear , Proteínas Nucleares , Progéria , Progéria/metabolismo , Progéria/tratamento farmacológico , Progéria/patologia , Progéria/genética , Animais , Lamina Tipo A/metabolismo , Lamina Tipo A/genética , Camundongos , Humanos , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Envelhecimento/metabolismo , Envelhecimento/efeitos dos fármacos , Cromatina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Modelos Animais de Doenças , Autofagia/efeitos dos fármacos

RESUMO

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disorder associated with features of accelerated aging. HGPS is an autosomal dominant disease caused by a de novo mutation of LMNA gene, encoding A-type lamins, resulting in the truncated form of pre-lamin A called progerin. While asymptomatic at birth, patients develop symptoms within the first year of life when they begin to display accelerated aging and suffer from growth retardation, and severe cardiovascular complications including loss of vascular smooth muscle cells (VSMCs). Recent works reported the loss of VSMCs as a major factor triggering atherosclerosis in HGPS. Here, we investigated the mechanisms by which progerin expression leads to massive VSMCs loss. Using aorta tissue and primary cultures of murine VSMCs from a mouse model of HGPS, we showed increased VSMCs death associated with increased poly(ADP-Ribosyl)ation. Poly(ADP-Ribosyl)ation is recognized as a post-translational protein modification that coordinates the repair at DNA damage sites. Poly-ADP-ribose polymerase (PARP) catalyzes protein poly(ADP-Ribosyl)ation by utilizing nicotinamide adenine dinucleotide (NAD+). Our results provided the first demonstration linking progerin accumulation, augmented poly(ADP-Ribosyl)ation and decreased nicotinamide adenine dinucleotide (NAD+) level in VSMCs. Using high-throughput screening on VSMCs differentiated from iPSCs from HGPS patients, we identified a new compound, trifluridine able to increase NAD+ levels through decrease of PARP-1 activity. Lastly, we demonstrate that trifluridine treatment in vivo was able to alleviate aortic VSMCs loss and clinical sign of progeria, suggesting a novel therapeutic approach of cardiovascular disease in progeria.

Assuntos

Modelos Animais de Doenças , Lamina Tipo A , Músculo Liso Vascular , Miócitos de Músculo Liso , Progéria , Animais , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Músculo Liso Vascular/efeitos dos fármacos , Progéria/patologia , Progéria/genética , Progéria/metabolismo , Camundongos , Humanos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Lamina Tipo A/metabolismo , Lamina Tipo A/genética , Aorta/patologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Poli ADP Ribosilação , Camundongos Endogâmicos C57BL , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia

RESUMO

Dendritic cells (DCs) play a crucial role in orchestrating immune responses, particularly in promoting IFNγ-producing-CD8 cytotoxic T lymphocytes (CTLs) and IFNγ-producing-CD4 T helper 1 (Th1) cells, which are essential for defending against viral infections. Additionally, the nuclear envelope protein lamin A/C has been implicated in T cell immunity. Nevertheless, the intricate interplay between innate and adaptive immunity in response to viral infections, particularly the role of lamin A/C in DC functions within this context, remains poorly understood. In this study, we demonstrate that mice lacking lamin A/C in myeloid LysM promoter-expressing cells exhibit a reduced capacity to induce Th1 and CD8 CTL responses, leading to impaired clearance of acute primary Vaccinia virus (VACV) infection. Remarkably, in vitro-generated granulocyte macrophage colony-stimulating factor bone marrow-derived DCs (GM-CSF BMDCs) show high levels of lamin A/C. Lamin A/C absence on GM-CSF BMDCs does not affect the expression of costimulatory molecules on the cell membrane but it reduces the cellular ability to form immunological synapses with naïve CD4 T cells. Lamin A/C deletion induces alterations in NFκB nuclear localization, thereby influencing NF-κB-dependent transcription. Furthermore, lamin A/C ablation modifies the gene accessibility of BMDCs, predisposing these cells to mount a less effective antiviral response upon TLR stimulation. This study highlights the critical role of DCs in interacting with CD4 T cells during antiviral responses and proposes some mechanisms through which lamin A/C may modulate DC function via gene accessibility and transcriptional regulation.

Assuntos

Células Dendríticas , Lamina Tipo A , Camundongos Endogâmicos C57BL , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Animais , Lamina Tipo A/metabolismo , Lamina Tipo A/genética , Camundongos , NF-kappa B/metabolismo , Vaccinia virus/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Camundongos Knockout , Vacínia/imunologia , Células Th1/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Sinapses Imunológicas/metabolismo , Sinapses Imunológicas/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo

RESUMO

Alterations in nuclear structure and function are hallmarks of cancer cells. Little is known about these changes in Cancer-Associated Fibroblasts (CAFs), crucial components of the tumor microenvironment. Loss of the androgen receptor (AR) in human dermal fibroblasts (HDFs), which triggers early steps of CAF activation, leads to nuclear membrane changes and micronuclei formation, independent of cellular senescence. Similar changes occur in established CAFs and are reversed by restoring AR activity. AR associates with nuclear lamin A/C, and its loss causes lamin A/C nucleoplasmic redistribution. AR serves as a bridge between lamin A/C and the protein phosphatase PPP1. Loss of AR decreases lamin-PPP1 association and increases lamin A/C phosphorylation at Ser 301, a characteristic of CAFs. Phosphorylated lamin A/C at Ser 301 binds to the regulatory region of CAF effector genes of the myofibroblast subtype. Expression of a lamin A/C Ser301 phosphomimetic mutant alone can transform normal fibroblasts into tumor-promoting CAFs.

Assuntos

Fibroblastos Associados a Câncer , Núcleo Celular , Lamina Tipo A , Receptores Androgênicos , Humanos , Receptores Androgênicos/metabolismo , Receptores Androgênicos/genética , Lamina Tipo A/metabolismo , Lamina Tipo A/genética , Fosforilação , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Núcleo Celular/metabolismo , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 1/genética , Fibroblastos/metabolismo , Membrana Nuclear/metabolismo , Masculino , Microambiente Tumoral

RESUMO

LMNA-related dilated cardiomyopathy (DCM) is an autosomal-dominant genetic condition with cardiomyocyte and conduction system dysfunction often resulting in heart failure or sudden death. The condition is caused by mutation in the Lamin A/C (LMNA) gene encoding Type-A nuclear lamin proteins involved in nuclear integrity, epigenetic regulation of gene expression, and differentiation. The molecular mechanisms of the disease are not completely understood, and there are no definitive treatments to reverse progression or prevent mortality. We investigated possible mechanisms of LMNA-related DCM using induced pluripotent stem cells derived from a family with a heterozygous LMNA c.357-2A>G splice-site mutation. We differentiated one LMNA-mutant iPSC line derived from an affected female (Patient) and two non-mutant iPSC lines derived from her unaffected sister (Control) and conducted single-cell RNA sequencing for 12 samples (four from Patients and eight from Controls) across seven time points: Day 0, 2, 4, 9, 16, 19, and 30. Our bioinformatics workflow identified 125,554 cells in raw data and 110,521 (88%) high-quality cells in sequentially processed data. Unsupervised clustering, cell annotation, and trajectory inference found complex heterogeneity: ten main cell types; many possible subtypes; and lineage bifurcation for cardiac progenitors to cardiomyocytes (CMs) and epicardium-derived cells (EPDCs). Data integration and comparative analyses of Patient and Control cells found cell type and lineage-specific differentially expressed genes (DEGs) with enrichment, supporting pathway dysregulation. Top DEGs and enriched pathways included 10 ZNF genes and RNA polymerase II transcription in pluripotent cells (PP); BMP4 and TGF Beta/BMP signaling, sarcomere gene subsets and cardiogenesis, CDH2 and EMT in CMs; LMNA and epigenetic regulation, as well as DDIT4 and mTORC1 signaling in EPDCs. Top DEGs also included XIST and other X-linked genes, six imprinted genes (SNRPN, PWAR6, NDN, PEG10, MEG3, MEG8), and enriched gene sets related to metabolism, proliferation, and homeostasis. We confirmed Lamin A/C haploinsufficiency by allelic expression and Western blot. Our complex Patient-derived iPSC model for Lamin A/C haploinsufficiency in PP, CM, and EPDC provided support for dysregulation of genes and pathways, many previously associated with Lamin A/C defects, such as epigenetic gene expression, signaling, and differentiation. Our findings support disruption of epigenomic developmental programs, as proposed in other LMNA disease models. We recognized other factors influencing epigenetics and differentiation; thus, our approach needs improvement to further investigate this mechanism in an iPSC-derived model.

Assuntos

Cardiomiopatia Dilatada , Diferenciação Celular , Haploinsuficiência , Células-Tronco Pluripotentes Induzidas , Lamina Tipo A , Miócitos Cardíacos , Transcriptoma , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Humanos , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Diferenciação Celular/genética , Haploinsuficiência/genética , Feminino , Transcriptoma/genética , Pericárdio/patologia , Pericárdio/metabolismo , Linhagem da Célula/genética , Análise de Célula Única , Regulação da Expressão Gênica , Mutação/genética , Adulto

Assuntos

Cardiomiopatia Dilatada , Lamina Tipo A , Transdução de Sinais , Vitamina D , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Transdução de Sinais/genética , Vitamina D/metabolismo , Masculino , Feminino , Mutação , Adulto

RESUMO

Lipodystrophic laminopathies are a group of ultra-rare disorders characterised by the presence of pathogenic variants in the same gene (LMNA) and other related genes, along with an impaired adipose tissue pattern and other features that are specific of each of these disorders. The most fascinating traits include their complex genotype-phenotype associations and clinical heterogeneity, ranging from Dunnigan disease, in which the most relevant feature is precisely adipose tissue dysfunction and lipodystrophy, to the other laminopathies affecting adipose tissue, which are also characterised by the presence of signs of premature ageing (Hutchinson Gilford-progeria syndrome, LMNA-atypical progeroid syndrome, mandibuloacral dysplasia types A and B, Nestor-Guillermo progeria syndrome, LMNA-associated cardiocutaneous progeria). This raises several questions when it comes to understanding how variants in the same gene can lead to similar adipose tissue disturbances and, at the same time, to such heterogeneous phenotypes and variable degrees of metabolic abnormalities. The present review aims to gather the molecular basis of adipose tissue impairment in lipodystrophic laminopathies, their main clinical aspects and recent therapeutic strategies. In addition, it also summarises the key aspects for their differential diagnosis.

Assuntos

Lamina Tipo A , Laminopatias , Lipodistrofia , Progéria , Humanos , Progéria/genética , Progéria/patologia , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Lipodistrofia/genética , Lipodistrofia/metabolismo , Lipodistrofia/patologia , Laminopatias/genética , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Fenótipo , Mutação

RESUMO

Hutchinson-Gilford Progeria Syndrome (HGPS) is an extremely rare genetic disorder that causes accelerated aging, due to a pathogenic variant in the LMNA gene. This pathogenic results in the production of progerin, a defective protein that disrupts the nuclear lamina's structure. In our study, we conducted a histopathological analysis of various organs in the LmnaG609G/G609G mouse model, which is commonly used to study HGPS. The objective of this study was to show that progerin accumulation drives systemic but organ-specific tissue damage and accelerated aging phenotypes. Our findings show significant fibrosis, inflammation, and dysfunction in multiple organ systems, including the skin, cardiovascular system, muscles, lungs, liver, kidneys, spleen, thymus, and heart. Specifically, we observed severe vascular fibrosis, reduced muscle regeneration, lung tissue remodeling, depletion of fat in the liver, and disruptions in immune structures. These results underscore the systemic nature of the disease and suggest that chronic inflammation and fibrosis play crucial roles in the accelerated aging seen in HGPS. Additionally, our study highlights that each organ responds differently to the toxic effects of progerin, indicating that there are distinct mechanisms of tissue-specific damage.

Assuntos

Modelos Animais de Doenças , Fibrose , Inflamação , Lamina Tipo A , Progéria , Animais , Progéria/genética , Progéria/patologia , Progéria/metabolismo , Camundongos , Inflamação/patologia , Inflamação/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Especificidade de Órgãos , Pulmão/patologia , Pulmão/metabolismo

RESUMO

Mutations in the nuclear envelope (NE) protein lamin A/C (encoded by LMNA), cause a severe form of dilated cardiomyopathy (DCM) with early-onset life-threatening arrhythmias. However, molecular mechanisms underlying increased arrhythmogenesis in LMNA-related DCM (LMNA-DCM) remain largely unknown. Here we show that a frameshift mutation in LMNA causes abnormal Ca2+ handling, arrhythmias and disformed NE in LMNA-DCM patient-specific iPSC-derived cardiomyocytes (iPSC-CMs). Mechanistically, lamin A interacts with sirtuin 1 (SIRT1) where mutant lamin A/C accelerates degradation of SIRT1, leading to mitochondrial dysfunction and oxidative stress. Elevated reactive oxygen species (ROS) then activates the Ca2+/calmodulin-dependent protein kinase II (CaMKII)-ryanodine receptor 2 (RYR2) pathway and aggravates the accumulation of SUN1 in mutant iPSC-CMs, contributing to arrhythmias and NE deformation, respectively. Taken together, the lamin A/C deficiency-mediated ROS disorder is revealed as central to LMNA-DCM development. Manipulation of impaired SIRT1 activity and excessive oxidative stress is a potential future therapeutic strategy for LMNA-DCM.

Assuntos

Cardiomiopatia Dilatada , Células-Tronco Pluripotentes Induzidas , Lamina Tipo A , Miócitos Cardíacos , Estresse Oxidativo , Espécies Reativas de Oxigênio , Sirtuína 1 , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Lamina Tipo A/metabolismo , Lamina Tipo A/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Humanos , Sirtuína 1/metabolismo , Sirtuína 1/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fenótipo , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/genética , Arritmias Cardíacas/patologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Mutação da Fase de Leitura , Cálcio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Membrana Nuclear/metabolismo , Mitocôndrias/metabolismo , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética

RESUMO

Our understanding of how fluid forces influence cell migration in confining environments remains limited. By integrating microfluidics with live-cell imaging, we demonstrate that cells in tightly-but not moderately-confined spaces reverse direction and move upstream upon exposure to fluid forces. This fluid force-induced directional change occurs less frequently when cells display diminished mechanosensitivity, experience elevated hydraulic resistance, or sense a chemical gradient. Cell reversal requires actin polymerization to the new cell front, as shown mathematically and experimentally. Actin polymerization is necessary for the fluid force-induced activation of NHE1, which cooperates with calcium to induce upstream migration. Calcium levels increase downstream, mirroring the subcellular distribution of myosin IIA, whose activation enhances upstream migration. Reduced lamin A/C levels promote downstream migration of metastatic tumor cells by preventing cell polarity establishment and intracellular calcium rise. This mechanism could allow cancer cells to evade high-pressure environments, such as the primary tumor.

Assuntos

Actinas , Cálcio , Movimento Celular , Humanos , Cálcio/metabolismo , Actinas/metabolismo , Linhagem Celular Tumoral , Trocador 1 de Sódio-Hidrogênio/metabolismo , Lamina Tipo A/metabolismo , Polaridade Celular/fisiologia , Microfluídica , Mecanotransdução Celular

RESUMO

Background: Laminopathy is a pathological manifestation observed in Alzheimer's disease (AD), leading to neuronal apoptosis. Objective: Our objective was to assess inhibitors of enzymes involved in laminopathy. Methods: The mRNA expression of the cathepsins L and B, caspases 3 and 6, lamins b1 and b2, granzymes A and B, and lamins A and C were extracted and analyzed from GSE5281 and GSE28146 datasets. A total of 145 ligands were selected for molecular docking. Subsequently, 10 ns and 100 ns atomistic molecular dynamics (MD) and Martini 3 were performed with NAMD for two selected ligands (PubChem id: 608841 and ChEMBL id: 550872). Results: The mRNA expression level highlighted caspase 6 and lamin A/C upregulation in the hippocampus of the AD samples, in contrast to cathepsin B, lamin b2, and caspase 3. Moreover, there was a strong correlation between the expression level of cathepsin B, lamin A/C, and caspase 6 in the AD group. The MD results suggested molecule with ChEMBL id of 550872 had higher free binding energy, while in longer simulation the molecule with PubChem id of 608841 was suggested to be more stable in complex with the receptor. Conclusions: Our findings suggest that lamins A/C, cathepsins B/L, caspase 6, and lamin B2 are associated with laminopathy as potential factors contributing to apoptosis in AD. We propose that simultaneous inhibition of caspases 6 and cathepsins L may decrease the rate of apoptosis triggered by lamin degradation. Nevertheless, further studies are required to confirm these observations due to the lack of in vivo findings.

Assuntos

Doença de Alzheimer , Apoptose , Caspase 3 , Caspase 6 , Catepsina B , Catepsina L , Humanos , Doença de Alzheimer/patologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Caspase 6/metabolismo , Caspase 6/genética , Apoptose/efeitos dos fármacos , Catepsina L/metabolismo , Catepsina L/genética , Catepsina B/metabolismo , Catepsina B/genética , Caspase 3/metabolismo , Laminopatias/genética , Simulação de Acoplamento Molecular , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Hipocampo/patologia , Hipocampo/metabolismo , Masculino

RESUMO

BACKGROUND: The nuclear envelope (NE), which is composed of the outer and inner nuclear membranes, the nuclear pore complex and the nuclear lamina, regulates a plethora of cellular processes, including those that restrict cancer development (genomic stability, cell cycle regulation, and cell migration). Thus, impaired NE is functionally related to tumorigenesis, and monitoring of NE alterations is used to diagnose cancer. However, the chronology of NE changes occurring during cancer evolution and the connection between them remained to be precisely defined, due to the lack of appropriate cell models. METHODS: The expression and subcellular localization of NE proteins (lamins A/C and B1 and the inner nuclear membrane proteins emerin and ß-dystroglycan [ß-DG]) during prostate cancer progression were analyzed, using confocal microscopy and western blot assays, and a prostate cancer cell system comprising RWPE-1 epithelial prostate cells and several prostate cancer cell lines with different invasiveness. RESULTS: Deformed nuclei and the mislocalization and low expression of lamin A/C, lamin B1, and emerin became more prominent as the invasiveness of the prostate cancer lines increased. Suppression of lamin A/C expression was an early event during prostate cancer evolution, while a more extensive deregulation of NE proteins, including ß-DG, occurred in metastatic prostate cells. CONCLUSIONS: The RWPE-1 cell line-based system was found to be suitable for the correlation of NE impairment with prostate cancer invasiveness and determination of the chronology of NE alterations during prostate carcinogenesis. Further study of this cell system would help to identify biomarkers for prostate cancer prognosis and diagnosis.

Assuntos

Lamina Tipo A , Lamina Tipo B , Proteínas de Membrana , Membrana Nuclear , Proteínas Nucleares , Neoplasias da Próstata , Masculino , Humanos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/genética , Membrana Nuclear/metabolismo , Linhagem Celular Tumoral , Proteínas de Membrana/metabolismo , Lamina Tipo B/metabolismo , Lamina Tipo A/metabolismo , Lamina Tipo A/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Distroglicanas/metabolismo , Regulação Neoplásica da Expressão Gênica , Núcleo Celular/metabolismo

RESUMO

Recent research into laminopathic lipodystrophies-rare genetic disorders caused by mutations in the LMNA gene-has greatly expanded our knowledge of their complex pathology and metabolic implications. These disorders, including Hutchinson-Gilford progeria syndrome (HGPS), Mandibuloacral Dysplasia (MAD), and Familial Partial Lipodystrophy (FPLD), serve as crucial models for studying accelerated aging and metabolic dysfunction, enhancing our understanding of the cellular and molecular mechanisms involved. Research on laminopathies has highlighted how LMNA mutations disrupt adipose tissue function and metabolic regulation, leading to altered fat distribution and metabolic pathway dysfunctions. Such insights improve our understanding of the pathophysiological interactions between genetic anomalies and metabolic processes. This review merges current knowledge on the phenotypic classifications of these diseases and their associated metabolic complications, such as insulin resistance, hypertriglyceridemia, hepatic steatosis, and metabolic syndrome, all of which elevate the risk of cardiovascular disease, stroke, and diabetes. Additionally, a range of published therapeutic strategies, including gene editing, antisense oligonucleotides, and novel pharmacological interventions aimed at addressing defective adipocyte differentiation and lipid metabolism, will be explored. These therapies target the core dysfunctional lamin A protein, aiming to mitigate symptoms and provide a foundation for addressing similar metabolic and genetic disorders.

Assuntos

Lamina Tipo A , Lipodistrofia , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Lipodistrofia/genética , Lipodistrofia/metabolismo , Lipodistrofia/terapia , Animais , Laminopatias/genética , Laminopatias/metabolismo , Progéria/genética , Progéria/metabolismo , Progéria/patologia , Mutação , Lipodistrofia Parcial Familiar/genética , Lipodistrofia Parcial Familiar/metabolismo , Lipodistrofia Parcial Familiar/terapia , Metabolismo dos Lipídeos/genética , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Resistência à Insulina/genética , Edição de Genes

RESUMO

Lamins are intermediate filament proteins that contribute to numerous cellular functions, including nuclear morphology and mechanical stability. The N-terminal head domain of lamin is crucial for higher order filament assembly and function, yet the effects of commonly used N-terminal tags on lamin function remain largely unexplored. Here, we systematically studied the effect of two differently sized tags on lamin A (LaA) function in a mammalian cell model engineered to allow for precise control of expression of tagged lamin proteins. Untagged, FLAG-tagged and GFP-tagged LaA completely rescued nuclear shape defects when expressed at similar levels in lamin A/C-deficient (Lmna-/-) MEFs, and all LaA constructs prevented increased nuclear envelope ruptures in these cells. N-terminal tags, however, altered the nuclear localization of LaA and impaired the ability of LaA to restore nuclear deformability and to recruit emerin to the nuclear membrane in Lmna-/- MEFs. Our finding that tags impede some LaA functions but not others might explain the partial loss of function phenotypes when tagged lamins are expressed in model organisms and should caution researchers using tagged lamins to study the nucleus.

Assuntos

Núcleo Celular , Lamina Tipo A , Membrana Nuclear , Lamina Tipo A/metabolismo , Lamina Tipo A/genética , Animais , Camundongos , Núcleo Celular/metabolismo , Membrana Nuclear/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética

RESUMO

In mammals, RNA interference (RNAi) was historically studied as a cytoplasmic event; however, in the last decade, a growing number of reports convincingly show the nuclear localization of the Argonaute (AGO) proteins. Nevertheless, the extent of nuclear RNAi and its implication in biological mechanisms remain to be elucidated. We found that reduced Lamin A levels significantly induce nuclear influx of AGO2 in SHSY5Y neuroblastoma and A375 melanoma cancer cell lines, which normally have no nuclear AGO2. Lamin A KO manifested a more pronounced effect in SHSY5Y cells compared to A375 cells, evident by changes in cell morphology, increased cell proliferation, and oncogenic miRNA expression. Moreover, AGO fPAR-CLIP in Lamin A KO SHSY5Y cells revealed significantly reduced RNAi activity. Further exploration of the nuclear AGO interactome by mass spectrometry identified FAM120A, an RNA-binding protein and known interactor of AGO2. Subsequent FAM120A fPAR-CLIP, revealed that FAM120A co-binds AGO targets and that this competition reduces the RNAi activity. Therefore, loss of Lamin A triggers nuclear AGO2 translocation, FAM120A mediated RNAi impairment, and upregulation of oncogenic miRNAs, facilitating cancer cell proliferation.

Assuntos

Proteínas Argonautas , Núcleo Celular , Proliferação de Células , Lamina Tipo A , MicroRNAs , Interferência de RNA , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Humanos , Núcleo Celular/metabolismo , Linhagem Celular Tumoral , Lamina Tipo A/metabolismo , Lamina Tipo A/genética , MicroRNAs/metabolismo , MicroRNAs/genética , Proliferação de Células/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Transporte Ativo do Núcleo Celular , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia

RESUMO

PURPOSE OF REVIEW: To highlight recent insights in different aspects of striated muscle laminopathies (SMLs) related to LMNA mutations. RECENT FINDINGS: Clinical and genetic studies allow better patient management and diagnosis, with confirmation of ventricular tachyarrhythmias (VTA) risk prediction score to help with ICD implantation and development of models to help with classification of LMNA variants of uncertain significance. From a pathophysiology perspective, characterization of lamin interactomes in different contexts revealed new lamin A/C partners. Expression or function modulation of these partners evidenced them as potential therapeutic targets. After a positive phase 2, the first phase 3 clinical trial, testing a p38 inhibitor targeting the life-threatening cardiac disease of SML, has been recently stopped, thus highlighting the need for new therapeutic approaches together with new animal and cell models. SUMMARY: Since the first LMNA mutation report in 1999, lamin A/C structure and functions have been actively explored to understand the SML pathophysiology. The latest discoveries of partners and altered pathways, highlight the importance of lamin A/C at the nuclear periphery and in the nucleoplasm. Modulation of altered pathways allowed some benefits, especially for cardiac involvement. However, additional studies are still needed to fully assess treatment efficacy and safety.

Assuntos

Lamina Tipo A , Laminopatias , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Laminopatias/genética , Animais , Mutação , Músculo Estriado/patologia , Músculo Estriado/metabolismo

RESUMO

Lamin A/C gene (LMNA) mutations contribute to severe striated muscle laminopathies, affecting cardiac and skeletal muscles, with limited treatment options. In this study, we delve into the investigations of five distinct LMNA mutations, including three novel variants and two pathogenic variants identified in patients with muscular laminopathy. Our approach employs zebrafish models to comprehensively study these variants. Transgenic zebrafish expressing wild-type LMNA and each mutation undergo extensive morphological profiling, swimming behavior assessments, muscle endurance evaluations, heartbeat measurement, and histopathological analysis of skeletal muscles. Additionally, these models serve as platform for focused drug screening. We explore the transcriptomic landscape through qPCR and RNAseq to unveil altered gene expression profiles in muscle tissues. Larvae of LMNA(L35P), LMNA(E358K), and LMNA(R453W) transgenic fish exhibit reduced swim speed compared to LMNA(WT) measured by DanioVision. All LMNA transgenic adult fish exhibit reduced swim speed compared to LMNA(WT) in T-maze. Moreover, all LMNA transgenic adult fish, except LMNA(E358K), display weaker muscle endurance than LMNA(WT) measured by swimming tunnel. Histochemical staining reveals decreased fiber size in all LMNA mutations transgenic fish, excluding LMNA(WT) fish. Interestingly, LMNA(A539V) and LMNA(E358K) exhibited elevated heartbeats. We recognize potential limitations with transgene overexpression and conducted association calculations to explore its effects on zebrafish phenotypes. Our results suggest lamin A/C overexpression may not directly impact mutant phenotypes, such as impaired swim speed, increased heart rates, or decreased muscle fiber diameter. Utilizing LMNA zebrafish models for drug screening, we identify L-carnitine treatment rescuing muscle endurance in LMNA(L35P) and creatine treatment reversing muscle endurance in LMNA(R453W) zebrafish models. Creatine activates AMPK and mTOR pathways, improving muscle endurance and swim speed in LMNA(R453W) fish. Transcriptomic profiling reveals upstream regulators and affected genes contributing to motor dysfunction, cardiac anomalies, and ion flux dysregulation in LMNA mutant transgenic fish. These findings faithfully mimic clinical manifestations of muscular laminopathies, including dysmorphism, early mortality, decreased fiber size, and muscle dysfunction in zebrafish. Furthermore, our drug screening results suggest L-carnitine and creatine treatments as potential rescuers of muscle endurance in LMNA(L35P) and LMNA(R453W) zebrafish models. Our study offers valuable insights into the future development of potential treatments for LMNA-related muscular laminopathy.

Assuntos

Animais Geneticamente Modificados , Carnitina , Creatina , Lamina Tipo A , Músculo Esquelético , Mutação , Peixe-Zebra , Animais , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/efeitos dos fármacos , Creatina/metabolismo , Carnitina/metabolismo , Modelos Animais de Doenças , Laminopatias/genética , Laminopatias/metabolismo , Natação , Transcriptoma , Humanos

RESUMO

Mesenchymal stem cells (MSCs) of placental origin hold great promise in tissue engineering and regenerative medicine for diseases affecting cartilage and bone. However, their utility has been limited by their tendency to undergo premature senescence and phenotypic drift into adipocytes. This study aimed to explore the potential involvement of a specific subset of aging and antiaging genes by measuring their expression prior to and following in vitro-induced differentiation of placental MSCs into chondrocytes and osteoblasts as opposed to adipocytes. The targeted genes of interest included the various LMNA/C transcript variants (lamin A, lamin C, and lamin A∆10), sirtuin 7 (SIRT7), and SM22α, along with the classic aging markers plasminogen activator inhibitor 1 (PAI-1), p53, and p16INK4a. MSCs were isolated from the decidua basalis of human term placentas, expanded, and then analyzed for phenotypic properties by flow cytometry and evaluated for colony-forming efficiency. The cells were then induced to differentiate in vitro into chondrocytes, osteocytes, and adipocytes following established protocols. The mRNA expression of the targeted genes was measured by RT-qPCR in the undifferentiated cells and those fully differentiated into the three cellular lineages. Compared to undifferentiated cells, the differentiated chondrocytes demonstrated decreased expression of SIRT7, along with decreased PAI-1, lamin A, and SM22α expression, but the expression of p16INK4a and p53 increased, suggesting their tendency to undergo premature senescence. Interestingly, the cells maintained the expression of lamin C, which indicates that it is the primary lamin variant influencing the mechanoelastic properties of the differentiated cells. Notably, the expression of all targeted genes did not differ from the undifferentiated cells following osteogenic differentiation. On the other hand, the differentiation of the cells into adipocytes was associated with decreased expression of lamin A and PAI-1. The distinct patterns of expression of aging and antiaging genes following in vitro-induced differentiation of MSCs into chondrocytes, osteocytes, and adipocytes potentially reflect specific roles for these genes during and following differentiation in the fully functional cells. Understanding these roles and the network of signaling molecules involved can open opportunities to improve the handling and utility of MSCs as cellular precursors for the treatment of cartilage and bone diseases.

Assuntos

Diferenciação Celular , Condrogênese , Células-Tronco Mesenquimais , Osteogênese , Placenta , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Feminino , Placenta/metabolismo , Placenta/citologia , Diferenciação Celular/genética , Condrogênese/genética , Gravidez , Osteogênese/genética , Biomarcadores/metabolismo , Senescência Celular/genética , Condrócitos/metabolismo , Condrócitos/citologia , Envelhecimento , Lamina Tipo A/metabolismo , Lamina Tipo A/genética

RESUMO

Progerin, the protein that causes Hutchinson-Gilford progeria syndrome, triggers nuclear membrane (NM) ruptures and blebs, but the mechanisms are unclear. We suspected that the expression of progerin changes the overall structure of the nuclear lamina. High-resolution microscopy of smooth muscle cells (SMCs) revealed that lamin A and lamin B1 form independent meshworks with uniformly spaced openings (~0.085 µm2). The expression of progerin in SMCs resulted in the formation of an irregular meshwork with clusters of large openings (up to 1.4 µm2). The expression of progerin acted in a dominant-negative fashion to disrupt the morphology of the endogenous lamin B1 meshwork, triggering irregularities and large openings that closely resembled the irregularities and openings in the progerin meshwork. These abnormal meshworks were strongly associated with NM ruptures and blebs. Of note, the progerin meshwork was markedly abnormal in nuclear blebs that were deficient in lamin B1 (~50% of all blebs). That observation suggested that higher levels of lamin B1 expression might normalize the progerin meshwork and prevent NM ruptures and blebs. Indeed, increased lamin B1 expression reversed the morphological abnormalities in the progerin meshwork and markedly reduced the frequency of NM ruptures and blebs. Thus, progerin expression disrupts the overall structure of the nuclear lamina, but that effect-along with NM ruptures and blebs-can be abrogated by increased lamin B1 expression.

Assuntos

Lamina Tipo A , Lamina Tipo B , Lâmina Nuclear , Lâmina Nuclear/metabolismo , Lamina Tipo A/metabolismo , Lamina Tipo A/genética , Lamina Tipo B/metabolismo , Lamina Tipo B/genética , Humanos , Progéria/metabolismo , Progéria/genética , Progéria/patologia , Animais , Precursores de Proteínas/metabolismo , Precursores de Proteínas/genética , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Camundongos

RESUMO

Genomic instability is not only a hallmark of senescent cells but also a key factor driving cellular senescence, and replication stress is the main source of genomic instability. Defective prelamin A processing caused by lamin A/C (LMNA) or zinc metallopeptidase STE24 (ZMPSTE24) gene mutations results in premature aging. Although previous studies have shown that dysregulated lamin A interferes with DNA replication and causes replication stress, the relationship between lamin A dysfunction and replication stress remains largely unknown. Here, an increase in baseline replication stress and genomic instability is found in prelamin A-expressing cells. Moreover, prelamin A confers hypersensitivity of cells to exogenous replication stress, resulting in decreased cell survival and exacerbated genomic instability. These effects occur because prelamin A promotes MRE11-mediated resection of stalled replication forks. Fanconi anemia (FA) proteins, which play important roles in replication fork maintenance, are downregulated by prelamin A in a retinoblastoma (RB)/E2F-dependent manner. Additionally, prelamin A inhibits the activation of the FA pathway upon replication stress. More importantly, FA pathway downregulation is an upstream event of p53-p21 axis activation during the induction of prelamin A expression. Overall, these findings highlight the critical role of FA pathway dysfunction in driving replication stress-induced genomic instability and cellular senescence in prelamin A-expressing cells.

Assuntos

Replicação do DNA , Instabilidade Genômica , Lamina Tipo A , Instabilidade Genômica/genética , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Humanos , Replicação do DNA/genética , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Senescência Celular/genética