Lamin B Research Articles

Impact of Nuclear Peripheral Chromatin Lamin LMNB1 Gene in the Proliferation and Migration of Glioma Cells.

The goal of this study is to explore the role of the LMNB1 gene in glioma. A cohort of 160 patients who underwent glioma surgery were randomly selected of this study. The LMNB1 expression was assessed employing immunohistochemical and real-time quantitative polymerase chain reaction methods. Initially, RNA interference technology was applied to suppress gene expression, followed by the evaluation of tumor cell proliferation, apoptosis, cell cycle dynamics, and migration. The underlying molecular mechanisms of LMNB1 function were examined by a human phospho-kinase array and immunoblotting. And we established the xenograft models to determine the effect of tumor growth as well as the degree of invasion in shLMNB1 mice. Elevated LMNB1 expression correlated with unfavorable overall survival and disease-free survival. A substantial inhibition in cell growth was observed subsequent to LMNB1 knockdown in SHG-44 and U251 glioma cells. SHG-44-shLMNB1 cells exhibited a reduction in the S phase population, along with an increase in cells in G1 and G2 phases. Similarly, shLMNB1 U251 cells showed fewer cells in the S phase and an elevation in cells in G1 phase. Notably, increased apoptosis was observed in U251-shLMNB1 cells and SHG-44-shLMNB1 cells. Wound healing and Transwell migration assays demonstrated a significant decrease in the migration rate of both SHG-44-shLMNB1 and U251-shLMNB1 cells. The phosphorylation levels of Akt1/2/3, as well as the expressions of PI3K, AKT, and p-AKT proteins, were reduced in the shLMNB1 group. Downregulation of LMNB1 repressed tumor progress in vivo. The silencing of LMNB1 was found to significantly reduce the proliferation of human glioma cells, induce apoptosis in tumor cells, impede the progression of the cell cycle, and inhibit the migration of tumor cells. Consequently, we hypothesize that LMNB1 promotes glioma cell proliferation through mechanisms involving the inhibition of tumor cell apoptosis, acceleration of the cell cycle, and enhancement of tumor cell migration. We found that LMNB1 exert critical roles in glioma progression may via regulation of PI3K/Akt signaling pathway. These observations suggest that LMNB1 holds clinical potential for diagnostic and prognostic applications in glioma, presenting novel targets for drug development.

Claudin-4 stabilizes the genome via nuclear and cell cycle remodeling to support ovarian cancer cell survival.

Alterations in the interplay between the nucleus and the cell cycle during cancer development lead to a state of genomic instability, often accompanied by observable morphological aberrations. Tumor cells can regulate these aberrations to evade cell death, either by preventing or eliminating genomic instability. In epithelial ovarian cancer (EOC), overexpression of claudin-4 significantly contributes to therapy resistance through mechanisms associated with genomic instability regulation. However, the molecular mechanisms underlying claudin-4 overexpression in EOC remain poorly understood. In this study, we modified claudin-4 expression and employed a unique claudin mimic peptide (CMP) to investigate claudin-4's function. Our findings show that claudin-4 supports ovarian cancer cell survival by stabilizing the genome through nuclear and cell cycle remodeling. Specifically, claudin-4 induced nuclear constriction by excluding lamin B1 and promoting perinuclear F-actin accumulation, thereby altering nuclear structure and dynamics. Similarly, cell cycle modifications due to claudin-4 overexpression resulted in fewer cells entering the S-phase and reduced genomic instability in tumors. Importantly, disrupting claudin-4's biological effects using CMP and forskolin increased the efficacy of PARP inhibitor treatment, correlating with alterations in the oxidative stress response. Our data indicate that claudin-4 protects tumor genome integrity by modulating the crosstalk between the nucleus and the cell cycle, leading to resistance to genomic instability formation and the effects of genomic instability-inducing agents.

Endothelial Cell Aging and Autophagy Dysregulation.

Entropy is a natural process that affects all living cells, including senescence, an irreversible physiological process that impairs cell homeostasis. Age is a significant factor in disease development, and the pathogenesis of endothelial cell aging is multifactorial. Autophagy dysfunction accelerates endothelial cell aging and cell death, while autophagy preserves endothelial cell youthfulness through intracellular homeostasis and gene expression regulation. Sirt, mTORC1, and AMPK are youthfulness genes that induce autophagy by inhibiting mTOR and upregulating FIP200/Atg13/ULK1. Aged endothelial cells have decreased levels of Lamin B1, γH2AX, Ki67, BrdU, PCNA, and SA β-Gal. Maintaining healthy young endothelial cells can prevent most cardiovascular diseases. Autophagy targeting is a potential future therapeutic strategy to modify endothelial cell age and potentially slow or reverse the aging process. This article provides state-of-the-art research on the role of autophagy in endothelial cell aging. Hypothesizing that autophagy dysregulation is associated with early endothelial cell dysfunction and further clinical sequelae, including atherosclerosis formation, leading to various cardiovascular diseases.

Pan-cancer TCGA analysis reveals the potential involvement of B-type lamins in dysregulating chromosome segregation in human cancer.

Lamins play a crucial role in maintaining nuclear structure and function. Our study investigates the expression patterns and clinical implications of B-type lamins with a special focus on lamin B2 across various cancer types using comprehensive RNA sequencing datasets. We found that high expression levels of lamin B1 and lamin B2 are associated with decreased overall and disease-free survival in cancer. This association is further pronounced when both lamins are co-expressed, indicating a compounded negative impact on patient prognosis. Additionally, we highlighted the relationship between B-type lamins and the tumor microenvironment. Lamin B1 mRNA expression shows a strong positive correlation with activated CD4+ T-cells and type 2 T-helper cells (Th2), suggesting its role in immune cell infiltration and response within tumors. Lamin B2 expression also correlates moderately with these immune cells, indicating a potential but lesser role in modulating the immune landscape. Notably, the epigenetic state of lamin B1 significantly affects the tumor microenvironment, suggesting a dual role in structural integrity and immune modulation. We have identified 9 lamin B2 interacting proteins that are co-expressed with B-type lamins in cancerous conditions and modulate cytokinesis and cell division pathways during tumorigenesis. Furthermore, we have identified specific molecular targets of B-type lamins that co-express with them in a range of human cancers and are potentially involved in dysregulating chromosome segregation and mRNA binding. Overexpression of these targets alongside B-type lamins correlates with poor prognosis in multiple cancers. These findings underscore the potential of B-type lamins as biomarkers for poor prognosis and as targets for cancer therapies.

Decreased DNA density is a better indicator of a nuclear bleb than lamin B loss.

Nuclear blebs are herniations of the nucleus that occur in diseased nuclei that cause nuclear rupture leading to cellular dysfunction. Chromatin and lamins are two of the major structural components of the nucleus that maintain its shape and function, but their relative roles in nuclear blebbing remain elusive. To determine the composition of nuclear blebs, we compared the immunofluorescence intensity of DNA and lamin B in the main nucleus body to the nuclear bleb across cell types and perturbations. DNA density in the nuclear bleb was consistently decreased to about half of the nuclear body while lamin B levels in the nuclear bleb varied widely. Partial Wave Spectroscopic (PWS) microscopy recapitulated significantly decreased likelihood of high-density domains in the nuclear bleb versus body, independent of lamin B. Time lapse imaging into immunofluorescence reveals that decreased DNA density marks all nuclear blebs while decreased lamin B1 levels only occur in blebs that have recently ruptured. Thus, decreased DNA density is a better marker of a nuclear bleb than lamin B level.

BRD7 regulates cellular senescence and apoptosis in ALS by modulating p21 expression and p53 mitochondrial translocation respectively

Cellular senescence is involved in the progression of neurodegenerative diseases. Motor neurons exhibit senescence-like alterations in ALS. BRD7, identified as a regulatory factor associated with cellular senescence, its function in ALS remains unclear. This study aims to investigate the potential role and mechanisms of BRD7 in ALS. We analyzed RNA levels using qRT-PCR, protein levels through immunofluorescence and western blot, and apoptosis via TUNEL staining. Cell transfection was conducted for in vitro experiments. The level of β-galactosidase was measured by β-galactosidase activity detection kit. ALS motor neurons exhibited senescence-like alterations, characterized by increased activity of p53, p21, and β-galactosidase, as well as reduced lamin B1 staining. Additionally, the expression of BRD7 was upregulated and induced cellular senescence and apoptosis. Downregulation of BRD7 alleviates the cellular senescence by inhibiting p21 rather than p53. Knockdown of BRD7 inhibited p53 mitochondrial translocation, leading to reduced apoptosis. Our results suggest that BRD7 plays an important role in the survival of ALS motor neurons. BRD7 knockdown can reduce cellular senescence and apoptosis by inhibiting p21 and p53 mitochondrial translocation.

Long-Axial-Range Double-Helix Point Spread Functions for 3D Volumetric Super-Resolution Imaging.

Single-molecule localization microscopy (SMLM) is a powerful tool for observing structures beyond the diffraction limit of light. Combining SMLM with engineered point spread functions (PSFs) enables 3D imaging over an extended axial range, as has been demonstrated for super-resolution imaging of various cellular structures. However, super-resolving structures in 3D in thick samples, such as whole mammalian cells, remains challenging as it typically requires acquisition and postprocessing stitching of multiple slices to cover the entire sample volume or more complex analysis of the data. Here, we demonstrate how the imaging and analysis workflows can be simplified by 3D single-molecule super-resolution imaging with long-axial-range double-helix (DH)-PSFs. First, we experimentally benchmark the localization precisions of short- and long-axial-range DH-PSFs at different signal-to-background ratios by imaging fluorescent beads. The performance of the DH-PSFs in terms of achievable resolution and imaging speed was then quantified for 3D single-molecule super-resolution imaging of mammalian cells by DNA-PAINT imaging of nuclear lamina protein lamin B1 in U-2 OS cells. Furthermore, we demonstrate how the use of a deep-learning-based algorithm allows the localization of dense emitters, drastically improving the achievable imaging speed and resolution. Our data demonstrate that using long-axial-range DH-PSFs offers stitching-free, 3D super-resolution imaging of whole mammalian cells, simplifying the experimental and analysis procedures for obtaining volumetric nanoscale structural information.

Lamin A and Emerin Protein Expression Remains Consistently Low and Nuclear Size is Unchanged in Normal Endometrium, Precancerous Lesions, and Endometrioid Carcinoma.

Nuclear laminar or inner nuclear membrane proteins, including lamin A, B1, and B2 and emerin, are involved in maintaining nuclear morphology. However, their expression patterns vary among tumors and remain incompletely understood. Endometrioid carcinoma (EC) exhibits mild nuclear atypia, although the underlying reasons have not been thoroughly explored. In this study, we quantitatively analyzed emerin and lamin A, B1, and B2 expression levels in normal endometrium (NE), precancerous lesions, and EC using computer-assisted image analysis to assess the proteins' roles in nuclear morphologic change during tumorigenesis. From NE to EC, nuclear size remained unchanged, and lamin A and emerin were consistently expressed at low levels, whereas lamin B1 and B2 expression gradually decreased. Given the association between lamin A and emerin as well as their roles in nuclear morphology, these results indicate that their consistent low expression may underlie the preservation of nuclear size and shape in EC relative to NE. Conversely, lamin B1 and B2 are implicated in tumor progression rather than nuclear morphology maintenance. As lamin A and emerin are expressed in many organs and tumors, the consistently low expression of these proteins from NE to EC highlights a notable feature of the endometrium and endometrial carcinogenesis.

H4K20me3-Mediated Repression of Inflammatory Genes is a Characteristic and Targetable Vulnerability of Persister Cancer Cells.

Anti-cancer therapies can induce cellular senescence, which is highly stable, or drug-tolerant persistence, which is efficiently reversed upon therapy termination. While approaches to target senescent cells have been extensively studied, further understanding of the processes regulating persistence is needed to develop treatment strategies to suppress persister cell survival. Here, we used mTOR/PI3K inhibition to develop and characterize a model of persistence-associated arrest in human cancer cells of various origins. Persister and senescent cancer cells shared an expanded lysosomal compartment and hypersensitivity to BCL-XL inhibition. However, persister cells lacked other features of senescence, such as loss of lamin B1, senescence-associated β-galactosidase activity, upregulation of MHC-I, and an inflammatory and secretory phenotype (SASP). Genome-wide CRISPR/Cas9 screening for genes required for the survival of persister cells revealed that they are hypersensitive to the inhibition of one-carbon (1C) metabolism, which was validated by the pharmacological inhibition of SHMT, a key enzyme that feeds methyl groups from serine into 1C metabolism. Connecting 1C metabolism with the epigenetic regulation of transcription, the repressive heterochromatic mark H4K20me3 was enriched at the promoters of SASP and interferon response genes in persister cells, while it was absent in proliferative or senescent cells. Moreover, persister cells overexpressed the H4K20 methyltransferases KMT5B/C, and their downregulation unleashed inflammatory programs and compromised the survival of persister cells. In summary, this study defined distinctive features of persister cancer cells, identified actionable vulnerabilities, and provided mechanistic insight into their low inflammatory activity.

Divergent regulation of long non-coding RNAs H19 and PURPL affects cell senescence in human dermal fibroblasts.

Cellular senescence is a permanent cell growth arrest that occurs in response to various intrinsic and extrinsic stimuli and is associated with cellular and molecular changes. Long non-coding RNAs (lncRNAs) are key regulators of cellular senescence by affecting the expression of many important genes involved in senescence-associated pathways and processes. Here, we evaluated a panel of lncRNAs associated with senescence for their differential expression between young and senescent human dermal fibroblasts (NHDFs) and studied the effect of a known senomorphic compound, resveratrol, on the expression of lncRNAs in senescent NHDFs. As markers of senescence, we evaluated cell growth, senescence-associated (SA)-β-Gal staining, and the expression of p21, Lamin B1 and γH2AX. We found that H19 and PURPL were the most altered lncRNAs in replicative, in doxorubicin (DOXO) and ionising radiation (IR)-induced senescence models. We then investigated the function of H19 and PURPL in cell senescence by siRNA-mediated silencing in young and senescent fibroblasts, respectively. Our results showed that H19 knockdown reduced cell viability and induced cell senescence and autophagy of NHDFs through the regulation of the PI3K/AKT/mTOR pathway; conversely, PURPL silencing reversed senescence by reducing (SA)-β-Gal staining, recovering cell proliferation with an increase of S-phase cells, and reducing the p53-dependent DNA damage response. Overall, our data highlighted the role of H19 and PURPL in the senescent phenotype and suggested that these lncRNAs may have important implications in senescence-related diseases.

Werner syndrome RECQ helicase participates in and directs maintenance of the protein complexes of constitutive heterochromatin in proliferating human cells.

Werner syndrome of premature aging is caused by mutations in the WRN RECQ helicase/exonuclease, which functions in DNA replication, repair, transcription, and telomere maintenance. How the loss of WRN accelerates aging is not understood in full. Here we show that WRN is necessary for optimal constitutive heterochromatin levels in proliferating human fibroblasts. Locally, WRN deficiency derepresses SATII pericentromeric satellite repeats but does not reduce replication fork progression on SATII repeats. Globally, WRN loss reduces a subset of protein-protein interactions responsible for the organization of constitutive heterochromatin in the nucleus, namely, the interactions involving Lamin B1 and Lamin B receptor, LBR. Both the mRNA level and subcellular distribution of LBR are affected by WRN deficiency, and unlike the former, the latter phenotype does not require WRN catalytic activities. The phenotypes of heterochromatin disruption seen in WRN-deficient proliferating fibroblasts are also observed in WRN-proficient fibroblasts undergoing replicative or oncogene-induced senescence. WRN interacts with histone deacetylase 2, HDAC2; WRN/HDAC2 association is mediated by heterochromatin protein alpha, HP1α, and WRN complexes with HP1α and HDAC2 are downregulated in senescing cells. The data suggest that the effect of WRN loss on heterochromatin is separable from senescence program, but mimics at least some of the heterochromatin changes associated with it.

Mouse polyomavirus infection induces lamin reorganisation.

The nuclear lamina is a dense network of intermediate filaments beneath the inner nuclear membrane. Composed of A-type lamins (lamin A/C) and B-type lamins (lamins B1 and B2), the nuclear lamina provides a scaffold for the nuclear envelope and chromatin, thereby maintaining the structural integrity of the nucleus. A-type lamins are also found inside the nucleus where they interact with chromatin and participate in gene regulation. Viruses replicating in the cell nucleus have to overcome the nuclear envelope during the initial phase of infection and during the nuclear egress of viral progeny. Here, we focused on the role of lamins in the replication cycle of a dsDNA virus, mouse polyomavirus. We detected accumulation of the major capsid protein VP1 at the nuclear periphery, defects in nuclear lamina staining and different lamin A/C phosphorylation patterns in the late phase of mouse polyomavirus infection, but the nuclear envelope remained intact. An absence of lamin A/C did not affect the formation of replication complexes but did slow virus propagation. Based on our findings, we propose that the nuclear lamina is a scaffold for replication complex formation and that lamin A/C has a crucial role in the early phases of infection with mouse polyomavirus.

Claudin-4 remodeling of nucleus-cell cycle crosstalk maintains ovarian tumor genome stability and drives resistance to genomic instability-inducing agents.

During cancer development, the interplay between the nucleus and the cell cycle leads to a state of genomic instability, often accompanied by observable morphological aberrations. These aberrations can be controlled by tumor cells to evade cell death, either by preventing or eliminating genomic instability. In epithelial ovarian cancer (EOC), overexpression of the multifunctional protein claudin-4 is a key contributor to therapy resistance through mechanisms associated with genomic instability. However, the molecular mechanisms underlying claudin-4 overexpression in EOC remain poorly understood. Here, we altered claudin-4 expression and employed a unique claudin-4 targeting peptide (CMP) to manipulate the function of claudin-4. We found that claudin-4 facilitates genome maintenance by linking the nuclear envelope and cytoskeleton dynamics with cell cycle progression. Claudin-4 caused nuclei constriction by excluding lamin B1 and promoting perinuclear F-actin accumulation, associated with remodeling nuclear architecture, thus altering nuclear envelope dynamics. Consequently, cell cycle modifications due to claudin-4 overexpression resulted in fewer cells entering the S-phase and reduced genomic instability. Importantly, disrupting biological interactions of claudin-4 using CMP and forskolin altered oxidative stress cellular response and increased the efficacy of PARP inhibitor treatment. Our data indicate that claudin-4 protects tumor genome integrity by remodeling the crosstalk between the nuclei and the cell cycle, leading to resistance to genomic instability formation and the effects of genomic instability-inducing agents.

Targeting Caspases 3/6 and Cathepsins L/B May Decrease Laminopathy-Induced Apoptosis in Alzheimer's Disease.

Laminopathy is a pathological manifestation observed in Alzheimer's disease (AD), leading to neuronal apoptosis. Our objective was to assess inhibitors of enzymes involved in laminopathy. 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). 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. 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.

Effect of lamins and emerin on nuclear morphology and histological architecture in lung adenocarcinoma

Emerin and lamins not only influence nuclear morphology but are also involved in differentiation. We herein examined 82 resected cases of invasive lung adenocarcinoma using computer-assisted image analysis of nuclear morphology on Feulgen-stained and immunohistochemical sections of lamin A, B1, B2, and emerin (four proteins) to calculate the rank sum of the cell positivity rates for these four proteins. The rank sum of four proteins showed weak negative correlations with the nuclear area and perimeter and a weak positive correlation with the nuclear shape factor. Interestingly, the top three cases with the highest rank sum were papillary adenocarcinoma, and the bottom three cases were acinar adenocarcinomas containing cribriform patterns. We compared the rank sum for grading (differentiation: G1, G2, and G3) and predominant histological subtypes and found that the rank sum of G3 was lower than that of G1 and G2. Furthermore, the rank sum was lower for acinar adenocarcinoma with >20 % cribriform pattern (acinar+cribri) and solid adenocarcinoma than for lepidic and papillary adenocarcinoma. Individual examination of the four proteins revealed that emerin expression was lower in G3 than in G1, and lamin B2 expression was lower in G3 than in G1 and G2. Compared with lepidic adenocarcinoma, acinar+cribri showed significantly lower expression of all four proteins among histological subtypes. These data indicated that the expression of lamin A, B1, B2, and emerin was markedly decreased in poorly differentiated adenocarcinoma (i.e., G3), especially in acinar+cribri. Our data suggested that changes in these four proteins can not only affect nuclear morphology but also histological structure in lung adenocarcinoma.

Altered expression and localization of nuclear envelope proteins in a prostate cancer cell system

BackgroundThe 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.MethodsThe expression and subcellular localization of NE proteins (lamins A/C and B1 and the inner nuclear membrane proteins emerin and β-dystroglycan [β-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.ResultsDeformed 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 β-DG, occurred in metastatic prostate cells.ConclusionsThe 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.

Unveiling the role of protein kinase A (PKA) activity in bovine oviductal epithelial cells: implications on apoptotic signaling pathways during the estrous cycle.

The complex interactome crucial for successful pregnancy is constituted by the intricate network of endocrine and paracrine signaling pathways, involving gametes, embryos, and the female reproductive tract. Specifically, the oviduct exhibits distinct responses to gametes and early embryos during particular phases of the estrus cycle, a process tightly regulated by reproductive hormones. Moreover, these hormones play a pivotal role in orchestrating cyclical changes within oviductal epithelial cells. To unravel the molecular mechanisms underlying these dynamic changes, our study aimed to investigate the involvement of protein kinase A (PKA) in oviductal epithelial cells throughout the estrus cycle and in advanced pregnancy, extending our studies to oviductal epithelial cell in primary culture. By a combination of 2D-gel electrophoresis, Western blotting, and mass spectrometry, we identified 17 proteins exhibiting differential phosphorylation status mediated by PKA. Among these proteins, we successfully validated the phosphorylation status of heat shock 70kDa protein (HSP70), aconitase 2 (ACO2), and lamin B1 (LMNB1). Our findings unequivocally demonstrate the dynamic regulation of PKA throughout the estrus cycle in oviductal epithelial cells. Also, analysis by bioinformatics tools suggest its pivotal role in mediating cyclical changes possibly through modulation of apoptotic pathways. This research sheds light on the intricate molecular mechanisms underlying reproductive processes, with implications for understanding fertility and reproductive health.

PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

Senescence of nondividing neurons remains an immature concept, with especially the regulatory molecular mechanisms of senescence-like phenotypes and the role of proteins associated with neurodegenerative diseases in triggering neuronal senescence remaining poorly explored. In this study, we reveal that the nucleolar polyglutamine binding protein 3 (PQBP3; also termed NOL7), which has been linked to polyQ neurodegenerative diseases, regulates senescence as a gatekeeper of cytoplasmic DNA leakage. PQBP3 directly binds PSME3 (proteasome activator complex subunit 3), a subunit of the 11S proteasome regulator complex, decreasing PSME3 interaction with Lamin B1 and thereby preventing Lamin B1 degradation and senescence. Depletion of endogenous PQBP3 causes nuclear membrane instability and release of genomic DNA from the nucleus to the cytosol. Among multiple tested polyQ proteins, ataxin-1 (ATXN1) partially sequesters PQBP3 to inclusion bodies, reducing nucleolar PQBP3 levels. Consistently, knock-in mice expressing mutant Atxn1 exhibit decreased nuclear PQBP3 and a senescence phenotype in Purkinje cells of the cerebellum. Collectively, these results suggest homologous roles of the nucleolar protein PQBP3 in cellular senescence and neurodegeneration.

Melatonin Improves Oxidative Stress Injury in Retinopathy of Prematurity by Targeting miR-23a-3p/Nrf2

Purpose Melatonin has promising protective effects for retinopathy. However, its roles in retinopathy of prematurity (ROP) and the underlying mechanisms remain unknown. We aimed to explore its roles and mechanisms in a ROP model. Methods Hematoxylin and eosin staining were used to observe the morphology of the retina. Immunofluorescence was used to detect positive (Nrf2+ and VEGF+) cells. Immunohistochemistry was used to detect the level of nuclear expression of PCNA in retinal tissue. Transmission electron microscope (TEM) was used to observe the morphology and structure of pigment cells. qRT-PCR was used to assay the expression of miR-23a-3p, Nrf2, and HO-1. Western blotting was used to detect the expression of Nrf2, HO-1, β-actin, and Lamin B1. Results Melatonin or miR-23a-3p antagomir treatment could ameliorate the Oxygen-induced pathological changes, increased the expression of Nrf2 and HO-1, SOD, and GSH-Px, and decreased the expression of VEGF, miR-23a-3p, MDA and the apoptosis in the ROP model. Further target prediction and luciferase reporter assays confirmed the targeted binding relationship between miR-23a-3p and Nrf2. Conclusion Our study showed that melatonin could ameliorate H2O2-induced apoptosis and oxidative stress injury in RGC cells by mediating miR-23a-3p/Nrf2 signaling pathway, thereby improving retinal degeneration.

Abstract We030: Microtubule Organizing Centers in Cardiomyocyte Proliferation and Maturation

Mammalian cardiomyocytes undergo a loss of proliferative capacity due to cell division failure, leading to polyploidy (genome duplication) during postnatal maturation. Centrosomes, primary microtubule organizing centers (MTOCs) in animal cells comprised of centrioles and pericentriolar matrix (PCM), play a crucial role in cell division. In differentiated cells, non-centrosomal (nc) MTOCs can reorganize microtubules, with cellular localization varying between cell types to mediate specialized functions. The specific role of ncMTOCs in cardiomyocyte division and maturation remains unknown. This study investigates the formation and function of ncMTOCs in cardiomyocytes, aiming to elucidate the regulating mechanism. Our results demonstrate that during the maturation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), centrosomes disassemble, and their PCM translocates to the nuclear envelope, establishing perinuclear ncMTOCs. Furthermore, maturing CMs with ncMTOCs often exhibit polyploidy, indicating incomplete cell division. Microtubule dynamic experiments reveal that ncMTOCs serve as organizing centers for microtubules, promoting their assembly around the nucleus in maturing cardiomyocytes. To understand the mechanism regulating the transition from centrosomes to ncMTOCs in cardiomyocytes, we identified a significant downregulation of the nuclear lamina protein Lamin B2 (LMNB2) during CM maturation. Our results indicate that LMNB2 is essential for mitotic spindle formation in CMs but not in stem cells, suggesting a distinct role for LMNB2 in CMs. Our results also suggest an increased centrosome to ncMTOC transition in LMNB2-depleted CMs at an early differentiation stage. Ongoing research aims to investigate the impact of perinuclear ncMTOCs on cardiomyocyte proliferation through pericentriolar matrix knockdown in hiPSC-CMs. Additionally, the study seeks to further elucidate the role of Lamin B2 in the transition of centrosome proteins and its underlying mechanism.