Lamins in development, tissue maintenance and stress

141st ENMC International Workshop Inaugural Meeting of the EURO-Laminopathies Project Nuclear Envelope-linked Rare Human Diseases: From Molecular Pathophysiology towards Clinical Applications 10–12 March 2006, Naarden, The Netherlands

A-type nuclear lamins, all encoded by the LMNA gene through alternative splicing, are targets for mutation in a wide range of rare diseases, including a dominant mutation that enhances production of lamin A splice variant progerin, causing Hutchinson-Gilford progeria syndrome (HGPS).1 Often ignored, however, is the complex and poorly understood connection between A-type lamins and cancer.2 Expression of the 2 main A-type lamins (Lamins A and C) vary in inconsistent patterns in a wide range of cancers. More recently, enhanced progerin expression has been associated with prostate cancer development, an intriguing finding, since this splice variant of lamin A occurs at low frequency in normal cells and has been associated with aging.1 A new study by Jung et al. reports a role for progerin in modulating p53 activity in renal cell carcinomas (RCCs), and, moreover, that progerin expression is controlled by the von Hippel-Lindau tumor suppressor (VHL).3 This is particularly interesting, since RCCs, like many cancers, are highly age-associated, and increasing influence of progerin with age could, at least in part, explain this phenomenon. Motivation for the study started with the puzzling observation that VHL inactivation is an early event in RCC tumor progression, inconsistent with its known role in promoting degradation of HIF-1α and thereby blocking the pro-angiogenic response to hypoxia.4 Putting together this observation with other clues, for instance that p53 declines functionally in RCCs generally without mutation, and that nuclear irregularities similar to those in HGPS (associated with progerin expression) are a common feature of RCCs, Jung et al. tested the hypothesis that VHL may regulate these pathways.3 Following these threads with extensive experimental studies, the authors found that RCCs lacking VHL have high progerin protein content in the absence of changes in LMNA mRNA levels, leading them to test and generate support for a model whereby the E3 ubiquitin ligase activity of VHL is required for progerin turnover.3 Excess progerin, in turn, binds to and sequesters p14/ARF, permitting p53 degradation through MDM2. Interestingly, this activity occurs independently of HIF-2α, another VHL regulated factor that can inhibit p53 function.5,6 Together, these findings connect VHL to the p53 pathway and ascribe to it additional tumor suppressor activities. Cellular production of normal lamin A involves the farnesylation of a cysteine residue in the C terminus of the protein.1 However, unlike other farnesylated proteins, such as Ras, the lamin A farnesylation event is transient, with proteolytic cleavage prior to the cysteine removing the modification in the mature protein. Progerin, however, lacks this cleavage site and remains permanently farnesylated, a feature that contributes to progeroid pathologies. Farnesyl transferase inhibitors, developed to inhibit Ras farnesylation, have been tested widely in cancer with limited success. The study by Jung et al. suggests that progerin may be another meaningful target of farnesyl transferase inhibitors in a subset of cancers.3 HGPS cells are reported to have both elevated DNA damage and an abnormal DNA damage response.1 However, the connection between progerin and p53 has not been made previously. This may be related to the nature of the cell being interrogated. For instance, primary fibroblasts engineered to express progerin have proliferation defects that can be suppressed through inhibition of p53 function by expression of HPV E6.7 Moreover, the progeroid phenotypes of a mouse HGPS model can be partially rescued by deletion of p53.8 The implication is that p53 remains at least partially active in these settings, even though progerin is expressed. One possibility is that the ability of progerin to interact with p14/ARF and suppress p53 depends on the immortalization status of the cells. Primary cells expressing progerin would maintain p53 inducibility, but not a subset of tumor cells, including RCCs. Consistently, Jung et al. identified a leukemia cell line where progerin expression was elevated, leading to inhibition of p53.3 However, they were also able to detect enhanced p53 expression in HGPS fibroblasts when progerin expression was reduced by siRNA, indicating that this regulatory pathway is at least partially intact. More studies will be needed to understand the complex interplay between progerin and p53 pathway components. Aging is the biggest risk factor for most forms of cancer, yet the molecular reasons underlying this phenomenon have yet to be ascertained. What changes in the aging organism enable cancer progression? While several possibilities, including dysregulated gene expression programs and enhanced inflammation, are likely factors, the study by Jung et al. raises a new possibility that progerin can interfere directly with normal events that mediate repair of DNA lesions.3 Further studies will be important to explore this intriguing possibility. (Fig. 1) Figure 1. Model for interactions between progerin and p53 pathway. This diagram, reproduced and enhanced from Jung et al.,3 demonstrates the proposed interactions between progerin and p53 in different settings. In normal cells (left), low progerin ...

Lamin B receptor (LBR) is an inner nuclear membrane (INM) protein that plays crucial roles in maintaining nuclear architecture and organization of peripheral heterochromatin. Lamins and LBR both contribute to chromatin tethering at the nuclear periphery, and the expression of LBR and A-type lamins is tightly regulated during development to ensure a faithful transition between different chromatin tethering modalities. Despite its well-established association with B-type lamins, the contributions of individual lamin isoforms to LBR localization and anchorage have not been systematically examined. Here, we used mouse embryonic fibroblasts (MEFs) lacking all endogenous lamins (triple lamin knockout: TKO) to assess how specific lamin isoforms and domains regulate LBR subcellular localization and mobility. Whereas ectopic expression of either lamin B1 or lamin B2 was sufficient to tether LBR to the nuclear envelope in TKO cells, expression of lamin A increased the lateral mobility of LBR at the nuclear membrane, resulting in its displacement from the nuclear envelope to the ER. The lamin A-induced displacement of LBR was mediated by phosphorylation of LBR. Overexpression of lamin A in wild-type MEFs similarly increased LBR phosphorylation and promoted its displacement from the nuclear envelope. Collectively, these findings define isoform-specific and antagonistic roles for A-type and B-type lamins in regulating LBR anchorage at the nuclear envelope. In addition, they indicate a lamin A-dependent mechanism that may reflect a broader developmental process, since LBR and lamin A sequentially tether peripheral heterochromatin during development.

Nuclear A- and B-type lamins are differentially expressed in tissues, depending on the degree of cellular differentiation and proliferative status. By studying lamin expression in testis parenchyma and testicular germ cell tumours, further insight may be gained into the degree of cellular differentiation in normal testis and into the whole spectrum of differentiation lineages found in testicular germ cell tumours. Frozen tissue sections of normal testis and the different types of testicular germ cell tumours were immunostained with monoclonal antibodies to distinct lamin subtypes. Lamin reactivity was evaluated in relation to the lineage and degree of cellular differentiation and the reactivity patterns were compared with each other and with those in normal testis. In normal testis, both A- and B-type lamins were expressed in Sertoli, Leydig, and peritubular cells, while in spermatogonia only B-type lamins were found and spermatocytes showed weak reactivity with the A-type lamin antibodies. Carcinoma in situ was most often positive for both of the B-type lamins and negative for the A-type lamins (lamins A and C). In testicular germ cell tumours, B-type lamins were always expressed, while A-type lamins were differentially expressed. Differentiated non-seminomas were positive for both of the A-type lamins, whereas embryonal carcinomas were positive for lamin C and negative for lamin A. Seminomas were negative for both of the A-type lamins, with the exception of seminomas containing a Ras mutation. Spermatogonia and seminoma cells, which follow a differentiation pathway along the spermatogenic lineage and show characteristics of germ cells, do not express A-type lamins. Non-seminomas, showing embryonal or extraembryonal differentiation, express A-type lamins to varying degrees, distinguishing embryonal carcinoma cells from other non-seminomatous components. This may aid in the evaluation of the percentage of embryonal carcinoma in non-seminomatous testicular germ cell tumours as a prognostic parameter.

Background and Aim: Hutchinson-Gilford Progeria Syndrome (HGPS) and progeroid laminopathies (PL) are rare genetic disorders characterized by accelerated aging and early onset cardiovascular complications. Despite recent advances in the genetic diagnosis of HGPS and PL and the advent of lonafarnib treatment, the epidemiology and clinical characteristics of these disorders in Asia remain unclear. This study aimed to assess the prevalence, clinical features, and diagnostic trends of the HGPS and PL in Japan.Methods: A nationwide two-step survey was conducted between July 2022 and January 2024, across 1,513 medical facilities.Results: The survey identified ten HGPS patients, including eight with a confirmed genetic diagnosis. Early onset features such as scleroderma-like skin changes, growth retardation, and joint contracture were important in facilitating an early and accurate diagnosis. Cardiovascular complications typically occurred during their teens, and abnormalities in lipid metabolism were frequently observed. Overlapping but distinct phenotypes have been noted in ZMPSTE24 deficiency and other laminopathies caused by LMNA pathogenic variants, such as Emery-Dreifuss muscular dystrophy. Four patients with definite HGPS and eight patients with progerin-related PL (definite HGPS, n = 4; uncertain HGPS, n = 2; ZMPSTE24 deficiency, n = 2) were reported alive on October 2023, and the prevalence of HGPS was estimated to be 1 in 15.5 to 31.1 million.Conclusions: This study provides updated epidemiological and clinical insights into HGPS and related laminopathies in Japan. The introduction of lonafarnib has the potential to extend survival, emphasizing the need to monitor for late-stage complications.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a disease of accelerated aging causing death in the mid-teens from myocardial infarction or stroke. The disease is caused by a point mutation in the gene encoding lamin-A. The mutated scaffolding protein is aberrantly farnesylated inducing a constellation of defects included nuclear abnormalities, genomic damage, and rapid senescence. Therapy targeting the abnormal farnesylation provides a modest extension of life, thus new insights and therapeutic approaches are urgently needed for these children. Consistent with previous morphological observations and new studies implicating YAP/TAZ mechanobiology as an important mechanical pathway for endothelial cell (EC) health under shear stress, we hypothesized that HGPS ECs have an innate mechanical disturbance rendering them unable to respond to external, atheroprotective cues. We used a microfluidic vessel-on-a-chip with channel geometries and fluid flow to precisely model the hemodynamic stimuli present in vasculature as we have previously described. We cultured iPSC-derived HGPS ECs in this system to study mechanoresponse to shear stress and YAP/TAZ signaling. HGPS ECs manifest a rounded, flattened appearance characteristic of senescent ECs, are unable to align in response to flow, and have aberrant YAP/TAZ activity despite unidirectional laminar flow. To explore the physical underpinnings of such biochemical disturbances, we used atomic force microscopy (AFM) to precisely characterize the shape of individual HGPS cells, and their deformation to a controlled force applied by the AFM cantilever. Preliminary measurements confirmed that HGPS cells have a reduced profile and are compositely stiffer (nuclear modulus + cytoskeletal modulus) than cells derived from the unaffected parent of the child. These data provide evidence of altered biophysical properties of senescent cells which we term “mechanical aging,” which is associated with aberrant signaling in response to hemodynamic stimuli. Further characterization of mechanical aging may lead to new therapeutic approaches for HGPS and other age-related diseases.

Lamins are type V intermediate filament proteins that support nuclear membranes. They are divided into A-type lamins, which include lamin A and C, and B-type lamins, which include lamin B1 and B2. In the rat brain, lamin A and C are expressed in relatively equal amounts, while the expressions of lamin B1 and B2 vary depending on the cell type. Lamins play important roles in normal morphogenesis and function. In the nervous system, their abnormal expression causes several neurodegenerative diseases such as peripheral neuropathy, leukodystrophy and lissencephaly. The retina belongs to the central nervous system (CNS) and has widely been used as a source of CNS neurons. We investigated the expression patterns of lamin subtypes in the adult rat retina by immunohistochemistry and found that the staining patterns differed when compared with the brain. All retinal neurons expressed lamin B1 and B2 in relatively equal amounts. In addition, horizontal cells and a subpopulation of retinal ganglion cells expressed lamin A and C, while photoreceptor cells expressed neither lamin A nor C, and all other retinal neurons expressed lamin C only. This differential expression pattern of lamins in retinal neurons suggests that they may be involved in cellular differentiation and expression of cell-specific genes in individual retinal neurons.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal disease manifested by premature aging and aging-related phenotypes, making it a disease model for aging. The cellular machinery mediating age-associated phenotypes in HGPS remains largely unknown, resulting in limited therapeutic targets for HGPS. In this study, we showed that mitophagy defects impaired mitochondrial function and contributed to cellular markers associated with aging in mesenchymal stem cells derived from HGPS patients (HGPS-MSCs). Mechanistically, we discovered that mitophagy affected the aging-associated phenotypes of HGPS-MSCs by inhibiting the STING-NF-ĸB pathway and the downstream transcription of senescence-associated secretory phenotypes (SASPs). Furthermore, by utilizing UMI-77, an effective mitophagy inducer, we showed that mitophagy induction alleviated aging-associated phenotypes in HGPS and naturally aged mice. Collectively, our results uncovered that mitophagy defects mediated the aging-associated markers in HGPS, highlighted the function of mitochondrial homeostasis in HGPS progression, and suggested mitophagy as an intervention target for HGPS and aging.

The majority of human cancer shows chromosomal instability (CIN). Although the precise mechanism remains largely uncertain, proper progression of mitosis is crucial. B-type lamins were suggested to be components of the spindle matrix of mitotic cells and to be involved in mitotic spindle assembly; thus, B-type lamins may contribute to the maintenance of chromosome integrity. Here, using a proteomic approach, we identified lamin B2 as a novel protein involved in CIN. Lamin B2 expression decreased in colorectal cancer cell lines exhibiting CIN, as compared with colorectal cancer cell lines exhibiting microsatellite instability (MIN), which is mutually exclusive to CIN. Importantly, lamin B2 knockdown in MIN-type colorectal cancer cells induced CIN phenotypes such as aneuploidy, chromosome mis-segregation and aberrant spindle assembly, whereas ectopic expression of lamin B2 in CIN-type colorectal cancer cells prevented their CIN phenotypes. Additionally, immunohistochemical analysis showed a lower expression of lamin B2 in cancer tissues extracted from patients with sporadic colorectal cancer (CIN-type) than that from patients with hereditary non-polyposis colorectal cancer (HNPCC; MIN type). Intriguingly, mitotic lamin B2 in MIN cancer cells was localized outside the spindle poles and mitotic lamin B2 localization was diminished in CIN cancer cells, suggesting an important role of lamin B2 in proper mitotic spindle formation. The obtained results suggest that lamin B2 maintains chromosome integrity by ensuring proper spindle assembly and that its downregulation causes CIN in colorectal cancer.

Gametogenesis and embryogenesis are dynamic developmental stages marked by extensive modifications in the organization of the genome and nuclear architecture. In the literature it is conveyed that only B-type lamins are required in these early stages of development and that A-type lamins are not present or required until differentiation of specific cell types associated with specialized tissue is initiated. To assess the presence of nuclear structures that are putatively involved in genome regulation, we investigated the distribution of lamin proteins throughout the early stages of porcine embryonic development, using testes tissue sections, oocytes and in-vitro fertilized (IVF) porcine embryos and employing anti-lamin antibodies. We have shown that anti-lamin A staining is present at the one-cell, two-cell, four-cell, and six- to eight-cell stages of early porcine embryo development, but diminishes at the morulae and blastocyst stages. Large intranuclear anti-lamin A foci are prominent in the early preimplantation stages. Both anti-lamin A/C and anti-lamin B staining were clearly present in all embryonic stages. Immature porcine oocytes revealed lamin rings using the monoclonal anti-lamin A/C antibody and many immature oocytes exhibited a pale rim staining pattern with anti-lamin A antibody. A-type lamins were not observed in sperm precursor cells. Thus, we have shown that A-type lamins and B-type lamins are present at the nuclear envelope in very early porcine embryos and that lamin A is also found in large intranuclear aggregates in two-cell to eight-cell embryos but is lacking from later embryonic stages.

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder caused by a de novo genetic mutation that leads to the accumulation of a splicing isoform of lamin A termed progerin. Progerin expression alters the organization of the nuclear lamina and chromatin. The life expectancy of HGPS patients is severely reduced due to critical cardiovascular defects. Progerin also accumulates in an age-dependent manner in the vascular cells of adults that do not carry genetic mutations associated with HGPS. The molecular mechanisms that lead to vascular dysfunction in HGPS may therefore also play a role in vascular aging. The vascular phenotypic and molecular changes observed in HGPS are strikingly similar to those seen with age, including increased senescence, altered mechanotransduction and stem cell exhaustion. This article discusses the similarities and differences between age-dependent and HGPS-related vascular aging to highlight the relevance of HGPS as a model for vascular aging. Induced pluripotent stem cells derived from HGPS patients are suggested as an attractive model to study vascular aging in order to develop novel approaches to treat cardiovascular disease.

Reduced adiponectin and HDL cholesterol without elevated C-reactive protein: Clues to the biology of premature atherosclerosis in Hutchinson-Gilford Progeria Syndrome

Lamin proteins are type V intermediate filament proteins (IFs) located inside the cell nucleus. They are evolutionarily conserved and have similar domain organization and properties to cytoplasmic IFs. Lamins provide a skeletal network for chromatin, the nuclear envelope, nuclear pore complexes and the entire nucleus. They are also responsible for proper connections between the karyoskeleton and structural elements in the cytoplasm: actin and the microtubule and cytoplasmic IF networks. Lamins affect transcription and splicing either directly or indirectly. Translocation of active genes into the close proximity of nuclear lamina is thought to result in their transcriptional silencing. Mutations in genes coding for lamins and interacting proteins in humans result in various genetic disorders, called laminopathies. Human genes coding for A-type lamin (LMNA) are the most frequently mutated. The resulting phenotypes include muscle, cardiac, neuronal, lipodystrophic and metabolic pathologies, early aging phenotypes, and combined complex phenotypes. The Drosophila melanogaster genome codes for lamin B-type (lamin Dm), lamin A-type (lamin C), and for LEM-domain proteins, BAF, LINC-complex proteins and all typical nuclear proteins. The fruit fly system is simpler than the vertebrate one since in flies there is only single lamin B-type and single lamin A-type protein, as opposed to the complex system of B- and A-type lamins in Danio, Xenopus and Mus musculus. This offers a unique opportunity to study laminopathies. Applying genetic tools based on Gal4 and in vitro nuclear assembly system to the fruit fly model may successfully advance knowledge of laminopathies. Here, we review studies of the laminopathies in the fly model system.

Inflammation is a hallmark of aging and accelerated aging syndromes such as Hutchinson–Gilford progeria syndrome (HGPS). In this study, we present evidence of increased expression of the components of the NLRP3 inflammasome pathway in HGPS skin fibroblasts, an outcome that was associated with morphological changes of the nuclei of the cells. Lymphoblasts from HGPS patients also showed increased basal levels of NLRP3 and caspase 1. Consistent with these results, the expression of caspase 1 and Nlrp3, but not of the other inflammasome receptors was higher in the heart and liver of Zmpste24−/− mice, which phenocopy the human disease. These data were further corroborated in LmnaG609G/G609G mice, another HGPS animal model. We also showed that pharmacological inhibition of the NLRP3 inflammasome by its selective inhibitor, MCC950, improved cellular phenotype, significantly extended the lifespan of progeroid animals, and reduced inflammasome‐dependent inflammation. These findings suggest that inhibition of the NLRP3 inflammasome is a potential therapeutic approach for the treatment of HGPS.

A-type Lamins Form Distinct Filamentous Networks with Differential Nuclear Pore Complex Associations