Lamin in inflammation and aging

Abstract

Aging is characterized by a progressive loss of tissue function and an increased susceptibility to injury and disease. Many age-associated pathologies manifest an inflammatory component, and this has led to the speculation that aging is at least in part caused by some form of inflammation. However, whether or not inflammation is truly a cause of aging, or is a consequence of the aging process is unknown. Recent work using Drosophila has uncovered a mechanism where the progressive loss of lamin-B in the fat body upon aging triggers systemic inflammation. This inflammatory response perturbs the local immune response of the neighboring gut tissue and leads to hyperplasia. Here, we will discuss the literature connecting lamins to aging and inflammation.

Introduction

Chronic systemic inflammation without apparent infection in elderly humans is often referred to as the “inflammaging” phenotype, and is primarily characterized by elevated levels of circulating pro-inflammatory cytokines[1–6]. Epidemiological studies have found a strong correlation between the inflammaging phenotype and the presence of several aging-associated pathologies[7–9]. Genome-wide association studies have implicated several genes that function in immune, inflammatory and stress responses as being modifiers of longevity and health span[10].

Understanding the contribution of inflammation to aging has been pursued for many years with much effort aimed at identifying biomarkers of aging and cellular events that might serve as triggers for inflammatory responses. While conceptually straightforward, identifying cellular events has been very difficult, and is still largely correlative. Further, understanding the consequences of inflammation on aging presents its own challenges because, in part, many age-related diseases such as cardiovascular disease and osteoarthritis invoke strong inflammatory responses[11].

Recent studies have implicated the involvement of the nuclear intermediate filament proteins, the lamins, in aging-related inflammation. Here we will first introduce the general functions of lamins and then discuss the studies connecting these proteins to inflammation and aging.

Nuclear Lamins

Nuclear lamins are classed as type V intermediate filaments. There are two lamin subtypes, A-type and B-type, which are distinguished by their protein sequences, physical properties and expression profiles. In humans, the A-type lamin is encoded by LMNA, while two separate genes, LMNB1 and LMNB2, encode the B-type lamins. In Drosophila, the subject of some discussion below, the A- and B-type lamins (LAMC and LAM) are encoded by LamC and Lam, respectively.

Alternative splicing of the human LMNA transcript produces the major lamin-A and lamin-C isoforms. Lamin-C lacks the final two exons found in lamin-A, and is not post-translationally processed like either lamin-A or the B-type lamins. Post-translational processing of this latter group involves a carboxyl-terminal farnesylation motif (CaaX). The cysteine residue of this motif is first farnesylated. A proteolytic event removes the “aaX” and then the farnesylcysteine is methylated. For B-type lamins, there is no further processing and the farnesylation is a permanent feature of the protein. In the case of lamin-A, however, the carboxyl-terminus undergoes a second proteolytic cleavage to produce a mature unfarnesylated protein[12]. A zinc metalloprotease known as Zmpste24 performs both proteolytic processing steps required for production of mature unfarnesylated lamin-A while the “aaX” in B-type lamin is removed by a protease known as Rce1[13–15].

The lamins are believed to assemble into a dense meshwork underneath the inner nuclear envelope. This meshwork can serve as an interaction node for chromatin and proteins of the nuclear periphery[16,17]. Considering the diversity of these interactions, it is not surprising that lamins function in different nuclear activities such as chromatin organization, DNA replication, transcriptional regulation, signal transduction, and nuclear shape maintenance[12,18–20]. Consequently, lamins are viewed as housekeeping proteins that are essential for cell viability.

More recent studies, however, have shown that mouse embryonic stem cells (mESCs) completely lacking lamin proteins can self-renew and differentiate in vitro[21]. Thus, lamins are not required for the survival of at least this cell type. The study of lamins in different model organisms suggests that lamins are required for the proper development of several organs (e.g., brain, the diaphragm, and the testis)[22–25]. This idea is also supported by the discovery of tissue-specific diseases caused by mutations in lamin genes. For example, different mutations in LMNA cause a spectrum of disorders ranging from dilated cardiomyopathy, to partial lipodystrophy, and to the segmental premature aging syndrome, Progeria[26–28]. To date, there are no reported mutations in LMNB1 that cause disease; however, duplication of this gene causes an adult-onset form of leukodystrophy[29]. LMNB2 has also recently been linked to a progressive form of epilepsy[30]. How lamins cause tissue-specific diseases remains unclear.

Lamin-A: aging and inflammation

Hutchinson-Gilford Progeria Syndrome (HGPS) is an exceptionally rare disorder that resembles premature aging. Most HGPS patients harbor the same LMNA mutation, a de novo C1824T change that enhances the use of a cryptic splice site and leads to the production of a permanently farnesylated form of lamin-A[31,32]. This aberrant form of lamin-A, termed progerin, causes nuclear blebbing, down-regulation of some nuclear envelope proteins, accumulation of DNA damage and accelerated cellular senescence[31,33,34]. Surprisingly, progerin mRNA has been detected at low level in both young and aged individuals, and some, but not all studies on the subject indicate that the progerin product appears more abundantly in select tissues from aged individuals[35–38]. While it is uncertain if there is any function for the progerin present in these tissues, progerin can be induced upon UV damage and might represent an extension of the DNA damage response[39].

Markers of inflammation have been examined in HGPS patient samples and mouse models for this disorder. Cells derived from HGPS patients show an elevated NF-κβ transcriptional response profile[40]. The overexpression of an atypical-HGPS LMNA mutant also increases mRNA levels of certain inflammatory cytokines[41]. Further, inflammatory markers are elevated in the arteries, liver and skin of Progeria mouse models (both Lmna and Zmpste24)[38,42–44]. While the basis of this is not well understood, inflammation appears to be a part of the accelerated aging phenotype caused by LMNA mutations.

Lamin B: aging and inflammation

Human and mouse primary cell lines have a finite replicative lifespan (Hayflick limit) when cultured in vitro. This phenomenon, more commonly called replicative senescence, is characterized by cessation of cell division, increased secretion of inflammatory factors and changes in cell morphology and chromatin organization[45,46]. Recent studies have found that the replicative senescence of cultured mammalian fibroblasts is accompanied by lamin-B1 reduction[47–49]. The induction of senescence in cell culture by expression of oncogenic Ras, or activation of the downstream Rb or p53 tumor suppressors also leads to lamin-B1 decline[47,48]. Additionally, lamin-B1 reduction was detected in senescence-prone fibroblasts derived from Progeria patients and in cells with shortened telomeres or other forms of DNA damage[34,48–51]. In vivo, the irradiation of mice leads to cell senescence and lamin-B1 loss in the liver[48]. The loss of lamin-B1 appears to be regulated at both mRNA and protein levels[48,49,52,53]. There are, however, studies that show the association of increased lamin-B1 protein level and senescence[49,54]. In these studies, the overexpression of lamin-B1 in primary fibroblasts was sufficient to drive cells towards senescence whereas depletion resulted in proliferative arrest[49,54]. While there is no clear rationale for why lamin-B1 levels change under any of these conditions, lamin-B1, whether it is a reduction or increase in protein level, appears to be a marker of cellular senescence and various forms of cellular stress[48,54].

The human skin is the only organ reported to naturally have an age-related loss of lamin-B1[49]. In an effort to explore this potential relationship in other tissues, we analyzed lamin levels in different organs from young and old Drosophila[55]. Drosophila is a tractable model for studies of aging, inflammation and lamins as this model is relatively short-lived and is historically important in the immunity field[56,57]. Further, the Drosophila system is armed with an extensive set of genetic tools and methods that facilitate tissue-specific in vivo experimentation[56]. We found that B-type lamin (LAM), but not A-type lamin (LAMC), was reduced in the brain (Figure 1A, B, unpublished observations) and the fat body (Figure 1C)[55]. Notably, not all tissues (e.g., gut, heart) lose LAM upon aging (Figure 1C). One possibility is that gut epithelium was continuously replaced by intestinal stem cells and so senescent cells were simply replaced. However, in post mitotic heart cells and oenocytes, which persist throughout the life of the fly, lamin loss was not apparent[55]. The simplest interpretation from these observations is that age-related loss of B-type lamin occurs in select tissues. What makes these tissues susceptible to lamin-B loss is unknown.

Figure 1. Reduction of lamin-B in the brain and fat body but not in the midgut upon aging.

(a) Lamin-B reduction in old fly brains. Brains were dissected from 15-day or 55-day fruit flies. Five brains were pooled for each sample. Alpha tubulin was used as a loading control. (b) Quantitation of lamin-B western blots. Lamin-B signal was normalized against alpha tubulin, and the levels in the 55-day sample were compared relative to the 15-day sample. (c) Lamin B immunostaining of the midgut (top) and fat body (bottom) from 10 and 50-day old flies. Cells from the 50-day old fat body show reduced and patchy lamin-B staining.

While there is some evidence that links B-type lamin and inflammation, this aspect has not been thoroughly explored. In cell culture, the induction of Peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1)-related coactivator (PRC)-mediated inflammatory stress by a topoisomerase inhibitor resulted in cell senescence and loss of lamin-B1[58]. Lamin-B1 mRNA was also stimulated by treatment of arthritic synovium macrophages with the anti-inflammatory cytokine, IL-10[59]. Using Drosophila, we showed that aged fat bodies with LAM reduction or young fat bodies with RNAi-mediated depletion of LAM had significant inflammation mediated by the up-regulation of peptidoglycan recognition proteins (PGRPs) of the immune deficiency (IMD) pathway. Elevated PGRPs repressed IMD activity in the midgut and contributed to over-proliferation of intestinal stem cells and gut hyperplasia. Thus, lamin loss in one tissue (fat body) can affect the function of other tissues (gut) and this is mediated in part by systemic inflammation (Figure 3) [55]. Further studies will be needed to investigate how the fat body secreted PGRPs influence the gut immune system and whether or not the old fat body cells are senescent.

Figure 3. Model for systemic inflammation induced by age-related lamin-B reduction.

Old fat bodies exhibit loss of LAM, which then increases the expression of inflammatory factor (PGRPs) secreted into the circulation. These factors then block the local immune response of the gut and lead to over-proliferation and hyperplasia of this tissue.

Lamin-B may maintain tissue homeostasis by controlling gene expression

To gain insight into the role of lamin-B in maintaining the fat body organ, we performed RNA-seq to identify gene expression differences in fat bodies from young (5 days), old (50 days) and 5-day fat bodies that had LAM prematurely depleted by a tissue-specific RNAi[55]. Gene ontology (GO) analyses revealed that age-associated LAM loss was characterized by changes in genes primarily belonging to immune, metabolic, proteolysis, and oxidative pathways (Figure 2A). Depletion of LAM in young fat body yielded a gene expression profile (Figure 2B) that was remarkably similar to old fat bodies (~86% concordance). While immune response genes were significantly up regulated, genes required for cell fate determination and cell-cell adhesion were significantly down regulated upon LAM loss in both old and LAM RNAi young fat bodies. This dichotomy indicates that lamin-B can either activate or repress genes important for the fat body organ.

Figure 2. Gene Ontology (GO) analyses of old wild type fat body and young fat body depleted of lamin-B.

GO terms were determined by DAVID Bioinformatics Resources 6.7. Shown are significant GO terms determined from genes that were up-regulated by equal or greater than 2 fold in (a) old fat bodies (50-day, wild type w¹¹¹⁸) or (b) young fat bodies depleted of lamin-B (5-day, Cg-Gal4/+;UAS-lamin-B RNAi/tub-Gal80^ts).

DNA adenine methyltransferase identification (DamID) and chromatin immunoprecipitation (ChIP)-sequencing methods have been used to map chromatin-lamin interaction in a number of cell types [22,60,61]. These lamin-B1 associated chromatin domains (also called LADs) were enriched for immune response genes and since lamins can modulate gene expression, it was feasible that LAM in the Drosophila fat body could repress these immune response genes in the absence of infection. Consistent with this idea, a global reduction of staining for heterochromatin protein 1 (HP1) and histone H3 lysine 9 trimethylation (H3K9me3) was observed both in old fat bodies and in young fat bodies depleted of LAM. Moreover, supplementing HP1 partially reduced the inflammatory response triggered by LAM reduction in the fat body. Direct analyses using ChIP-qPCR of fat body tissue revealed that depletion of LAM reduced H3K9me3 on several immune response genes. Thus, lamin-B represses the expression of immune response genes and inflammation in fat bodies by maintaining the heterochromatic state of these genes[55].

Conclusions and future considerations

Aging is multifactorial, and different theories that center on cellular damage have been proposed to explain this phenomenon[62]. While the relationship between the nuclear lamina and various forms of cellular damage has not been extensive explored, there are examples in the literature that suggest that protein homeostatic mechanisms, such as autophagy and the Ubiquitin Proteasome System, are particularly relevant. For instance, autophagy activation with either rapamycin or temsirolimus improved the cell morphology of HGPS fibroblasts, and heart function of LMNA mutant animals[63–65]. Two recent studies showed that autophagy is involved in the turnover of lamin-B1 upon induction of senescence[52,53]. Further, proteasome activity appears to be reduced in dermal fibroblasts derived from Progeria patients[66]. Maintaining the homeostasis of cellular proteins, a process often referred to as proteostasis, appears to be intertwined with many known pathways of lifespan extension, including dietary restriction, enhanced autophagy, and reduced insulin signaling[67]. Studies have shown the decline in a cell’s ability to manage the misfolded and/or aggregated proteins occurs upon aging[67]. It is possible that the nuclear proteins such as B-type lamin and some nuclear pore complex (NPCs) components, which are particularly long-lived, accumulate damage and decline as a result[68,69]. Since both lamin-B and the components of NPCs can regulate gene expression, even subtle damage to these proteins may lead to positive feedback that increases aging-related factors, and perhaps furthers the aging process[70]. Considering the emerging connections, future research on the various roles of lamins may provide us with a clearer picture as to why we age.