Nuclear Envelope Diseases and Chromatin Organization

Towards a Drosophila model of Hutchinson–Gilford progeria syndrome

Gemma S. Beard, Joanna M. Bridger, Ian R. Kill, David R.P. Tree

Abstract

The laminopathy Hutchinson–Gilford progeria syndrome (HGPS) is caused by the mutant lamin A protein progerin and leads to premature aging of affected children. Despite numerous cell biological and biochemical insights into the basis for the cellular abnormalities seen in HGPS, the mechanism linking progerin to the organismal phenotype is not fully understood. To begin to address the mechanism behind HGPS using Drosophila melanogaster, we have ectopically expressed progerin and lamin A. We found that ectopic progerin and lamin A phenocopy several effects of laminopathies in developing and adult Drosophila, but that progerin causes a stronger phenotype than wild-type lamin A.

  • Drosophila melanogaster
  • Hutchinson–Gilford progeria syndrome (HGPS)
  • lamin A
  • nuclear structure
  • progerin

Introduction

Hutchinson–Gilford progeria syndrome (HGPS) is a childhood premature aging disease and affected individuals die at an average age of 13.5 years from cardiac infarction [1]. A number of characteristics of aging are accelerated in HGPS, including the development of sclerotic skin, alopecia, skeletal hypoplasia and dysplasia, osteoporosis, lipodystrophy and progressive cardiac dysfunction [1]. HGPS is a sporadic autosomal dominant disease caused by de novo mutations in the LMNA gene, making HGPS a laminopathy [2,3]. The nuclear lamina is a filamentous structure that provides structural support for the nucleus, organizes protein complexes within the nucleus and directs the interaction of these complexes with chromatin [4]. Its primary components are A-type and B-type lamins. B-type lamins are found in most cells, whereas A-type lamins are expressed mainly in differentiating cells. Both forms contain an N-terminal head domain, a C-terminal tail domain and a central rod domain through which they form homodimers [5]. These homodimers polymerize and interact to form a higher-order filamentous network juxtaposed to the inner nuclear membrane. Additionally, lamins are also localized to foci in the nuclear interior and as part of the interior nuclear matrix [68].

The human LMNA gene encodes lamins A and C. Lamin A contains a C-terminal CaaX box (where a is an aliphatic residue) which lamin C lacks. The CaaX box allows the transfer of a farnesyl group to lamin A that is subsequently removed by proteolytic cleavage. Some 90% of the mutations found in HGPS lead to a splicing defect in the C-terminus of the LMNA gene, resulting in a 50-amino-acid deletion in the mutant protein, progerin [3]. This deletion eliminates the cleavage site used to remove the farnesyl group. Progerin is thus predicted to retain its farnesyl group and, in the nuclear lamina, it displaces a pool of lamin A from homopolymers to form heterotypic structures containing B-type lamins and progerin [5]. Accumulation of these structures leads to defects in nuclear composition, organization and dynamics. The mechanical properties of HGPS nuclei are altered, and they display defects in mitosis and the cell cycle [9,10]. HGPS cells also show misregulation of cell-signalling pathways, including Notch, Wnt [11,12] and the p53 DNA-damage-response pathway [13,14]. It has been shown that progerin is produced sporadically in wild-type cells and its accumulation is seen in normal primary older cells, linking progerin to physiological aging [15,16]. Ultimately, progerin accumulation affects aging by changing the viability, identity and function of cell populations. How these molecular and cellular changes lead to aging in tissues and organisms remains unknown. The fruitfly Drosophila melanogaster is a powerful system in which to study the mechanisms linking the abnormal progerin-containing lamina to the HGPS phenotype.

The Drosophila genome contains two lamin genes: lamC and Dm0 which have been classed as A- and B-type lamins respectively on the basis of their expression patterns [17,18]. Molecular analysis, however, reveals that Drosophila and vertebrate lamins have evolved from a single gene in a common ancestor, so their expression patterns are a consequence of convergent evolution [19]. Null mutations in the Drosophila A-type lamin cause complete pre-metamorphosis lethality [20], whereas null mutations in the Drosophila B-type lamin are pupal lethal [19]. The levels of the lamin proteins are also critical for their function, as ectopic overexpression of either lamin causes lethality in a stage- and tissue-specific manner [21]. The Drosophila A-type lamin does not contain a CaaX motif and so is not thought to be farnesylated. Consequently, engineering progerin-like mutations in the Drosophila LamC is problematic. There is a strong history of using ectopic expression of human disease genes to solve biological problems, e.g. in neurodegenerative disease [22]. We have examined the effect of ectopic expression of either progerin or human lamin A on nuclear morphology and adult lifespan in Drosophila. Although ectopic expression of progerin and lamin A both lead to abnormal nuclei in developing Drosophila that strongly resemble laminopathy nuclei, progerin leads to a much stronger phenotype than lamin A, causing extensive nuclear breakdown. Ectopic expression of progerin or lamin A in adult Drosophila also strongly reduces their lifespan.

Nuclear defects in developing Drosophila expressing lamin A and progerin

We tested the hypothesis that ectopic expression of progerin in Drosophila melanogaster will phenocopy HGPS. We also ectopically expressed wild-type human lamin A as a control to test for the specificity of the effect of ectopic progerin. This analysis is complicated by reports that increases in the levels of wild-type human lamin A also lead to a progeroid phenotype in human cells. We ectopically expressed lamin A and progerin in developing Drosophila using the bipartite UAS (upstream activating sequence) Gal4 system [23]. Ubiquitous ectopic expression of progerin during development using the actin-5c-Gal4 line resulted in lethality during the larval–pupal transition of the life cycle, whereas lamin A expression produced phenotypically normal adults. This was unexpected, as ectopic expression of wild-type Drosophila lamin C during larval and pupal development is lethal [21]. As the nuclear phenotype of HGPS and aged human cells is well characterized, we studied the nuclei of the larval CNS (central nervous system) and salivary glands of progerin- and lamin A-expressing third instar larvae. The larval CNS is highly mitotic and diploid, whereas salivary glands are non-mitotic and highly polyploid. Salivary gland nuclei are very large with extensive nuclear membranes. The structure of the nuclear envelope and distribution of lamins were analysed by indirect immunofluorescence and laser-scanning confocal microscopy. Control nuclei of the CNS are slightly ellipsoid, whereas wild-type salivary gland nuclei are almost perfectly spherical. They share a smooth morphology and an even distribution of the Drosophila B-type lamin, Dm0. In the cells ectopically expressing lamin A, the nuclei were characterized by the presence of numerous micronuclei, each containing DNA (reminiscent of human laminopathy cells described previously) (Figures 1B, 1D and 1E). This is consistent with recent reports that overexpression of wild-type human lamin A has been shown to lead to a progeroid phenotype in cultured cells [24]. In contrast, ectopic expression of progerin led to considerable nuclear breakdown resulting in the presence of herniations and invaginations at the nuclear periphery and severe fragmentation of nuclei (Figures 1C–1E), similar to that observed in HGPS cells. We scored the fraction of abnormal nuclei present in these two tissues in wild-type, and progerin- and lamin A-expressing third instar larvae. The nuclei were scored as normal, bearing micronuclei juxtaposed to the main nuclear body, containing herniations and invaginations and nuclei undergoing total fragmentation. Interestingly, the effect of ectopic lamin A and progerin was more pronounced in salivary glands than in the CNS. Additionally, the ectopic production of both lamin A and progerin led to minor mislocalization of wild-type Drosophila lamins where it is found in punctae-containing human proteins (Figures 2A–2C, arrows).

Figure 1 Phenotype of ectopic expression of progerin and lamin A in Drosophila

(AC) Third instar larval salivary glands stained for the presence of Drosophila Dm0: (A) wild-type salivary gland nuclei; (B) lamin A-expressing salivary gland nuclei displaying micronuclei (arrows); and (C) progerin-expressing salivary gland nuclei showing herniations and invaginations (arrows) and nuclear fragmentation (arrowheads). Scale bars, 20 μm. (D, E) Quantification of the proportions of nuclei showing defects in wild-type, and lamin A- and progerin-expressing salivary glands (D) and CNS (E).

Figure 2 Effect of ectopic expression of progerin and lamin A on localization of endogenous Drosophila Dm0 and on the lifespan of adult fruitflies

(AC) Third instar larval CNS ectopically expressing lamin A stained for the presence of lamin A (A), Drosophila Dm0 (B) and overlay (C) showing relocalization of Dm0 to punctate structures containing ectopic human lamin A (arrows). Magnification, ×60 with ×2 digital zoom. (D) Survival curves for adult Drosophila expressing progerin, lamin A, and non-expressing controls. Ectopic expression was driven by actin-5c-GeneSwitch and controls are Drosophila containing the same chromosomes as the progerin-expressing cohort grown without RU286.

Reduced lifespan in adults expressing lamin A and progerin

We expressed progerin and lamin A ubiquitously in adult Drosophila using the conditional GeneSwitch system [25,26]. This removes any effect the genetic background may have on the fitness of Drosophila, as control and experimental fruitflies have the same genetic background and only differ with respect to the introduction of the inducing agent, RU286, into the food. No significant alteration in lifespan was observed in Drosophila grown on the level of RU286 used in any of the parental Drosophila lines, ensuring that the presence of the drug or the GeneSwitch protein was not responsible for any reduction in lifespan. When progerin or lamin A was ubiquitously expressed during adult life, the median adult lifespan of Drosophila was reduced compared with genetically identical controls (Figure 2D). In our hands, non-expressing control fruitflies have a median lifespan of 38 days, whereas progerin-expressing fruitflies have a median lifespan of 11 days, and lamin A-expressing fruitflies have a median lifespan of 15 days. This represents a 71% decrease for progerin and a 61% decrease for lamin A. Although we cannot conclude that this decreased lifespan is due to accelerated aging rather than a pathological process leading to reduced fitness, it does phenocopy the effect of imbalances in lamin A metabolism in human cells.

Concluding remarks

Nuclei showed differential abnormalities when ectopically expressing lamin A or progerin, with progerin eliciting a stronger cellular phenotype than lamin A. The effect of either human protein was more pronounced in larval salivary glands than in the larval CNS. This may be due to the larger nuclear lamina of these large polyploid nuclei and reveals that mitosis is not a strict prerequisite for nuclear defects caused by progerin accumulation. In terms of their effect on nuclear structure, ectopic progerin and lamin A in Drosophila phenocopies their effects on human cells. Both progerin and lamin A have negative effects on the lifespan of adult Drosophila, although the effect of progerin is stronger than that of lamin A. The effect of lamin A on the lifespan of adult Drosophila is consistent with the finding that increased levels of wild-type lamin A in human cells results in a progeroid cellular phenotype. It could also be due to abnormal processing of the human proteins in Drosophila. To validate our Drosophila model of HGPS, we are currently testing the processing of progerin and human lamin A in Drosophila by biochemical means.

Footnotes

  • Nuclear Envelope Diseases and Chromatin Organization: Independent meeting held at New Hunt's House, King's College, Guy's Campus, London, U.K., 23–24 April 2008. Organized and Edited by Juliet Ellis (King's College London, U.K.).

Abbreviations: CNS, central nervous system; HGPS, Hutchinson–Gilford progeria syndrome

References

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