Progeria Research Day at Brunel University

Abstract

Hutchinson-Gilford Progeria Syndrome (HGPS) is a severe premature aging syndrome that affects children. These children display characteristics associated with normal aging and die young usually from cardiovascular problems or stroke. Classical HGPS is caused by mutations in the gene encoding the nuclear structural protein lamin A. This mutation leads to a novel version of lamin A that retains a farnesyl group from its processing. This protein is called Progerin and is toxic to cellular function. Pre-lamin A is an immature version of lamin A and also has a farnesylation modification, which is cleaved in the maturation process to create lamin A.

In April 2011 the Brunel Institute for Aging Studies (BIAS) hosted a Progeria Research Day at Brunel University, West London, which highlighted the syndrome to interested UK scientists and the public. The keynote speaker was Prof. Nico Lévy from Marseilles who spoke about his research and the on-going clinical trial he and his group have initiated in Europe (Fig. 1). Other speakers from around the UK were invited to share their research on both HGPS and similar diseases and pre-lamin A’s involvement in normal aging. These were Prof. Cathy Shanahan from Kings College London, Prof. Chris Hutchison from Durham University and Dr. David Vaux from Oxford University. The Brunel Progeria Research Team also presented their work on HGPS encompassing whole model organisms, in vitro cell cultures, global genome affects, chromosome behavior, transcription and splicing regulation, with a view to understand the disease at every level, find and assay treatments and develop novel interventions.

Figure 1. Prof. Nico Lévy presenting his keynote lecture to the Progeria Research Day at Brunel University.

The day was made extra special by a short presentation from Hayley Okines, a young lady with HGPS, and a candid and insightful talk from James Routh from Rabbit Productions who has made the television programmes that have followed Hayley’s life so far (Fig. 2).

Figure 2. Miss Hayley Okines speaking to the Progeria Research Day audience.

On Friday the 15th April attendees from across the country arrived at Brunel University in West London for a special “Progeria Research Day” organized by Dr. Ian Kill, head of the Progeria Research Team at Brunel University. One of the main objectives of the day was to bring together scientists across the UK interested in Hutchinson Gilford Progeria Syndrome (HGPS), premature and normal aging to highlight the work ongoing in the UK and at Brunel University on Progeria. The attendees were not only scientists interested in hearing about progress in Progeria research in the UK, but also families with children suffering from HGPS, members of the public associated with the Brunel Institute of Aging Studies (BIAS) and a TV film crew filming the fifth instalment of the life of Hayley Okines, a young lady with HGPS.

HGPS is a severe premature aging syndrome that results in a fore-shortened life-span with children and young adults typically dying from heart attacks and strokes caused by atherosclerosis.¹ The children also suffer from growth impairment, sclerotic skin, bone abnormalities, alopecia, joint contractures and lipodystrophy, however, intelligence is normal.

The most common gene mutated in HGPS is LMNA,^2-4 a gene that codes for nucleoskeleton proteins lamins A and C. This places HGPS into a family of disease termed laminopathies that have been found to have a wide range of pathologies and with mutations all along the LMNA gene (see ref. 5 for comprehensive overview of diseases). Both lamins A and C arise form the LMNA gene by alternative splicing but only lamin A goes through elaborate processing to create the mature lamin A protein. Pre-Lamin A is initially transcribed with a C-terminal CaaX motif (C = cysteine, a = any aliphatic amino acid, X = any amino acid). Farnesyl pyrophosphate is added to the terminal cysteine residue by the action of farnesyltransferase. The three terminal amino acids are cleaved by ZMPSTE24 followed by methylation of the C-terminal cysteine by isoprenylcysteine carboxyl methyltransferase. Finally, the terminal 15 amino acids are cleaved, including the farnesylated cysteine residue, by ZMPSTE24 leaving mature lamin A as the product. The most common mutation in HGPS is a silent mutation that creates a cryptic splice site in the mutant gene resulting in a 50-amino acid deletion producing a shortened protein that is missing the ZMPSTE24 endoprotease site. The resultant protein called “Progerin” retains its farnesylation (reviewed in ref. 5). It is becoming clear that not only accumulation of progerin is toxic to cells but also accumulation of unprocessed pre-lamin A can pose a threat to nuclear health and genomic stability. In HGPS the toxicity of progerin is most likely caused by the farnesylation then it follows that if this farnesylation could be prevented then this would lower the toxicity of progerin. Thus the idea was put forward for using farnesyl transferase inhibitors to interfere with the farnesylation of proteins within cells. FTI drugs were tested using in vitro and in vivo models^6-8 and were quickly identified as candidates in the clinic as the main basis of a clinical trial.^9,10 However, the broad spectrum of proteins that may be affected by FTIs is cause for concern¹¹ and alternatives such as statins and bisphosphanates have both been suggested¹² and are now in use in the clinic pioneered by Prof. Nico Lévy (http://clinicaltrials.gov/ct2/show/NCT00731016).

Prof. Lévy was invited to the Progeria Research Day as the keynote speaker so that the assembled audience could hear of the progress the Marsailles team are making on their open-label single arm non-placebo clinical drug trial treating HGPS children with a combination of bisphosphonates and statins. Prof. Lévy gave a thoroughly candid and open talk about the progress of the children on his European trial. In overview, he indicated the modifications in weight, bone density and cardiovascular risk parameters. The drugs used in this trial were previously used in a mouse pre-clinical model and not only showed a decrease in the number of DNA double-strand breaks but in addition rescued nuclear shape deformaties and inhibited global prenylation. Prof. Lévy also described other diseases that have problems with nuclear lamin expression and processing, such as Reynold’s Syndrome, which highlighted the importance of lamin C in cell function, given that in some cells both B-type lamins were missing as was lamin B receptor and lamin A.¹³ Prof. Lévy also addressed how and where prelamin A matures into lamin A, showing that this may occur in the endoplasmic reticulum and not in the nucleus as is dogma. Furthermore, he discussed how the farnesyl group on lamin A is required for lamin A being anchored initially at the nuclear envelope but once it is severed then lamin A is no longer attached to the nuclear envelope.

Prof. Christopher Hutchinson from Durham University presented his groups’ data on Reactive Oxygen Species (ROS) in cells derived from HGPS and Restrictive Dermopathy (RD) patients demonstrating that these cells contain more ROS induced double strand breaks. It seems that the HGPS and RD cells cannot repair ROS induced DSB unless the cells were pre-treated with n-acetyl cysteine (NAC), a ROS scavenger.¹⁴ This treatment of cells with NAC also improved the HGPS and RD cell growth dynamics. Prof. Hutchison then changed tack to discuss the behavior of 53BP1 DNA repair protein and demonstrated how interestingly it is a lamin A binding protein. Indeed, in cells with mutations that result in an absence of A-type lamins, 53BP1 is found in the cytoplasm and DNA repair capability is lost. This was confirmed by siRNA knockdown of A-type lamins. A-type lamin binding was found to be to the Tudor domain in 53BP1. It was hypothesized by Prof. Hutchison that A-type lamins maintain genome integrity by keeping 53BP1 in the nucleus so that it can be quickly deployed to sites of DNA damage.

Although the symposium focused on HGPS research, the link between normal aging and wild type farnesylated pre-lamin A is now beginning to be explored and so two eminent UK researchers were invited to present their data on pre-lamin A accumulation in cells and the consequences.

Dr. David Vaux from the University of Oxford drew our attention to the drug Saquinavir that is prescribed for HIV patients. This drug is an inhibitor of Zmpste24/FACE1 preventing the cleavage of the mature lamin A resulting in accumulation of farnesylated pre-lamin A. Cells treated with Saquinavir¹⁵ or in which Zmpste24 is knocked-down by RNA interference displayed abnormal morphological changes to the nucleus, with convolutions of the nuclear envelope and invaginations into the nucleoplasm of either the inner nuclear membrane alone or both the inner and outer nuclear membranes together.¹⁶ SREBP1 is a transcription factor that binds to lamin A and is important in differentiation of precursor cells into adipocytes. In cells where pre-lamin A is accumulating, SREBP changes both its location and solubility leading reduced nuclear SREBP1 availability and downregulation of genes regulated by SREBP1. These cells then have an altered metabolic activity and modified metabolomic profile.

Prof. Cathy Shanahan from Kings College London also talked about the importance of pre-lamin A and its connection to normal cell aging. Prof. Shanahan works with vascular smooth muscle cells (VSMC) that are important for maintaining the integrity of blood vessels in the body. When these cells start to fail they become osteogenic in phenotype, resulting in calcification of the vasculature, which in turn leads to atherosclerosis and cardiovascular problems including infarction and stroke. As stated earlier, these are the main causes of death in children with HGPS. Indeed, premature atherosclerosis and the calcification of vessels is seen in HGPS children. Most interestingly, aging VSMCs in vitro and in vivo accumulate pre-lamin A which is due to downregulation of ZMPSTE24 as the VSMC aged.¹⁷ Prof. Shanahan also investigated DNA damage in aged VSMC and found that both in vivo and in vitro there was persistent DNA damage as measured by γH2AX foci. Furthermore, manipulating the accumulation of pre-lamin A by knocking down ZMPSTE24 leads to increased DNA damage and cell cycle defects and some forms of genome instability. Prelamin A accumulation induces the expression of genes involved in the calcification and also changes the nuclear distribution of proteins such as nesprin 2 and ERK.

The day was hosted by the Brunel Institute for Ageing Studies as one in a number of aging research related symposia to be held throughout the year at Brunel University. The day also highlighted the progress and expansion of the Brunel Progeria Research Team, whose research covers HGPS in whole model organisms, in vitro cell cultures, global genome affects, chromosome behavior, transcription and splicing regulation all with a view to understand the disease at every level, find and assay treatments and develop novel interventions.

The day was started by Dr. Ian Kill who provided a general introduction to HGPS, including the molecular biology of the disease and clinical outcomes. He went on to describe some of his ongoing cell biological research into HGPS looking at how progerin accumulation can affect transcription, wound healing, locomotion, proliferation and stress responses. In particular he described some experiments where progeria and wild type fibroblasts were subjected to physiological levels of stretching. Stressing progeria cells in this way caused an increase in the fraction of cells with abnormal nuclear morphologies whereas wild type cells were virtually unaffected. This correlated well with increased levels of expression of NO in progeria cells but not in wild type.

Dr. David Tree described his model of HGPS in the fruit fly Drosophila melanogaster.¹⁸ He demonstrated how ubiquitous ectopic expression of progerin reduces the median lifespan of adult flies by 22% whereas ectopic expression of wild-type human lamin A only reduced the lifespan by 11%, in keeping with findings that mis-expression of normal human lamin A expression negatively affects human cells. Using tissue specific ectopic expression the Tree group found that expression of progerin in adult muscle resulted in a 27% reduction in the median lifespan of adult flies whereas pan-neuronal expression gives only a modest reduction in lifespan. Expression in the adult fat body, the fly organ that functions like the mammalian liver and also has extensive immune functions, has no discernable affect on lifespan at all. Dr. Tree described how ectopic co-expression of mutant forms of p53 which have been well-characterized to act as dominant-negatives, ameliorated the life-span reduction caused by Progerin expression. In comparison preventing apoptosis, using ectopic expression of DIAP1 or p35, both of which block apoptosis in flies, had no effect on the median lifespan of Progerin expressing flies.

Dr. Joanna Bridger informed the audience of how chromosomes were positioned non-randomly in normal proliferating human dermal fibroblasts in a gene-density distribution and how this changes to a more size-related distribution in non-proliferating HDFs.^19-21 When the Bridger group assessed chromosome positioning in actively proliferating HGPS cells it was found that chromosomes were positioned as if they were in a normal quiescent nuclei.^22,23 This mislocalization may affect how genes on specific chromosomes are regulated changing gene expression profiles. Dr. Bridger also described how chromosome territories can relocalize very rapidly when placed in low serum in control cells and that this is affected in the HGPS cells. This correlated with an aberrant distribution of nuclear myosin 1β that is responsible for aiding chromosome movement when cells are placed in low serum.^21,24,25 When the HGPS cells were treated with FTIs chromosome positioning was restored as was the distribution of nuclear myosin 1β and the ability of chromosomes to move when serum was removed.

Dr. Christopher Eskiw also referred to the massive mislocalization of chromosomes within HGPS cells as compared with normal cells,²³ emphasizing that even though this reorganization could translate to the dramatic and devastating phenotypes seen throughout many tissues due to changes in gene expression, it remains unclear how altering chromatin organization influences gene expression. Dr. Eskiw informed the audience how previous studies have indicated that gene expression is influenced by changes in the chromatin organization e.g., the 3-dimensional organization of the globin locus control region and the globin genes is fundamental for proper regulation of these genes in developing erythrocytes.^26,27 Disruption of the spatial organization between the LCR and the globin genes prevents developmental switching as well as severely retarding expression. Similarly, correct conformational organization of the genome has been demonstrated to be critical for V(D)J recombination in B cells,²⁸ T helper cell differentiation,²⁹ and odorant gene selection in mouse olfactory receptor cells.³⁰ These studies indicate that there is a universal role for genome organization and stressed the point that proper organization of genome folding, either directly or indirectly involving LMNA, likely leads to changes in gene expression and to HGPS. Dr. Eskiw revealed his on-going studies using next generation sequencing to perform RNA-Seq experiments to identify which genes have changed expression in HGPS cells compared with unaffected controls. Correlating the data with changes in genome organization will indicate how lamin A protein influences genome folding and highlight the importance of genome conformation maintenance in regulating gene expression patterns. Furthermore, this type of study will identify genes that are not only critical for the progression of HGPS but also for normal aging, thus identifying mechanisms leading to aging at the cellular level.

Dr. Evgeny Makarov then described recent progress in the understanding of molecular mechanisms of pre-mRNA splicing of human LMNA gene, encoding lamin A and C proteins, and especially, its aberrant splicing that causes the premature aging of HGPS patients. His goal is to identify the proteins that play a key role in modulating the splicing outcomes of two alternative splicing events: (1) aberrant production of progerin, and (2) alternative LMNA splicing generating lamin A and C proteins that have partially overlapping functions. The latter event is important to study because progerin can be only produced from the pre-mRNA encoding lamin A protein and therefore, an ability to force a splicing outcome in favor of lamin C over lamin A can be potentially employed to decrease the amount of progerin that, in turn, should lead to slowing the premature aging of HGPS patients. To identify the key proteins responsible for progerin or alternative lamin A/C production, Dr. Makarov is employing a comparative proteomics approach. The different in vitro splicing systems have been established which mimic both splicing events in a test tube containing the cell extracts and synthesized pre-mRNAs encompassing alternatively spliced LMNA exons. Then, using a novel purification procedure, Dr. Makarov has already isolated in parallel the spliceosomes that are assembled on the normal and HGPS-mutated pre-mRNA, and their protein composition is currently under investigation by mass spectrometry. The proteins that are present in one of two compared counterpart complexes, but absent in the other, will be considered as candidates for being the crucial LMNA splicing regulators and will be further investigated by a number of different techniques to prove their functional roles. The identification of the proteins that are responsible for the aberrant splicing outcome will provide new pharmaceutical targets for the screening of drugs which can eventually be used to treat children with HGPS.

One of the highlights of the day was the talk given by James Routh of Rabbit Productions, who has made four documentaries following the life of Hayley Okines. James’s documentaries have been highly influential in bringing awareness of HGPS to a wide public audience both within the UK and internationally. In his presentation, James was able to give the audience a very personal and objective insight into how Hayley’s life has changed over the years how this has affected her family life. James’s unique perspective allowed the hard science to be counter-balanced by the human view and the impact that the breakthroughs in science had on Hayley, her family and other friends of Hayley with progeria. Just before the lunch break, Hayley was brave enough to stand up and present a short prepared speech to thank all the scientists and clinicians working together to find and apply treatments against progeria.

Figure 3. Speakers at the Progeria Research Day. From left to right Prof. Nicolas Lévy, Dr. Joanna Bridger, Prof. Cathy Shanahan, Dr. Ian Kill, Dr. David Vaux, Prof. Christopher Hutchison, Dr. Christopher Eskiw and Dr. David Tree.

Glossary

Abbreviations:

BIAS

Brunel Institute of Ageing Studies

DNA

deoxyribose nucleic acid

DSB

double strand breaks

FTI

farnesyltransferase inhibitors

HGPS

Hutchinson-Gilford Progeria Syndrome

mRNA

messenger RNA

NAC

n-acetyl cysteine

NO

nitric oxide

RD

restrictive dermopathy

ROS

reactive oxygen species

siRNA

small interference RNA

SREBP1

sterol regulatory element-binding protein 1

VSMC

vascular smooth muscle cells