Understanding Cell Aging/Death (Page 3)

Due to Hutchinson-Gilford progeria syndrome being a disease associated with premature aging and cell death it is important to understand how and why this process is occurring in the cells of HGPS patients in order to better understand the disease and develop therapeutic treatments. When researchers began to look for what genes were upregulated in HGPS patients, they found Ankyrin G (ANK3) to be specifically overexpressed when compared to normal non-HGPS fibroblasts. Authors hoped that this specific gene and the genes co-clustered with ANK3 would provide clues to the mechanism of cell death in HGPS cells and patients (Wang et al. 2006).

Another aspect that relates to cellular aging that researchers decided to investigate is the telomere. Telomere shortening is common of cellular aging and replicative senescence. Telomers are protected against machinery that recognizes DNA managed by telomere repeat-binding factors (TRFs) which bind duplex telomeric DNA as homodimers. TRF2 is responsible for facilitating strand invasion of single-stranded overhangs on chromosome ends and turning them into duplex telomeric DNA called t-loops. Without TRF2 function, the DNA damage response pathway activity is increased and so is the level of cellular senescence. T-loops can also form at interstitial telomeric sequences, which form an interstitial t-loop, which authors found to be stabilized by lamin A interacting with TRF2. Progerin levels increasing and reduction of lamin A levels, main factors in HGPS, leads to reduced t-loop formation and telomere loss. This suggests that cellular ageing is associated with the interaction of TRF2 and lamin A (Wood et al. 2014).

Rapamycin (mTOR) kinase is another aspect linked to aging and various different diseases, causing researchers to gain an interest. mTOR is made up of 2 complexes (mTOR complex 1 and mTOR complex 2) and has two main substrates, S6k1 and 4E-BP1 which are both involved in the initiation of translation. The mTOR complex signaling cascades lead to the phosphorylation of these substrates. It was demonstrated that rapamycin reverses elevated mTORC1 signaling and rescues the abnormal pathologies. Reduction in S6K1 activity in the muscle indicates that altered skeletal muscle function contributes to the mortality of mice with HGPS and other lamin related diseases, extending the lifespan of the mice tested. In contract, 4E-BP1 reduced survival of the mice. This knowledge can be used to implement rapamycin as a therapeutic approach for HGPS and other laminopathies (Liao et al. 2017).

When induced pluripotent stem cells were generated from fibroblasts obtained from HGPS patients, these cells showed the absence of progerin, lack of a nuclear envelope, and epigenetic alterations normally associated with premature aging, interesting scientists. Differentiation of HGPS induced pluripotent stem cells to smooth muscle cells leads to the appearance of premature senescence phenotypes associated with vascular aging. DNA-dependent protein kinase catalytic subunit was also found as a downstream target of progerin and the absence of it is correlated with premature aging. Somatic cells from HGPS patients can therefore be maintained in a pluripotent state. This study created an induced pluripotent stem cell model to study aging, specifically in diseases such as HGPS (Liu et al. 2011).

Figure 1.  Immunostaining of calponin and lamin A in iPSC-derived SMCs. Arrowheads show dysmorphic nuclei(Liu et al. 2011).

Another pathway examined in regards to premature aging and accelerated senescence is p53 signaling. Increased progerin activates the p53 signaling pathway in which certain isoforms of p53 regulate senescence in normal cells. The role of p53 signaling in premature aging was previously unknown. Though, a study reported that p53 isoforms were expressed in fibroblasts of HGPS patients (Muhlinen et al. 2018). In addition, p53 isoforms in HGPS patients are associated with their accelerated senescence and their manipulation can restore the fibroblast’s ability to replicate. Specific isoforms are able to modulate signaling to promote the repair of spontaneous progerin-induced DNA double strand breaks in addition to extending the replicative lifespan. Restoration of the expression of certain isoforms can therefore act as a therapeutic strategy for HGPS patients in extending their lifespan (Muhlinen et al. 2018).

Figure 2. Model of Δ133p53 effects on DNA damage and senescence in HGPS fibroblasts (Muhlinen et al. 2018)

It is well known that defective DNA repair increases the susceptibility to senescence in HGPS patients, but the mechanisms of this predisposal is unknown. It was discovered that defective DNA repair extends the time of the Chk1-dependent G2 checkpoint activation and therefore sensitized the cells to senescence through enhancing mitosis skipping. The activation of p53 results in the degradation and repression of mitotic regulators (Johmura et al. 2016). The checkpoint is extended by the introduction of the TopBP1 activation domain and the nondegradable mutant of claspin. The contrast was also found in which the checkpoint is shortened by activating the expression of SIRT6 or depleting OTUB2, reducing the cell’s susceptibility to senescence. In addition to various possible targets being found in this study, the knowledge of how defective DNA repair affects the extension of the G2 checkpoint is essential for understanding the predisposition to senescence in HGPS patients (Johmura et al. 2016).

Progerin in HGPS patients is known to distort nuclei and sequester nuclear proteins, so the protein homeostasis in the disease began to be explored by researchers. Progerin levels increasing and lamin A levels decreasing can drive nucleolar expansion. In addition, HGPS patients have increased levels of reactive oxygen species and less cellular ATP, contributing to exhausting the resources of mesenchymal stem cell pools, which in turn limits tissue renewal (Buchwalter and Hetzer 2017). The overall global protein synthesis and protein turnover in HGPS patients is elevated due to activated nucleoli and enhanced ribosome biogenesis and activity in the HGPS fibroblasts, and therefore a biomarker of premature aging (Buchwalter and Hetzer 2017). In addition, there were increased protein translation and toxic depletion of intracellular energy stores. Nucleolar size and regulation of its activity can then be used as a therapeutic target for HGPS patients and premature aging (Buchwalter and Hetzer 2017).

Progerin in HGPS patients was also shown to cause the loss of subcutaneous white adipose tissue, which is common in normal aging. Researchers showed that when the expression of progerin is sustained in preadipocytes and adipocytes of subcutaneous white adipose tissue, the results showed significant tissue pathology over a period of time, which included fibrosis and lipoatrophy (impairment of subcutaneous white adipose tissue to store energy) (Revêchon et al. 2017). This caused ectopic fat deposition in visceral depots of  non-adipose sites. Adipose tissue’s sensitivity to progerin helps to better understand how HGPS patients experience aging pathologies (Revêchon et al. 2017).

It is known that HGPS can cause loss and dysfunction of smooth muscle cells. A three-dimensional model of HGPS was made, replicating an arteriole-scale tissue engineered blood vessel using induced pluripotent stem cells from smooth muscle cells taken from an HGPS patient (Atchison et al. 2017). This can therefore be used as a helpful model in understanding aging in HGPS patients.

References

4 thoughts on “Understanding Cell Aging/Death (Page 3)

  1. Hey Brittany, thank you for sharing your amazing theme pages. You mentioned in your first page that a SNP is what causes the mutation in LMNA and that there is the use of oligonucleotide that could be a treatment. Is this the same as genetic engineering like CRISPR and if this is not, has there been work done in mouse models suggesting that genetic engineering could be a solution to this problem? Also, you mention that the mutated LMNA causes a toxic protein. Is this because the protein forms aggregates (similar to Alzheimer’s) or does the early stop codon like mutation leave out an active site or an allosteric binding location?

    1. Thank you for the comment, Calvin! So, no it is not like CRISPR, this paper is a bit older. The oligonucleotide is complementary to the mutation region in exon 11. It sterically blocks the splicing machinery and the event in lamin A. (Here is a link to the paper if you are interested: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1351119/) Interestingly, in all of my research I have not come across any gene editing done to treat progeria, but it seems like that would be a useful solution.
      As for the toxicity of progerin, the LMNA gene codes for lamins, which make up the nuclear lamina , it functions in retention of proteins in the inner nuclear membrane, chromatin organization, DNA replication, gene expression, and even more things. So, no it is not about the protein aggregates. The lack of lamin A harms various processes. The alternative transcript due to the splicing event, yields the toxic form, progerin, which when farnesylated leads to the various symptoms related to the nuclear lamina. I hope I answered your question!

  2. Hi Brittany,

    Great job on your project! I appreciate your discussion of the several different mechanisms by which the mutated lamin A is implicated in cellular aging pathways. I have a few questions:

    1. What does the literature describe as the most pertinent mechanisms by which the symptoms premature aging are made manifest? Are all of the aforementioned pathways expected to be equal contributors?
    2. I’m interested in the interactions among TRF2, lamin A, and the T-loops. What kinds of assays have been utilized to model this interaction? When Lamin A is mutated, does it prevent any interaction at all with TRF2 or does it prevent the TRF2-lamin A complex from stabilizing the T-loops? Additionally, what roles do T-loops play in maintaining telomere integrity?

    Thank you!

    1. Hi Rebecca! Thank you for your comment and questions. Your question about what mechanisms are most pertinent in premature aging is a difficult one because the literally doesn’t specify as to what is the most pertinent. I think this is due to the still lack of knowledge about the subject. Though, I noticed a few themes across my findings. For example, signaling of p53 isoforms can have several effects on aging, including being involved in G2 mitosis checkpoint, so I can speculate that this role is much more relevant than is already known. In addition, there seems to be a theme of loss of stored energy, specifically in fat tissues.
      ChIP identified binding sites of TRF2 and found the sites of lamin A are very close. Using ChIP-qPCR the authors found lamin A associates with interstitial t-loops (ITLs). Lamin A knockdown shows lamin A decrease leads to TRF2 binding decrease at ITS that associate with lamin A. A co-immunoprecipitation analysis of TRF2 and lamin A found a specific interaction with TRF2. Authors also found that TRF2 and progerin do not precipiate, meaning only wildtype lamin interacts stably with TRF2, using co-immunoprecipitation. The authors also tested lamin A knockdown and found frequency of inverted chromosome ends was reduced for all three chromosomes tested and that reduction was also seen with the progerin mutation. As for ITL integrity, authors found that reduction in telomeric signaling by FISH was due to a genomic loss of telomeres, as they saw a decrease in signal and telomeric shortening in HGPS cells. The authors just suggested that disruption of inverted chromosome ends, which should have similar effects as t-loops, may contribute to HGPS.
      Hope this was helpful!

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