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Nuclear Architecture: Part of the Genomic Instability Hallmark of Aging

The nuclear lamina is a component of the cellular nucleus with critical roles in the maintenance of nuclear architecture and stability, and also genome organization and function. Mutations in the genes that encode for cellular structures have been found to be key components in aging syndromes such as the Hutchinson-Gilford and the Néstor-Guillermo premature aging syndromes.

Summary of Hallmark: Before 2013

Before 2013 and based on the López-Otín et. al "Hallmarks of Aging" review, defects in nuclear architecture have been pointed to as a known cause of aging by various experts in the field. To understand the importance of nuclear lamins, it is key to also understand their role within the cellular nucleus. Nuclear lamins not only protect the DNA from outside factors, but it also has other important purposes, which include "providing a scaffold for tethering chromatin and protein complexes that regulate genomic stability."

Because nuclear lamins are such a major component of the nuclear envelope, their mutations can result major phenotypic effects on the organism. This is seen, for example, in accelerated-aging syndromes. The Hutchinson-Gilford progeria syndrome is caused by a mutation of the gene LMNA, which results in a build up of the aberrant lamin A isoform named progerin. This mutated isoform results in misshapen nuclei.

Update since the publication of the López-Otín et al. Hallmarks of Aging review: 2013 to 2017

To understand the advances that have been made since the release of the López-Otín review, it is important to understand the role of the different types of lamins in the nuclear membrane, the results of protein malfunctions and mutations, the research that has been done in order to characterize the cause of the mutations and ultimately finding possible cures, and lastly the connection between the health and architecture of the nuclear lamina and other components that have been also known to also accelerate aging.

Lamins

The nucleoplasm contains a complex, peripheral network known as the nuclear matrix. The peripheral region of the nuclear matrix is known as the nuclear lamina. This region is of high importance for the integrity of the cell because it interacts with inner nuclear membrane proteins, nuclear pore complexes, and peripheral chromatin. The nuclear membrane does not only contain lamins as its main or only component, it is also made up by other scaffolding proteins such as nuclear mitotic apparatus protein, known as NuMA, acting and several actin-binding proteins and less-known matrins or matrin-domain containing proteins.

A very important component of the nuclear envelope, in fact, are lamins; a type V intermediate filaments. In the human species, there are four types of lamins: A, B1, B2, and C. There are four types of lamin proteins, but only three genes in the human genome that encode for these four proteins. Through alternative splicing, the gene LMNA encodes for lamin A and lamin C; these two proteins are expressed in differentiated cells. The genes LMNB1 and LMNB2 encode for the B1 and B2 proteins, respectively, and at least one form of lamin B is expressed in every somatic cell.

Just like other proteins that perform specific roles in the cell, lamins undergo a series of post-translational modifications that allow them to be directed to the nuclear membrane including:

1. Farnesylation: necessary for protein localization
2. Advanced glycation end-products: seen in intima and media cells from large elastic arteries
3. Phosphorylation of lamins
4. Ubiquitylation and deubiquitylation: protein quality control in the endoplasmic reticulum
5. Symoylation and desumoylation: a covalent and reversible lysine modification.

Even though lamins do have an important role in creating a stable nuclear envelope, it is not their only role within the cell. Besides proving a structural framework to the nucleoplasm, peripheral and nucleoplasmic lamins along with interacting protein partners and the many other nuclear membrane components play a number of important roles in the following:

1. Epigenetics
2. Chromatin organization
3. DNA replication and repair
4. Transcription

Hutchinson-Gilford Progeria Syndrome: A Disease Caused by Laminopathies

Alterations to the LMNA gene through mutations have been known to cause cellular decline, which ultimately lead to severe phenotypic characteristics. Degenerative disorders caused by mutations of the lamin genes are known as laminopathies. Some of the phenotypes seen in patients diagnosed with nuclear morphological abnormalities include:

1. Neuropathies
2. Muscular dystrophies
3. Lipodystrophies
4. Premature aging diseases

Even with thorough understanding of the phenotypes seen in patients with defective lamin genes, the specific relationship between the gene mutation itself and different phenotypes is poorly understood. It has been difficult to determine this relationship because different mutations of the LMNA gene can lead to the same phenotype. Another factor that has intrigued research scientists is to try to understand why laminopathies have been seen to affect a single or few tissues, even when lamin A and lamin C are expressed throughout.

Back in 2003, two different laboratories published their findings relating the mutation of the LMNA gene to be the cause of the Hutchinson-Gilford Progeria Syndrome (HGPS). In this case, the research groups were able to determine the specific cause of this accelerated-aging syndrome. Both lamin A and lamin C are transcribed from the same gene through alternative splicing, which creates two very different lamins. Lamin C possesses five unique C-terminal residues, and lamin A is synthesized as a 664-residue prelamin A precursor that eventually becomes mature lamin A through a series of post-translational modifications.

Within these very important post-translational modification lies the mechanism that results in a faulty lamin A protein, known as progerin. It is not the mechanism itself that produces an erroneous lamin A protein, it is the mutation within the gene that does not allow the modifications to produce a properly mature lamin A protein.

A single base substitution within the LMNA exon 11 causes an activation of a cryptic splice site, which leads to an inframe deletion of 50 amino acids. Normally, the lamin A protein is farnesylated and carboxymethylated. In case of the abnormal lamin A, within the 50 amino acids that were deleted was a site for endoproteolytic cleavage, which renders the lamin permanently farnesylated and carboxymethylated. This single-base mutation has been seen to cause cellular decline through a series of mechanisms including epigenetic changes, telomere shortening, DNA repair defects, misregulated gene expression, and the characteristic phenotype of abnormal nuclear morphology.

This abnormal lamin A is known as progerin which is not only seen in accelerated-aging syndrome, but also in senescent cells and cells from old individuals. This suggests that the production of progerin could a factor in aging.

Technology Advancements for the Study of Lamins

To be able to study lamins, these structures have to be purified from the rest of the other components that share the same nuclear environment without causing artifacts. Even though lamins are part of such complexes structures, new methods have been introduced since 2013 that facilitate laminal purification in order to understand laminal cellular organization.

For example, Ce-lamin was ectopically expressed ex vivo in Xenopus laevis oocytes and then purified using minimal disruptive methods. The samples were then visualized using cryo-electron tomography. Analysis showed that Ce-lamin "assembles into flexible protofilaments that interacts with each other and exhibit a diameter of 5–6 nm. These data show that protofilaments are the basic assembly units in vivo and that they can assemble into thicker, higher order, filaments. Therefore, the 10 nm intermediate filament-like lamin filament structure represents only one form of assembly out of several assembly possibilities."

Even though significant movement toward a future of full understanding of how lamins interact with each other and the other components of the nuclear lamina, there is still ample room for improvement. Gruenbaum et al. argue that new tools in "biochemistry, imaging and structural techniques, such as high-resolution detectors for cryo-ET and super-resolution fluorescence microscopy, in combination with established methods, will likely provide unprecedented view of lamins in the near future."

Research Articles Published in Relation to Laminopathies: 2013-2017

Since 2013, there has been an abundant number of research laboratories trying to further characterize the relationship between disruptions of the nuclear lamina and its role in physiological aging in order to develop treatments for various, known diseases.

Chemical inhibition of NAT10 corrects defects of laminopathic cells

This article was published on May 2, 2014 in the Science Magazine journal which is under the American Association for the Advancement of Science (AAAS) organization.

In this article, the researchers aimed the study at finding a way to fix the misshapen nuclei and altered chromatin organization characteristic of diseases associated with cancer and laminopathies, especially the Hutchinson-Gilford Progeria Syndrome (HGPS).

The authors understood that a misshapen nucleus can be detrimental to the cell because it renders the cell fragile and also because such altered shape leads to a downstream effects on chromatin structure, gene expression, DNA replication, or DNA repair. Therefore, their hypothesis was that by restoring nuclear shape, this would improve chromatin structure and ameliorate all of the other side effects, and ultimately see an improved organismal phenotype.

To start the experiment, Homo sapiens bone osteosarcoma (U2OS) cells were transfected by small interfering RNA (siRNA) that silenced the LMNA gene (siLMNA). This change in the cell's genome was seen to cause nuclear shape defects, global chromatin relaxation, and increased nuclear area when compared to a healthy nucleus.

The authors turned their attention into finding out the specific acetyltransferase responsible for the translation of the erroneous copy of the LMNA gene. Based on compound screening, they found that 4-(4- chlorophenyl)-2-(2-cyclopentylidenehydrazinyl) thiazole, named compound (1), was able to restore nuclear circularity and global chromatin compaction through its inhibition of a lysine acetyltransferase (KAT).

A couple of factors of compound (1) made it a particularly important molecule to study including:

1. Molecule (1) was identified in Saccharomyces cerevisiae as a GCN5 network inhibitor, but its effects were independent of this network.
2. Molecule (1) was seen to improve the nuclear morphology of several cancer cell lines: this indicated that the effects of the compound were not specific to siLMNA cells.

From compound (1), the researchers used "click-chemistry," a method that taggs a small molecule to a specific drug-associated protein, in order to make it easier to find out the acetyltransferase targeted by molecule (1). From this experiment, the scientists found that N-acetyltransferase 10 (NAT10) was the target of compound (1).

Because of its instability, compound (1) was used to create a homolog named "Remodelin." Remodelin was then tested on siLMNA and HGPS-derived patient cells, and it was found to produce the same results as compound (1) regarding its ability to remodel nuclear shape.

This experiment used chemical, cellular, and genetic approaches to try to find a possible cure to laminopathies, using HGPS-cells as a real-world example for future applications.

Disruption of PCNA-lamins A/C interactions by prelamin A induces DNA replication fork stalling

This article was published online on September 27, 2016, in the journal Nucleus.

The article did not look into lamin gene mutation; its focus was the accumulation of prelamin A, the precursor to mature lamin A before it has undergone important post-translational modifications, and its effects in cellular health.

According to the authors, evidence suggests that prelamin A accumulates in cells as an organism ages, specifically in vascular smooth muscle cells. The accumulation of prelamin A has cytotoxic effects, including DNA damage. The relationship between the accumulation of the protein with DNA damage is poorly understood, however.

It is known that lamins interact with components of the DNA replication machinery, including the sliding-clamp proliferating cell nuclear antigen (PCNA). PCNA provides a sliding clamp for DNA polymerase δ. Its presence is also important in maintaining the connection between the polymerase and the DNA. The interactions between lamins and PCNA actually serve the purpose of positioning PCNA on nuclear chromatin. If the LMNA gene is mutated, then this interaction between PCNA and lamins is compromised which results in reduced binding affinity.

The authors used U2OS cells to test how expression of prelamin A affected the PCNA.

It was found that the expression of prelamin A caused mono-ubiquitination of PCNA and increased formation of Pol η foci. These two results are indicative of DNA replication fork stalling. The authors concluded that prelamin A was involved in the stalling of the DNA replication fork, and thus of the whole process of DNA replication. The study finds that for the first time prelamin A was shown to directly induce DNA damage via DNA replication.

The overall conclusion of the researchers stated that the DNA replication fork stalling by prelamin A eventually induced double strand breaks of the DNA which hinders the ability of the cell to maintain homeostasis. When focused on cardiovascular tissue, the authors presented a possible relation between prelamin A accumulation and natural aging.

Cofilin Regulates Nuclear Architecture through a Myosin-II Depeasdfsdfssddhanotransduction Module

This research article was published on January 19, 2017, on the journal Scientific Reports.

The authors wanted to explore the mechanism that regulates nuclear shape, and they found a relationship between F-acting cytoskeletal organization and nuclear morphology.

The study finds that cofilin/ADF-family-F-acting remodeling proteins are essential for normal nuclear architecture in different cell types.

By using siRNA to silence the gene encoding Cofilin/ADF, the researchers saw a series of striking nuclear defects including shapes, nuclear lamina disruption, and reductions to peripheral heterochromatin.

From these observations, the experiments focused on how the Cofilin/ADF modulates other components in the cellular nucleus. It was found that Cof/ADF modulates myosin-II activity through competitive inhibition for binding to F-actin.

The researches concluded that nuclear shape was not completely controlled by general cytoplasmic F-actin organization, but through intracellular actomyosin forces.

This study thoroughly concludes by stating that a faulty cofilin and nucleo-cytoskeletal mechanocoupling can result in abnormal nuclear shape, and the importance of this finding for the field of laminopathies research.

Studies of the Correlations between the Nuclear Architecture Hallmark of Aging to Other Hallmarks From the Same Review

The task of understanding the mechanisms underlying physiological aging is no simple one. This is because biological systems are part of complex networks that influence each other in direct and indirect ways. Therefore, to understand how the many mechanisms are connected is a large portion of current studies trying to determine the factors that underlie physiological aging.

A review article published on April 11, 2016, in the journal Nucleus focused on the relationship between mammalian telomeres and lamins.

According to the review, numerous aspects at the biochemical, cellular and organismal level suggest that telomeres and lamins are more related than previously thought.

Telomeres interact with lamins through telomeric proteins and telomere-associated factors. If an alteration happens to either or both of the structure, cellular decline is bound to be observed.

Through this interaction, the review determines that the study of cellular senescence, a hallmark of aging, can also be brought into the complex mix of interacting factors that cause aging.

The authors state that the most striking link between lamins, telomeres, and senescence is the ability of telomerase reverse transcriptase (TERT) to rescue senescence induced by both lamins and telomere alterations.

Upon damage to the laminal and telomere structure of the cell, it also been seen that stem cell exhaustion is induced.

This review is an example of how the scientific community is starting to develop more interdisciplinary approaches to understanding the mechanisms of aging.