Ageing-related disorders Flashcards
Define ageing. What are the nine hallmarks of molecular ageing?
Aging is a universal process in biological organisms that is characterized by a time-dependent progressive decline in cellular and tissue function. At the molecular and cellular level, nine hallmarks have been proposed to contribute to the extremely complex, multifactorial process of aging:
- genomic instability and defects in nuclear architecture;
- telomere attrition;
- epigenetic alterations and chromatin remodeling;
- loss of proteostasis;
- deregulated nutrient sensing;
- mitochondrial dysfunction;
- cellular senescence;
- stem cell exhaustion and;
- altered intercellular communication.
Outline HGPS as a splicing disease.
Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature-ageing disorder arising from mutations in either genes encoding A-type lamins, or genes encoding lamin-processing enzymes. It is autosomal-dominant and affects only 1 in 4-8 million live births. The most common cause of HGPS is silent point mutations in the LMNA gene, which encodes lamin A, leading to aberrant splicing of the prelamin A mRNA and production of the lamin A variant, progerin.
Lamins, being either A- or B-type, are filament proteins that make up the nuclear lamina, a complex mesh network on the inner-face of the nuclear membrane. They have many integral cellular roles, including nuclear support and gene regulation, meaning it is no surprise that the effects of HGPS are so severe, with death at an average age of 13 years. A-type lamins are encoded by LMNA, which gives rise to four isoforms through alternative splicing.
Eriksson, Brown et al. (2003) revealed that LMNA mutations are the main cause of HGPS, in particular the substitution G608G (GGC>GGT) which they found in 90% of classical cases with mutant LMNA. This substitution is found in exon 11 of LMNA, and although the mutation is considered silent, it changes the nucleotide sequence so that it more closely resembles the consensus 5’SS. This has the effect of activating a cryptic donor site in the middle of exon 11 that is a stronger splice site than the WT, resulting in a deletion of 150 nt in the lamin A mRNA. This deletion does not disrupt the reading frame of the protein, so results in a 50 aa deletion from lamin A causing production of the truncated pathogenic protein progerin. This leads to aberrant function of the nuclear lamina, including altered gene regulation.
What are the different types of progeroid syndromes? Give examples
Progeroid or premature aging syndromes are a class of rarely occurring genetic disorders. They can be broadly classified into unimodal progeroid syndromes (affecting only one tissue type) and segmental progeroid syndromes (affecting several tissues and displaying some but not all symptoms of normal physiological aging).
- Familial Alzheimer’s disease and Parkinson’s disease fall under the first category.
- The segmental progeroid syndromes largely comprise of monogenic disorders with malfunction arising from single gene mutations in the affected individuals. Some of the most widely studied examples are Cockayne syndrome, Werner syndrome, HGPS and Bloom’s syndrome. They can be further categorized into four groups based on the type of genes being mutated:
What are the different types of lamins found in human cells? What are their function(s)?
The major nuclear lamin proteins expressed in humans are lamins A, C, B1, B2 and B3 encoded by the genes LMNA (for both lamins A and C), LMNB1 and LMNB2 (for both lamins B2 and B3), respectively. These proteins form the only class of intermediate filament proteins in the nucleus which:
- support the structure and shape of the nucleus;
- anchor chromatin;
- and also tether nuclear pore complexes in their appropriate functional positions.
How is lamin A made?
Lamin A (LA) is processed from its precursor prelamin A which contains a CaaX motif at its C terminal. This Cysteine residue undergoes farnesylation which leads to the cleavage of the aaX group followed by a methyl esterification of the Cysteine residue by isoprenylcysteine carboxyl methyltransferase (ICMT). This activates cleavage of an additional 15 amino acids of the precursor by the metalloproteinase, ZMPSTE24, to generate mature lamin A.
How can studying premature ageing disorders like HGPS help our understanding of ageing?
HGPS and normal ageing share many cellular phenotypes, such as abnormal nuclear shape, loss of epigenetic marks and increased DNA damage, as well as tissue pathologies including reduced bone density and cardiovascular disease.
Thus, better understanding of the molecular pathogenesis underlying progeroid syndromes can lead to a better understanding of the normal human ageing process.
Outline lamins.
Lamins are type V intermediate filament proteins expressed in all metazoan cells. They are the major building blocks of the nuclear lamina, a complex filamentous meshwork underneath the inner nuclear membrane (INM). Lamins share with their cytoskeletal counterparts the domain organization, encompassing a ~45-nm-long central α-helical rod domain flanked by two globular domains, but they contain additional lamin-specific motifs and domains in the C-terminus, such as a nuclear localization signal, a highly conserved immunoglobulin (Ig)-like fold and in most cases a CaaX box (C = cysteine, a = aliphatic residue, X = any amino acid).
Lamins are structural components, providing mechanical support for the nucleus, and recent reports showed that lamins define the mechanochemical properties of the nucleus; lamin A is responsible for nuclear stiffness, and B-type lamins for nuclear elasticity. Besides their mechanochemical role, lamins have a multitude of additional functions, including chromatin organization, gene regulation, DNA repair, and (mechano-) signaling.
What are the symptoms of HGPS?
Children with HGPS appear normal at birth but start to exhibit many distinctive clinical features within the first year of life. Classical progeria symptoms include severe growth retardation, loss of hair and subcutaneous fat, prominent eyes and scalp veins, aged-looking skin, joint stiffness and reduced bone density. As children get older they suffer from osteoporosis, atherosclerosis and cardiovascular diseases as the most severe aspect of the disease. HGPS patients die at an average age of 14 years due to myocardial infarction, heart failure or progressive atherosclerosis.
What is the consequence of cells expressing progerin?
Progerin is expressed in multiple tissues, mostly of mesenchymal origin including skin, bone, skeletal muscle, adipose tissue, heart and large and small arteries. Expression of progerin induces various cellular defects in a dominant-negative manner, including highly lobulated nuclei with thickened lamina, loss of peripheral heterochromatin, accumulation of DNA damage, telomere aberrations and mitochondrial dysfunction, leading to differentiation defects and premature cellular senescence. In addition, progerin expression leads to decreased expression levels of lamin B1, heterochromatin protein 1 α (HP1α) and LAP2α, and loss of nucleoplasmic lamins. These changes together with the abnormal nuclear morphology are often used as cellular disease markers to test therapeutic strategies in cell and mouse models.
Some HGPS patients display mosaicism, what does this mean?
Some HGPS sufferers display germinal mosaicism, with some cells displaying a HGPS genotype, and others displaying a normal genotype- this shows that the mutation happens after fertilisation.
Outline lamin A structure.
The nuclear lamins are type V intermediate filament proteins featuring a central α-helical rod domain that is flanked by non-helical head and tail domains. The large carboxy-terminal globular domain of about 200–300 amino acid residues is organized as an immunoglobulin (Ig)-like β-fold and invariably contains a nuclear localization sequence.
- Head
- Alpha-helical rod domain (site of dimerisation)
- Tail
The head and the rod tend to be the same between different lamins, with variation happening in the tail. The rod domain consists of four regions (1A, 1B, 2A, and 2B). Lamin A exists as dimers, and individual dimers are called parallel in register, i.e. they line up with one another. These lamin dimers exist in two conformations: anti-parallel, and head-to-tail. Through many of these interactions, a filament can be formed. The generation of the fibre, however is unknown, but it is likely that these filaments form sheets which join to form fibres. Somehow the lamins form cross connections to form a network.
What is the importance of lamins being phosphorylated?
Lamins are phospho-proteins, meaning they can exist in phosphorylated forms. Phosphorylation at some of the many phospho-sites is important in lamina assembly and disassembly. The enzymes that are capable of phosphorylating lamin A, these include protein kinase C (PKC) and Cdk1/Cyclin B. Lamins are highly phosphorylated during mitosis, which is the period where lamins are the most phosphorylated. This is important for their assembly state, as hyperphosphorylation of the lamin filaments causes them to disintegrate (lamins exist as dimers, and become soluble when phosphorylated). This allows for the physical separation of chromosomes to separate into two new daughter cells.
Only three amino acids can be phosphorylated: Tyrosine (Y) and Serine (S) and Threonine (T). Phosphorylation sites exist either side of the helical domain (important for dimerisation). Mutated phosphorylation sites can cause problems in disassembly of the nuclear lamina.
- Cdk1/Cyclin B: A single mutation (19 or 22) causes 37% of failed disassembly to occur, and 70% when a double mutation occurs (19 or 22 with 392). Phosphorylation either side of the helical domain by Cdk1 is needed in order for proper disassembly.
- PKC: A single PKC mutation has no effect, but a double mutation (e.g. 403/404) causes the accumulation of lamin A on the cytoplasmic surface. PKC is probably involved in nuclear import. Phosphorylation of different sites on the same protein has very different impacts, including nuclear import and regulating nuclear assembly.
How does the presence of farnesyl on progerin alter the cell? What are the pathological consequences?
The presence of the farnesyl group in progerin is thought to be a predominant toxic feature in the pathogenesis of the disease. In healthy cells, A- and B-type lamins form distinct homopolymers and micro-domains at the nuclear periphery, but this segregation may be lost in HGPS cells due to the stable association of permanently farnesylated progerin with the membrane.
- Progressive progerin accumulation at the INM during cellular aging of HGPS cells leads to immobilization of wildtype lamin A at the lamina, thickening and increased stiffness of the lamina, prominent lobulation of the nuclear envelope, and clustering of nuclear pores.
Overall, these alterations disrupt the structural and functional integrity of the nuclear lamina and may render cells more susceptible to damage through physical stress.
While in wild-type cells nuclei respond to shear stress by up-regulation and re-distribution of A-type lamins, this rearrangement does not work properly in HGPS cells. This defect is of particular importance in tissues that are exposed to mechanical stress such as vasculature, bone and joints, which also present some of the most prominent pathologies in HGPS.
How does lamin A interact with DNA? What are the effects of progerin on the normal interactions?
A-type lamins can directly interact with DNA and histones, and together with a number of lamin A-binding chromatin proteins, such as members of the LEM domain protein family, they have been implicated in higher-order chromatin organization, heterochromatin formation and epigenetic regulation.
A-type lamins contribute to the tethering of heterochromatic genomic regions, termed lamina associated domains (LADs) to the nuclear lamina but they also interact with promoter regions of genes, thereby affecting gene expression during cell differentiation. It is therefore not surprising that progerin-expressing HGPS nuclei display significant changes in chromatin, such as
- loss of peripheral heterochromatin;
- a decrease in the repressive histone marks H3K9me3 and H3K27me3, and;
- an increase in H4K20me3.
How does the expression of progerin contribute to the senescence phenotype?
Progerin expression also affects the expression and localization of the nucleotide excision repair protein XPA (Xeroderma Pigmentosum complementation group A) at DNA lesions, resulting in persistent activation of DNA damage response checkpoint kinases ATM and ATR. Persistent DNA damage in turn activates tumor suppressor p53 and promotes senescence, one of the phenotypes described as hallmarks of HGPS.
Humans express ____ major lamin proteins: lamin B1 (LB1), lamin B2 (LB2), and lamins A and C (LA, LC), encoded by _____, _____, and ____ genes, respectively. Lamins A, LB1, and LB2 are expressed as _________ that contain a carboxyl-terminal CaaX motif. The cysteine in the CaaX motif undergoes _____________, followed by cleavage of the last three amino acids (aaX) and _____ esterification. While LB1 and LB2 remain farnesylated, LA undergoes additional cleavage of the last 15 amino acids and becomes nonfarnesylated.
Humans express four major lamin proteins: lamin B1 (LB1), lamin B2 (LB2), and lamins A and C (LA, LC), encoded by LMNB1, LMNB2, and LMNA genes, respectively. Lamins A, LB1, and LB2 are expressed as prelamins that contain a carboxyl-terminal CaaX motif. The cysteine in the CaaX motif undergoes farnesylation, followed by cleavage of the last three amino acids (aaX) and methyl esterification. While LB1 and LB2 remain farnesylated, LA undergoes additional cleavage of the last 15 amino acids and becomes nonfarnesylated.
What is the most common mutation causing HGPS?
The most frequent mutation in Hutchinson–Gilford progeria syndrome (HGPS), affecting approximately 90% of patients, is a de novo autosomal dominant, single base substitution mutation in LMNA (C1824T). This mutation activates a cryptic splice site, which produces a mutant LA protein with an internal deletion of 50 amino acids that disrupts the last cleavage step of prelamin A. This truncated LA, termed progerin, is permanently farnesylated, toxic to cells and displays altered structural and biochemical properties.
- 18/21 mutations occur in LMNA exon 11 (Lamin A)
- 1/21 in exon 2 (Lamin A and C)
