vinay @ cell aging & death

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Presentation on Cell aging & death:

Presentation on Cell aging & death Presented by- Vinay k. Dubey M. Pharm. 1 st year ( p’cology ) Jeanne Calment

Cell aging :

Cell aging Cellular aging is the result of a progressive decline in the proliferative capacity and life span of cells and the effects of continuous exposure to exogenous factors that cause accumulation of cellular and molecular damage. Ageing refers to the increased impairment of physiological function with age. Chronological age - number of years lived Physiologic age - age by body function Functional age - ability to contribute to society

Characteristics:

Characteristics Decline in functional efficiency of specialized non dividing cells. e.g. – Nerve cells Decline of division capacity of actively dividing cells (k/a Replicative senescence/ heyflick effect). e.g. – Lymphocytes ,epithelial cells After a fixed number of divisions, cells are arrested in a terminally non- dividing state, known as replicative senescence.

Cell homeostasis- :

Cell homeostasis- The normal cell is confined to a fairly narrow range of function and structure by its genetic programs of metabolism, differentiation, and specialization; by constraints of neighboring cells; and by the availability of metabolic substrates. It is nevertheless able to handle normal physiologic demands, maintaining a steady state called homeostasis. Cell homeostasis is important for proper functioning of organism.

Cellular Responses to Stress and Noxious Stimuli:

Cellular Responses to Stress and Noxious Stimuli More severe physiologic stresses and some pathologic stimuli may bring about a number of physiologic and morphologic cellular adaptations . If injury is beyond to adaptation & reached to the point of no return it will result in death of cell.

Hypertrophy Hypertrophy is an increase in the size of cells resulting in increase in the size of the organ.:

Hypertrophy Hypertrophy is an increase in the size of cells resulting in increase in the size of the organ. Physiologic hypertrophy of the uterus during pregnancy. A, Gross appearance of a normal uterus ( right ) and a gravid uterus ( left ) that was removed for postpartum bleeding. B, Small spindle-shaped uterine smooth muscle cells from a normal uterus. Compare this with ( c ) large, plump hypertrophied smooth muscle cells from a gravid uterus

Atrophy Shrinkage in the size of the cell by the loss of cell substance is known as atrophy.:

Atrophy Shrinkage in the size of the cell by the loss of cell substance is known as atrophy. A, Normal brain of a young adult. B, Atrophy of the brain in an 82-year-old male with atherosclerotic disease. Atrophy of the brain is due to aging and reduced blood supply.

Metaplasia Metaplasia is a reversible change in which one adult cell type (epithelial or mesenchymal) is replaced by another adult cell.:

Metaplasia Metaplasia is a reversible change in which one adult cell type (epithelial or mesenchymal ) is replaced by another adult cell. Metaplasia of normal columnar ( left ) to squamous epithelium ( right ) in a bronchus, shown

PowerPoint Presentation:

hyperplasia The adaptive response may consist of an increase in the number of cells, called hyperplasia. It takes place if the cellular population is capable of synthesizing DNA, thus permitting mitotic division. e.g. removal of liver part. The two types of physiologic hyperplasia are- hormonal hyperplasia , exemplified by the proliferation of the glandular epithelium of the female breast at puberty and during pregnancy; compensatory hyperplasia , that is, hyperplasia that occurs when a portion of the tissue is removed or diseased.

CELL INJURY :

CELL INJURY cell injury results when cells are stressed so severely that they are no longer able to adapt or when cells are exposed to inherently damaging agents or suffer from intrinsic abnormalities. Injury may progress through a reversible stage and irreversible stage culminate in cell death. REVERSIBLE CELL INJURY Injury is manifested as functional and morphologic changes that are reversible if the damaging stimulus is removed. Adaptive states are compatible with homeostasis. Since a cell that is in an adaptive state can still be in homeostasis, albeit on a different level , the morphological appearance of the cell will change over time to reflect its adaptive change. e.g. ischemic injury.

Mechanism of ischemia:

Mechanism of ischemia

Irreversible cell injury:

Irreversible cell injury When the cell can not adapt an injury or unable to maintain its homeostasis & have passed the "point of no return“, it is in the phase of irreversible stage. The cell death can occurs at this phase. Early morphologic changes of irreversible injury include : mitochondrial swelling and vacuolization plasma membrane damage calcium entry into the cell lysosomal swelling amorphous densities in the mitochondria

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Changes during injury:

Changes during injury

Theories of cell aging:

Theories of cell aging Stochastic (Damage-Based) Theories of Aging Free Radical Theory CELL Damage Theory Telomere Theory Protein Cross-Linking Theory Programmed Theories of Aging Canges in gene expression concerning repair enzymes, hormonal levels, immune system function, etc. Evolutionary Theory of Aging

 Free radical theory:

Free radical theory

DNA damage:

DNA damage Except ROS, reactive nitrogen species (RNS), such as peroxynitrites and nitrogen oxides, have also been implicated in DNA damage. Upon reaction with guanine, peroxynitrite has been shown to form 8-nitroguanine. Due to its structure, this adduct has the potential to induce G:C→T:A transversions . Fig.- Reaction of guanine with hydroxyl radical

Lipid peroxidation:

Lipid peroxidation

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gaseous pentane (a good marker of lipid peroxidation .) Lipid propagation

Protein damage:

Protein damage Reduction of the ferryl complex by Tyr is described by the reaction: Fe(IV) O + Tyr OH + H+→ Fe(III) + H2O + Tyr O• and alternatively by other amino acids leads to the subsequent formation of other amino acid radicals via an electron-transfer process that occurs throughout the protein. The oxidation of methionine (Met) residues of proteins leads to the formation of a mixture of the S– and R– isomers of methionine sulphoxide , Met–(S)–SO & Met–(R)–SO: Met + ROS → Met–(S)–SO +Met–(R)–SO + products

Protein damage:

Protein damage The amino acid residue side chains that are most vulnerable to attack by various ROS and RNS lead to the formation of the following products: arginine→glutamic semialdehyde glutamate→4-hydroxy-glutamate histidine→2-oxo-histidine tyrosine→3,4-dihydroxy phenylalanine valine→3,4-hydroxy valine cysteine→cys– S–S– cys , cys –S–S–R disulphied proline→glutamic semialdehyde , 2-pyrrolidone-4-hydroxy-proline methionine→methionine sulphone and sulphoxide

CELL Damage Theory:

CELL Damage Theory The theory explains that errors in cell homeostasis may lead to cancer, apoptosis, or cellular dysfunction. Different organelles of a cell performs differ work, any damage will affect their function. It can occurs in different ways- Damage by Influx of intracellular ca 2+ Damage in membrane permeability Damage by free radicals Mitochondrial damage

Damage by Influx of intracellular ca2+:

Damage by Influx of intracellular ca 2+

Damage in membrane permeability:

Damage in membrane permeability

Damage by free radicals:

Damage by free radicals .

Mitochondrial damage:

Mitochondrial damage

Telomere Theory:

Telomere Theory Repetitive DNA sequences at the ends of all human chromosomes. They contain thousands of repeats of the six-nucleotide sequence, TTAGGG Telomeres are also thought to be the "clock" that regulates how many times an individual cell can divide. Telomeric sequences shorten each time the DNA replicates.

- a cell has divided and when telomeres are short, cellular senescence (growth arrest) occurs.:

- a cell has divided and when telomeres are short, cellular senescence (growth arrest) occurs. - Human telomeres are composed of a repetitive hexameric sequence [TTAGGG]n about 10-15,000 bp in length. - The bulk of telomeric DNA is double-stranded [TTAGGG/ CCCTAA]n, but the end consists of a 3’ overhang of single stranded repeats [TTAGGG]n. - Telomeres shorten by about 50-150 base pairs at each division because conventional DNA polymerases cannot replicate the ends of linear DNA. - Once the telomere shrinks to a certain level, the cell can no longer divide. Its metabolism slows down, it ages, and dies.

Repairing of telomere:

Repairing of telomere Germ cells (along with certain immortal cells e.g. cancer cells) have an enzyme, telomerase, which repairs shortened telomeres. In humans (e.g. stem cells), telomerase is a complex of proteins including a cellular reverse transcriptase ( hTERT ) and an RNA ( hTR ), which acts as a template for catalyzing DNA addition at the telomere. Somatic cells (but not germ cells and many tumour cells) lack telomerase, and thus telomere shortening could be a “clock” that eventually stops somatic cell division.

MOA of telomerase:

MOA of telomerase

However, 1. There is no correlation b/w telomere length & the lifespan of an animal (e.g. shorter telomeres of humans than mice). 2. There is no correlation between telomere length and a person’s age.:

However, 1. There is no correlation b/w telomere length & the lifespan of an animal (e.g. shorter telomeres of humans than mice). 2. There is no correlation between telomere length and a person’s age. Telomeres Signal to the Cell- In normal cells the telomere signal is transduced via the p53 tumour suppressor protein, which is activated by acetylation . This activates p21CIP1/WAF1, a cyclin -dependent kinase inhibitor that shuts down the cell cycle and leads to senescence. Cells with inactive p53 (e.g. deacetylation ) become immortal and can progress to a cancerous state.

Aging & cancer:

Aging & cancer

The Hayflick Limit (M1):

The Hayflick Limit (M1) The Hayflick limit (or Mortality Stage 1), in which cells stop dividing and become senescent Replicative (or Cellular) Senescence is a Block to Tumour Formation. Cells from a human fetus go through ~60 doublings, whereas those of an 80 year old go through ~30 ( replicative senescence) while Cells of an adult mouse go via about 12-15 doublings. Cells can pass through the Hayflick Limit (M1)..?? Inactivation of the tumour suppressors p53 and RB by viral oncoproteins such as: • human papillomavirus type 16 E6 (inactivates p53) and E7 (inactivates RB) or SV40 Large T antigen (inactivates both) allows cells to pass through the Hayflick Limit (M1).

Tumor cell replication:

Tumor cell replication Overcome the M1 block (generally by inactivating tumoursuppressors ). 2. Bypass M2 ( by reactivating endogenous telomerase). Immortalization always requires the reactivation of telomerase to preserve genomic stability by maintaining chromosome ends. E2F Rb P Rb P P P P P Cyclin D cdk S-phase Gene p16 p53 E2F

Protein Cross-Linking Theory:

Protein Cross-Linking Theory The glycosylation theory of aging suggests that the aerobic binding of sugars to proteins impairs protein function, leading to the inefficiencies in cellular function associated with aging. Protein Cross-Linking is encouraged by high blood glucose levels, so a diet with moderate to low intake of carbohydrates is recommended to avoid the theoretical aging effects of glycosylation .

Cell death:

Cell death Cells are mortal in nature. By injury or other reasons it will die in a manner. If the cell is no longer needed, it goes for apoptosis. Cell death can occur in following ways- 1- Apoptosis (cell suicide) 2- Necrosis (cell murder)

Difference between necrosis & apoptosis:

Difference between necrosis & apoptosis

Necrosis & apoptosis:

Necrosis & apoptosis

Apoptosis:

Apoptosis Apoptosis is a pathway of cell death that is induced by a tightly regulated suicide program in which cells destined to die. Activate enzymes capable of degrading the cells' own nuclear DNA and nuclear and cytoplasmic proteins The plasma membrane of the apoptotic cell remains intact, but the membrane is altered in such a way that the cell and its fragments become avid targets for phagocytes. The dead cell is rapidly cleared before its contents have leaked out, and therefore cell death by this pathway does not elicit an inflammatory reaction in the host. Thus, apoptosis differs from necrosis.

Morphology of apoptosis These features characterize better in electronic microscope. :

Morphology of apoptosis These features characterize better in electronic microscope. Cell shrinkage Chromatin condensation Formation of Apoptotic bodies Phagocytosis

Biochemical features of apoptosis:

Biochemical features of apoptosis Apoptotic cells exhibited a distinctive biochemical modification that underlie the structural changes- Protein cleavage Protein cross linking DNA breakdown Phagocytic recognition

Protein cleavage:

Protein cleavage Hydrolysis of protein occurs by activation of several members of a family of cysteine protease named caspases . It need to be activated to induce apoptosis. Ced-3 is the prototype of a family of dozen protease, known as Caspases . It activate DNAse , which causes fragmentation of DNA. Caspases cleave nuclear lamin (cellular protein) fragmentation of nucleus, cytoskeletal protein disruption. Mammalian contain family of atleast 7 caspases , classified as either initiator or effector .

Caspase activation :

Caspase activation The mammalian initiator caspase-9 is activated as a complex with Apaf-1 and cytochrome c in the apoptosome . Caspase-9 then cleaves and activates effector caspases , such as caspase-3. The effector caspases cleave a variety of cell proteins, including nuclear lamins , cytoskeletal proteins, and an inhibitor of DNase , leading to death of the cell.

Genetic regulation of apoptosis:

Genetic regulation of apoptosis The third gene identified as a key regulator is called Bcl-2. The Bcl-2 family - The Bcl-2 family of proteins is divided into three functional groups. 1- Antiapoptotic proteins (e.g., Bcl-2 and Bcl-xL ) have four Bcl-2 homology domains (BH1-BH4). 2- The multidomain proapoptotic proteins (e.g., Bax and Bak ) have three homology domains (BH1-BH3), whereas 3- the BH3-only proapoptotic proteins (e.g., Bid, Bad, Noxa , Puma, and Bim ) have only one homology domain (BH3).

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Mechanism of apoptosis:

Mechanism of apoptosis Extrinsic pathway of apoptosis- Many cells express surface molecules, called death receptors, that trigger apoptosis. Also k/a death receptor pathway . Most of these are members of the tumor necrosis factor (TNF) receptor family that contain in their cytoplasmic regions a conserved "death domain,”. Mainly death receptors are the type I TNF receptor and Fas (CD95). - When cytotoxic T cells recognize (bind to) their target, - they produce more FasL at their surface & bind with the Fas on the surface of the target cell. - The clustered Fas proteins then activate proteins that bind and activated procaspase-8 molecules, which cleave and activate one another.

- The activated caspase-8 molecules then activate downstream procaspases to induce apoptosis. - caspase 8 initiates a cascade of caspase activation leading to phagocytosis of the cell TNF receptor and Fas (CD95). :

- The activated caspase-8 molecules then activate downstream procaspases to induce apoptosis. - caspase 8 initiates a cascade of caspase activation leading to phagocytosis of the cell TNF receptor and Fas (CD95).

Intrinsic pathway of apoptosis-:

Intrinsic pathway of apoptosis- Also k/a mitochandrial pathway . Mitochondria contain several proteins that are capable of inducing apoptosis. These proteins include cytochrome c and anti-apoptotic protein (Bcl-2 and Bcl -x). The choice between cell survival and death is determined by the permeability of mitochondria, which is controlled by a family of more than 20 proteins, the prototype of which is Bcl-2. When cells are deprived of survival signals or subjected to stress, Bcl-2 and/or Bcl -x are lost, and replaced by pro-apoptotic members of family, such as Bax , Bak and Bim .

- When Bcl-2 and/or Bcl-x level decreases, mitochondrial permeability increases and release several protein that can activates caspases. :

- When Bcl-2 and/or Bcl-x level decreases, mitochondrial permeability increases and release several protein that can activates caspases.

Mechanism of apoptosis:

Mechanism of apoptosis

Formation of apoptotic bodies:

Formation of apoptotic bodies

phagocytosis:

phagocytosis

Alternative pathways:

Alternative pathways Autophagy - It refers lysosomsl digestion of the cells own components. Intracellular orgenells first covered in an autophagic vacuole formed from ribosome free regions of rough ER. Now vacuole fuses with lysosomes or golgi elements to form an autophagolysosome .

Lysosomes contain various digestive enzymes, including proteases. Lysosomes take up cellular proteins by fusion with autophagosomes, which are formed by the enclosure of areas of cytoplasm or organelles(mitochondrion) in fragments of the endoplasmic reticulum. This fusion yields a phagolysosome, which digests the contents of the autophagosome. :

Lysosomes contain various digestive enzymes, including proteases. Lysosomes take up cellular proteins by fusion with autophagosomes, which are formed by the enclosure of areas of cytoplasm or organelles(mitochondrion) in fragments of the endoplasmic reticulum. This fusion yields a phagolysosome , which digests the contents of the autophagosome .

Examples of apoptosis:

Examples of apoptosis The resorption of the tadpole tail at the time of its metamorphosis into a frog occurs by apoptosis. The formation of the fingers and toes of the fetus requires the removal, by apoptosis, of the tissue between them. The sloughing off of the inner lining of the uterus (the endometrium ) at the start of menstruation occurs by apoptosis. The formation of the proper connections (synapses) between neurons in the brain requires that surplus cells be eliminated by apoptosis

NECROSIS:

NECROSIS Morphological changes that follow cell death in living tissue largely resulting from degradative action of enzymes on lethally injured cell. Morphology- 1- Cytoplasmic Changes- necrotic cell shows increased eosinophilia attribute in part to loss of the normal basophilia imparted by the RNA in the cytoplasm & in part to increased binding of eosin to denatured intracytoplasmic proteins. It is shown in image which illustrates cytoplasmic hypereosinophilia of the necrotic cardiac myocytes . Notice absence of striations and the homogeneous, dense pink staining of the cytoplasm.

Ischemic necrosis of the myocardium. A, Normal myocardium. B, Myocardium with coagulation necrosis (upper two thirds of figure), showing strongly eosinophilic anucleate myocardial fibers.:

Ischemic necrosis of the myocardium. A, Normal myocardium. B, Myocardium with coagulation necrosis (upper two thirds of figure), showing strongly eosinophilic anucleate myocardial fibers.

2- Nuclear Changes :

2- Nuclear Changes Nuclear changes assume one of three patterns, all due to breakdown of DNA and chromatin. The basophilia of the chromatin may fade ( karyolysis ), presumably secondary to deoxyribonuclease ( DNase ) activity. A second pattern is pyknosis , characterized by nuclear shrinkage and increased basophilia ; the DNA condenses into a solid shrunken mass. In the third pattern, karyorrhexis , the pyknotic nucleus undergoes fragmentation. In 1 to 2 days, the nucleus in a dead cell completely disappears.

Types of Necrosis :

Types of Necrosis Coagulative Necrosis – A form of tissue necrosis in which the component cells are dead but the basic tissue architecture is preserved for at least several days. Presumably the injury denatures not only structural proteins but also enzymes and so blocks the proteolysis of the dead cells ; as a result, eosinophilic , anucleate cells may persist for days or weeks. the necrotic cells are removed by phagocytosis of the cellular debris by infiltrating leukocytes and by digestion of the dead cells by the action of lysosomal enzymes of the leukocytes.

Coagulative necrosis is characteristic of infarcts (areas of ischemic necrosis) in all solid organs except the brain. :

Coagulative necrosis is characteristic of infarcts (areas of ischemic necrosis) in all solid organs except the brain.

Liquefactive Necrosis :

Liquefactive Necrosis It is seen in focal bacterial or, fungal infections, because microbes stimulate the accumulation of inflammatory cells and the enzymes of leukocytes digest ("liquefy") the tissue. If the process was initiated by acute inflammation, the material is frequently creamy yellow and is called pus. E.g.- hypoxic death of cells within the central nervous system often evokes liquefactive necrosis.

An infarct in the brain, showing dissolution of the tissue. :

An infarct in the brain, showing dissolution of the tissue.

gangrenous necrosis:

gangrenous necrosis It is not a distinctive pattern of cell death, the term is still commonly used in clinical practice. It is usually applied to a limb, generally the lower leg, that has lost its blood supply and has undergone coagulative necrosis involving multiple tissue layers. When bacterial infection is superimposed, coagulative necrosis is modified by the liquefactive action of the bacteria and the attracted leukocytes (so-called wet gangrene ).

Caseous necrosis:

Caseous necrosis Caseous necrosis is encountered most often in foci of tuberculous infection. The term " caseous " (cheese-like) is derived from the friable yellow-white appearance of the area of necrosis. On microscopic examination, the necrotic focus appears as a collection of fragmented or lysed cells with an amorphous granular appearance. Unlike coagulative necrosis, the tissue architecture is completely obliterated and cellular outlines cannot be discerned. Caseous necrosis is often enclosed within a distinctive inflammatory border; this appearance is characteristic of a focus of inflammation known as a granuloma .

A tuberculous lung with a large area of caseous necrosis containing yellow-white and cheesy debris.:

A tuberculous lung with a large area of caseous necrosis containing yellow-white and cheesy debris.

Fat necrosis:

Fat necrosis This occurs in the calamitous abdominal emergency known as acute pancreatitis. In this disorder, pancreatic enzymes that have leaked out of acinar cells and ducts liquefy the membranes of fat cells in the peritoneum, and lipases split the triglyceride esters contained within fat cells. The released fatty acids combine with calcium to produce grossly visible chalky white areas (fat saponification ), On histologic examination, the foci of necrosis contain shadowy outlines of necrotic fat cells with basophilic calcium deposits, surrounded by an inflammatory reaction.

Fat necrosis in acute pancreatitis. The areas of white chalky deposits represent foci of fat necrosis with calcium soap formation (saponification) at sites of lipid breakdown.:

Fat necrosis in acute pancreatitis. The areas of white chalky deposits represent foci of fat necrosis with calcium soap formation ( saponification ) at sites of lipid breakdown.

Fibrinoid necrosis:

Fibrinoid necrosis Fibrinoid necrosis is a special form of necrosis usually seen in immune reactions involving blood vessels. This pattern of necrosis is prominent when complexes of antigens and antibodies are deposited in the walls of arteries. Deposits of these "immune complexes," together with fibrin that has leaked out of vessels, result in a bright pink and amorphous appearance in H&E stains, called " fibrinoid " (fibrin-like) by pathologists. The immunologically mediated diseases (e.g., polyarteritis nodosa ) in which this type of necrosis is seen.

Fibrinoid necrosis in an artery in a patient with polyarteritis nodosa. The wall of the artery shows a circumferential bright pink area of necrosis with protein deposition and inflammation.:

Fibrinoid necrosis in an artery in a patient with polyarteritis nodosa . The wall of the artery shows a circumferential bright pink area of necrosis with protein deposition and inflammation.

The Progerias:

The Progerias The progerias are characterized by the early onset of complex senescent phenotypes. Werner Syndrome (WS) is an autosomal recessive disease of genomic instability characterized by premature onset of age-related diseases, including graying of hair and hair-loss, atherosclerosis, osteoporosis, type II diabetes mellitus, cataracts and cancer. Average diagnosis age is 30ies & the mean age of death is 47( most by cancer or atherosclerosis). They usually do not develop Alzheimer-type dementia.

Hutchinson-Gilford Progeria Syndrome (HGPS) it is a very rare (1in 4 million) premature ageing disorder.:

Hutchinson-Gilford Progeria Syndrome (HGPS) it is a very rare (1in 4 million) premature ageing disorder. Life expectancy of HGPS individuals is short (13 years) Death almost invariably from atherosclerosis. Majority of HGPS cases due to a de novo mutation in the lamin A gene ( LMNA). Lamins (type V intermediate filament proteins) are the main structural components of the nuclear lamina. - They exist as A and B types and are required for: nuclear shape, mechanical stability, assembly & positioning chromatin organization transcription regulation DNA replication

LMNA gene (12 exons) encoding lamin A and lamin C proteins.C-terminus of pre-lamin A bears a CaaX motif (C is cysteine, a is any aliphatic (non-polar, hydrophobic) aa, and X is any aa). :

LMNA gene (12 exons ) encoding lamin A and lamin C proteins. C -terminus of pre- lamin A bears a CaaX motif (C is cysteine , a is any aliphatic (non-polar, hydrophobic) aa , and X is any aa ). Farnesol , The cysteine is the site of post-translational farnesylation allowing it to be included into the nuclear membrane. The C-terminal region is then removed by proteolysis, yielding the mature form of lamin A.

Aberrant nuclear shape in HGPS:

Aberrant nuclear shape in HGPS In HGPS, a mutation in exon 11 creates a novel splice donor site yielding progerin (LAΔ50), which lacks 50 aa near the C terminus. This results in the loss of the proteolytic site so that progerin is permanently farnesylated . Predicted to yield mislocalized nuclear membrane complex that alters nuclear structure and function.

Treatment of progeria:

Treatment of progeria Farnesyl transferase inhibitors (FTIs) inhibit farnesylation and may be useful in the treatment of HGPS, since they could inhibit the permanent farnesylation of progerin .

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