Apoptosis and Intracellular Accumulations Dr Bikramajit Singh Saroya Windsor University SOM

Apoptosis Programmed cell death Suicide program Cells destined to die activate activate enzymes that degrade the cells' own nuclear DNA and nuclear and cytoplasmic proteins Intact plasma membrane Apoptosis eliminates cells that are genetically altered or injured beyond repair and does so without eliciting a severe host reaction, thereby keeping the extent of tissue damage to a minimum

Physiologic Apoptosis Programmed destruction of cells during embryogenesis Involution of hormone dependent tissues upon hormone withdrawal Ex: Endometrial cell breakdown during menstrual cycle, Ovarian follicular atresia in menopause Cell loss in proliferating cell populations to maintain homeostasis Ex: Immature lymphocytes in the bone marrow and thymus that fail to express useful antigen receptors Elimination of potentially harmful self-reactive lymphocytes Elimination of cells that have served their useful purpose

Apoptosis in Pathologic Conditions DNA damage - radiation, cytotoxic anticancer drugs, extremes of temperature, and even hypoxia Accumulation of misfolded proteins Cell injury in certain infections - especially viral Pathologic atrophy in parenchymal organs after duct obstruction

Mechanism of Apoptosis The Mitochondrial (Intrinsic) Pathway of Apoptosis The Death Receptor (Extrinsic) Pathway of Apoptosis Activation and Function of Caspases Clearance of Apoptotic Cells

Mechanism of Apoptosis The two pathways of apoptosis differ in their induction and regulation, and both culminate in the activation of caspases. In the mitochondrial pathway, proteins of the BCL2 family, which regulate mitochondrial permeability, become imbalanced and leakage of various substances from mitochondria leads to caspase activation. In death receptor pathway, signals from plasma membrane receptors lead to the assembly of adaptor proteins into a “death-including signaling complex,” which activates caspases, and the end result is the same.

Anti-apoptotic genes (BCL-2 gene) and Pro-apoptotic genes (BAX, BAK genes) BCL-2 proteins maintain mitochondrial membrane integrity and prevent leakage of mitochondrial proteins that can trigger apoptosis (e.g., cytochrome c) BAX and BAK - proapoptotic genes activated by damage to DNA, misfolded proteins, viral infections, and other injurious events produces protein products that form channels in the mitochondrial membrane that cause leakage of cytochrome c into the cytosol

Intrinsic(Mitochondrial) Pathway A: Survival signals induce anti apoptotic proteins like BCL-2 that maintain integrity of mitochondrial membranes, prevent leakage of mitochondrial proteins B: Loss of survival signals, DNA damage, other insults - activate the pro-apoptotic proteins BAX and BAK, which form channels in the mitochondrial membrane causing leakage of cytochrome c and other proteins leading to caspase activation and apoptosis

Extrinsic (Death Receptor) Pathway Responsible for elimination of self-reactive lymphocytes and damage by cytotoxic T lymphocytes Initiated by engagement of plasma membrane death receptors Death receptors - members of the TNF receptor family FAAD- Fas-associated death domain; FasL - Fas ligand)

Biochemical Features of Apoptosis Activation of caspases Caspases : Initiator and Executioner Initiator caspases – caspase 8 and 9 Executioner caspases – caspase 3 and 6 DNA and Protein Breakdown Membrane alteration to promote recognition by phagocytes : Movement of some phospholipids (notably phosphatidylserine) from the inner leaflet to the outer leaflet of the membrane, Binding of a protein called annexin V Annexin V staining is commonly used to identify apoptotic cells

Morphologic Changes Cell shrinkage: dense cytoplasm, tightly packed organelles Chromatin condensation and DNA fragmentation (most characteristic feature of apoptosis) Cytoplasmic blebs and apoptotic bodies Phagocytosis of apoptotic cell bodies by macrophages Intact plasma membranes

Apoptosis of an epidermal cell in an immune reaction Apoptosis of an epidermal cell in an immune reaction. The cell is reduced in size and contains brightly eosinophilic cytoplasm and a condensed nucleus.

Disorders of Apoptosis Defective apoptosis and increased cell survival. Cancer Autoimmune disorders Increased apoptosis and excessive cell death. Neurodegenerative diseases, Death of virus-infected cells

Autophagy Cell eats its own contents Survival mechanism in times of nutrient deprivation. Autophagolysosome = autophagic vacuole + lysosome Mechanism of cell loss in : degenerative disorders of CNS.

Intracellular Accumulations 2 main pathways of abnormal intracellular accumulations Inadequate removal of normal substance (defects in packaging and transport) FATTY LIVER Failure to degrade a metabolite (inherited enzyme deficiencies) STORAGE DISORDERS Based on duration - Transient/ permanent Based on Location - Cytoplasm/ nucleus

Mechanism of Intracellular accumulation A normal endogenous substance is produced at a normal or increased rate, but the rate of metabolism is inadequate to remove it ex: Fatty change in the liver Defects in protein folding and transport and an inability to degrade the abnormal protein efficiently ex: Accumulation of mutated alpha 1- antitrypsin in liver cells. Inherited defects of enzymes required for metabolism of a particular substance ex: Lysosomal storage disorders Exogenous substance ex: Carbon particles, Silica

Abnormal metabolism, as in fatty change in the liver Mutations causing alterations in protein folding and transport, so that defective molecules accumulate intracellularly

(3) Deficiency of critical enzymes for breaking down certain compounds, causes substrates to accumulate in lysosomes (lysosomal storage diseases) (4) An inability to degrade phagocytosed particles, as in carbon pigment accumulation

Intracellular Accumulations Fatty Change (Steatosis) - mainly in the liver Cholesterol and Cholesteryl Esters - Atherosclerosis Proteins - Mallory hyaline, Russell bodies, Neurofibrillary tangles Glycogen Pigments Carbon (Anthracosis) Lipofuscin (Brown atrophy) Melanin Hemosiderin

Steatosis ( Fatty change) Abnormal accumulation of triglycerides within parenchymal cells Most common cause of fatty change in the liver in developed nations – alcohol abuse & non alcoholic fatty liver disease(NAFLD/NASH) is associated with malnutrition, obesity, diabetes. Seen in liver and the heart commonly Clear vacuoles within parenchymal cells Stains: Sudan IV / Oil red O – Orange red color

Steatosis Morphology Gross – enlarged bright yellow soft greasy Histological features : Small vacuoles in the cytoplasm around the nucleus Vacuoles coalesce Clear spaces with Nucleus displaced to periphery Fatty cysts – enclosed fat globules coalesce after rupture of contiguous cells

Cholesterol and Cholesterol Esters Atherosclerosis: In atherosclerotic plaques, smooth muscle cells and macrophages are filled with lipid vacuoles, made up of cholesterol and cholesterol esters Xanthomas: Clusters of foamy cells in sub-epithelial connective tissue of skin and in tendons, producing masses Cholesterolosis: focal accumulations of cholesterol-laden macrophages in the lamina propria of the gallbladder Niemann-Pick disease, type C: mutations affecting an enzyme involved in cholesterol trafficking resulting in cholesterol accumulation in multiple organs

A: Achilles tendon xanthoma - slightly yellow nodular lesions at the distal end of the Achilles. B: Xanthelasma - Yellow, raised lesions on the lower left eyelid C: Palmar xanthomas - yellow macules on the palm accentuated in the creases D: Eruptive xanthomas - numerous small yellow papular lesions distributed over the buttocks

Proteins Reabsorption droplets in proximal renal tubules – in renal diseases associated with proteinuria Normal secreted proteins produced in excessive amounts – Russell bodies(abnormal immunoglobulins) Defective intracellular transport and secretion of critical proteins - α1-antitrypsin deficiency Accumulation of cytoskeletal proteins – Mallory Hyaline(keratin intermediate filaments), Nuerofibrillary tangles(Alzeimer’s) Aggregation of abnormal proteins - Amyloidosis

Bone marrow core biopsy showing numerous plasma cells, some with eccentric nuclei and round cherry red inclusions (Russell bodies)

Mallory's hyaline or "alcoholic" hyaline – globular red hyaline material within hepatocytes. The globules are aggregates of intermediate filaments in the cytoplasm resulting from hepatocyte injury

Neurofibrillary "tangle" of Alzheimer's disease

Hyaline Change Descriptive histologic term An alteration within cells or in the extracellular space that gives a homogeneous, glassy, pink appearance in routine histologic sections stained with hematoxylin and eosin In long-standing hypertension and diabetes mellitus, the walls of arterioles, especially in the kidney, show hyaline change

Accumulation of Glycogen Readily available energy source stored in cytoplasm of healthy cells Excessive intracellular deposits seen in patients with genetic disorders collectively referred to as the “glycogen storage diseases”, or “glycogenoses” Rose-to-violet color with PAS stain

Pigments Exogenous Pigments Endogenous Pigments Anthracosis: carbon (coal dust) in lungs and lymph nodes Tattooing Endogenous Pigments Lipofuscin ( wear and tear pigment, Brown Atrophy) Melanin ( Melanoma, Addison Disease) Homogentisic acid, a black pigment accumulates in patients with alkaptonuria (Oochronosis) Hemosiderin ( Hemosiderosis, Heart failure cells) Bilirubin (Jaundice, kernicterus

Pathologic calcification Abnormal tissue deposition of calcium salts, smaller amounts of iron, magnesium & other mineral salts 2 types: Dystrophic calcification Metastatic calcification

Dystrophic calcification Normal serum levels of calcium Normal calcium metabolism Deposition of calcium in dying tissues Encountered in areas of necrosis Seen in aging or damaged heart valves Atheromas of advanced atherosclerosis Dystrophic calcification of the aortic valve. View looking down onto the unopened aortic valve in a heart with calcific aortic stenosis. It is markedly narrowed (stenosis). The semilunar cusps are thickened and fibrotic, and behind each cusp are irregular masses of piled-up dystrophic calcification

Dystrophic calcification Pathogenesis : Calcium enters the necrotic cells and binds to phosphate producing calcium phosphate, which is basophilic in the hematoxylin-eosin stain Gross : Fine, white granules or clumps, felt as gritty stones Histological appearance: basophilic amorphous, granular, sometimes clumped Calcification in areas of caseous necrosis in tuberculosis Calcified atherosclerotic plaques Calcification of aortic valves in elderly Calcification in chronic pancreatitis Periventricular calcification in congenital cytomegalovirus infection Psammoma bodies (Papillary cancer of thyroid, Meningioma)

Metastatic Calcification In normal tissues when there is HYPERCALCEMIA Causes of hypercalcemia: Hyperparathyroidism Destruction of bone tissue Vitamin D–related disorders Renal failure Aluminum intoxication Milk-alkali syndrome.

Metastatic Calcification Principally affects the interstitial tissues of the gastric mucosa kidneys, lungs, systemic arteries, and pulmonary veins Noncrystalline amorphous deposits or as hydroxyapatite crystals