Mechanisms of Cell Death Lecture Notes PDF

MECHANISMS OF CELL DEATH Dr. C.L. Neary Mechanisms of Disease MBS00609 Pathology Terminology Etiology: cause of a disease Pathogenesis: biochemical and molecular mechanisms of disease development Morphology: appearance of cells/tissues/organs Clinical features (...

MECHANISMS OF CELL DEATH Dr. C.L. Neary Mechanisms of Disease MBS00609 Pathology Terminology Etiology: cause of a disease Pathogenesis: biochemical and molecular mechanisms of disease development Morphology: appearance of cells/tissues/organs Clinical features (manifestation): functional consequences of morphological changes CELL DEATH Why do cells die? Lack of resources – Hypoxia – Nutrient deficiency – Growth factor withdrawal Exposure to toxins Removal of aging/ineffective cells Attack by immune system – Infection – Autoimmunity Cellular level: Signs of Injury Intracellular accumulations Fatty deposits Lipofuscin Protein Robbins PBoD Fig. 2-30 Robbins Fig. 2-25 Robbins PBoD Fig. 2-32 Modes of Cell Death Unregulated (pathological) – Necrosis (signaling) – cell breaks down/explodes and contents are released Regulated (physiological) – Apoptosis (multiple) – cell disassembles and packages contents for phagocytosis Alternatives – Necroptosis (regulated necrosis) – Anoikis (detachment-induced cell death) – Ferroptosis Necrosis Normal Stressed Necrotic Cuboidal Epithelium Blebbing Loss of Nuclei Even Size Eosinophilia Breakdown of Even Staining Swelling Membranes Robbins Fig. 2.4 Types of Necrosis Coagulative (next slide) – Loss of cell architecture but not tissue architecture Liquefactive – Digestion of cells results in viscous mass Caseous – Fragmented cells and granular debris surrounded by inflammation Fibrinoid (next slide) – Immune complexes and fibrin in walls of blood vessels  Tissue Level: Necrotic damage Vessel lumen Coagulative necrosis Fibrinoid Necrosis Robbins Fig. 2-6 Robbins Fig. 2-10 Coagulative necrosis Fibrinoid necrosis N: Normal Pink area = necrosis I: Infarct Numerous nuclei = neutrophils (inflammation) Immune cell infiltrate Causes of Necrosis Overwhelming damage Toxins Excessive Calcium More on calcium later Damage to the ER and mitochondria Reactive Oxygen Species (ROS) Ischemia Membrane damage Nutrient withdrawal APOPTOSIS Physiological Cell Death Specific instances of physiological cell death: Embryogenesis origin of the term ‘programmed cell death’ death of specific cells at specific times, i.e. during digit development Tissues that produce new cells as part of their function immature lymphocytes in the bone marrow and thymus that fail to express useful antigen receptors epithelial cells in intestinal crypts, so as to maintain a constant number (homeostasis) Physiological Cell Death (cont.) Loss of hormone-dependent tissues when hormone levels fall endometrial cell breakdown during the menstrual cycle ovarian follicular atresia in menopause Immune function Pyroptosis Destruction of self-reactive lymphocytes to prevent auto- immunity Death of cells that have served their purpose neutrophils in an acute inflammatory response lymphocytes at the end of an immune response Apoptotic Initiators Viral infections Ionizing radiation Chemical damage to cells Cytokines (TNF, Fas ligand) Mitochondrial damage UPR Calcium influx Unresolved stress Major Morphological Features of Apoptosis Cell rounding/condensation Nuclear condensation/fragmentation – Visible with DAPI staining Membrane blebbing – Visible with light microscopy Formation of apoptotic bodies – Packaging of cell contents into vesicles What it looks like Robbins PBoD Condensed Epidermal cell Condensed/Fragmented Nuclei Membrane blebbing Nuclear Fragmentation - DAPI Caspase activation APOPTOTIC SIGNALING Two Different Starting Points Extrinsic – Death receptors on the plasma membrane are activated and transduce a signal through intracellular signaling pathways to activate caspases Intrinsic – Mitochondrial signals induce release of pro-apoptotic proteins that activate caspases What are Caspases? – Specific proteases that disassemble the cell – Biochemical markers of apoptosis Caspases Cysteine Aspartases Consensus Sequences – Cleave after aspartic acid residue Inflammatory Involved in NFκB signaling Apoptotic: Initiator v. Executioner – Initiator caspases (2, 8, 9, 10) – Executioner caspases (3, 6, 7) Extrinsic Apoptotic Signaling TNF/TNFR v. FasL/Fas TNF FasL extracellular TNFR Fas intracellular TRADD FADD DISC Complex I RIP C8/10 TRAF RIP TRADD Complex II FADD C8/10 Caspase 8/10 Activation Death Domain Superfamily Four subfamilies, all involved in assembly of protein complexes: 1. Death Domain (DD) subfamily 2. Death Effector Domain (DED) subfamily 3. Caspase Recruitment Domain (CARD) subfamily 4. Pyrin Domain (PYD) subfamily Through Homotypic binding Selected signaling complexes involving the DD superfamily Signaling Domains Protein components complexes DISC DD, DED Fas, FADD, caspase-8 or -10 Complex I DD TNFR1, TRADD, RIP, TRAF Complex II DD, DED TRADD, RIP, FADD, caspase-8 or -10 Apoptosome CARD Apaf-1, cytochrome c, ATP/dATP, caspase-9 PIDDosome DD, CARD PIDD, RAIDD, caspase-2 Inflammasome PYD NALP1, ASC, caspase-1, caspase-5 Adapted from Table 1, Park HH et al, AnnuRevImmunol (2007) 25:561–86 Death Domain Family Members  Caspases Initiators – Autocatalytic Executioners – Cleavage DED – Extrinsic CARD – Intrinsic Inflammation Salvesen&Ashkenazi 2011  Intrinsic Apoptotic Signaling Apoptosome  First member: Bcl-2 Bcl-2 protein – B cell lymphoma 2 family – 4 conserved domains (BH1-4) Tait&Green 2010 Mitochondrial Pro-Apoptotic Factors Activated by caspases through tBid Bid is truncated by multiple different proteases including calpains and caspases Bid will sequester anti- apoptotic BCL-2 family members or activate pro- apoptotic members (Bax and Bak) Kilbride&Prehn 2012 Mitochondrial Apoptotic Factors Transmembrane Protein Family Kilbride&Prehn 2012 Apoptosome formation Damaged Mitochondria WD Cyt c APAF-1 ATP APAF-1 oligomerization APAF-1 Cyt c APAF-1 Pro-C9 CARD autocatalysis Caspase-9 STRESS AND DEATH Other Mechanisms Failed Stress Response p53-induced cell death – DNA damage (genotoxic stress) ER stress/UPR Ferroptosis – Halts protein translation and upregulates chaperone expression Calcium signaling Bid and others p53: the anti-oncogene p53 is critical in DNA damage repair – Lack of p53 increases cancer susceptibility – Overexpression promotes aging Transcription factor – Negative regulators of cell cycle progression – Apoptosis promoting genes (Bax, Bak) PUMA – p53 upregulated modulator of apoptosis Balance is necessary – Cell type specific responses PIDDosome PIDD = p53-induced death domain Genotoxic PIDD Stress RIP p53 PIDD RAIDD C2 IKK Also: cell cycle control DNA repair (unique PIDDosomes) NF-κB (cell survival) Intrinsic pathway (through Bid cleavage) Death Domain Family Members ER stress-induced cell death ER stress – accumulation of misfolded proteins Unfolded Protein Response – stress response that promotes degradation of proteins and increased chaperone production to improve folding Not resolved – cell death is induced Related to autophagy-induced cell death Exact pathway still being determined ER participates in death signaling Release of ER calcium can prime mitochondria (intrinsic pathway) ER propagates death-inducing stress signals through Bcl-2 family members Contributes to: Fas-induced cell death (BAP31 cleavage by Caspase 8) p53-induced cell death Cellular Calcium Fig 1. Hajnoczky et al (2003) Cellular calcium overload is a known factor in necrosis Bcl-2 family members affect calcium homeostasis Calcium toxicity: 3 separate points ER calcium depletion induces UPR ER calcium release may activate specific enzymes (precise mechanisms under investigation) o Calpain (protease) – Bid/Bax/Bcl-2 cleavage; Caspase 12 activation o Calcineurin (phosphatase) – Bad dephosphorylation Excessive mitochondrial calcium o Impairs mitochondrial function (depolarization) o Increased ROS generation o Induces release of proapoptotic factors Smac/DIABLO, AIF, cyt c, OMI/HtrA2 Tissue Damage: Calcification Basophilic deposits Dystrophic – normal calcium levels; sites of stress/injury Metastatic – hypercalcemia (usually due to hormonal change) Review Questions 1. Compare and contrast the major morphological characteristics of necrosis and apoptosis. How would you differentiate them in a histopathological slide? 2. Compare and contrast extrinsic and intrinsic apoptotic signaling. How do these pathways interact? 3. What are the advantages and disadvantages of complicated cell death signaling pathways? 4. Compare and contrast the DISC, the apoptosome, and the PIDDosome.  References Book: Cell Death: Apoptosis and Other Means to an End D. R. Green. Cold Spring Harbor Laboratory Press, 2nd ed. 2018 Publications: Breckenridge DG, Germain M, Mathai1 JP, Nguyen M and GC Shore. Regulation of apoptosis by endoplasmic reticulum pathways. Oncogene (2003) 22:8608–8618 [doi: 10.1038/sj.onc.1207108] Hajnoczky G, Davies E and M Madesh. Calcium signaling and apoptosis. Biochem. Biophys. Res. Commun. (2003) 304:445-454 [doi: 10.1016/S0006-291X(03)00616-8] Janssens S, and A Tinel. The PIDDosome, DNA-damage-induced apoptosis and beyond. Cell Death Differ. (2012) 19:13–20 [doi: 10.1038/cdd.2011.162] Kilbride SM and JHM Prehn. Central roles of apoptotic proteins in mitochondrial function. Oncogene (2013) 32:2701-2711 [doi: 10.1038/onc.2012.348] References (cont.) Publications (cont.): Munoz-Pinedo C, Guio-Carrion A, Goldstein JC, Fitzgerald P, Newmeyer DD, and DR Green. Different mitochondrial intermembrane space proteins are released during apoptosis in a manner that is coordinately initiated but can vary in duration. Proc. Natl. Acad. Sci. USA (2006) 103(31):11573–11578 [doi: 10.1073_pnas.0603007103] Park HH, Lo Y-C, Lin S-C, Wang L, Yang JK, and H Wu. The Death Domain Superfamily in Intracellular Signaling of Apoptosis and Inflammation Annu. Rev. Immunol. (2007) 25:561–86 [doi: 10.1146/annurev.immunol.25.022106.141656] Qin S, Yang C, Li S, Xu C, Zhao Y, and H Ren. Smac: Its role in apoptosis induction and use in lung cancer diagnosis and treatment. Cancer Lett. (2012) 318:9–13 [doi: 10.1016/j.canlet.2011.12.024] Van Opdenbosch N, and M Lamkanfi. Caspases in Cell Death, Inflammation, and Disease. Immunity (2019) 50(6):1352-1364 [doi: 10.1016/j.immuni.2019.05.020] INFECTIOUS AND SKIN DISEASES Part 2, Week 1 Mechanisms of Disease Infectious and Skin Diseases Immune Response Basics Meningitis Parasitic diseases o Trichinosis Skin disorders o Psoriasis o Verrucae o Pemphigus Clinical Presentation: Immune Response Inflammation Clinically – redness, swelling, pain Histologically – edema, WBC Edema is mediated by mast cells (immediate) and eosinophils (later response) Pyogenic – pus production (dead bacteria, WBC) Granuloma – macrophages surrounded by T cells Tumor necrosis factor (TNF) is important mediator (cytokine) Response ends when phagocytes clear all antigen; lack of T cell stimulation results in apoptosis Acute v. Chronic Inflammation Acute – Dilation of small blood vessels – Increased microvasculature permeability – Migration and activation of immune cells Chronic – Infiltration by macrophages, lymphocytes, plasma cells – Tissue destruction – Attempts at healing Acute Inflammation: Lymphocytic Infiltration Initial – neutrophils Later - mononuclear Congested blood vessels Robbins Fig. 3.5  Robbins Fig. 3.12 Acute: Serous Inflammation Skin blister  Robbins Fig. 3.13 Exudate: Fibrinous Inflammation F = Fibrin -deposit from exudate due to large vascular leakage P = pericardium  Robbins Fig. 3.14 Acute: Purulent Inflammation Neutrophils and cell debris Congested blood vessels visible nearby  Robbins Fig. 3.17 Tissue level: Chronic Inflammation * Chronic inflammatory cells Tissue destruction Attempted repair Acute for contrast  Robbins Fig. 3.22 Chronic Inflammation: Granulomas Central necrosis Giant cells Epithelioid cells Lymphocytes Summary of Immune Pathology Basics Acute v. Chronic Inflammation – Associated cells – Different types Visible Necrosis – Different types Congested blood vessels Edema Fibrosis Tissue Destruction MENINGITIS Infectious Disease  Meninges 3 layers: 1. Dura mater (tough mother) 1. Outermost covering 2. Dense CT 2. Arachnoid (spider-like) 1. Middle layer 2. Subarachnoid space is fluid filled with many projections 3. Pia mater (tender mother) 1. Innermost covering (immediately next to the nerve tissue) 2. Loose CT and small blood vessels Ross 12.31 Meningitis Bacterial or Viral infection Similar Presentation Acute onset fever Headache Stiff neck Photophobia Confusion Image from WebMD Neuronal Injury Inflammation in the subarachnoid space Substantial infiltration by neutrophils May breach blood-brain barrier and cause localized inflammation in neural tissue Damage to blood vessels can cause hemorrhage into the brain Most damage is due to pressure Image from WebMD  Meningitis Pathogenesis A. Colonization of the Nasopharynx B. Evade Opsonization in the Bloodstream C. CSF access through endothelium of Blood-brain barrier Hammer Fig. 4-7 Meningitis Pathogenesis: Defense Pathogenetic Sequence of Bacterial Neurotropism Neurotropic Stage Host Defense Strategy of Pathogen 1. Colonization or mucosal Secretory IgA IgA protease secretion invasion Ciliary activity Ciliostasis Mucosal Adhesive pili epithelium 2. Intravascular survival Complement Evasion of alternative pathway by polysaccharide capsule 3. Crossing of blood-brain Cerebral Adhesive pili barrier endothelium 4. Survival within CSF Poor opsonic Bacterial replication activity Reproduced, with permission, from Quagliarello V, Scheld WM. Bacterial meningitis: Pathogenesis, pathophysiology, and progress. N Engl J Med. 1992;327:864. Table 4–6 From Hammer’s Pathophysiology of Disease Immunoglobulin A Produced by plasma cells associated with mucosa Epithelium lining oral cavity, nasopharynx, bronchial tubes, gastrointestinal system First line of defense Not an inflammatory Ig; opsonization Primarily acts through exclusion, binding and cross- linking Extensive glycosylation to prevent degradation by proteases Ciliostasis Prevent movement of bacteria out of bronchial tubes Attachment of bacteria to cilia impedes movements Toxins May damage axoneme May deplete ATP Adhesive Pili Some bacteria have projections (pili) that can bind to non-ciliated mucosal cells Once bound, they can cross the epithelium Must also be able to cross the basement membrane Infection occurs most easily in simple epithelia Nasopharynx Intestines Bacterial Toxins Exotoxins (excreted by cell) Highly antigenic (antitoxin neutralizes) Highly toxic (fatal in microgram quantities) Usually do not induce fever Usually bind to specific receptors Endotoxins (part of cell wall) Weakly immunogenic Toxic at 10-100’s micrograms Induce fever No specific receptors PARASITIC DISEASES Parasitic Diseases Can be caused by single-celled and multicellular organisms Damage can be caused by consumption by parasite e.g. hookworms consume blood; can cause anemia Damage may actually result from immune response Trichinosis Trichinella Spiralis (nematode) Obtained by ingestion of undercooked meat, usually pork Infects skeletal muscle Symptoms Fever Myalgia Periorbital edema Life cycle of Trichinella spiralis 1. Adult in intestines produce larva 2. Larva infiltrate blood 3. Exit blood vessels in skeletal muscle 4. Infect muscle fibers 5. Adults die and muscle fiber calcifies Fig. 3 from Mitreva and Jasmer (2006) Enteric Phase Strong immune response to larvae T helper cells produce cytokines Eosinophil and Mast cell activation Increased intestinal mobility T helper cytokines Mast cell granules Expel larvae from gut in animal models Inflammatory response to larvae elsewhere can cause widespread destruction Muscle Phase Muscle cell is co-opted as a nurse Disruption of myofibrils Enlarged/central Robbins & Cotran Fig 8-56 nuclei Collagen capsule formation Clinical Presentation Enteric Stage Typical of enteric disease Diarrhea and nausea Vomiting, pain, low grade fever Muscle Stage Typical of infection/muscle damage Myalgia and paralysis Fever, headache, skin rash Edema and conjunctivitis INTEGUMENTARY DISORDERS Disorders of the Skin Two Types: Growths and Rashes Growths Cyst Malformation Benign/malignant neoplasm Dermatitis (rashes) Non-neoplastic PSORIASIS  Psoriasis Inflammatory skin disease Scaling skin condition Robbins & Cotran Fig. 25-26A Pathology of Psoriasis Thickened epidermis Elongated rete ridges Neutrophil infiltration Hammer Fig 8-10 Excessive epidermal proliferation Shortened cell cycle 2X proliferative population Accumulation of nucleated cells in the stratum corneum (parakeratosis) Endothelial cell proliferation Pathogenesis of Psoriasis Immunologic abnormalities T helper lymphocytes (MHCs) Cytokine overexpression (TNF, IFNγ,IL-2) Presence of unique dendritic cells Precise mechanism unknown Antigen unknown Genetic link to HLA-C Sensitized T cells accumulate in epidermis (IFNγ) Also induced by localized trauma Angiogenesis Angiogenic factors found in psoriatic lesions TNFα TGFβ IL8 VEGF Released from keratinocytes Stimulate epidermal hyperplasia, vascular growth, leukocyte infiltration Regulates psoriatic keratinocyte activity VERRUCAE Verrucae (warts) Squamoproliferative proliferation of squamous cells Caused by human papillomaviruses Generally regress (self-limited) Virus transmitted by contact Viral typing can confirm if problematic infection (poor prognosis -> cancer)  Robbins & Cotran Fig 25-38 Verruca Pathology Epidermal Hyperplasia is uneven (verrucous or papillomatous)  Robbins & Cotran Fig 25-38 Verruca Pathology Cytoplasmic vacuolization (halos) Increased keratohyalin granules Eosinophilic keratin aggregates in cells  Robbins & Cotran Fig 25-38 Verruca Development IHC reveals HPV viral proteins in keratinocytes E6 of HPV may interfere with maturation PEMPHIGUS Blisters Acantholysis Dissolution of intercellular bridges Which ones determine where blister forms Robbins & Cotran Fig 25-27  Robbins & Cotran Fig 25-28 Pemphigus Autoimmune formation of blisters Autoantibodies attack the intercellular junctions In epidermis In mucosa  Robbins & Cotran Fig 25-32 Types of pemphigus Foliaceus o subcorneal lesion Vulgaris o suprabasal lesion Bullous Pemphigoid Eosinophils o subepidermal, nonacantholytic lesion Lymphocytes Occasional neutrophil  Autoantibodies attack junctions TEM: BP antigen (BPAG) associates with hemidesmosome (HD) in base of IHC for IgG in Bullous Pemphigoid keratinocyte LL: lamina lucida AF: anchoring fibrils LD lamina densa Robbins & Cotran Fig 25-33 Review Questions 1. What is the benefit of homotypic binding in DD family members? Why are there such a large number of them (think of p53 signaling)? 2. Consider the benefits v. the costs of the immune response. 3. What is the purpose of the meninges? How does this have a negative impact in meningitis? 4. Why are early psoriatic plaques characterized by redness and swelling? 5. Compare and contrast blisters with warts. References Murray: Medical Microbiology (eBook) Chapter 14: Mechanisms of Bacterial Pathogenesis Ross: Histology (eBook) Chapter 12: Nerve System Hammer & McPhee: Pathophysiology of Disease (eBook) Chapter 4: Infectious Diseases Mitreva M and DP Jasmer. Biology and genome of Trichinella spiralis. WormBook (2006) 23:1-21. http://www.wormbook.org/chapters/www_genomesTrichinella/genomesTrichinella.html Coimbra S, Figueiredo A, Castro1 E, Rocha-Pereira P, and A Santos-Silva. The roles of cells and cytokines in the pathogenesis of psoriasis. Int. J. Dermatol. (2012) 51:389–398 [doi:10.1111/j.1365-4632.2011.05154.x] Next: Week 2 Genetic Diseases Basic Pathology of Genetic Diseases Chromosomal Abnormalities Complex Multigenic Disorders Monogenic Genetic Disorders