Sub-cellular Pathology

CELL ORGANELLES AND DISEASE

Fiona McKie-BellBOD

Subcellular Pathology

- ‘Identification of the primary lesion in any pathological process in terms of the organelles and metabolic processes that are involved’

Contents

• Endocytosis and lysosomes• Organelle disorders• Named diseases• Exam questions

Disorders• PM

– Permeability– Enzymes– Cytolysins– C’– Inherited Disorders

• Nucleus– Chromosomal

abnormalities– Single gene defects– Toxic damage– Nutritional deficiencies

• Mitochondrion• Endoplasmic reticulum• Cytoskeleton• Lysosomes

– General sig. In pathology– Resistance– Inappropriate cellular release

(intra and extra)– Storage diseases

Differential Centrifugation

•Involve stepwise increases in the speed of centrifugation.

•At each step, more dense particles are separated from less dense particles

•Successive speed of centrifugation is increased until the target particle is pelleted out.

The Plasma Membrane

• 'Fluid mosaic model’• Chemically distinct from most other membranes

in the cell– contains more sphingomyelin and cholesterol.

• Many extrinsic proteins have carbohydrate moieties attached to them– important in determining antigenicity of cells e.g.

blood group antigens

Structure of a typical cell membrane Figure 10.10: (Mathews, Van Holde Biochem 3rd ed)

Effects on Permeability of the Plasma Membrane

– Affect the sodium/potassium ATPase (sodium pump)• Maintains electrolyte balance

– Ouabain (a plant glycoside) Digitalis (a plant steroid), Tetrodotoxin, (puffer fish) Saxitoxin (microscopic algae - 'the red tide')

• Can be used in the treatment of cardiac conditions– Increase the force of contraction of heart muscle by altering the

excitability of the tissue (a function of the sodium and potassium concentrations across the membrane).

– http://highered.mcgraw-hill.com/sites/0072495855/student_view0/chapter2/animation__how_the_sodium_potassium_pump_works.html

Effects on Permeability of the Plasma Membrane

– Binds to the sodium channel in the plasma membrane and blocks ion movement.

– Tetrodotoxin - neurotoxin found in some organs of the puffer fish

– Saxitoxin is produced by microscopic algae ('the red tide')

• One of only two naturally occurring toxins to be classified as a Schedule 1 Chemical Warfare agent

...and the waters that were in the river were turned to blood. And the fish that were in the river died; and the river stank and the Egyptians could not drink of the water of the river... Exodus 7: 20-21

Chemical & biological warfare

• 1950s – US experiments with saxitoxin as chemical weapon

• 1000x more toxic than the nerve gas, sarin• Used as suicide pill for U-2 spyplane pilots• 1970 – Richard Nixon ordered destruction of all

stocks• Now used in research into sodium ion channels

and nervous disorders

Effects of Enzymes on the Plasma Membrane - Antigenicity

• Affecting antigenicity of cells by removing components from the extracellular surface of the plasma membrane e.g. – Neuraminidase (removes carbohydrate sialic acid

groups) • Influenza (H1N1) Haemaggluttinin, Neuramaminidase -plays its

major role after virus leaves an infected cell - ensures virus doesn't get stuck on the cell surface by clipping off the ends of these polysaccharide chains.

– Trypsin (removes glycopeptides).

Effects of enzymes on the plasma membrane - Lysis

• Phospholipases – Hydrolyse phospholipid components of the plasma

membrane– Lead to the formation of lysophosphatides (very

potent surfactant molecules)– Cell lysis occurs– Found in snake venoms, some cytolytic pathogenic

bacteria (e.g.Clostridium perfringens.)

• Cytolysin e.g. Streptolysin O – toxin produced by Streptococci – A cholesterol-dependent cytolysin (CDC)– causes cell lysis by interacting with the cholesterol of

the host plasma membrane, disrupting the membrane and forming a large pore

Membrane Phospholipids

Sphingosine – alcohol amine

Action of Phospholipases

Found inSnake Venoms

Complement-related membrane injury

• When complement becomes bound to a cell surface, its activation leads to the formation of a 'membrane attack complex'

• Causes lysis of the cell – e.g. red cells in

incompatible blood transfusions

Inherited membrane disorders

• Familial Hypocholesterolaemia (see later)• Hartnup Disease

– defect in the transport proteins for uptake of the essential amino acid tryptophan by intestinal epithelial cells.

– Trp is an important precursor of the vitamin nicotinic acid

• clinical signs of Hartnup disease are similar to those of the vitamin deficiency disease, pellegra (3Ds)

• (Dementia, Dermatitis, and Diarrhoea)

Cystic Fibrosis

• Due to an inherited defect in the cystic fibrosis trans-membrane conductance regulator (CFTR)

• CFTR is involved in chloride ion transport across the plasma membrane

• Important in creating sweat, mucus and digestive juices.

• Multiple theories on Cl transport changes and symptoms

Damage to the plasma membrane - diagnostic aspects

• Changes in the permeability of the plasma membrane can be detected by – histochemical methods– biochemical methods

• very useful in the diagnosis of certain diseases

The Nucleus

• Contains the genetic material of the cell

• DNA can be affected in a number of ways.– Chromosomal abnormalities– Single gene defects– Toxic damage– Nutritional deficiencies

Chromosomal abnormalities

• Alteration in the diploid number– e.g. Down syndrome (trisomy 21) - congenital

abnormalities

• Chromosomal breakages– e.g. ataxia-telangiectasia syndrome– ATM Gene regulates cell cycle checkpoints, repair dsDNA, regulating

p53, BRCA1 and CHEK2, telomere repair– Symptoms

• Dysfunction of the cerebellum• ataxia (unsteadiness or incoordination of limbs, posture, and

gait)• telangiectasia of the eyes and skin (a complex of abnormally

and permanently dilated blood vessels)• severe immunological deficiency• ‘radiosensitivity’ leading to leukaemia

Down’s SyndromeTrisomy 21

Single Gene Defects

• No obvious morphological abnormality in the nucleus or chromosomes

• Mutation (even single aa change)– e.g. PKU, SCD (val-glu sub.)

Toxic Damage

• Chemical carcinogens– e.g. alkylating agents such as the nitrogen

mustards

• Cancer chemotherapy– e.g. 5-fluorouracil, methotrexate - interfere

with synthesis of DNA precursor– vincristine - damages the mitotic spindle (not

DNA)

Nutritional Damage

• Folic acid or Vitamin B12– deficiencies of these vitamins affect DNA

synthesis• result in large nuclei, but with less DNA content

than optimal for mitosis• mainly evident in red cell precursor cells in bone

marrow

The Mitochondrion

• Prime target in cellular damage caused by hypoxia

• Affected by ‘uncouplers' of electron transport such as dinitrophenol

• The enzymes of the electron transport chain are inhibited by poisons such as rotenone and cyanide

Mitochondrial diseases• Can affect any organ at any age

• Severely debilitating and often fatal

• In USA, 50 million adults suffer from diseases involving mitochondrial dysfunction– Cancer, infertility, diabetes, heart disease, blindness,

deafness, kidney disease, liver disease, stroke, migraine

– Ageing, Parkinson’s disease, Alzheimer’s disease

• Of 4 million children born in US per year, 4,000 develop mitochondrial disease

• Many diseases still being discovered

• No cure

Mitochondrial DNA

• The mitochondrion is the only organelle apart from the nucleus that contains DNA

• Circular DNA (linear in nucleus) • Mitochondrial DNA maternal inheritance

– important implications in genetic analysis involving family histories

Disorders of mitDNA

• Some inherited diseases are due to defects in genes in mitochondrial DNA

• e.g. Leber's Hereditary Optic Neuropathy (LHON). – Rare disease - results in blindness (by 15-35 years)– Due to defect in the NADH dehydrogenase (in the

electron transport chain). – Reduced ATP formation.

LHON

• Rare disorder - maternal transmission• Males never transmit LHON to offspring• Females less frequently affected than males• Results in degeneration of the optic nerve

– (nerves very dependent on ox phos)

• Main mutation in the NADH dehydrogenase gene is at bp11778

• his - arg mutation in the enzyme

The Endoplasmic Reticulum

• Smooth endoplasmic reticulum is the site of cytochrome P450 mixed function oxidase (CYP)– enzyme essential in the detoxification of many

substances.

• Exposure of liver cells to certain drugs can result in proliferation of the SER

• This can have serious consequences for individuals who are taking more than one drug.

Induction of CYP

• CYP in the SER can be induced by several substances– ethanol, acetone, polycyclic hydrocarbons, and

phenobarbital

• May give rise to an increased rate of hydroxylation and subsequent excretion of therapeutic drugs

• Induction of CYP by one drug may stimulate the metabolism of other drugs that are also substrates for the enzyme

Example 1

• Rifampicin (anti-tuberculosis agent) – induces CYP– Increases clearance of oral contraceptives

• Increased incidence of pregnancy in women given both drugs.

Example 2

• Phenobarbital (a barbiturate) induces CYP• Warfarin (anticoagulant) require caution with

other meds.• V sensitive to being metabolised rapidly by

induced CYP• If CYP- Inducing drugs are withdrawn, CYP

levels fall• If the warfarin dose is not also reduced

– Haemorrhage– In some cases DEATH!

The Cytoskeleton

• Microtubules (20 – 25 nm diameter)

• Actin filaments (6 – 8 nm)

• Myosin filaments (15 nm)

• Intermediate filaments (10 nm)

Cytoskeletal Lesions

• Defects in proteins of the cytosleleton affect -• Cell locomotion

– e.g. leukocyte migration– Phagocytosis– sperm motility (leading to infertility)

• Movement of intracellular organelles – e.g. Chediak-Higashi syndrome, in which there is

impaired fusion of lysosomes with phagosomes containing bacteria.

Accumulations/Disorders of cytoskeletal proteins

• Alzheimer’s disease– neurofibrillary tangle - composed of

neurofilaments and microtubular proteins• Mallory body is characteristic of Alcoholic Liver

Disease. – composed of intermediary filaments (mainly

prekeratin)• DMD

– Gene (1987) ‘Dystrophin’ – Structurally related to spectrin– Plays key role in anchoring cytoskeleton to

extracellular matrix– Such anchorage may enable the plasma

membrane to withstand the stress of muscle contraction

LYSOSOMES• Structure and formation• Enzymes• Mechanisms

– Heterophagy– Exocytosis– Autolysis– Crinophagy

• Sig. in pathology– Toxin– Micro-organisms– Inappropriate release and disease states– Storage disorders

See also lysosome slides

Lysosomes• Vesicle surrounded by single membrane

– Distinct from mitochondria and microbodies

• Isolated by differential centrifugation• Over 60 hydrolytic enzymes (acid pH optima)

known to be lysosomal in origin. Include –– Peptide hydrolases– Glycosidases– Nucleases– Phospholipases– Phosphatases– Sulphatases

Lysosomal enzymes

• Most are glycoproteins with acid pH optim– Some (e.g. cathepsin G, and elastase) are active at

neutral pH

• The bacteriocidal enzymes lysosyme and myeloperoxidase are found in polymorphonuclear leucocytes

• Lysosomes - capable of degrading almost any material of biological origin

Lysosomal membrane

• Contains cholesterol and sphingomyelin

• Also present in the plasma membrane– but not to any extent in other membranes in

the cell

• Reflects exchange of material between the lysosomal amd plasma membranes during endocytosis and exocytosis

Origin of lysosomes

• Synthesis of lysosomal protein takes place in the rough endoplasmic reticulum

• The enzymes are packages into vesicles by the Golgi apparatus and become PRIMARY LYSOSOMES

• Primary lysosomes fuse with vesicles of various origin to become SECONDARY LYSOSOMES

Secondary lysosomes

• Fusion of a primary lysosome with a vesicle containing material to be digested means that the hydrolytic enzymes are not released into the cytoplasm (where they could cause damage)

• Secondary lysosomes have a heterogeneous appearance because of the variety of material contained in endocytotic vesicles or phagosomes

Mechanisms involving lysosomes

• Heterophagy– Breakdown of extracellular material taken up by

endocytosis or phagocytosis

• Exocytosis– Release of the contents of an intracellular vesicle

without loss of cytoplasm (reversal of endocytosis)

• Autophagy– Digestion of intracellular material (cytoplasm and

organelles) within a secondary lysosome

• Crinophagy– Disposal of excess secretory proteins

Importance of Lysosomes in Pathology

• Lysosomes have particular importance in pathology because they are:– involved in reactions to toxic substances– involved in digestion of foreign

microorganisms– contain many hydrolytic enzymes,which, if

released inappropriately may cause both intracellular and extracellular damage

1. Autolysis of Tissues

• a) Programmed Cell Death (Apoptosis)– interdigital cell death in foetus - resulting in 5

fingers– involution of the uterus and ovaries post

menopause– atrophy of breast tissue following weaning

Role of Lysosomes in Autolysis

• b) Cell Death• Autolysis = digestion of cell contents• Stages

– rupture of lysosomes (as a secondary response to the primary injury)

– release of hydrolytic enzymes into the cytosol– digestion of intracellular material

2. Turnover of Cell and Tissue Components

• Turnover = constant synthesis and degradation of tissue components

• Circumstantial evidence that lysosomes may be involved in turnover – The necessary degradative enzymes are

present– autophagy can be observed.

Lysosomes in Turnover?

• Some observations do not support lysosomal involvement in turnover – i.e. most proteins have different turnover rates

and it is difficult to account for this if there is random uptake of proteins by autophagy.

3. Heterophagy

• Heterophagy = Intracellular digestion of potentially pathogenic microorganisms

• Bacteria, viruses, and fungi are ingested by macrophages and neutrophils.

Lysosomes in Neutrophils

• The neutrophil is packed with large numbers of lysosomal granules– rich in enzymes capable of digesting both cells and

extracellular matrix materials.

• Neurophils can actively digest pathogens, which are then destroyed-– by the generation of FRs and hypochlorous acid– by lysosomal enzymes.

Neutrophils

• Neutrophils have membrane receptors – for the Fc portion of antibodies– for complement components bound to foreign

particles– to bacterial polysaccharides.

• Foreign material is targeted• Neutrophils do not take up material to which they

do not bind. • Phagosomes (containing internalised material)

fuse with primary lysosomes, and digestion of the foreign material ensues

Resistance to lysosomal digestion

• In some bacterial infections, the lysosomal system is the vehicle by which the infection is propagated.

• The bacteria resistant to lysosomal digestion include: • Mycobacterium tuberculosis, M. leprae, M.Brucella• Legionella pneumophilia• Salmonella typhi

• Listeria monocytogenes• Shigella flexneri

• These microorganisms can survive and multiply in the lysosomes of phagocytic cells, and as a result are spread throughout the body (and may produce toxins).

Mechanisms of resistance

• M.tuberculosis, M.leprae, Legionnela pneumophilia– Inhibit fusion of phagosome with primary lysosome

• Salmonella typhimurium– Cell wall resists digestion by lysosomal enzymes

• Listeria monocytogenes, Shigella flexneri– Lyse the phagosome membrane

4. Intracellular release of hydrolases producing cellular damage

• This occurs in mineral-induced lung disease (pneumoconiosis) such as silicosis and asbestosis.

Pneumoconiosis

• Arises from exposure to several minerals. – Coal dust

• relatively inert• large amounts must be deposited in the lungs

before the onset of disease.

– Silica and asbestos are more reactive and result in fibrosis of the lung at lower levels of exposure.

Silicosis

• Silica makes up the greater part of the earth's crust.

• Various forms– sand

• too coarse to pass into the periphery of the lung• represents little danger to humans

– silica dust (2-3m diameter) is very dangerous

High risk occupations

• Mining, quarrying, tunnelling, sandblasting, etc.

• About 10 years exposure required for silicosis to occur.

• In high risk occupations, 10 - 15% are affected after 30 years.

Cause of silicosis

• Most inhaled dust is removed by entrapment in mucus

• Followed by expulsion from lung through ciliary movement

• If some dust reaches the alveolar duct bifurcations - taken up by macrophages and accumulates

Involvement of macrophages

• Uptake by macrophages is followed by – an inflammatory response– fibroblast proliferation– collagen deposition (fibrosis).

Involvement of lysosomes

• Inhaled particles that are taken up by alveolar macrophages enter the lysosomal system.

• Interactions between the silica particles and the lysosomal membrane (hydrogen bonding??) results in – disruption of the lysosome– release of hydrolytic enzymes

Cycle of damage

• Release of hydrolytic lysosomal enzymes results in– death of the macrophage– re-release of the silica particles,

• The particles are in turn taken up by other macrophages.

• This results in another round of damage

Lung damage and fibrosis

• Thus a vicious cycle is set up resulting– indirect lung damage through lysosomal

enzyme activity and Free Radicals – fibrosis.

• The lung tissue becomes less elastic and this impairs gaseous exchange.

5. Extracellular Release of Hydrolases Producing Damage to Connective Tissue

Matrix

• Rheumatoid arthritis (RA)

• RA is an autoimmune disease that causes chronic inflammation of connective tissue, primarily in the joints

Rheumatoid arthritis (RA)

• The first tissue to be affected in RA is the synovial membrane which lines the joint cavity.

• Eventually, inflammation may spread to – the articular cartilage– the fibrous joint capsule– the surrounding tendons and ligaments

• This causes pain, deformity, and loss of function.

Incidence of RA

• RA affects 1 - 2% of adults

• Like most autoimmune diseases, is most frequent in women (3:1).

Aetiology of RA

• Despite intensive research. the cause of RA remains obscure.

• It is probably a combination of genetic, environmental, hormonal, and reproductive factors.

Leukocyte infiltration

• The synovial fluid may become turbid due to the number of cells neutrophils and macrophages present

• These phagocytes ingest the immune complexes

• In the process of digestion, hydrolytic enzymes are released

• These enzymes degrade synovial tissue• Blood vessels become occluded

Involvement of lysosomes

• Occlusion of blood vessels decreases blood flow to the synovial tissue

• This results in hypoxia and metabolic acidosis. • The reduction in pH results in the release of

hydrolytic lysosomal enzymes from the synovial cells into the surrounding tissue

• This initiates erosion of the articular cartilage.

Evidence for involvement of lysosomes in RA

• Lysosomal enzymes can be detected in synovial fluid

• RA-like symptoms can be produced if lysosomal enzymes are injected into joints

• Vitamin A renders lysosomal membranes unstable. Rabbits injected with Vit.A lose their ability to prick up their ears due to loss of proteoglycan from the ear cartilage.

Evidence for involvement of lysosomes in RA

• Digestion of limb bone cartilage in tissue culture can be inhibited by an antibody to Cathepsin D (a lysosomal protease)

• Several agents used in therapy of RA (e.g. gold, hydrocortisone) are known to have direct effects on the function and stability of the lysosomal membrane.

6. Lysosomal Storage Diseases

• Pompes disease– A glycogen storage disease

• Due to inherited deficiency of 1-4 glucosidase (acid maltase).

– Glycogen that has been ingested by autophagy cannot be degraded, and accumulates in the lysosomes.

– Cell death and degeneration of tissue occurs due to release of hydrolytic enzymes from lysosomes that have disrupted due to overloading

Lysosomal storage diseases

• Tay-Sachs Disease– absence of -N-hexosaminidase involved in

glycolipid metabolism

• Metachromatic Leukodystrophy– absence of arylsulphatase A, leading to

accumulation of sulphatides in the brain

Gaucher’s Disease

• The most common lysosomal storage disease• Failure to degrade a substance normally subject

to lysosomal digestion• Results in an accumulation of substance• Increase in the size and number of lysosomes• Leads to cellular malfunction and effects on

organs

Gaucher’s disease

• Incidence = 1:2,500• Three types

– Type 1 (most common- found mainly in Jews)• Spleen enlargement• Bone lesions• CNS not involved• No serious symptoms and normal lifespan

– Type II• Extensive CNS involvement and early death

– Type III• Intermediate between I & II• Dementia and spasticity by 10 years

Cause of Gaucher’s disease

• Deficiency of glucocerebrosidase• Degrades the glycolipid glucocerebroside to

glucose and the lipid, ceramide

• Gene cloned in 1985– 30 different mutations – Severity predicted by nature of the mutation

• Ser for asn Type I• Pro for leu Type II or III

Type I

• Macrophages are the only cells affected

• Macrophages in human spleen and liver degrade 1011 red blood cells per day

• In Gaucher’s disease the greatest effect of undigested glucocerebroside is seen in these tissues

Treatment

• Enzyme replacement therapy– Often impractical because of problems with

impermeability of cell membranes to extracellular protein

• Feasible in GD because macrophages can take up exogenous protein (e.g. glucocerebrosidase from placenta)– If the enzyme is modified to expose mannose

residues, this increases uptake (macrophages recognise mannose)

Treatment

• Treatment of GD with placental enzyme costs £250,000 per annum

• Costs may fall if genetically engineered enzyme becomes available

• Gene therapy is a possibility