LAQ questions Flashcards
Neurodegenerative diseases (Alzheimers)
Common features
pathological hallmarks
amyloid hypothesis
tau hypothesis
risk factors
Neurodegenerative diseases are highly heterogeneous : umbrella term- different phenotypes have different causes. Pleiotropic- different symptoms experienced by different people
Common features:
Impairment somewhere in the cell
Decreased transmission at the synapse
Dying back of neurites
Cell death
Alzheimers is the most common neurodegenerative disease and the most common cause of dementia. Onset is usually 65>. Not a normal part of ageing, it is a disease.
The pathological hallmarks of alzheimer’s disease is:
Brain shrinkage
Proteinopathies- aggregation of proteins
Amyloid plaque- extracellular protein aggregates- enriched in AB peptides
Neurofibrillary tangles- intracellular protein aggregates- enriched in tau protein
AB is cleaved from a transmembrane protein called amyloid beta precursor protein (APP) by proteases.
Formation of amyloid plaques - cleavage by beta secretase , -cleavage by gamma secretase, -amyloid beta fragments are released and accumulate to form amyloid plaques outside the cell.
Amyloid hypothesis for alzheimer’s is that mutations in 3 proteins that are important in the formation of AB peptide cause rare early onset forms of alzheimers: APP, PSEN1, PSEN2 (the last 2 are components of gamma secretase)
Tau normally binds microtubules on axons
Hyperphosphorylated tau is displaced causing tangles and destabilised microtubules.
Importance of microtubules in neurites- shape and structure of cell, - positioning of organelles, - transporting vesicular cargo
Tau hypothesis for alzheimer’s is that in typical late onset of AD, neurofibrillary tangles are seen before amyloid plaques and well correlated with cell death and progression. (Suggests Tau is upstream of AB)
Risk factors for AD:
Downs syndrome (APP is present on chromosome 21)
gender (female)
Smoking
Head injury
Lower education
Neurodegenerative diseases (Parkinsons)
Parkinsons is a movement disorder with symptoms such as:
Resting tremor
Bradykinesia (slow movement)
Rigidity
Postural instability (falling over)
Non motor symptoms such as depression and anxiety, sleep disorders, loss of smell
Pathological hallmarks of parkinsons are:
Loss of dopaminergic neurons of the substantia nigra
Proteinopathy - lewy bodies
Intracellular protein aggregates
Enriched in α-synuclein protein- involved in neurotransmitter release
Lewy bodies not pathogenic but increased α-synuclein is
Categories of genes that cause parkinson’s disease:
Early onset recessive mitochondrial conditions
Mitochondria have a finite lifespan due to oxidative stress
Damaged mitochondria removed by mitophagy- autophagy of mitochondria
Loss of function mutations in 2 proteins central to activating mitophagy- PINK1 and Parkin- cause early onset PD
Late onset- autosomal dominant PD
Some genetic causes found but more limited:
SNCA (α-synuclein) gene amplification- (confirms this is pathogenic)
LRRK2 gain of function
VPS35 gain of function
GBA loss of function
Mutations that cause “PD-plus” conditions
GBA is linked to α-synuclein because GBA encodes GCase which is a lysosomal enzyme. Α-synuclein is degraded in the lysosome.
In a healthy person- GCase goes into lysosomes- degrades α-synuclein- there is no accumulation, no lewy bodies, no PD
In PD: There is a GCase mutation- less GCase- impaired lysosomes- can’t break down α-synuclein- accumulating
Also if there is an increased α-synuclein, thes excess inhibits GCase into lysosomes, so there is no breakdown.
MAPT (gene coding for lau protein) has links to PD
Neurofibrillary tangles can be found in PD brains (even in same cells as Lewy bodies)
Risk factors of PD:
Gender (men)
Red hair (~2x risk)
Head injury
Not smoking, not consuming caffeine
Herbicides, pesticides
Apoptosis
Apoptosis can be defined as “a selective process for the deletion of superfluous, infected or transformed cells”. It is irreversible. There are 2 types of apoptosis: intrinsic (caused by DNA damage (via p53 dependent pathway), interruption of the cell cycle, inhibition of protein synthesis and viral infection (once virus is in the cell) and extrinsic (caused by withdrawal of survival factors, extracellular signals like TNF).
Caspases are used- these function by forming an activation cascade where they cleave the next to activate it so there is signal amplification.
Inactive procaspase Y is cleaved in 2 sites and will release 2 subunits (a large and a small) and a prodomain. The 2 subunits will dimerise to form the active caspase.
The extrinsic pathway is induced by ligand binding to receptors= receptor dimer or multimer-isation of the receptor leading to activation of the caspase.
Death ligands such as Fas ligand (FasL) or tumour necrosis factor (TNF) bind to their respective death receptors (Fas, TNFR) on the cell surface. This allows for similar domains to dimerise/ multimerise in the receptor.
in this case the death domain is called FADD and it dimerises with TNFR death domains
the death effector domains will then dimerise with death effector domains on procaspase 8
this complex is called DISC
The intrinsic pathway is induced by cytochrome c released from mitochondria.
Cytochrome C binds to its binding site on APAF-1. Which then dimerises with procaspase-9 on the CARD domain.
There are pro (Bax) and anti (BCL-2) apoptotic BCL-2 proteins which either suppress or facilitate cytochrome c release.
Necrosis
Necrosis is the removal of damaged cells from an organism. Failure to do so could lead to chronic inflammation. It is caused by a lack of blood supply to the affected area due to injury, infection, cancer, inflammation. The higher the distance from a blood vessel, the lower the pH and O2.
In a necrotic cell when there is a lack of O2, this prevents the production of ATP causing the cell to swell due to water influx. This is because the ion channels require ATP to function, and so without it osmolarity changes.
A change in osmolarity causes lysosomes to rupture releasing enzymes which degrade organelles and destroy nuclear material.
The cell is irreversibly damaged when if it were to be reoxygenated, it would not be able to produce ATP again.
The nucleus of a necrotic cell would look like:
Chromatin condensation
Fragmentation of nucleus
Dissolution of chromatin by DNAse enzyme
The chromosome would look like
Becomes more opaque as there is protein denaturation and aggregation like when you fry an egg white
Cell liquefies- liquefactive necrosis
Difference between necrosis and apoptosis
The difference between necrosis and apoptosis is that necrosis affects a whole group of cells, whereas apoptosis is more specific and affects 1 or a few cells. Another difference is that apoptosis is irreversible and is energy dependent (requires ATP), whereas in necrosis, the initial steps are reversible. Cell size increases in necrosis and decreases in apoptosis due to disassembly of cytoskeleton.
What are cell cultures?
Advantages of cell cultures.
Differences between the 2 types.
Producing primary tissue cell.
Disadvantage of primary tissue cell.
Immortalised cell lines characteristics.
Producing cell lines
Cell cultures- laboratory technique where cells are grown under controlled conditions outside of their natural environment.
Advantages of cell cultures
Control of physio-chemical (pH, temperature) and physiological conditions (nutrients, hormones)
Control of micro- environments of the cell (cell-cell interactions)
Cells can be stored in liquid nitrogen for long periods (cryopreservation)
Cells can be easily quantified
Reduces animals in scientific experiments
Cheaper to maintain
Differences between primary tissue cells and immortal cell lines:
Primary tissue cells are taken from a tissue directly from patient (e.g. biopsy from a patients tumour), They are good for personalised medicine
They are not immortalised
They are ready to use
Finite lifespan (6-7 divisions)
Immortal cell lines
Derived from primary tissue cells
They are immortal
They are less heterogeneous because they are derived from 1 cell
To isolate 1 type of cell from a biopsy of a tissue in primary tissue cells:
Cells are allowed to migrate out of an explant- this allows for the maintenance of cell morphology
Mechanical- mincing, pipetting, sieving and/or Enzymatic dissociation- trypsin, collagenase protease, DNAse
Disadvantages of primary tissue cells:
Inter-patient variation
Limited number collected and limited lifespan
Hard to maintain
Difficult molecular manipulation
Phenotypic instability
Variable contamination
Immortalised cell lines characteristics
They may still have a limited number of cell divisions (around 30)
Phenotypically stable
Good reproducibility
Are easy to grow
Producing cell lines:
There are 2 main methods:
-Isolating the cells from a cancerous tissue like HeLa cells that are from cervical cancer
-Immortalisation of healthy primary cells usually through genetic manipulation (The main genetic targets are p53, pRB and telomerase (expression of TERT))
A cell line might die after a certain number of divisions due to telomeres shortening, after a critical value when it is too short, the cell will apoptose with the use of p53 and pRB.
We may inhibit p53 and pRB by using oncoviruses such as HPV and SV40, which have viral oncoproteins that target p53 and pRB.
The mechanism of action of SV40 is that viral oncoproteins produce a large T antigen and a small T antigen. The large T antigen targets p53 and pRb (binds to the DNA)
The mechanism of action of HPV is that viral oncoproteins produce E6 and E7. E6 targets p53 and degrades it. E7 binds to pRb and inactivates it.
Some cells need both the introduction of the telomerase gene and inactivation of pRB/p53 for immortalisation.
Telomerase insertion into cell lines- circular plasmid taken, selection marker neomycin gene selected, growth promoting factor is selected, transfection, treated with neomycin as a selection pressure, only the colonies that survive are selected- these grow with the telomerase.
HeLa are cells taken from a cervical tumour and produce an endless amount of identical cells. These cells are immortal. Usually after multiple divisions, cell lines tend to apoptose (multiple possible mechanisms inc telomerase)
Difference between 2D and 3D cells.
Explain further about 3D cell cultures.
Most common in the lab e.g.
2D
Forced apical-basal polarity
High stiffness
Results not relevant to human physiology
Limited communication between cells
Simple
Affordable
3D
More relevant to human physiology
Adhesion in all 3 dimensions
Variable stiffness
No forced polarity
More complex
Added expense
Spheroids are:
Generated from cell lines
A 3D cellular aggregate
Usually homogeneous
Cannot differentiate
Organoids are:
Generated from primary tissue cells
Derived from stem cells or progenitor cells
Contain multiple cells types
Cells organised into differential cell types- resemble their in vivo counterpart
Organoids can be used in the lab: By taking a biopsy, grow then in 3D to form organoids, treat them with different drugs- check which are most affected, make treatment decisions
Describe Cell transfection
Cell transfection - introduction of dna material into a eukaryotic cell using non viral techniques such as physical (electroporation and nucleofection) and chemical (lipofection)
Electroporation- an electric field is applied to the cells of interest in order to increase their permeability, this allows material to be inserted into the cell.
Lipofection - transfection technique where dna material is introduced in the cell via liposomes. Liposomes fuse easily with the cell membrane because they are made from phospholipids
Nucelofection - combination of both lipo and electro. Method is secret and patented
Cancer, what is it?
Proto-oncogenes, tumour suppressor genes
5 models
Cancer is a disease of the genome at the cellular level. It is characterised by:
Abnormal cell proliferation
Tumour formation
Invasion of neighbouring normal tissue
Metastasis to form new tumours at distant sites
Some hallmarks of cancer are:
Evading growth suppressors
Induction of angiogenesis
Avoiding immune destruction
Resisting cell death
Genome instability and mutation
The 3 causes of cancer are:
Infectious agents (viruses e.g. HPV, EBV, HBV)
External carcinogens (smoking, uv radiation, heavy metals)
Genetics (chromosomal alterations, aneuploidy)
Multistep process of carcinogenesis is initiation, promotion and progression
Proto-oncogenes- normal genes that can be activated to become oncogenic
Oncogene- a proto-oncogene that has mutated in a way that leads to signals that cause uncontrolled growth.
Tumour suppressor genes- inhibit both growth and tumour formation
5 models of cancer:
1- mutational- (chemical, physical (radiation), viral, genetic (inherited germline mutation))
2- genome instability
3- non genotoxic (epigenetic modifications- not through structural changes of DNA but functional)
4- darwinian (cell selection- cells with selective advantage) (based on the role of environment in selecting cells that have some acquired advantage)
5- tissue organisation
Ames test- to determine the mutagenic activity of chemicals by observing whether they cause mutations in sample bacteria.
Somatic mutation theory- cancer is derived from a single somatic cell that has successfully accumulated multiple DNA mutations
Tissue organisation field theory- carcinogenesis is a problem of tissue organisation. Carcinogenic agents destroy the normal tissue architecture, disrupting cell to cell signalling and compromising genomic integrity.
Chromosomal translocation
A portion of chromosome 9 containing the ABL protooncogene is translocated to chromosome 22, where it fuses with BCR to form fusion protein BCR-ABL. This has constitutive tyrosine kinase activity which causes abnormal cell proliferation. This causes chronic myeloid leukaemia.
Gene amplification
HER2 located on chromosome 17 is amplified which causes overexpression of the HER2 protein (a receptor tyrosine kinase). This contributes to breast cancer. There are now drugs that target HER2, e.g. herceptin
Tumour suppressors and oncogenes
Gain in function mutations turns proto oncogenes into oncogenes. Proto- oncogenes become activated into active oncogenes by translocation, gene amplification and point mutation.
The majority of oncogene proteins are involved in signalling pathways that regulate cell proliferation and survival in response to growth factor stimulation.
Tumour suppressor genes act to stop uncontrolled growth, promotes differentiation and trigger apoptosis. Loss of function mutations cause inactivation of the gene. P53 is known as the guardian of the genome and a transcription factor. It is mutated in 50% of cancers.
Normal tissues have a balance of cell proliferation and cell death. Cancer tissues have an imbalance.
Oncogenes are involved in cell growth
Tumour suppressors are involved in inhibiting cell growth
