Week 6B: Apoptosis, Fatty acid oxidation disorders, adrenoleukodystrophy Flashcards
HC 33, 34, 35, 36
How is the regulation mechanism of apoptosis called?
Trigger-decision-execution
Apoptosis concept. And what happens to the debris made?
Proteolytic breakdown of nucleus, organelles and membrane
> Clearance debris by surrounding tissue
Pathology of failing apoptosis
Immune system defect
> Cancer
Which cells are prone to apoptosis
-Damaged cells
-Stressed cells
-Cells signalled by body signals
Process of apoptosis
-Initiation
-Shrinkage, form blebs, proteins for breakdown activated
-Enzymes break down nucleus and cell emits signals to attract macrophages
-Cell fragments with smaller pieces containing cell components and destroyed nucleus
-Macrophages recognize cell parts and removal
Which molecule is part of the electron-transport chain and apoptosis signalling?
Cytochrome c
> central sensor in apoptosis
Cytochrome c as sensor in apoptosis
-Mitochondria respond to cell stress
-Intermembrane space leak into cytosol, and there Cyt C becomes co-factor in apoptosis
Major players apoptosis (4)
- Caspases
- Death receptors: TNF/TNFR family
- Bcl-2 family: Bcl-2 like, Bax like, BH3-only
- Mitochondria as central sensor
Cyt C in cytosol is the cofactor for …
Apoptotic complex of Caspase9-Apaf1 > casp-9 generates more caspases and creates apoptosis
Caspase types
Initiator and executioner caspases
What kind of molecules are caspases?
Proteases which cleave proteins
> Highly specific functions
> Limited substrates
> Limited and directed process
Recognition sequence of caspases
They are cysteine proteases with unique specificity for cleavage after Asp residue
Caspases are made as zymogens, how are they activated?
Cleavage after internal Asp-residues
> can activate each other
Active caspase structure
Active heterodimer
Initiator caspases numbers and activation
Casp-8, Casp-9, Casp-10, Casp-2
> Long prodomain involved in activation
Executioner caspases numbers and activation
Casp-3, Casp-6, Casp-7
> Short prodomains, and cleave specific cellular proteins during apoptosis
In which processes are caspases involved?
-Some types in apoptosis initiator
-Some types in apoptosis effector
-Some types in cytokine maturation
Which caspase is the most active executioner caspase?
Casp-3
ICE
Interleukin converting enzyme, a cytokine maturation caspase
Death receptors (TNF-TNFR family) contain receptors present at this location:
Cell surface
Fas is a …
TNFR family member
Structure TNFR-family
Trimeric receptors
> trimeric ligands such as TNF, FasL and TRAIL
Result interaction ligand with TNFR-family (FasL binds Fas)
Trimerization of intracellular part: signalling
> intracellular interactions occur via specific domains and adapter proteins
> In Fas signalling: FADD (adapter) recruited which permits Casp-8 transactivation (protein-protein interactions)
> Casp-8 activation by induced proximity > apoptosis
Are all TNF-TNFR family members for cell death? How do they differ?
No, some for co-stimulation and survival
> Divergence in intracellular domains: bind different signalling molecules
> cysteine rich domains
> ligands are similar
Bcl-2 is a oncogene in …
Follicular lymphoma
Bcl-2 as oncogene
T(14:18) translocation juxtaposes IgVh enhancer (very active immunoglobin heavy chain enhancer in B cells) with Bcl-2
> oncogenic rearrangement
> Bcl-2 inhibits cell death as opposed to inducing proliferation
Role Bcl-2
Prevent mitochondrial Cyt C release when mitochondria are damaged
> Pro-survival state by expressing Bcl-2: inhibit apoptosis
Pro-apoptotic Bcl-2 family members
Bax-like: Bax, Bak
Anti apoptotic Bcl-2 family members
Bcl-2-like: Bcl-2, Bcl-xL, A1/Bfl-1
BH3-only members (pro-apoptotic)
Bid, Bim, Bad, Noxa, Puma etc
How is threshold for apoptosis determined
Balance between pro- and anti-apoptotic members of Bcl-2 family
What happens after activation Bax/Bak
Form oligomers and a pore in the membrane: leakage Cyt C
Homology Bcl-2 family
1-4 Bcl homology domains (BH1/2/3/4)
BH3-only subfamily activation types
-Transcriptionally induced: Puma, Noxa
-Present in dormant form: Bid, Bim, Bmf, Bad
BH3 peptides insert into …
Bcl-2-like or Bax-like proteins into hydrophobic groove
> selective interaction of Bcl-2 family members
> Some are promicuous like Bim and others more selective like Noxa
-Bim and Puma inhibit Bcl-2, Bcl-xL, Bcl-w and Mcl-2
-Bad inhibits Bcl-2/xL/w
-Noxa inhibits Mcl-1
Finetuning BH3 peptides by
expression certain members
Pro-apoptotic signalling
Activation BH3 peptides like Puma, Noxa, Bim and Bad by hypoxia, steroids, growth factor withdrawal, UV, Taxols or other drugs
> Inhibit Bcl-2-likes
Bax/Bak form complex to drill hole in mitochondrial membrane
Bcl-2-likes inhibit apoptosis by binding
Bax/Bak
> Bcl-2, Bcl-xL, Bcl-w, Mcl-1
Apoptosis pathways in lymphocytes
-Intrinsic pathway: Cyt C activates Casp-9
> Bcl-2 inhibits Cyt C release out of mitochondria
> DNA damage, cytostatic drugs, cytokine deprivation and metabolic stress induce Cyt-C release by activating BH3 pepides
> Activation ProCasp-9 bound by Apaf1: release and active.
-Death receptors/ extrinsic pathway: activation Casp-8 through FasL/Fas and recruitment FADD
> Casp-8 activates Casp-3 which causes apoptosis
> Flip inhibits activation Casp-8
-Cytotoxic T cells
> recognizing peptide on cell surfuce
> perforin release to make pore
> Granules with proteolytic enzymes: granzyme B into cell
> Bid to t-Bid by granzyme B
> t-Bid induces Bax?
CD95 is
Fas
And CD95L is FasL
What happens after Casp-3 activation?
-Cleavage ROCK1 > phoshatidyl serine exposure on outer leaflet after flip flop
-Cleavage of NDUFS1: subunit of respiratory Complex 1: shutdown
-CAD/ICAD cleavage: DNA fragmentation
-PARP cleavage: shutting down DNA repair
-Cleavage cytoskeletal proteins: actin, tubulin, fodrin and vimentin > membrane blebbing
Results Casp-3 activation globally
-Shutdown essential functions and repair mechanisms
-Disassembly of the cell into small fragments that are recognized and cleared by phagocytotic cells.
Necrosis
Passive cell death: damaged and blow up: swelling, membrane rupture and inflammatory response because contents are released
Necroptosis
Regulated form cell death
> Death receptor triggering and TNFa synthesis
> Necroptosome/RIPoptosome formation
> not details needed to learn
Name a form of cell death which involves iron
Ferroptosis
The neat form of cell death
Apoptosis
> cell fragmentation and clearance by macrophages
> immunologically silent > necroptosis is not silent
Necroptosis cascade
TNFa binds TNFR
> activation apoptosis through Casp-8
Or
phosphorylation and activation RIPK1/3 which activate MLKL by phosphorylation
> MLKL forms channel to release cytokines and inflammatory mediators and permeabilise the membrane
HC34: Explain the Ramos Burkitt lymphoma cell model
Model to detect apoptosis in the lab
> B cell line known for chromosomal translocations which changes apoptotic activity
How are Ramos Burkitt lymphoma cells triggered for apoptosis
aCD95: antibodies for Fas death receptor
aBCR: target B cell receptor
> trigger them
> detect properties using flow cytometry
Cell membrane change in apoptotic cells
Phosphatidyl serine on outer leaflet
Annexin V and PI for detecting apoptosis
-Annexin V binds phosphatidyl serine on outer leaflet and can be labeled by fluorescent label green (FITC)
-In late apoptotic cells: damage in PM, PI: propidium ionide goes in and binds DNA: once bound red fluorescence.
> Early apoptotic cells: Annexin V + and PI-
> Late apoptotic cells: double positive
Track phases of apoptosis with Annexin V and MitoTracker. Why is zVAD used in an extra analysis for apoptosis?
Shift to higher level Annexin V and then also lower MitoTracker.
> zVAD is a specific inhibitor of caspases
zVAD function in apoptosis detection
Specific inhibitor of caspases
aCD95 leads to early activation of caspase …
casp-8
aBCR + zVAD reaction in Ramos Burkitt lymphoma cells
drop in MitoT but not Annexin V
> mitochondria still damaged, but caspases not activated.
Detection apoptosis using Western Blot
-Western Blot cyt C release and casp-3 processing after aCD95 and aBCR with leukemic B cells
> many fragments casp-3: processed and activated
> antibodies on blot
Mitochondria are not part of lysate in Western Blot apoptosis detection. Is casp-3 activation faster in aCD95 or aBCR treatment of the leukemic B cells?
In aCD95 treatment
> cleavage, multiple fragments
Detecting by blocking casp-9 activity genetically. How?
Dominant negative mutant casp-9
The apoptosome contains:
Apaf, Casp-9, cyt C (heptameric structure)
In the activation of caspases, the cysteine active centre is changed to ..
alanine
How are CD95 and BCR triggering changed by dominant negative mutant of casp-9?
Blocking of the apoptotic pathway and mitochondrial damage
> casp-9 also involved in BCR triggered apoptosis
What are JeKo cells and how are they used in apoptosis detection?
T-cells directed malignant B cells (are killed)
> different necroptotic and apoptotic JeKo cells
> Necroptotic morphology is different to apoptotis
-apoptosis: condensed nucleus
-necroptosis: blown nucleus
Most apoptotic cells have … DNA. Which assay is used to detect this?
Sub-G1 DNA: fragmented
> shown with PI assay (propidium iodide)
Detect apoptosis with enzymatic assays: Fluorometric protease assay
-DEVD: aspartic acid: D, cleavage site for caspase (3 for example)
> Cleavage DEVD-pNA > release fluorescent pNA molecule (or other chosen fluorescent molecule)
-Fluorometric protease assay
Clonogenic survival assay for long-term survival
-Grow slowly, colonies to look at
-Long-term survival cells affected, or not
-Growth potential limited when less colonies formed
> detect if there is long-term survival!
Assays for cell death detection (not apoptosis specifically)
-Metabolism - ATP -mitochondrial function
-MTT assays and variations
Druggable target apoptosis
Inhibitors of Bcl-2 > specific > kill cancer cells which overexpress Bcl-2
How to target resisting cell death cells directly or indirectly
Direct: Triggering pro-apoptotic BH3-only mimetics
Indirect: Blocking pro-survival signals
Blocking prosurvival signals in cancer: how do cancer cells rely on signals from environment? (for which functions)
-To drive proliferation
-To prevent apoptosis
-To maintain adhesion
BH3-only mimetics
-Pharmacological compounds that act like BH3-only proteins, and bind to prosurvival Bcl-2 members
> very effective against cancers that rely on Bcl-2
ABT-737 function
BH3 mimetic that inhibits Bcl-2 and Bcl-xL
Problem ABT-737
-Too effective > patients get bleeding because platelets need Bcl-xL
- Dose limiting toxicity: also active against healthy cells
BH3 mimetic anti cancer strategy sequence
-Give activators of Bax/Bak: the BH3-only peptides
> get bound by Bcl-2 likes
> BH3 mimetics bind Bcl-2 likes
> Activators like Bim and Puma can bind and activate Bax/Bak
> Cytochrome C release > activation intrinsic apoptosis through caspases
ABT-199 function
-Target cells which depend on Bcl-2 survival like cancer cells
-Little toxicity
> But: selection towards other Bcl-2-likes
Chronic lymphocytic leukemia (CLL) cells traffic and growth
-Between lymph nodes and blood
-Cannot grow in blood > cycle back (because vulnerable for apoptosis)
> massive cell death in peripheral blood compartment
-In lymph nodes: protected from apoptosis
> 10,000 fold reduction sensitivity for ABT-199 after CD40-CD40L signalling in LNs
> increased resistance to novel drugs like BH3 mimetics
> Cells are flexible in apoptotic balance
CLL cells in LNs which are resistant to drug treatment are … to the drug
refractory
Inhibit pro-survival signals through BCR pathway
-Activate BCR
> Activates Bruton’s tyrosine kinase (BTK) in signalling cascade
> drug against BTK (essential in oncogenic signalling) in B cell malignant cell
PCI-32765 function as drug (ibrutinib)
Abrogate BCR controlled signalling and adhesion
> inhibit fibronectin signalling (immunoglobin driven adhesion to fibronectin is inhibited)
Effect BTKi (inhibitor) like PCI-32765 (ibrutinib)
CLL Cells cannot cycle anymore: adhesion prevention > cells flow from LNs to blood
> cannot go back because of BTKi
> slowly perish in blood
Which chemokine promotes BTK
CXCR4
Effect active BTK
Promote integrins for adhesion in lymph node
> proliferation and survival
CLL combination treatment
BTKi + ABT-199
> impared proliferation + survival and induced apoptosis by forcing cells in blood and blocking Bcl-2-likes
How can drug resistance against a BTKi occur?
Mutation at the site where the BTKi binds
HC35: Where does translation of genes from mtDNA occur
In ribosomes in the mitochondria
Which important organs rely on beta oxidation to gain energy
Heart, liver, skeletal muscles, the brain
Chain reactions beta-oxidation
-Oxidation by chain length specific acyl dehydrogenases
-Hydration
-Oxidation
-Thiolysis: cleave acetyl-CoA off
VLCADD
Deficiency in VLCAD
> inborn error of metabolism
VLCADD is among the …. deficiencies
FAO deficiencies (FOAD), FA oxidaion
Types of FOADs in expression
Severe neonatal onset or milder later onset
> newborn screening programs important
Common complications of FOADs
-Cardiomyopathies
-Rhabdomyolysis (breakdown skeletal muscle)
-Hypoglycemia
-Muscle weakness and pain
-Fatigue
FAO flux assay in VLCADD
Long chain FA oxidation flux variable between patients and can be increased when culturing at 30 degrees celsius
> FAO flux depends on FAO deficiency type in patients genes
Which step is defective in VLCADD?
Breakdown long-chain fatty acids in oxidation reaction in beta oxidation in mitochondria
> Mutation ACADVL gene
Consequences VLCADD
-Energy shortage (ATP)
-Accumulation of acylcarnitines and acyl-CoAs
Clinical features liver, heart and muscle in VLCADD
Liver; hypoglycemia
Heart: Cardiomyopathy and/or arrhythmia
Muscle: myopathy, muscle weakness, possible hypotonia
How is myopathy measured in the blood
Levels of Creatine kinase > 250 U/L while reference is 70-170
Why newborn screening programs for VLCADD?
Prevent early-life complications
> know the patients and use cells for research
However: what can’t the newborn screening program do for VLCADD patients?
-Cannot prevent late-life complications
-No prediction about when they will get sick
> But can give advice for specific diet
Neonatal VLCADD characteristics
-Hypotonia
-Hypoketotic hypoglycemia
-Liver failure
-Cardiomyopathy
-Lipid accumulation in organs
Late onset VLCADD characteristics
-Dark urine
-Recurrent rhabdomyolysis (breakdown muscle) after prolonged exercise
-High creatine kinase in blood»_space;170 U/L (rhabdomyolysis detected)
Why is it not possible to accurately investigate VLCADD in animals?
It does not correlate well with human disease
> different metabolisms
> humans use more FAs for fuel
Disease triggers of VLCADD
-Infections
-Fasting
-Fever
-Prolonged exercise
> use of beta-oxidation
Dietary advice in VLCADD
Eat lots of glucose but restrict Long-chain FAs
Alternative disease model for VLCADD
Human induced pluripotent stem cells: hiPSCs
> from skin fibroblast sample: make hiPSCs and let them differentiate to specific tissue type like cardiomyocytes
Disease markers VLCADD in hiPSCs
-Accumulation acyl-CoA
-Accumulation acylcarnitines (FAO does not proceed for long chain FAs)
-Low ATP
-Increased [Ca2+] flux
Why are cardiomyocytes the preferred disease model for VLCADD?
Measure membrane potential
> decreased action potential compared to control cell line
VLCADD cardiomyocytes have an increased Ca2+ transient, what does this mean
Higher amplitude of Ca2+ flux
Effect VLCADD patient when giving resveratrol (RSV)
Upregulate mitochondrial content and therefore its functions
> more energy made
> more efficient VLCAD, upregulated
> increased membrane potential like the control
» resue abnormal action potential in mild VLCADD CM (cardiomyocytes)
Effect etomoxir (ETO) in VLCADD
Block the CPT-1 enzyme and therefore FAO
> Prevent acylcarnitine accumulation in patients
> not overloading the FAO system
> Rescue membrane potential in CM
Substrate FA lengths VLCAD
C12-16
Why is the immune response and its crosstalk with metabolism relevant for FOADs?
It is a disease trigger
> Immune processes cost a lot of energy: FAs needed
Which cytokine response is impaired in VLCADD?
IL-6 response to LPS (in bacterial cell wall) of skin fibroblasts
Is the IL-6 response always impaired in VLCADD?
No, only when triggered using LPS
> when triggered with Poly(I:C) (molecule from RNA virus) > IL-6 synthesis and release
LPS pathway
LPS binds TLR4
> signalling pathway to activate transcription of IL-6
Which components of the LPS to IL-6 pathway are downregulated in severe VLCADD?
TLR4 mRNA and JunB expression
Newborn screening in VLCADD can …
be life-saving when early diagnosis
HC36: Adrenoleukodystrophy is a … disease and therefore more … get it
X-linked, more males
Adrenoleukodystrophy (ALD) characteristics in prevalence and affected organs
Rare disease: 1:15,000
> X-linked (ABCD1 gene)
> VLCFAs
> affects brain, spinal cord, adrenal cortex and testes
How are VLCFAs made (very-long chain)
By elongation of LCFAs like palmitate
VLCFA breakdown in the ..
peroxisomes
Which transporter is defect in ALD and what is its function?
ABCD1, transport VLCFAs to peroxisome
> VLCFA accumulation
ALD is a ….genic disease
monogenic
Is the onset of ALD predictable? How many males are affected by adrenal insufficiency before age of 10 y?
No, males can develop adrenal insufficienct from six months age or later in life
> affects 50% male patients before 10 y
What deficiency is developed in hormones in ALD because of adrenal deficiency (males >6 months)
Glucocorticoid deficiency: no cortisol
> Life threatening
> Insufficient immune system because deficient cortisol production: too slow response
> well treatable with hormone replacement
Cerebral leukodystrophy
Water accumulation in (back of) brain (ALD in males > 3 y)
> 30% males <10y affected
> diagnosis based on neurological symptoms: too late for HSCT (bone marrow stem cell transplant), only possible before neurological symptoms.
> most severe type
How is adrenal insufficiency a risk factor for cerebral leukodystrophy?
Blood barrier may be open
Which ALD patients are affected by myeloneuropathy (spinal cord disease)
Males: >18 y
Females >40 y
Treatment myeloneuropathy
No disease-modifying therapy, only supportive
Rational for ALD newborn screening
Preventive treatment, for example the bone marrow transplant prior to the neurological symptoms
> follow up and monitoring
Wilson and Jungner Criteria for Newborn Screening and effect for ALD NBS in Netherlands
-Early diagnosis must be directly beneficial to the neonate (treatment)
> only the case for males in ALD
> no treatment myeloneuropathy, but treatment for adrenal deficiency and leukodystrophy
> only male newborns in the newborn screening program
Biomarker ALD in NBS (newborn screening)
C26”0-lysophosphatidylcholine (C26”0-lysoPC)
> heel prick of blood
What was a challenge of the SCAN pilot study for sex specific NBS for ALD
> identify males using dry blood spots
do not identify other untreatable diseases: lower quality of life)
X-counter in NBS
Identify males
> Measure for chromosomes 3, 4 and 7
> Gene on autosomal chromosome and X (but not Y) chromosome
> same expression level: XX
> If 2/3 chromosome comparisons show same level: XX > exclusion because female
Dutch 4 Tier ALD-NBS algorithm
Stop testing after a negative result in one of sequential tiers
Tier 1: C26:0-lysoPC: cutoff at 0.32 uM for positive
Tier 2: X-counter: XX excluded
Tier 3: C26:0-lysoPC test with less false positives (cutoff 0.150 uM)
Tier 4: ABCD1 gene sequencing
What happens with newborns classified as ALD patient after ALD-NBS?
Referral to pediatric neurologist
Long-term follow up ALD diagnosis after NBS
MRI every 6 months until 12 y/o and then every year
