Full summary - Cell death in pathophysiology (part 2)

FROM CELL DEATH
TOORGAN FAILURE
(PART 2)
1e master Biomedische wetenschappen (moleculaire mechanismen van ziekten)




UAntwerpen

,TABLE OF CONTENTS

INTRODUCTION TO PART 2 OF THIS SUMMARY ..........................................................................3

CH13: PRECLINICAL RESEARCH ...............................................................................................4

1. PHASES OF TRANSLATIONAL HEALTH RESEARCH ................................................................................ 4
2. PASSENGER MUTATIONS (PAMU’S) CONFOUND INTERPRETATION OF CONGENIC KO MICE ............................. 5
3. CASE STUDY 1: CASP11 PASSENGER MUTATION – AFFECTS >86 KNOCKOUT MODELS .................................. 8
4. CASE STUDY 2: PANX1-NULL MICE ................................................................................................ 9
5. CASE STUDY 3: SERPINA3I MUTATION ........................................................................................... 10
6. CASE STUDY 4: AOX4 MUTATION ................................................................................................. 10
7. CONCLUSIONS AND SOLUTIONS ................................................................................................. 10
8. TAKE HOME MESSAGES ............................................................................................................. 11


CH14: NECROPTOSIS IN DISEASE ........................................................................................... 12

1. TYPES OF CELL DEATH AND IMMUNOLOGICAL CONSEQUENCES ........................................................... 12
2. CDAMPS (CELL DEATH ASSOCIATED MOLECULAR PATTERNS) ............................................................... 12
3. TOOLS TO STUDY NECROPTOSIS IN VIVO ........................................................................................ 13
4. CELL DEATH (NECROPTOSIS) IN PATHOLOGY .................................................................................. 14
5. RIPK1 IN THE INTESTINAL EPITHELIUM ........................................................................................... 19


CH15: PYROPTOSIS IN DISEASE (IN ANTI-INFLAMMATORY DISEASES (AIDS)) ............................ 25

1. INFLAMMASOME-INDUCED PYROPTOSIS ....................................................................................... 25
2. ROLE OF GSDMD IN PYROPTOSIS ................................................................................................. 25
3. NLR SENSOR PROTEINS CAPABLE OF INDUCING INFLAMMASOME-INDUCED PYROPTOSIS ............................ 26
4. INFLAMMASOPATHIES: AUTO-INFLAMMATORY DISEASE DUE TO INFLAMMASOME HYPERACTIVATION ............... 28
5. NLRP3-INDUCED INFLAMMASOPATHIES AND ROLE OF GSDMD-MEDIATED PYROPTOSIS .............................. 28
6. NLRP1-INDUCED INFLAMMASOPATHIES AND ROLE OF GSDMD-MEDIATED PYROPTOSIS .............................. 30
7. PYRIN-INDUCED INFLAMMASOPATHIES AND ROLE OF GSDMD-MEDIATED PYROPTOSIS ............................... 31


CH16: FERROPTOSIS IN DISEASE............................................................................................ 33

1. INTRODUCTION ...................................................................................................................... 33
2. FERROPTOSIS INDUCTION TO TREAT THERAPY-RESISTANT CANCER....................................................... 34
3. BLOCK FERROPTOSIS AS TREATMENT FOR ORGAN INJURY AND DEGENERATION ......................................... 36


GUEST LECTURE 1: VERVAET – NEPHRECTOMY INDUCED KIDNEY REPAIR................................ 43

1. BASICS ABOUT THE KIDNEY ....................................................................................................... 43
2. INTERPLAY BETWEEN CKD AND AKI ............................................................................................... 43

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,3. ROLE OF ISCHEMIA AND REPERFUSION INJURY ................................................................................ 43
4. PARADOXICAL BENEFIT OF NEPHRECTOMY ..................................................................................... 44
5. INNOVATIVE GFR MEASUREMENT TECHNIQUE ................................................................................. 44
6. EXPERIMENTAL OBSERVATIONS ON KIDNEY RECOVERY ...................................................................... 44
7. PATHOMICS AND AI INTEGRATION ................................................................................................ 44
8. HISTOLOGICAL AND FUNCTIONAL FINDINGS ................................................................................... 44
9. DEBATED MECHANISM OF REPAIR ................................................................................................ 44
10. EPITHELIAL BEHAVIOR – CLONAL AND CELL CYCLE ANALYSES .............................................................. 45
11. CURRENT AND FUTURE RESEARCH DIRECTIONS (NB) ....................................................................... 45
12. CONCLUSION ........................................................................................................................ 45


GUEST LECTURE 3: HOSTE - CELL DEATH IN THE SKIN ............................................................. 46

1. MAIN OBJECTIVES ................................................................................................................... 46
2. THE SKIN: STRUCTURE & FUNCTION ............................................................................................. 46
3. STRUCTURE OF THE SKIN & KERATINOCYTE DIFFERENTIATION .............................................................. 47
4. CONCEPTUAL DISTINCTION: PCD – TERMINAL DIFFERENTIATION........................................................... 48
5. CELL DEATH IN SKIN BIOLOGY..................................................................................................... 48
6. HAIR FOLLICLE CYCLING: A MODEL OF CELL DEATH .......................................................................... 49
7. OTULIN: A KEY DEUBIQUITINASE IN KERATINOCYTES .......................................................................... 49
8. DELETION OF LUBAC COMPONENTS IN KERATINOCYTES ..................................................................... 49
9. PHENOTYPE OF OTULIN-DEFICIENT MICE (ΔKEROTULIN) ...................................................................... 50
10. ABBERANT CELL DEATH DRIVES INFLAMMATION ............................................................................... 50
11. SCRNA-SEQ ANALYSIS OF OTULIN-DEFICIENT SKIN ........................................................................... 50
12. OTULIN AND WNT SIGNALING: A CRITICAL LINK ................................................................................ 51
13. THERAPEUTIC RESCUE BY B-CATENIN STABILIZATION ........................................................................ 51
14. OTULIN REGULATES TCF3/TCF4 STABILITY ..................................................................................... 51
15. OTULIN IN WNT-DRIVEN TUMORS ................................................................................................ 52
16. FINAL CONCLUSIONS............................................................................................................... 52


EXAM QUESTIONS PREVIOUS YEARS ...................................................................................... 52

1. QUESTIONS REGARDING CH13 ................................................................................................... 52
2. QUESTIONS REGARDING CH14 ................................................................................................... 53
3. QUESTIONS REGARDING CH15 ................................................................................................... 54
4. QUESTIONS REGARDING CH16 ................................................................................................... 55
5. QUESTIONS GUEST LECTURES .................................................................................................... 55




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,INTRODUCTION TO PART 2 OF THIS SUMMARY

This summary is a comprehensive, elaborate and structured overview based on all the lecture slides and notes provided during
Part 2 of the course Cell death in pathophysiology (formerly known as ‘From Cell Death to Organ Failure’).

Part 2 is only followed by Biomedical Sciences students.

Please note that Part 1 of the course is also available on Stuvia.

This summary is intended to help you understand the material and prepare confidently for the oral exam.




With this summary, and by studying the exam ques=on I obtained a 17/20 on the oral exam !




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,CH13: PRECLINICAL RESEARCH
1. PHASES OF TRANSLATIONAL HEALTH RESEARCH


Divided into 5 progressive phases, each moving scienPfic discoveries closer to pracPcal use in human health:

• T0: Basic Science Research
o Basic biomedical research, no intervenPons with human subjects
o Fundamental exploraPon of biological mechanisms, molecular targets, and disease processes.
o Lays the groundwork for idenPfying potenPal therapeuPc targets.
• T1: TranslaBon to Humans
o Early-stage human tesPng.
o Involves moving promising targets and compounds from the lab into clinical tesBng in human subjects.
o Example: First-in-human studies to test safety, dosage.
• T2: TranslaBon to PaBents
o Focuses on efficacy in a clinical seEng (e.g., Phase II/III trials).
o OpPmizaPon of treatments and delivery to individual paBents.
• T3: TranslaBon to PracBce
o ImplementaPon of findings into clinical guidelines or health pracPces.
o Includes post-markeBng surveillance and broader disseminaPon among pracPPoners.
• T4: TranslaBon to Community
o ApplicaPon on a populaBon level (e.g., public health strategies).
o Involves policy development, community educaPon, and broad-scale impact assessment.


DRUG DISCOVERY AND DEVELOPMENT PIPELINE

1. Discovery of Targets
o UPlizes genomics and proteomics:
§ Genomics: Analysis of gene expression.
§ Proteomics: Study of protein profiles in diseased vs. healthy Bssue.
o Also involves modulaBon of targets in animal and cell models to validate relevance.

2. Assay Development: CreaPng reliable and reproducible biological tests (assays) to measure drug-target interacPon.

3. High-Throughput Screening (HTS): Screening 1000s of compounds to find ones that interact with the target (aka “hits”).

4. Hit-to-Lead OpBmizaBon
o Further tesPng of iniPal hits for efficacy, selecBvity, and safety.
o Reduces pool to a few promising candidates (“leads”).

5. Lead OpBmizaBon: Refine the chemical structure for potency, specificity, bio-availability, reduced toxicity
o Typically narrows to 2–3 strong candidates.

6. Preclinical Development
o Involves in vivo tesBng (animal models) to assess: efficacy, ADME, toxicity
o Ensures drug readiness before human tesPng.

7. Clinical Development
o Human trials through Phases I–III.
o Tests for safety, dosing, efficacy, side effects.

8. Market Approval: Afer successful trials and regulatory approval, 1 lead compound is selected for release to the market.


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,KEY GENETIC APPROACHES IN DRUG DEVELOPMENT

Forward GeneBcs
• Goal: Discover gene funcBon by observing phenotypic outcomes.
• Method:
o Start with random mutaBons (chemically, radiaPon, etc.).
o IdenPfy observable changes in phenotype.
o Work backwards to idenBfy the mutated gene responsible.
• Insight: How unknown genes affect development, disease, or behavior.

Reverse GeneBcs
• Goal: InvesBgate phenotypes by intenBonally modifying specific genes.
• Method:
o Start with a known gene or sequence.
o Use techniques like knockout (KO) or knockdown (KD).
o Observe resulBng phenotypic changes to infer gene funcPon.
• Insight: How manipulaPng a known gene affects biological pathways.


STEP IN PRECLINICAL DEVELOPMENT: TRANSGENIC MOUSE LINES

• Knockout (KO) or Knockin (KI) mice are generated to:
o Remove a gene (KO) or overexpress/modify a gene (KI).
o Study gene funcPon and relevance in disease models.
• These models are put into a disease context (e.g., inflammaPon, cancer, neurodegeneraPon) to test therapeuPc
hypotheses.
• However …
o Many Pmes, what works in transgenic mice fails in clinical trials.
o Historical issue: KO mice were not always reliable, leading to uncertain conclusions due to limitaPons in
genePc modeling techniques (to be discussed later in the chapter).


2. PASSENGER MUTATIONS (PAMU’S) CONFOUND INTERPRETATION OF CONGENIC KO MICE


INTRODUCTION

• GeneBcally modified mice are commonly used in preclinical research to invesPgate gene funcPon & disease
mechanisms.
• Most gene targePng is performed using embryonic stem cells (ESCs) from the 129 mouse strain, which are then
backcrossed onto recipient strains such as C57BL/6J.
• This process introduces unintended "passenger mutaBons" – genePc variaPons from the donor ESC strain that flank the
targeted gene.
• These passenger mutaPons can lead to changes in gene expression or protein funcPon, potenPally confounding the
interpretaPon of phenotypic outcomes.
• Nearly all transgenic mice derived from 129 ESCs carry mulPple passenger mutaPons, which can interfere with
experimental conclusions.




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,CREATING TRANSGENIC KNOCKOUT (KO) MICE: THE STANDARD PROCEDURE

• 129 strain embryonic stem (ES) cells are used to create KO mice:
o Reason: 129 cells are uniquely capable of reliably incorporaPng genePc modificaPons.
o ~80% of all KO mice are made using 129 ES cells.


BASIC STEPS IN TRANSGENIC MOUSE CREATION

1. Step 1: A gene construct is introduced into 129 ES cells, creaPng genePcally modified cells.
2. Step 2: Modified ES cells are injected into C57BL/6 (BL6) blastocysts à then implanted into pseudopregnant mothers.
3. Step 3: The offspring are chimeras, having traits from both:
o C57BL/6 (black coat)
o 129 strain (grey coat)
o The more mixed their coat color, the more likely their germ cells carry the KO mutaBon.


BREEDING STRATEGIES AFTER CHIMERA CREATION

• OpBon 1: Breed with 129 mice
o Produces F1 mice (WT or heterozygous).
o F2 generaPon includes WT, heterozygous, and KO mice.
o ConPnued inbreeding of KOs leads to a pure 129 background with the modificaPon (= coisogenic strain)
o Problem: 129 strain is disease-resistant, limiPng relevance for many disease models

• OpBon 2: Breed with C57BL/6 (BL6) mice (preferred in disease research, since they are suscepPble to various diseases)
o Requires mulBple backcrosses with BL6 to replace most of the 129 genome.
o Goal: Create a congenic strain that retains the KO but otherwise has a BL6 background
§ The targeted mutaPon is introduced via 129-derived ES cells
§ The mice are backcrossed to C57BL/6 to transfer the mutaPon into that background.
o Risk: SPll retains a small segment of 129 DNA around the mutaPon—where passenger mutaPons may exist




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,THE PROBLEM: HIDDEN INFLUENCE OF PASSENGER MUTATIONS FROM 129 STRAIN

• Even afer backcrossing (typically 10x), genes, near the target mutaBon, from the 129 strain ofen remain:
o In other words: the region closely flanking the targeted gene remains of donor orgigin (129 genome)
§ Because the gene recombinaPon frequency decreases near the targeted locus
§ A recombinant frequency (RF) of 1% is equivalent to 1 cM (cenPMorgan)
o This is due to geneBc linkage—genes close to the KO mutaPon are unlikely to be separated by homologous
recombinaBon.
• The issue is especially problemaBc when genes in this region differ between 129 and BL6, leading to unexpected
phenotypes unrelated to the intended KO.

Example:
• Casp11 is non-funcBonal in 129 mice due to a natural mutaPon:
o → 129 mice show resistance to sepBc shock.
o A KO model using 129-derived DNA might lack Casp11 by default, confounding the interpretaBon.


COMPARATIVE GENOMICS BETWEEN 129 AND BL6 MICE: KEY FINDINGS

• Total of 1084 genes differ between 129 and BL6 mice.
o This represents about 4% of the mouse genome.
• These differences include:
o Sequence alteraPons
o FuncPonal variaPons
o Differences in gene expression


BACKCROSSING AND RECOMBINATION: WHAT GETS RETAINED?

• Goal: Reduce 129 genePc influence by backcrossing with BL6 mice.
• With each generaPon, the BL6-derived region around the target expands, reducing surrounding 129 DNA.

StaBsBcal Insights Ager 10 Backcrosses:

• Within 10 cenBmorgans (cM) of the target (approx. 400 genes):
o 40% chance the DNA sPll comes from the 129 strain.
• Within 5 cM of the target (approx. 200 genes):
o 60% chance of 129 origin.
• Within 1 cM of the target (approx. 40 genes):
o 90% chance the sequence is sPll from 129.
o These regions are almost impossible to separate from the KO mutaPon via recombinaPon.



CONSEQUENCES FOR RESEARCH AND INTERPRETATION

• These passenger mutaBons can significantly alter phenotype, misleading researchers about the effect of the actual KO.
• Common issue: Researchers assume a phenotype is due to gene delePon, when it could actually be due to an adjacent
129-specific gene with different funcPon.
• Example again: Loss of casp11 funcPon in 129 may be wrongly interpreted as a result of a nearby gene KO.




7

,HISTORICAL CONTEXT AND ONGOING CHALLENGES

• First warnings emerged in 1997, but despite this:
o A large number of KO models were developed without addressing the passenger mutaBon issue.
• These complicaPons sPll pose a major challenge in interpreBng data from transgenic mouse models, parPcularly in
immunology and disease studies.


3. CASE STUDY 1: CASP11 PASSENGER MUTATION – AFFECTS >86 KNOCKOUT MODELS




ORIGINAL HYPOTHESIS AND ASSUMPTION

• Researchers originally hypothesized:
o Caspase-1 (Casp1) plays a criBcal role in inflammasome funcBon.
o Therefore, Casp1 knockout (KO) mice would be protected against sepBc shock.
• Casp1 KO mice did indeed show protecBon from sepPc shock, seemingly supporPng this hypothesis.


THE UNEXPECTED CONFOUNDER: CASP11 PASSENGER MUTATION

• However, further invesPgaPon revealed:
o The real reason for the observed protecPon was not Casp1 deleBon, but a passenger mutaBon in Caspase-11
o Casp1 and Casp11 genes are located very close together in the genome (genePcally closely linked)
§ Due to this proximity, they cannot be separated by standard backcrossing, resulPng in unintended
double knockout (Casp1 and Casp11 KO).


EXPERIMENTAL CONFIRMATION: REINTRODUCING CASP11

• To validate this new insight à Casp11 was reintroduced into Casp1 KO mice.


➤ Result: ProtecBon against LPS-induced sepBc shock disappeared, confirming:
• The absence of Casp11, not Casp1, was the true protecBve factor.




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, EXPANSION OF THE PROBLEM: MMP KNOCKOUTS AND CASP11

• The MMP gene family (Matrix Metalloproteases), also located near Casp11, became a focus:
o Researchers observed that MMP KO mice were protected against LPS-induced shock.
o However, this protecPon was only present if the Casp11 mutaBon was also present.


➤ When MMPs were knocked out on other chromosomes, no protecBon occurred in mice with wild-type Casp11.
➤ Suggests the MMP-KO protecBon was an arBfact of co-exisBng Casp11 mutaBon.


GENETIC MAPPING AND ELIMINATION OF THE CASP11 PASSENGER MUTATION

• A separate laboratory conducted further backcrossing to remove the Casp11 mutaPon:
o They compared mice with vs. without the Casp11 mutaBon:
§ Mice with Casp11 mutaBon: Protected against sepPc shock.
§ Mice with WT Casp11: Not protected.
o This ruled out MMPs as contributors to shock resistance.
o The only determinant was the presence or absence of Casp11 mutaBon.


SCIENTIFIC CONSEQUENCES: MISINTERPRETATION RISK

• This case study highlights a criBcal issue in transgenic research:
o Misinterpreted phenotypes can arise from unrecognized passenger mutaBons.
o In this case, more than 86 KO models were affected by the Casp11 passenger mutaBon, potenPally leading to
erroneous scienBfic conclusions.


KEY LESSONS FROM THE CASP11 CASE

• Always consider geneBc background, especially for genes located close together.
• Passenger mutaBons can invalidate conclusions, even in well-designed studies.
• Proper geneBc controls and genotyping for nearby regions are essenBal to ensure accurate ayribuPon of phenotypes.


4. CASE STUDY 2: PANX1-NULL MICE


• Panx1 (Pannexin 1) is a hemichannel protein believed to play a role in:
o InteracPng with the NLRP3 inflammasome
o Delivering LPS (lipopolysaccharide) to the cytosol
• Nlrp3-null mice were known to be resistant to LPS-induced sepBc shock, suggesPng a link between Panx1 and
inflammatory pathways.


EXPERIMENTAL OBSERVATIONS

• 129 strain Panx1 KO mice:
o Showed survival and were protected against sepBc shock.
• C57BL/6 ES cell-derived coisogenic Panx1 KO mice:
o Showed no survival or protecBon, indicaPng no role of Panx1 alone in protecPon.


➤ This discrepancy highlighted the Casp11 passenger mutaBon as the true factor conferring resistance to LPS-induced shock,
not the Panx1 delePon itself.




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