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Pathomechanismen > Julian Großkreutz Summary > Flashcards

Julian Großkreutz Summary Flashcards

1
Q

Parkinson Syndrome

A
  • alpha Synucleine
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2
Q

Amyotrophic Lateral Sclerosis

A
  • TDP43
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3
Q

Genetics ALS - Frontotemporal Dementia (20 % fam.)

A

Analysis strategies:
- linkage analysis, candidate gene analysis
- GWAS
- Exome sequencing
- genome sequencing

Gene associated with ALS: C9orf72

FTD: MAPT, GRN
ALS: SOD1, hnRNP A1, NEK1, others

KIF5A virants with the respective allele
- loss of function: 2
- missense: 88
- missense: 4
- SNV: 123

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4
Q

C9orf72 ALS: miRNA, nuclear pore and TDP43 pathology

A
  • RAN translation
  • FUS mutations
  • alternative splicing defects
  • altered microRNA biogenesis
  • disrupted binding of long introns
  • protein aggregates
  • aging
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5
Q

What are microRNAs?
-> key data

A

Active (mature) microRNA (miRNA) molecules:

Feature: single stranded (ss) non-coding RNAs (20-25 nucleotides)
Occurence: almost all eukaryotes encode miRNAs
Function: RNA silencing: Suppress gene expression in a sequence-specific manner at post-transcriptional level

-RNA interference (RNAi)
- Posttranscriptional gene silencing (PTGS)

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6
Q

What are microRNAs?
-> RNA silencing

A
  • components of ribonucleoprotein complexes: RNA-induced silencing complex (RISC)
  • miRNAs bind to complementary target mRNAs
  • complementary in part or in whole to target mRNAs
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7
Q

Biogenesis of miRNAs: from genes to functional miRNAs

A

Intergenic & intronic miRNA genes
pri-miRNA (primary miRNA transcript)
-> Large primary transcripts contain one or more
hairpin structures consisting
- an incomplete base-paired stem (~35 nts)
- an apical loop of variable size
- flanking single-stranded 5’ and 3’ segments
- the length of these structures is ~100-120 nts

pre-miRNA (precursor miRNA)
- Length: 70 nts
- Stem-loop structure (see above)
- Length of the stem region: ~20-25 nts
- two nucleotide 3’ overhang
Note: mirtron transcripts = pre-miRNAs!

miRNA duplex (miR/miR)
- ~20-25 nucleotides per strand
- RNA with incomplete stem complementarity
- two nucleotide 3’ overhangs on each strand

Mature miRNA (miR)
- single-stranded miRNA which can bind to various target mRNAs by base pairing

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8
Q

Drosha-dependent miRNA biogenesis pathway in the nucleus

A

Transcription of intergenic and intronic miRNA genes:
- long pri-mRNA with 5’ cap and poly(A)

Processing of pri-miRNAs into pre-miRNAs:
- Microprocessor:
nuclear heterotrimeric protein complex consisting of:
- Drosha a RNase III endonuclease
- Two DGCR8 (dsRNA-binding protein)

Export:
- The Exportin-5/Ran-GTP complex recognizes the RNA stem together with the short 2 nucleotides 3’ overhang of the pre-miRNA
- Transportation of the pre-miRNA from the nucleus to the cytoplasm.

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9
Q

Processing of pre-miRNAs into miRNA duplexes: Dicer

A

Helicase
- autoinhibitory module
- binds the apical loop of the pre-miRNA
- binds cofactors
- contributes to processivity (long dsRNAs)

Central domain (DUF, Platform, PAZ)
- binding to 5’-phosphate 3’-overhang

Nase IIIa/Rnase IIIb
- intramolecular dimer
- create the catalytic centers

dsRBD
- binding of RNA substrate

Dicker 218 kDA
Nase type: RNAse III endonuclease
Localization: cytosolic
Substrates: long dsRNAs or pre-miRNAs
Cleavage products: dsRNA duplexes (siRNA, miRNA)
Cofactors: dsRBD1, dsRBD2, dsRBD3
dsRNA-binding proteins (dsRBP) TRBP or PACT: improve processing efficiency and accuracy

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10
Q

Formation of the RNA-induced silencing complexes (RISC)

A

Loading of the miRNA duplex
- Composed of one of the four human Argonaute proteins (AGO1-4), Dicer, and a dsRNA binding protein (e.g., TRBP or PACT)
- Loading of miRNA duplex onto Argonaute

Selection of the mature miRNA strand
- Pre-RISC consists of a Argonaute and a miRNA duplex
- Removal of the passenger strand leads to the formation of an activated RISC
- The active RISC (miRISC) contains a single stranded miRNA (guide strand) which directs the complex to the target mRNA for posttranscriptional gene silencing

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11
Q

Model of microRNA (miRNA)-mediated gene silencing

A
  • miRISC binds to the 3’ UTR of the target mRNA
  • AGO recruits the scaffold protein GW182
  • GW182 interacts with poly(A)-binding protein and de-adenylase complexes

1) Repression of cap-dependent translation via CCR4-NOT (de-adenylation independent)
2) De-adenylation by de-adenylase complexes
3) Removement of 5’ cap by de-capping proteins
4) mRNA degradation by 5’-3’ exonuclease

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12
Q

Target binding of microRNAs

A
  • a single miRNA (seed region!) might bind to more than 200 target mRNAs (3’UTR)
  • several miRNAs can bind to a single mRNA
  • a single miRNA can bind to multiple binding sites on a single target mRNA

-> determining the function of miRNAs is: to identify their target mRNAs

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13
Q

How can we confirm computational predicted miRNA targets?

Functional studies: Altering endogenous level of miRNAs in cells

A

1) Transfection of synthetic Dicer substrate dsRNA (27-30 nts) which enter the pathway of endogenous miRNAs and increase their activity -> gain of miRNA function
2) Transfection of synthetic antisense oligonucleotides which reduce the endogenous miRNA activity -> loss of miRNA function

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14
Q

Mechanisms of deregulated expression of microRNAs

A

Epigenetics: DNA Methylation, Histone Acetylation, Histone Methylation
Transcription Factor: p53, p73, Snai, PPARgamma, Samd4, p63, ETS-1, Ascl2, Klf9
miRNA biogenesis and processing: Dicer, Drosha, DGCR8
CeRNA: competing endogenous RNA
Gene Sequence Alteration: Gene deletion (mir-15a, mir-16-1), Gene Amplification (miRNA-17 ca 92 cluster)

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15
Q

miRNAs can cast as oncogenes or tumor suppressors in cancer cells

A

GAIN OF FUNCTION
-Gene amplification
- Translocation
- Epigenetic regulation (DNA methylation down regulated)
- Inhibition of ceRNA expression

LOSS OF FUNCTION
- Deletion
- Mutations
- Epigenetic regulation (DNA methylation unregulated)
- Overexpression of ceRNA

PROMOTION OF TUMORUGENESIS BY
- upregulated proliferation, genetic instability, invasion, angiogenesis
- down regulated cell death

ceRNA = competing endogenous RNAs

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16
Q

miRNAs: New biomarkers in cancer disease
-> miRNA profiling

A

miRNA profiling methods:
- Reverse Transcription-quantitative PCR (RTqPCR)
- Hybridization-based microarrays
- NGS-based RNA-seq

Tumor biopsy (invasive)
Patient serum (non-invasive)

Diagnostic biomarker
- Discriminate between tumor and normal
- Discriminate between benign and malignant disease
- Classification of different subtypes
- Detection in an early stage

Predictive biomarker
- Information on therapy response
- Treatment decision

Prognostic biomarker
- Predict disease outcome
- Predict disease-free survival
- Monitor disease recurrence

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17
Q

Cell death: necrotic and apoptotic cell disintegration

A

NECROSIS
1. Cell swelling
2. Later. clumping and random degradation of nuclear DNA
3. Cell rupture
4. Release of cellular contents
5. Inflammation and damage of neighboring tissue

APOPTISIS
1. Cell shrinkage
2. Condensation and fragmentation
3. Formation of beds and apoptotic bodies
4. No release of cellular contents
5. Phagocytosis, no inflammation, individual cell death

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18
Q

Essential function during embryogenesis

A

Morphogenesis
- Shaping many developing structures
- Tissue and organ development
- Removal of transient structures

Examples:
-> Formation of
- interdigital spaces of fingers and toes
- middle ear spaces
- nostrils
- vaginal opening
etc.

Elimination of about 50% of all neurons
Important function during sexual differentiation
-> In the male, the müllerian ducts are lost
-> In the female, the wolffian ducts are lost

19
Q

Maintain tissue and organ homeostasis in adult humans

A

TISSUE/ORGAN AND THE CORRESPONDING AVERAGE LIFESPAN
- Small intestine epithelium -> 1.4 days
- Stomach -> 2-9 days
- Large intestine (crypt cells) -> 3-4 days
- Skin epidermis cells -> 10-30 days
- Olfactory cells -> 60 days
- Urinary bladder (epithelium) -> ca 2 months
- Osteoblasts (bone cells) -> 3 months
- Erythrocytes (red blood cells) -> 4 months
- Liver hepatocyte cells -> 0.5 - 1 year
- Lymphocytes - Effector cells -> days - weeks
- Lymphocytes - Memory cells -> years
- Oocytes (female gametes) -> no regeneration

20
Q

Analysis of mitochondrial transmembrane potential

A
  • Cyanine dye JC-1: lipophilic, cationic, and fluorescent dye
  • Pass through membranes
  • Intracellular localization & fluorescence emission of the dye depend on the mitochondrial membrane potential
    » Normal transmembrane potential (~140 mV): accumulation & aggregation of JC-1 into mitochondria
    -> red-fluorescent
    » Depolarization: enrichment of the monomeric dye in the cytoplasm
    -> green-fluorescent
21
Q

Extracellular exposure of PS can be used to detect apoptotic cells

A
  • Annexin V (Ca++ binding protein) specifically binds with high-affinity (Kd = 0.1 nM) to PS in the presence of calcium
  • Annexin V labeled with a fluorescent dye (e.g., fluorescein, phycoerythrin (PE), Cy5) can be used to analyze apoptotic cells
  • This method serves as an indicator of early stages of apoptosis
  • Application: particularly suitable for suspension cells
22
Q

TUNEL, a technique to detect apoptotic cell death at single-cell level in tissues
-> terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling

A

Principles of the TUNEL Assay
During apoptosis:
Double-strand breaks and single-strand breaks (“nicks”) into DNA » Generation of f􏰆ee 3􏰏-OH termini
TdT (terminal deoxynucleotidyl transferase)
- Recognition of free 3’-OH termini
- Template-independent DNA polymerase
- Attachment of labeled nucleotides to all 3’OH ends

Measurement
- Flow cytometry
- Fluorescence microscopy

23
Q

Formation of apoptotic bodies

A

DISASSEMBLY OF AN APOPTOTIC CELL
1. Apoptotic membrane blebbing
2. Apoptotic membrane protrusion formation
3. Cell fragmentation

Blebs, protrusions and apoptotic bodies

24
Q

Hallmarks of apoptosis and useful methods

A
  1. Decrease of the mitochondrial transmembrane potential -> JC1 dye
  2. Activation of specific proteins (e.g. cascades, nucleases)
  3. Exposure of phosphatidylserine (PS) on the cell’s surface -> Annexing V
    -> Biochemical/molecular features
  4. Chromatin condensation, association of chromatin bodies to the nuclear membrane, nucleus fragmentation as well as some fragmentation of DNA -> DNA laddering TUNEL
  5. Cytoskeleton collapse, disruption of cell-cell adhesion and cell-matrix interaction, and cell shrinkage
  6. Membrane blebbing and formation of apoptotic bodies -> DAPI staining
    -> Morphological features
  7. Phagocytosis by neighboring cells or macrophages without inflammation
  8. Elimination of a single cell
  9. If apoptotic bodies are not disposed of, they rupture and undergo secondary necrosis
    -> Physiological effects
25
Q

C. elegant shares many cellular/mo,ecular structures and biological characteristics with higher organisms

A
  • Organized with mouth and brain at the anterior end and anus at the posterior end
  • Same basic tissues as in other animals (nerve, muscle, gut, skin)
  • It develops from an embryo to adult using developmental processes (e.g.: proliferation & differentiation)
  • It grows, reproduces, gets old and dies.
  • Coding genes: 20,191 and Human coding genes: 20, 465
26
Q

Short life cycle - Embryogenesis

A
  • Eggs are laid at about the 30 cell stage (roughly 2.5 hrs post fertilization at 20 C)
  • from fertilized egg (zygote) to hatching (small worm consisting of 558 cells = L1) takes about 14 hours (20 C)
27
Q

Waves of programmed cell death during C. elegant hermaphrodite development

A
  • Embryonic cell death (1st wave) -> 4-7.5 h
  • Laval cell deaths (2nd wave) -> 30 h
  • Germ-cell deaths (3rd wave) -> 60-120 h
28
Q

Physiological cell death during C. elegant development

A
  • During the development of the hermaphrodite, 131 of 1090 somatic cells are eliminated by programmed cell death (PCD).
  • 959 cells remain in the adult hermaphrodite.
  • 105 of the 131 cells that undergo PCD are embryonic neurons. (Others: muscle, hypodermal, and pharyngeal cells)
  • In the adult hermaphrodite, germ cells (only in female gonads) are also eliminated by PCD.
  • Note: Regenerative cell division occurs only in female germ cells
29
Q

Molecular regulation of apoptosis in C.elegans - Execution

A

Regulation through a series of direct protein-protein interactions (PPI)

EGL-1 (Inhibitor pro-apoptotic -> BH3-only)
-> binding and inhibition of CED-9
-> release of CED-4
CED-9 (Inhibitor, anti-apoptotic -> BH1-4-domain)
-> binding and inhibition of CED-4
CED-4 (Activator -> Apaf-1-like)
-> oligomerization, binding and activation of CED-3 proenzyme
CED-3 (protease (caspase))
-> Substrate cleavage and death

30
Q

Molecular regulation of apoptosis in C.elegans
-> Summary

A

1) The general apoptosis machinery consists of an activator (CED-4) & a protease (CED-3) (recruitment and autoactivation)
2) In healthy cells, the inhibitor CED-9 blocks the function of the activator CED-4
3) In dying cells, EGL-1 binds to and inhibits CED-9 activity
4) The activity of EGL-1 is regulated at the transcriptional level. Depending on the cell type, transcription factors can be activators or repressors

31
Q

Mitochondria-dependent and receptor-dependent apoptosis

A

EXTRINSIC PATHWAY (RECEPTOR-DEPENDENT APOPTOSIS)
- binding of ligands to specific death receptors
- activation of caspases

INTRINSIC PATHWAY (MITOCHONDRIA-DEPENDENT APOPTOSIS)
- activation of pro-apoptotic proteins
- release of pro-apoptotic factors from mitochondria
- formation of apoptosomes
- activation of caspases

32
Q

Effectors in mammalian cell death (execution) - intrinsic pathway -

A

Pro-apoptotic proteins of the BCL-2 family (EGL-1)
-> inhibiert
Anti-apoptotic proteins of the BCL-2 family (CED-9)
-> inhibiert
Activator Apaf-1 (CED-4)
-> aktiviert
Proteases Caspases (CED-3)
-> aktiviert
Substrate cleavage and cell death

33
Q

The Bcl-2 protein family

A

The death machinery in mammals is very complex and involves many apoptosis- regulating proteins (anti & pro)
Boxes: conserved Bcl-2 homology (BH) domains
TM: Most members contain a hydrophobic C-terminal transmembrane domain

Note:
One additional subgroup of pro-apoptotic proteins
-> BH1-3 multidomain proteins

34
Q

Activation of BAX

A

Binding of the tBID BH3 domain to the trigger site leads to conformational changes in BAX
- Freeing of the TM (a9) -> insertion into MOM
- Exposing the BH3 domain & freeing the BH3 binding groove
- Dissociation of tBID, BAX homodimerization/homooligomerization
- Symmetric or asymmetric autoactivation

Inactivate BAK/BAX:
The BH3 domain (alpha2) is covered by alpha1 and alpha8

35
Q

Effects of anti- and pro-apoptotic proteins III

A

Activation of apoptosis
Indirect activation of pro-apoptotic BH1-3 proteins
- Binding of BH3-only sensitizers (BAD, BMF) to anti-apoptotic BH1-4 proteins (BCL2, BLC-XL) displaces the bound BH3-only activators (BID, BIM).
- The released activators bind transiently to pro-apoptotic multidomain proteins.
-> activation & homooligomerization of BAX, BAK
- Binding of BH3-only protein to anti-apoptotic BH1-4 proteins (BCL2, BLC-XL) displace bound pro-apoptotic BH1-3 proteins which can be activated by BH3-only activators and inhibit anti-apoptotic BH1-4 proteins.

36
Q

BH3-only proteins can be activated by diverse death signals

A
  • Loss of cell-ECM contact -> Translocation from actin cytoskeleton -> BFM sensitizer
  • Growth factor deprivation -> Dephosphorylation -> Bad sensitizer
  • Death receptor activation -> Caspase 8 activation -> Bid -> Cleavage -> t-Bid Activator
  • Cytokine deprivation -> Dissociation from microtubules -> Translocation -> Bim Activator
  • DNA damage -> p53 transcription activator -> Puma Activator
37
Q

BAX/BAK-mediated mitochondrial outer membrane permeabilization (MOMP)

A
  1. BAX/BAK
    -> Shuttling between Cytosol and OMM
    -> BAX/BAK activation and stabilization at the OMM
    -> BH3-only proteins
  2. Dimerization
    -> higher order oligomers
  3. Lipid pore formation and MOMP
    -> Release of cytochrome c, SMAC and others from the IMS
    -> Cristae remodelling
    -> mtDNA release
    -> IMM herniation
    -> BAX/BAK acuumulation and macropore formation
38
Q

Formation of the apoptosome and activation of initiator caspase-9

A

CARD + Propeller (Closed configuration, ADP-bound, autoinhibited, compact monomer
-> binding of cytochrome c
-> exchange: ADP -> (D)ATP
open configuration: extended Apaf-1 subunit
-> Oligomerization
Formation of the apoptosome complex mediated by NOD
-> Recruitment of initiator pro-caspases-9 (CARD-CARD)
Close proximity leads to auto-activation of initiator caspase 9 (proximity-induced homodimerization; cleavage site)
Activated initiator caspase-9 remains bound to the apoptosome where it activates effector capsizes (e.g. caspase-3, -7)

39
Q

Effects of caspases

A

DESTRUCTION OF PROTEIN FUNCTION
- Paxillin: Component of the focal adhesion complex; collapse of the cytoskeleton and loss of adhesion
- AKT: Survival-Kinase; loss of kinase activity; activation of pro-apoptotic proteins (sensitizer BAD)

ACTIVATION OF PROTEIN FUNCTION
-Ca2+-indpendent Phospholipase A2: Caspase-3-activated enzyme converts phosphatidylcholine to lysopghosphatidylcholine (LPC); important for attraction of phagocytes
- Pannexin: Hexameric membrane channel; caspase-mediated cleavage results in ATP and UTP release that attracts phagocytes to the dying cell
- Helicard: CARD-containing RNA helicase; loss of CARD results in nuclear translocation; new function: reorganization of chromatin, accelerates DNA degradation

40
Q

Mitochondrial pro-apoptotic factors

A
  1. Cytochrome c
    - Binding to Apaf-1 to form the oligomeric apoptosome
  2. Smac/Diablo
    - Contains an IAP-binding motif (IBM; Ala-Val-Pro-Ile) which binds to the BIR of XIAPs
    - Competitively disrupts the XIAP-mediated inhibition of caspase
  3. Omi/HtrA2, protease
    - Contains an IBM
    - Disrupts the interaction between XIAPs and caspases and degrades XIAPs
  4. Endonuclease G
    - Chromosomal fragmentation (LMW-DNA) and generation of single-stranded nicks
  5. AIF (apoptosis-inducing factor)
    - Condensation of chromosomal DNA; supports DNase-mediated DNA fragmentation

Smac/Diablo: second mitochondria-derived activator of caspases / direct IAP-binding protein with low pI Omi/HtrA2 : Omi stress-regulated endoprotease/High temperature requirement protein A2

41
Q

Neurodegenerative, neuromuscular and motoneuron diseases (ND/NMD/MND)

A
  • Spinocerebellar ataxias
  • Atypical Parkinson-Syndromes
  • Huntington disease
  • Frontotemporal dementia
  • Amyotrophic lateral sclerosis
  • Spinale muscular atrophy
  • Hered. spastic paralysis
  • Myopathies
  • Myositis
  • Myasthenia gravis
42
Q

Exon-Skipping in Duchenne Muscular Dystrophy

A
  • Healthy: Dystrophin produced
  • DMD patient ex49-50 -> Premature stop codon (out-of-frame) -> no dystrophin produced
  • Eteplirsen-treated DMD patient ex49-50 -> reading frame restored -> shortened, functional dystrophin produced
43
Q

Primary endpoint in amyotrophic lateral sclerosis trials: ALSFRS-R
ALSFunctionalRatingScale-R (12 Questions 4…0pts Healthy 48 -> 0 no function)

A

Bulbar (#1-#3)
- Speech
- Saliva
- Swallowing

Upper Extr. (#4-#6)
- Writing
- Eating
- Hygiene

Torso/lower Extr. (#7-#9)
- Turning in bed
- Walking
- Climbing stairs

Breathing (#10-#12)
- Dyspnoe
- Orthopnoe
- respirat.Insufficiency

Pathomechanismen (13 decks)

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