Unusual Suspects Unusual Suspects of of Amyotrophic Lateral - - PowerPoint PPT Presentation

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Unusual Suspects Unusual Suspects of of Amyotrophic Lateral - - PowerPoint PPT Presentation

Dec 02, 2022 97 likes •452 views

Unusual Suspects Unusual Suspects of of Amyotrophic Lateral Sclerosis (ALS) Amyotrophic Lateral Sclerosis (ALS) An Investigation for the Mechanism of the Motor Neuron Degeneration Neurodegenerative Diseases Each neurodegenerative

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Unusual Suspects Unusual Suspects

  • f
  • f

Amyotrophic Lateral Sclerosis (ALS) Amyotrophic Lateral Sclerosis (ALS)

An Investigation for the Mechanism

  • f the Motor Neuron Degeneration
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SLIDE 2

Neurodegenerative Diseases

  • Each neurodegenerative disease is

characterized by the death of specific populations of neurons in defined regions of the brain, spinal cord, and peripheral nerves Alzheimer's, Alzheimer's,   Cerebral Cortex Cerebral Cortex Parkinson's, Parkinson's,   Basal Ganglia Basal Ganglia Huntington's diseases Huntington's diseases, ,   Striatum Striatum ALS ALS  Motor neurons Motor neurons

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SLIDE 3
  • However neuropathological features are similar
  • Protein aggregation

Protein aggregation

  • Mitochondrial dysfunction

Mitochondrial dysfunction

  • Disrupted cellular transport

Disrupted cellular transport

  • Inflamation

Inflamation

  • Mutations in the same

gene may result to several different neurodegenerative disease Hence a common mechanism? Hence a common mechanism?

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How to reveal this mechanism?

1. Search for new genes – Genome-wide association studies – Linkage analysis – Protein-protein interactions 2. Functional analysis

Hanabusa Itchō (1652-1724)

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SLIDE 5

Yeast Two Hybrid Screens Validation Homogeneous and Heteregeneous Mammalian Cell Cultures X

Negative Positive

Drosophila Homologue of Alsin CG7158 “Knock-out” Mutant CG7158 “Knock-in” Transgenic Drosophila cells and tissues Mutation search in the selected genes Drosophila Cell Cultures

ALS Phenotype

Yes/No No Yes/No

Conclusions

No Yes

Mutations Characterisation in Mammalian Cell Cultures

Project: 108T179 Project: 108T179

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SLIDE 6

 “Dying back Dying back” of the motor neuron  Degeneration and death of upper upper and lower lower motor neurons in the brain and spinal cord  Progressive muscle weakness  Atrophy and spasticity  Denervation of the respiratory muscles and diaphragm is the fatal event

Amyotrophic Lateral Sclerosis

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Incidence: 1-2 / 100 000 / year Prevalence: 4 - 8 / 100 000 Estimated ALS patients:

World ~ 90 000 – 100 000 Turkey ~ 5000 – 7500

Responsible for 1/1000 deaths

Responsible for 1/1000 deaths

ALS is the most common motor neuron disease

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SLIDE 8

Most ALS Cases Are Isolated Incidences  

% 90 % 90

 

% 10 % 10

sALS

fALS

 Familial ALS : fALS

(AD inheritance)

 Sporadic ALS : sALS

(no documented family history, genetic contribution?)

fALS and sALS are clinically similar!! fALS and sALS are clinically similar!!

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SLIDE 9

FALS : Genetically Heteregeneous

Autosomal Autosomal Recesive Recesive X linked X linked Dominant Dominant

* *ALS1 (SOD1, chr.21)

ALS1 (SOD1, chr.21) ALS3 (?, chr.18) ALS3 (?, chr.18) ALS4 (SETX, chr.9) ALS4 (SETX, chr.9) [J] [J]

* *ALS6 (?, chr.16)

ALS6 (?, chr.16)

* *ALS7 (?, chr.20)

ALS7 (?, chr.20)

* *ALS8 (VAPB, chr.20)

ALS8 (VAPB, chr.20) Dynactin Dynactin ALS10 (TDP-43) ALS10 (TDP-43) FUS/TLS FUS/TLS ALS2 (Alsin, chr.2) [J] ALS2 (Alsin, chr.2) [J] ALS5 (?, chr.15) [J] ALS5 (?, chr.15) [J]

* * ALSX (?, X chr.)

ALSX (?, X chr.)

* * Typical ALS

Typical ALS

* *ALS-M

ALS-M (COX1, mt (COX1, mt DNA) DNA)

* *ALS-M

ALS-M (IARS2, mt (IARS2, mt DNA) DNA)

* * Single Families

Single Families

Autosomal Autosomal Dominant Dominant Maternal Maternal

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SLIDE 10

Genes involved in ALS

 SOD1  Alsin  Senataxin  VAPB  Dynactin  TDP-43  FUS FUS/TLS /TLS

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Unusual Suspects Unusual Suspects

  • f
  • f

Amyotrophic Lateral Sclerosis (ALS) Amyotrophic Lateral Sclerosis (ALS)

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Alsin (ALS2)

Three small GTPase regulator homologous domains: Three small GTPase regulator homologous domains:

  • The regulator of choromosome condensation 1 (RCC1)

The regulator of choromosome condensation 1 (RCC1)

  • Rho guanine exchange factor (Rho GEF)

Rho guanine exchange factor (Rho GEF)

  • Vacuolar protein sorting 9 (VSP9)

Vacuolar protein sorting 9 (VSP9) (Endosomal (Endosomal trafficking) trafficking)

May be the first step for the elucidation of a common May be the first step for the elucidation of a common mechanism of motorneuron degeneration! mechanism of motorneuron degeneration!

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SLIDE 13
  • Alsin mutations lead related

Alsin mutations lead related but clinically distinct motor but clinically distinct motor neuron degenerative neuron degenerative diseases: diseases: – ALS ALS – PLS PLS – HSP HSP – SMA SMA

Alsin Mutations

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SLIDE 14

Yeast Two Hybrid Screen

  • A popular technique to discover

protein-protein interactions by testing for physical interactions.

  • Provides an important first hint for

the identification of interacting partners.

  • Current Applications
  • Determination of sequences

crucial for interaction

  • Drug and poison discovery
  • Determination of protein

function

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SLIDE 15

True-False Interaction Test in Yeast Cells

  • LT
  • LT
  • AHLT
  • AHLT

Colony Lift X-Gal Assay Colony Lift X-Gal Assay

+ + (Bait+Prey)

(Bait+Prey)

  • (0+Prey)

(0+Prey)

+ Control + Control

  • Control
  • Control

Bait+0 Bait+0

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Yeast Two Hybrid Screens Origins

  • SRPK2

SRPK2

  • UXT

UXT

  • NDUFV1

NDUFV1

  • PMM1

PMM1

  • VARS2

VARS2

  • PSMB4

PSMB4 Continues Continues

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SLIDE 17

Senataxin Screens

  • Snapin

Snapin   Exocytosis, Calcium Channel Modulation Exocytosis, Calcium Channel Modulation

  • Sorcin

Sorcin   Calcium Channel Modulation Calcium Channel Modulation

  • Aborted

Aborted

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Why DH/PH Domain?

  • DH and PH domains are connected to each other

with elastic regions.

  • mSOD1 interacts with Alsin through DH/PH region
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Two New Candidates of ALS

  • SRPK2 (Serine Rich Protein Kinase 2)

– Phosphorylates many key proteins involved in cell survival

  • Serine rich splicing factors
  • Apoptosis related protein (Acinus)
  • Cell cycle related protein (Cyclin A1)
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SLIDE 20

Revealing the first helices in the chain?

Alsin SRPK2 SRPK2 SFRS2 FUS/TLS NDUFV1 NDUFV1 EWSR1

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SLIDE 21

UXT (Ubiqutiously Expressed Transcript)

  • UXT (Ubiqutiously Expressed Transcript)

– Coactivator of Androgen Receptor – Essential cofactor in NF-kB transcriptional enhanceosome

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Proteins are Interacting with DBL Proteins are Interacting with DBL Homology Domain Homology Domain

+ + + + +

P r e f o l d i n - 

+ + + + +

P r e f o l d i n -  P r e f o l d i n - 

S R P K 2 U X T

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SLIDE 23

Validation of Interaction in vivo

IP of SRPK2

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Cell Culture Models

  • Aim:

– To simulate the natural environment where the protein interactions occur without the obstacles

  • f using animal models.
  • Cost
  • Time
  • Difficult methodology
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Method of Action for Cell Culture Models

– Differentiation of immortalized cell lines

  • Neuroblasts (SH-SY5Y)  neuron like cells
  • Myoblasts (C2C12)  muscle like cells
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Procedure for Neuroblast/Myoblast Differentiation

SH-SY5Y  Neuron like

  • Grow

a week in 10%FBS/DMEM/30M RA

  • Trypsinize and seed and

grow a week in 1%HS/DMEM/30M C2C12  Muscle like Proceeding to confluency grow a week in 1%HS/DMEM

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Training of SH-SY5Y cells for differentiation

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SLIDE 28

SHSY5Y neuronlike differentiation

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– Establishment of differentiated heterogeneous cell cultures

  • SH-SY5Y-C2C12

SH-SY5Y-C2C12

JBC 2003 Vol. 278 No: 46 p45435-45444

Cocultures

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Coculture Trial

No need to add RA No need to add RA

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Available Techniques Available Techniques

  • Gene regulation
  • Cytological

– Cellular structures – Cellular processes

Observations Observations

  • qPCR
  • CAT Assays
  • Immunolocalization
  • Realtime observation of
  • Cellular structures
  • Cytoplasmic transport
  • Apoptosis
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SLIDE 32

UXT vs Alsin

  • Overexpression of mAlsin in human SH-SY5Y cells
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Animal Models Animal Models

  • Drosophila M. Cell Cultures

– Aim: Characterise insect homologues of mammalian proteins under investigation

  • Transgenic Drosophila M. Models

– Aim: Further approach to real life processes of the disease

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SLIDE 34