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 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 diseasesHuntington's diseases, , Striatum StriatumALSALS Motor neurons Motor neurons

• However neuropathological features are similar• Protein aggregationProtein aggregation• Mitochondrial dysfunctionMitochondrial dysfunction• Disrupted cellular transportDisrupted cellular transport• InflamationInflamation

•Mutations in the same gene may result to several different neurodegenerative disease

Hence a common mechanism?Hence a common mechanism?

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)

Yeast Two Hybrid Screens

Validation

Homogeneous and Heteregeneous Mammalian Cell

Cultures

XNegative

Positive

Drosophila Homologue of Alsin CG7158 “Knock-out”

Mutant CG7158 “Knock-in”

Transgenic Drosophila cells and tissues

Mutation search in the selected genesDrosophila Cell

Cultures

ALS Phenotype

Yes/No

No

Yes/No

Conclusions

No

Yes

Mutations Characterisation in Mammalian Cell Cultures

Project: 108T179Project: 108T179

“Dying backDying back” of the motor neuron

Degeneration and death of upperupper and lowerlower 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

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 deathsResponsible for 1/1000 deaths

ALS is the most common motor neuron disease

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!!

FALS : Genetically Heteregeneous

Autosomal Autosomal RecesiveRecesive

X linked X linked DominantDominant

**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)

DynactinDynactin

ALS10 (TDP-43)ALS10 (TDP-43)

FUS/TLSFUS/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 ALSTypical ALS

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

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

* * Single FamiliesSingle Families

Autosomal Autosomal DominantDominant

MaternalMaternal

Genes involved in ALS

SOD1 Alsin Senataxin VAPB Dynactin TDP-43

FUSFUS/TLS/TLS

Unusual Suspects Unusual Suspects of of

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

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!

• Alsin mutations lead related Alsin mutations lead related but clinically distinct motor but clinically distinct motor neuron degenerative neuron degenerative diseases:diseases:

– ALSALS– PLSPLS– HSPHSP– SMASMA

Alsin Mutations

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 ApplicationsDetermination of sequences crucial for interactionDrug and poison discoveryDetermination of protein function

True-False Interaction Test in Yeast Cells

- LT- LT

- AHLT- AHLT

Colony Lift X-Gal AssayColony Lift X-Gal Assay

+ + (Bait+Prey)(Bait+Prey)-- (0+Prey)(0+Prey)

+ Control+ Control

- Control- Control Bait+0Bait+0

Yeast Two Hybrid ScreensOrigins

• SRPK2SRPK2• UXTUXT• NDUFV1NDUFV1• PMM1PMM1• VARS2VARS2• PSMB4PSMB4

ContinuesContinues

Senataxin Screens

• Snapin Snapin Exocytosis, Calcium Channel Modulation Exocytosis, Calcium Channel Modulation• Sorcin Sorcin Calcium Channel Modulation Calcium Channel Modulation• AbortedAborted

Why DH/PH Domain?

• DH and PH domains are connected to each other with elastic regions.

• mSOD1 interacts with Alsin through DH/PH region

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)

Revealing the first helices in the chain?

Alsin

SRPK2SRPK2 SFRS2

FUS/TLS

NDUFV1NDUFV1

EWSR1

UXT (Ubiqutiously Expressed Transcript)

• UXT (Ubiqutiously Expressed Transcript)– Coactivator of Androgen Receptor– Essential cofactor in NF-kB transcriptional enhanceosome

Proteins are Interacting with DBL Proteins are Interacting with DBL Homology DomainHomology 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

Validation of Interaction in vivo

IP of SRPK2

Cell Culture Models

• Aim:– To simulate the natural environment where the

protein interactions occur without the obstacles of using animal models.

• Cost• Time• Difficult methodology

Method of Action for Cell Culture Models

– Differentiation of immortalized cell lines• Neuroblasts (SH-SY5Y) neuron like cells

• Myoblasts (C2C12) muscle like cells

Procedure for Neuroblast/Myoblast

DifferentiationSH-SY5Y Neuron like

• Grow a week in 10%FBS/DMEM/30M RA

• Trypsinize and seed and grow a week in 1%HS/DMEM/30M

C2C12 Muscle like

Proceeding to confluency grow a week in 1%HS/DMEM

Training of SH-SY5Y cells for differentiation

SHSY5Y neuronlike differentiation

– Establishment of differentiated heterogeneous cell cultures

• SH-SY5Y-C2C12SH-SY5Y-C2C12

JBC 2003 Vol. 278 No: 46 p45435-45444

Cocultures

Coculture Trial

No need to add RANo need to add RA

Available TechniquesAvailable Techniques

• Gene regulation

• Cytological– Cellular structures

– Cellular processes

ObservationsObservations

• qPCR• CAT Assays

• Immunolocalization

• Realtime observation of • Cellular structures• Cytoplasmic transport

• Apoptosis

UXT vs Alsin

• Overexpression of mAlsin in human SH-SY5Y cells

Animal ModelsAnimal 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