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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