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

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  Cerebral Cortex  Alzheimer's, Cerebral Cortex Alzheimer's,  Basal Ganglia  Parkinson's, Parkinson's, Basal Ganglia  Striatum ,  Huntington's diseases Huntington's diseases, Striatum  Motor neurons ALS  Motor neurons ALS

• 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?

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 Project: 108T179 Project: 108T179 Screens Negative Validation X Conclusions Positive Mutations Characterisation in Homogeneous and No Mammalian Cell Cultures Heteregeneous Mammalian Cell Yes Cultures Mutation search in the Drosophila Cell selected genes Cultures Drosophila Homologue of Alsin Transgenic Drosophila Yes/No CG7158 “ Knock-out ” cells and tissues ALS No Phenotype Yes/No Mutant CG7158 “ Knock-in ”

Amyotrophic Lateral Sclerosis  “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

ALS is the most common motor neuron disease  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

Most ALS Cases Are Isolated Incidences  Familial ALS : fALS   % 10 % 10 (AD inheritance)  Sporadic ALS : sALS   % 90 % 90 (no documented family history, genetic contribution?) fALS sALS fALS and sALS are clinically similar!! fALS and sALS are clinically similar!!

FALS : Genetically Heteregeneous Autosomal Autosomal Maternal Autosomal Autosomal X linked Maternal X linked Dominant Recesive Dominant Dominant Recesive Dominant * ALS1 (SOD1, chr.21) * ALS-M * * ALS-M ALS1 (SOD1, chr.21) ALS2 (Alsin, chr.2) [J] ALS2 (Alsin, chr.2) [J] * ALSX (?, X chr.) * ALSX (?, X chr.) (COX1, mt (COX1, mt ALS3 (?, chr.18) ALS3 (?, chr.18) DNA) DNA) ALS5 (?, chr.15) [J] ALS5 (?, chr.15) [J] ALS4 (SETX, chr.9) ALS4 (SETX, chr.9) [J] [J] * ALS-M * ALS-M * ALS6 (?, chr.16) * ALS6 (?, chr.16) (IARS2, mt (IARS2, mt DNA) DNA) * ALS7 (?, chr.20) * ALS7 (?, chr.20) * ALS8 (VAPB, chr.20) * ALS8 (VAPB, chr.20) Dynactin Dynactin * Typical ALS * Typical ALS ALS10 (TDP-43) ALS10 (TDP-43) * Single Families * Single Families FUS/TLS FUS/TLS

Genes involved in ALS  SOD1  Alsin  Senataxin  VAPB  Dynactin  TDP-43  FUS FUS/TLS /TLS

Unusual Suspects Unusual Suspects of of Amyotrophic Lateral Sclerosis (ALS) Amyotrophic Lateral Sclerosis (ALS)

Alsin (ALS2) 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! 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)

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

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

- - + (Bait+Prey) + (Bait+Prey) (0+Prey) (0+Prey) True-False Interaction Test - LT - LT in Yeast Cells + Control + Control - Control Bait+0 - Control Bait+0 - AHLT - AHLT Colony Lift X-Gal Assay Colony Lift X-Gal Assay

Yeast Two Hybrid Screens Origins • SRPK2 SRPK2 • UXT UXT • NDUFV1 NDUFV1 • PMM1 PMM1 • VARS2 VARS2 • PSMB4 PSMB4 Continues Continues

Senataxin Screens • Snapin  Exocytosis, Calcium Channel Modulation Snapin  Exocytosis, Calcium Channel Modulation • Sorcin  Calcium Channel Modulation Sorcin  Calcium Channel Modulation • Aborted Aborted

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? SRPK2 SFRS2 SRPK2 FUS/TLS Alsin NDUFV1 NDUFV1 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 Domain Homology Domain S R P K 2 + + + + + U X T + + 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 - 

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 Differentiation SH-SY5Y  Neuron like C2C12  Muscle like • Grow a week in Proceeding to confluency 10%FBS/DMEM/30  M RA grow a week in 1%HS/DMEM • Trypsinize and seed and grow a week in 1%HS/DMEM/30  M

Training of SH-SY5Y cells for differentiation

SHSY5Y neuronlike differentiation

Cocultures – Establishment of differentiated heterogeneous cell cultures • SH-SY5Y-C2C12 SH-SY5Y-C2C12 JBC 2003 Vol. 278 No: 46 p45435-45444

Coculture Trial No need to add RA No need to add RA

Observations Available Techniques Observations Available Techniques • Gene regulation • qPCR • CAT Assays • Cytological • Immunolocalization – Cellular structures • Realtime observation of – Cellular processes • Cellular structures • Cytoplasmic transport • Apoptosis

UXT vs Alsin • Overexpression of mAlsin in human SH-SY5Y cells

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