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

Unusual Suspects of 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, Cerebral Cortex Parkinson's, Basal Ganglia Huntington's diseases, Striatum ALS Motor neurons

However neuropathological features are similar Protein aggregation Mitochondrial dysfunction Disrupted cellular transport Inflamation Mutations in the same gene may result to several different neurodegenerative disease Hence a common mechanism?

How to reveal this mechanism? 1. 2. Search for new genes Genome-wide association studies Linkage analysis Protein-protein interactions Functional analysis Hanabusa Itchō (1652-1724)

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

Amyotrophic Lateral Sclerosis Dying back back of the motor neuron Degeneration and death of upper and 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

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

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

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

Unusual Suspects of Amyotrophic Lateral Sclerosis (ALS)

Alsin (ALS2) May be the first step for the elucidation of a common mechanism of motorneuron degeneration! Three small GTPase regulator homologous domains: The regulator of choromosome condensation 1 (RCC1) Rho guanine exchange factor (Rho GEF) Vacuolar protein sorting 9 (VSP9) (Endosomal trafficking)

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

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

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

Senataxin Screens Snapin Exocytosis, Calcium Channel Modulation Sorcin Calcium Channel Modulation 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 FUS/TLS Alsin 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 Homology Domain SRPK2 ++ ++ + UXT P r e f o ld in - P re fo ld in - P re fo ld in - ++ ++ +

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 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

Training of SH-SY5Y cells for differentiation

SHSY5Y neuronlike differentiation

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

Coculture Trial No need to add RA

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

UXT vs Alsin Overexpression of malsin in human SH-SY5Y cells

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