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

Transcription

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

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

3 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?

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

5 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

6 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

7 ALS is the most common motor neuron disease Incidence: 1-2 / / year Prevalence: 4-8 / Estimated ALS patients: World ~ Turkey ~ Responsible for 1/1000 deaths

8 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!!

9 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

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

11 Unusual Suspects of Amyotrophic Lateral Sclerosis (ALS)

12 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)

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

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

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

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

17 Senataxin Screens Snapin Exocytosis, Calcium Channel Modulation Sorcin Calcium Channel Modulation Aborted

18 Why DH/PH Domain? DH and PH domains are connected to each other with elastic regions. msod1 interacts with Alsin through DH/PH region

19 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)

20 Revealing the first helices in the chain? SRPK2 SFRS2 FUS/TLS Alsin NDUFV1 EWSR1

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

22 Proteins are Interacting with DBL Homology Domain SRPK UXT P r e f o ld in - P re fo ld in - P re fo ld in

23 Validation of Interaction in vivo IP of SRPK2

24 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

25 Method of Action for Cell Culture Models Differentiation of immortalized cell lines Neuroblasts (SH-SY5Y) neuron like cells Myoblasts (C2C12) muscle like cells

26 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

27 Training of SH-SY5Y cells for differentiation

28 SHSY5Y neuronlike differentiation

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

30 Coculture Trial No need to add RA

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

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

33 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

34