Benevolent Platform Precision Medicine

Multiple assays used for target validation in ALS, similar approaches being employed for Parkinson's disease 1. Autophagy assay in Hela cells 2. Glutamate induced excitotoxicity in sporadic ALS patient iPSC MNs Glutamate induced excitotoxicity assay (Annexin V ALS Motor Neurons) TR-FRET between LC3-Tb and LC3-D2 or p62- D2 and p62-Tb LC31 is found in the cytoplasm and is conjugated to the lipid phosphatidyl-ethanolamine to form LC311 LC311 binds to the autophagic vacuoles' membrane marker of autophagic vacuoles • SQSTM1/p62 -selective autophagy receptor -sequesters ubiquitinated proteins into AVS by interacting with LC3 - p62 is a substrate for autophagic degradation → its degradation can be used as a marker of autophagic clearance PAGE 2 Astrocytes 3. SITraN ALS iAstrocyte mouse motor neuron co-culture assay Lactate efflux transporters tp75 NGF receptor The University Sheffield Pathophysiologically Relevant in vitro Model of ALS SITraN Human Astrocytes HD9-GFP+ mouse motor neurons % motor i Con155 Con3050 ConAG Pat 78 Pat183 Pat201 Pat009 Pat 12 iAstrocyte line Pat17 Control C9ORF72-ALS Sporadic ALS ALS Patient Derived iAstrocytes are Toxic to Motor Neurons in Co-Culture Impaired axonal transport ALS mechanisms Reduced lactate release Motor neuron Apoptosis Mitochondrial dysfunction NO BAX Glutamate transport Excitotoxicity Ca ROS PGE2 Altered RNA processing Complement activation C1q Autophagy Pro-NGF PGE2 NO Inflammation MCP-1 ER stress Distal axonopathy Microglia Misfolded proteins BEN-3765 Time() + Glbra 10 M 1.M 1000- o Sam Co Annexin V Oy ANOVA The ALS PSC motor neurons were recovered and seeded onto a Matrigel-coated 96-well plate at 15.000/well for 4 days On Day 0, the were treated with vehicle controls or smal molecule at afferent doses for 7 days, and then treated with bet Glutamate for 3 days. The Annexin V red dye (Butun were added to visualize the apoptotic cells using IncuCyte 53 software (red object count 5 images in one wel of each condition were analysed. The average of the Total Red Object Area Well was shown Left panell Dynamic data (Right pane Dyda iXCells BIOTECHNOLOGIES Proteasome impairment 4. Tunicamycin induced motor neuron toxicity Controll Neurite Length pic 6.7 In-house hiPSC- MN assay (% Control) DIC 7.0 -10 -10 · Log [Tunic amycin] (M) Log [Tunic amycin] (M) CYTOPLAN Tunicamycin 1 96h 144 h Perturbation of ER homeostasis Accumulation of RES PERK ATF6 Unfolded Protein Response (UPR) Unfolded Benevolent 47

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