Investigation Of ER-mitochondria Complex And MtDNA In Neurodegeneration Using OMM-ER Linker And UL12.5 Protein

Neuritic degeneration is an important early pathological step in neurodegeneration,and mitochondria play an important role in this process.This project includes two parts for studying the function of mitochondria in neural degeneration:part one is the function of ER-Mito complex in neural degeneration,and part 2 is the relationship of mitochondrial DNA(mtDNA)depletion and neural degeneration.In part one,we established a Charcot-Marie-Tooth type 2A(CMT2A)iPSCs-derived neurons disease model.In iPSCs-derived neurons from a CMT2A patient harboring a mutation in MFN2,we observed the neuritic degeneration with dysfunctional mitochondria,fragmented ER and ER-Mito complex formation.To illustrate the mechanisms connecting neuritic degeneration to the functional and morphological remodeling of ER and mitochondria,we set up in vivo and in vitro neuritic degeneration models by spared nerve injury in rat and neurite-cutting induced neural degeneration in human induced-pluripotent stem cell derived neurons.We found that neuritic ER becomes fragmented and forms complexes with mitochondria at sites with high local Ca2+ concentration during neuritic degeneration.Furthermore,mitochondrial membrane potential is required for ER fragmentation and mitochondrial Ca2+ elevation during neuritic degeneration,IP3R inhibitor Xestospongin C or mitochondrial uniporter inhibitor Ru360 inhibit ER fragmentation and protect neurite from degeneration.Mechanically,tightening of theER-mitochondria associations by expression of a short "synthetic linker" and ER Ca2+releasing together could promote mitochondrial dysfunction,increased reactive oxygen species generation and neuritic degeneration.Finally,our study reveals a dynamic remodeling of the ER-mitochondria interface underlying neuritic degeneration.In part two,we expressed UL12.5-a mitochondrial isoform of the viral alkaline nuclease,to directly degrade mtDNA of human neurons(rhoO neurons).We show that UL12.5 located in neural mitochondria and depleted neural mtDNA,which increase ryanodine receptor(cyc)expression,cytoplasmic Ca2+ concentration and ROS level,decrease ATP production.In mitochondria,mtDNA depeletion cause mPTP open,mitochondrial dynamic dysfunction and ??m maintained,while oligomycin depolarized ??m in rhoO neurons.Further studies found the dysfunction of the mitochondria's oxidative phosphorylation system(OXPHOS)and abnormal electrophysiology activity.2-Dexoy-D-Glucose(2-DG,inhibitor of glucose metabolism)which could model caloric restriction caused rhoO neurons degeneration and apoptosis.Lastly,we used the mouse model with POLG mutation,which cause mtDNA mutation,to study the affection of caloric restriction.We found 2-DG cause motor performance defection much more severe than physiological saline treatment in mtDNA mutated mouse.Collectively,this is the first report of the model of rho0 neuron,and this project provides a systematic study in rhoO neurons.Mouse behavior experiments further confirm that caloric restriction worse exercise intolerance in the mouse with POLG mutation.