Characterizing Secretory Mitophagy in Lund Human Mesancephalic (LUHMES) Cells as a Model of Parkinson’s Disease

Abstract

Parkinson’s disease is characterized by the progressive loss of dopamine-producing neurons. Production of dopamine induces oxidative stress that damages mitochondria. Therefore, mitochondrial damage is an important feature of this neurodegeneration. Secretory mitophagy (SM) is a noncanonical mitophagy process triggered by high levels of oxidative stress, where damaged mitochondria are packaged into extracellular vesicles (EVs) and released from cells when the normal lysosome removal process is overwhelmed. This project investigates whether SM may serve as a protective mechanism in Parkinson’s disease. LUHMES, conditionally immortalized midbrain-derived human neuronal precursor cells, are capable of differentiation into dopamine producing neurons. We collected and characterized EVs from untreated early-passage LUHMES cells. EVs were isolated through centrifugation and analyzed using mass spectrometry. Protein classes and signaling pathways were examined to characterize the baseline protein cargo in unstressed stem cells before neuronal differentiation. Western blotting was used to confirm EV- and neuron-associated markers. Proteomic analysis identified proteins involved in ATP synthesis, p53 signaling, and Parkinson’s and Alzheimer’s disease-presinilin pathways. These pathways regulate cellular energy production, DNA repair and cell cycle control, and neuronal signaling and are essential for normal neuronal development and function. However, their levels may be elevated in pathological conditions. The presence of these proteins in EVs of untreated neuronal stem cells suggests that a baseline level of secretory mitophagy may occur naturally in LUHMES cells. This baseline will provide a reference for determining whether oxidative stress increases secretory mitophagy in future experiments.