Profiling Lysosomal and Mitochondrial Dysfunction in Neurodegenerative Diseases Using Human Fibroblasts for Translational Therapeutic Screening

Abstract

Lysosomal dysfunction and mitochondrial health are intricately connected, playing essential roles in cellular homeostasis. Lysosomes are acidic membrane-bound organelles responsible for degrading and recycling cellular waste, while mitochondria generate the energy required for cellular functions. Growing evidence implicates roles for lysosomal and mitochondrial dysfunction in neurodegenerative diseases, including Alzheimers and Parkinsons disease. With novel therapeutics targeting both the lysosomal and mitochondrial functions, robust assays for compound screening are becoming critical to evaluate modulation of both organelles in disease-relevant cellular models. Here, we investigated human fibroblasts derived from healthy donors, as well as patients with Alzheimers and Parkinsons disease, to assess their capacity to model key aspects of lysosomal and mitochondrial dysfunction. Lysosomal function was evaluated using various assays, including quantification of lysosomal proteins (TMEM175 and LAMP1), LysoTracker staining, measurement of lysosomal pH, and lysosomal enzymatic activity. Autophagic flux was assessed by measuring p62 levels as a marker of autophagy. Mitochondrial function was investigated by measuring mitochondrial calcium levels, membrane potential, oxidative stress, and mitochondrial content using MitoTracker. To explore the potential of using human fibroblasts for in vitro compound screening, we validated these assays in a 384-well high-throughput format using compounds such as chloroquine and ammonium chloride. Our findings demonstrate that human fibroblasts faithfully recapitulate lysosomal and mitochondrial dysfunctions characteristic of neurodegenerative diseases. Moreover, the use of robust assays positions these cells as a valuable platform for high-throughput screening to identify novel therapeutics targeting lysosomal and mitochondrial pathways.