SOD1 mediates lysosome-to-mitochondria communication and its dysregulation by amyloid-ß oligomers.

Altered mitochondrial DNA (mtDNA) occurs in neurodegenerative disorders like Alzheimer's disease (AD); how mtDNA synthesis is linked to neurodegeneration is poorly understood. We previously discovered Nutrient-induced Mitochondrial Activity (NiMA), an inter-organelle signaling pathway where nutrient-stimulated lysosomal mTORC1 activity regulates mtDNA replication in neurons by a mechanism sensitive to amyloid-ß oligomers (AßOs), a primary factor in AD pathogenesis (Norambuena et al., 2018). Using 5-ethynyl-2'-deoxyuridine (EdU) incorporation into mtDNA of cultured neurons, along with photoacoustic and mitochondrial metabolic imaging of cultured neurons and mouse brains, we show these effects being mediated by mTORC1-catalyzed T40 phosphorylation of superoxide dismutase 1 (SOD1). Mechanistically, tau, another key factor in AD pathogenesis and other tauopathies, reduced the lysosomal content of the tuberous sclerosis complex (TSC), thereby increasing NiMA and suppressing SOD1 activity and mtDNA synthesis. AßOs inhibited these actions. Dysregulation of mtDNA synthesis was observed in fibroblasts derived from tuberous sclerosis (TS) patients, who lack functional TSC and elevated SOD1 activity was also observed in human AD brain. Together, these findings imply that tau and SOD1 couple nutrient availability to mtDNA replication, linking mitochondrial dysfunction to AD.

A novel lysosome-to-mitochondria signaling pathway disrupted by amyloid-ß oligomers.

The mechanisms of mitochondrial dysfunction in Alzheimer's disease are incompletely understood. Using two-photon fluorescence lifetime microscopy of the coenzymes, NADH and NADPH, and tracking brain oxygen metabolism with multi-parametric photoacoustic microscopy, we show that activation of lysosomal mechanistic target of rapamycin complex 1 (mTORC1) by insulin or amino acids stimulates mitochondrial activity and regulates mitochondrial DNA synthesis in neurons. Amyloid-ß oligomers, which are precursors of amyloid plaques in Alzheimer's disease brain and stimulate mTORC1 protein kinase activity at the plasma membrane but not at lysosomes, block this Nutrient-induced Mitochondrial Activity (NiMA) by a mechanism dependent on tau, which forms neurofibrillary tangles in Alzheimer's disease brain. NiMA was also disrupted in fibroblasts derived from two patients with tuberous sclerosis complex, a genetic disorder that causes dysregulation of lysosomal mTORC1. Thus, lysosomal mTORC1 couples nutrient availability to mitochondrial activity and links mitochondrial dysfunction to Alzheimer's disease by a mechanism dependent on the soluble building blocks of the poorly soluble plaques and tangles.

Isolation, growth, and characterization of human renal epithelial cells using traditional and 3D methods.

The kidney is a highly heterogeneous organ that is responsible for fluid and electrolyte balance. Much interest is focused on determining the function of specific renal epithelial cells in humans, which can only be accomplished through the isolation and growth of nephron segment-specific epithelial cells. However, human renal epithelial cells are notoriously difficult to maintain in culture. This chapter describes the isolation, growth, immortalization, and characterization of the human renal proximal tubule cell. In addition, we describe new paradigms in 3D cell culture which allow the cells to maintain more in vivo-like morphology and function.