Alterations in mitochondrial function, as observed in neurodegenerative diseases, lead to disrupted energy metabolism and production of damaging reactive oxygen species. Here, we demonstrate that mitochondrial dysfunction also disrupts the structure and function of lysosomes, the main degradation and recycling organelle. Specifically, inhibition of mitochondrial function, following deletion of the mitochondrial protein AIF, OPA1, or PINK1, as well as chemical inhibition of the electron transport chain, impaired lysosomal activity and caused the appearance of large lysosomal vacuoles. Importantly, our results show that lysosomal impairment is dependent on reactive oxygen species. Given that alterations in both mitochondrial function and lysosomal activity are key features of neurodegenerative diseases, this work provides important insights into the etiology of neurodegenerative diseases.A prominent feature of neurodegenerative diseases, including Parkinson disease (PD) 3 and Alzheimer disease, is the accumulation of undigested protein aggregates (1, 2). Although the underlying mechanisms are complex, several cellular alterations can cause aggregate accumulation, including impaired quality control pathways and the generation of reactive oxygen species (ROS) as a consequence of mitochondrial damage (1, 3).In healthy cells, protein aggregates and damaged cellular components are delivered to lysosomes to be degraded through a process termed autophagy (1, 2, 4, 5). As such, autophagy plays an important neuroprotective role. Nevertheless, the defects in degradation pathways observed in neurodegenerative diseases extend well beyond alterations in autophagy. Specifically, disruption of lysosomal function has been linked to neuronal loss in several neurodegenerative diseases. For example, mutations in the lysosomal ATPase ATP13A2 cause PD, whereas lysosomal dysfunction in Gaucher disease leads to Parkinsonism and the appearance of Lewy bodies (6, 7). In addition, the ␣-synuclein-containing Lewy bodies found in PD are positive for lysosomal markers, suggesting that they represent lysosomes that failed to degrade their content (8). In fact, lysosomal alterations are a common feature of PD (8 -11). Nevertheless, the pathological consequences of a loss of lysosomal function extend well beyond PD, because a wide variety of mutations that impair lysosomes and cause the accumulation of intracellular material (lysosomal storage diseases) show features of neurodegeneration (12).A second key metabolic pathway required for neuronal survival is mitochondrial activity. In fact, mitochondrial dysfunction is a common feature of neurodegenerative diseases. For example, decreased activity of complex I of the electron transport chain (ETC) is present in a number of PD cases (13, 14), whereas amyloid ␤, Tau tangles, and Htt aggregates all cause mitochondrial dysfunction (15-17). In addition, several genes mutated in PD (including PINK1, Parkin, and DJ-1) affect mitochondrial function and turnover (18 -21), whereas deregulation of mitochon...

Mitochondria exist as a highly interconnected network that is exquisitely sensitive to variations in nutrient availability, as well as a large array of cellular stresses. Changes in length and connectivity of this network, as well as alterations in the mitochondrial inner membrane (cristae), regulate cell fate by controlling metabolism, proliferation, differentiation, and cell death. Given the key roles of mitochondrial dynamics, the process by which mitochondria constantly fuse and fragment, the measure of mitochondrial length and connectivity provides crucial information on the health and activity of various cell populations. However, despite the importance of accurately measuring mitochondrial networks, the tools required to rapidly and accurately provide this information are lacking. Here, we developed a novel probabilistic approach to automatically measure mitochondrial length distribution and connectivity from confocal images. This method accurately identified mitochondrial changes caused by starvation or the inhibition of mitochondrial function. In addition, we successfully used the algorithm to measure changes in mitochondrial inner membrane/matrix occurring in response to Complex III inhibitors. As cristae rearrangements play a critical role in metabolic regulation and cell survival, this provides a rapid method to screen for proteins or compounds affecting this process. The algorithm will thus provide a robust tool to dissect the molecular mechanisms underlying the key roles of mitochondria in the regulation of cell fate.

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