Search

Mitochondrial dysfunction has long been implicated in the neurodegenerative disorder Parkinson's disease (PD); however, it is unclear how mitochondrial impairment and α-synuclein pathology are coupled. Using specific mitochondrial inhibitors, EM analysis, and biochemical assays, we report here that intramitochondrial protein homeostasis plays a major role in α-synuclein aggregation. We found that interference with intramitochondrial proteases, such as HtrA2 and Lon protease, and mitochondrial protein import significantly aggravates α-synuclein seeding. In contrast, direct inhibition of mitochondrial complex I, an increase in intracellular calcium concentration, or formation of reactive oxygen species, all of which have been associated with mitochondrial stress, did not affect α-synuclein pathology. We further demonstrate that similar mechanisms are involved in amyloid-β 1-42 (Aβ42) aggregation. Our results suggest that, in addition to other protein quality control pathways, such as the ubiquitin-proteasome system, mitochondria per se can influence protein homeostasis of cytosolic aggregation-prone proteins. We propose that approaches that seek to maintain mitochondrial fitness, rather than target downstream mitochondrial dysfunction, may aid in the search for therapeutic strategies to manage PD and related neuropathologies., Competing Interests: Conflict of interest—The authors declare no conflict of interest., (© 2020 Lautenschläger et al.)

The function of intact organelles, whether mitochondria, Golgi apparatus or endoplasmic reticulum (ER), relies on their proper morphological organization. It is recognized that disturbances of organelle morphology are early events in disease manifestation, but reliable and quantitative detection of organelle morphology is difficult and time-consuming. Here we present a novel computer vision algorithm for the assessment of organelle morphology in whole cell 3D images. The algorithm allows the numerical and quantitative description of organelle structures, including total number and length of segments, cell and nucleus area/volume as well as novel texture parameters like lacunarity and fractal dimension. Applying the algorithm we performed a pilot study in cultured motor neurons from transgenic G93A hSOD1 mice, a model of human familial amyotrophic lateral sclerosis. In the presence of the mutated SOD1 and upon excitotoxic treatment with kainate we demonstrate a clear fragmentation of the mitochondrial network, with an increase in the number of mitochondrial segments and a reduction in the length of mitochondria. Histogram analyses show a reduced number of tubular mitochondria and an increased number of small mitochondrial segments. The computer vision algorithm for the evaluation of organelle morphology allows an objective assessment of disease-related organelle phenotypes with greatly reduced examiner bias and will aid the evaluation of novel therapeutic strategies on a cellular level.

Motor neurons vulnerable to the rapidly progressive deadly neurodegenerative disease amyotrophic lateral sclerosis (ALS) inherently express low amounts of calcium binding proteins (CaBP), likely to allow physiological motor neuron firing frequency modulation. At the same time motor neurons are susceptible to AMPA receptor mediated excitotoxicity and internal calcium deregulation which is not fully understood. We analysed ER mitochondria calcium cycle (ERMCC) dynamics with subsecond resolution in G93A hSOD1 overexpressing motor neurons as a model of ALS using fluorescent calcium imaging. When comparing vulnerable motor neurons and non-motor neurons from G93A hSOD1 mice and their non-transgenic littermates, we found a decelerated cytosolic calcium clearance in the presence of G93A hSOD1. While both non-transgenic as well as G93A hSOD1 motor neurons displayed large mitochondrial calcium uptake by the mitochondrial uniporter (mUP), the mitochondrial calcium extrusion system was altered in the presence of G93A hSOD1. In addition, ER calcium uptake by the sarco-/endoplasmic reticulum ATPase (SERCA) was increased in G93A hSOD1 motor neurons. In survival assays, blocking the mitochondrial sodium calcium exchanger (mNCE) by CGP37157 as well as inhibiting SERCA by cyclopiazonic acid showed protective effects against kainate induced excitotoxicity. Thus, our study shows for the first time that the functional consequence of G93A hSOD1 overexpression in intact motor neurons is indeed a disturbance of the ER mitochondria calcium cycle, and identified two promising targets for therapeutic intervention in the pathology of ALS., (Copyright © 2013 Elsevier Inc. All rights reserved.)