Mitochondrial respiratory complex II inhibition plays a central role in Huntington's disease (HD). Remarkably, 3-NP, a complex II inhibitor, recapitulates HD-like symptoms. Furthermore, decreases in mitochondrial fusion or increases in mitochondrial fission have been implicated in neurodegenerative diseases. However, the relationship between mitochondrial energy defects and mitochondrial dynamics has never been explored in detail. In addition, the mechanism of neuronal cell death by complex II inhibition remains unclear. Here, we tested the temporal and spatial relationship between energy decline, impairment of mitochondrial dynamics, and neuronal cell death in response to 3-NP using quantitative fluorescence time-lapse microscopy and cortical neurons. 3-NP caused an immediate drop in ATP. This event corresponded with a mild rise in reactive oxygen species (ROS), but mitochondrial morphology remained unaltered. Unexpectedly, several hours after this initial phase, a second dramatic rise in ROS occurred, associated with profound mitochondrial fission characterized by the conversion of filamentous to punctate mitochondria and neuronal cell death. Glutamate receptor antagonist AP5 abolishes the second peak in ROS, mitochondrial fission, and cell death. Thus, secondary excitotoxicity, mediated by glutamate receptor activation of the NMDA subtype, and consequent oxidative and nitrosative stress cause mitochondrial fission, rather than energy deficits per se. These results improve our understanding of the cellular mechanisms underlying HD pathogenesis. Huntington's disease (HD) is a fatal progressive neurodegenerative disorder with autosomal dominant inheritance. An abnormal CAG expansion coding for a polyglutamine stretch in the N-terminal region of the huntingtin gene causes HD. Disease results when the polyglutamine stretch contains 40 or more residues, and repeats of 36-39 residues have reduced penetrance. The clinical symptoms of HD include progressive motor, cognitive, and emotional deficits due to the changes in the cortex and striatum. How mutant huntingtin (mtHtt) triggers neurodegeneration is not clear. Among the proposed mechanisms are transcriptional dysregulation, axonal and dendritic transport defects, protein aggregation, and excitotoxic pathways mediated by glutamate receptors.In addition to these pathways, there is strong evidence that deficits in energy metabolism and mitochondria play a pivotal role in HD pathogenesis. [1][2][3][4][5][6] For example, brain tissue of HD patients 1,2 and transgenic mice expressing the mtHtt gene 5 exhibit reduced activity of the mitochondrial respiratory complexes II, III, and IV. In addition, striatal neuronal cultures expressing mtHtt exhibit decreased expression of respiratory complex II. 7 Furthermore, HD patients lose weight despite normal or increased calorie intake and their cortex and basal ganglia have increased lactate levels, indicative of a metabolic defect. 8 Moreover, mitochondria isolated from the lymphoblasts of HD patients, brain tissue of mtHtt mice,...

Mitochondrial morphology and length change during fission and fusion and mitochondrial movement varies dependent upon the cell type and the physiological conditions. Here, we describe fundamental wide-field fluorescence microcopy and 3D imaging techniques to assess mitochondrial shape, number and length in various cell types including cancer cell lines, motor neurons and astrocytes. Furthermore, we illustrate how to assess mitochondrial fission and fusion events by 3D time-lapse imaging and to calculate mitochondrial length and numbers as a function of time. These imaging methods provide useful tools to investigate mitochondrial dynamics in health, aging and disease.

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