Technical Reference #1691

1691.

Impaired Balance of Mitochondrial Fission and Fusion in Alzheimer's Disease Xinglong Wang; Bo Su; Hyoung-gon Lee; Xinyi Li; George Perry; Mark A. Smith; and Xiongwei Zhu, Case Western Reserve University, The Journal of Neuroscience, 29(1691), (2009)
Link To Paper

Abstract:
Mitochondrial dysfunction is a prominent feature of Alzheimer’s disease (AD) neurons. In this study we explored the involvement of an abnormal mitochondrial dynamics by investigating the changes in the expression of mitochondrial fission and fusion proteins in AD brain and the potential cause and consequence of these changes in neuronal cells. We found that mitochondria were redistributed away from axons in the pyramidal neurons of AD brain. Immunoblot analysis revealed that levels of DLP1 (also referred to as Drp1) OPA1 Mfn1 and Mfn2 were significantly reduced whereas levels of Fis1 were significantly increased in AD. Despite their differential effects on mitochondrial morphology manipulations of these mitochondrial fission and fusion proteins in neuronal cells to mimic their expressional changes in AD caused a similar abnormal mitochondrial distribution pattern such that mitochondrial density was reduced in the cell periphery ofM17cells or neuronal process of primary neurons and correlated with reduced spine density in the neurite. Interestingly oligomeric amyloid- -derived diffusible ligands (ADDLs) caused mitochondrial fragmentation and reduced mitochondrial density in neuronal processes. More importantly ADDL-induced synaptic change (i.e. loss of dendritic spine and postsynaptic density protein 95 puncta) correlated with abnormal mitochondrial distribution. DLP1 overexpression likely through repopulation of neuronal processes with mitochondria prevented ADDL-induced synaptic loss suggesting that abnormal mitochondrial dynamics plays an important role in ADDL-induced synaptic abnormalities. Based on these findings we suggest that an altered balance in mitochondrial fission and fusion is likely an important mechanism leading to mitochondrial and neuronal dysfunction in AD brain.

Materials & Methods:
Time-lapse imaging. Neurons were seeded in glass-bottom dishes (MatTek) and then transfected with Mito-Dendra2. Forty-eight hours after transfection cells were placed in a well-equipped live imaging station (Zeiss CTI-Controller 3700) with controlled CO2 content humidity and temperature of stage objective and the air. Images were captured with a Zeiss LSM 510 inverted laser-scanning confocal fluorescence microscope. Images of red signal were collected using 543nmexcitation light from an argon laser and a 560nmlong-pass filter; those of green fluorescence were collected using 488 nm excitation light from an argon laser and a 500–550 nm bandpass barrier filter. During time-lapse imaging frames were captured every 10 s for at least 1 h without apparent phototoxicity or photobleaching. Image analysis was also performed with open-source image-analysis programs WCIF ImageJ (developed by W. Rasband; NIH).

Microscopic Technique
Electron Microscopy

Cell Type(s)
M17