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Schaefer 2016 Abstract MitoFit Science Camp 2016

From Bioblast
Prominent role of amyloid beta and its intracellular localization in Alzheimer´s disease associated mitochondrial dysfunction.

Link:

Patrick Schaefer

Schaefer PM, von Einem B, Walther P, Calzia E, Rueck A, von Arnim CAF (2016)

Event: MitoFit Science Camp 2016 Kuehtai AT

Mitochondrial disturbances are known as an early feature of Alzheimer´s disease, result in an energy imbalance in the brain and consequently are discussed to be one underlying feature of neuronal cell death. The amyloid precursor protein (APP) as well as amyloid beta (Aβ), one of its cleavage products, were shown to play a key role in the mitochondrial toxicity.

To further elucidate the role of both proteins in mitochondrial impairment, we performed metabolic characterizations of intact cells which were modified as to the respective protein levels. This encompassed high-resolution respirometry in an Oroboros Oxygraph-2k as well as the determination of mitochondrial mass, mitochondrial membrane potential and lactic acid release. In addition we performed electron microscopy to investigate APP/ Aβ localization and established a multimodal metabolic imaging based on NADH autofluorescence lifetime.

We could show that APP overexpression reduced mitochondrial respiration. Interestingly the reduced respiration could be rescued by addition of a gamma secretase inhibitor (GSI), which lowered especially the intracellular/mitochondrial pool of Aβ.

Consequently Aβ rather than APP is likely to convey the mitochondrial toxicity. Furthermore, as the toxic potential of intracellular Aβ underlines the rational of a selective vulnerability of different cell types to Aβ-induced mitochondrial defects, we established a multimodal optical system to measure cell metabolism on the single cell level. Relying on NADH fluorescence lifetime imaging microscopy (NADH FLIM), here we demonstrate that our optical metabolic imaging system is able to quantitatively confirm the results obtained in the Oroboros Oxygraph-2k.

Our results demonstrate the importance of assessing energy metabolism on the single cell level to shed light onto Alzheimer´s disease associated mitochondrial dysfunction, highlighting the potential of combining respirometry and NADH FLIM for metabolic characterization.


O2k-Network Lab: DE Ulm Radermacher P


Labels: MiParea: Respiration, mt-Membrane  Pathology: Alzheimer's 

Organism: Human  Tissue;cell: Nervous system, HEK  Preparation: Intact cells 



HRR: Oxygraph-2k  Event: B3  MitoFit Science Camp 2016 

Affiliations

1-Inst Neurology; 2-Central Facility Electron Microscopy; 3-Inst Anästhesiol Pathophysiol Verfahrensentwicklung; 4-Core Facility Confocal and Multiphoton Microscopy; Ulm Univ, Germany. - [email protected]

Figure 1

MitoFit Training Camp 2016 Schaefer Figure.jpg

Figure 1. NADH FLIM of HEK293 cells overexpressing Alzheimer´s disease related protein

Representative image sections of NADH autofluorescence lifetime in HEK293 cells transduced with an empty control vector (mock) or an APP overexpressing vector (HEK APP) ± gamma secretase inhibitor treatment. In the upper panel the whole cells were analysed, the lower panel shows the mitochondria-rich region. NADH lifetime is coded in falsecolors with the corresponding color-palette to be shown below the images. Lifetimes were calculated using a biexponential decay with lifetimes fixed to 400ps and 2500ps in SPCImage 4.9. The white bar has a length of 10μm.