Antioxidants Rescue Mitochondrial Transport in Differentiated Alzheimer's Disease Trans-Mitochondrial Cybrid Cells

Abstract

Mitochondrial dysfunction and axonal degeneration are early pathological features of Alzheimer's disease (AD)-affected brains. The underlying mechanisms and strategies to rescue it have not been well elucidated. Here, we evaluated axonal mitochondrial transport and function in AD subject-derived mitochondria. We analyzed mitochondrial transport and kinetics in human trans-mitochondrial "cybrid" (cytoplasmic hybrid) neuronal cells whose mitochondria were derived from platelets of patients with sporadic AD and compared these AD cybrid cell lines with cybrid cell lines whose mitochondria were derived from age-matched, cognitively normal subjects. Human AD cybrid cell lines, when induced to differentiate, developed stunted projections. Mitochondrial transport and function within neuronal processes/axons was altered in AD-derived mitochondria. Antioxidants reversed deficits in axonal mitochondrial transport and function. These findings suggest that antioxidants may be able to mitigate the consequences of AD-associated mitochondrial dysfunction. The present study provides evidence of the cause/effect of AD specific mitochondrial defects, which significantly enhances our understanding of the AD pathogenesis and exploring the effective therapeutic strategy for AD.

Keywords: Alzheimer’s disease; antioxidants; cybrid cells; mitochondrial dysfunction; mitochondrial transport.

Fig. 1

Comparison of differentiation status and mitochondrial density in neuronal processes between non-AD and AD cybrid cells during differentiation. A) Representative morphological images from non-AD and AD cybrid cells under undifferentiated conditions or induced by staurosporine (14 days after SAT treatment). Scale bar = 50 μm. B) Quantification of neuronal process length of cybrid cells using the image J program. Black columns denote the process length of cybrid cells following SAT treatment in both non-AD and AD groups. White open columns represent process lengths of undifferentiated cybrid cells without SAT treatment. C) Images show morphology of mitochondria in cybrid cells with MitoRed staining. In the processes of differentiated non-AD cybrid cells (I) and the enlarged images for processes (II), more mitochondria were observed than in the AD cells (III, and the enlarged images for processes in IV). Mitochondrial density (D, E) in the processes of differentiated non-AD and AD cybrid cells were quantified. Total numbers of mitochondria were counted within the processes per cybrid cell (D) and the numbers of mitochondria were counted per 100 μm process length (E). Data were collected from 20–25 processes from each non-AD or AD cybrid cell line.

Fig. 2

Comparison of mitochondrial function, mitochondrial membrane potential, and reactive oxygen species (ROS) levels between differentiated non-AD and AD cybrid cells. Enzymatic activity of complex I (A), IV (B), and cellular ATP levels (C) were determined in cell lysates from indicated cell groups. Data are expressed as fold increase relative to non-AD cybrid cells. Generation of ROS was detected by electron paramagnetic resonance (EPR) spectra (D, E) and mitochondrial ROS levels were measured by MitoSox staining intensity (F, G). Mitochondrial membrane potential was measured by TMRM staining intensity (H, I). Quantifications were determined of the signal intensity of EPR (D), immunofluorescent intensity for MitoSox (F) and TMRM (H) in mitochondria of the indicated cybrid cells. The representative EPR and staining images were shown for EPR (E), and red fluorescence for Mitosox (G) and TMRM (I), respectively. Data were collected from 20–25 processes from each non-AD or AD cybrid cell line.

Fig. 3

Comparison of mitochondrial movement parameters in the neuronal process of differentiated non-AD and AD cybrid cells. A, B) The percentage of movable (A) and stationary mitochondria (B) in the processes of cybrid cells. C) Average mitochondrial travel velocity and (D) mitochondrial travel distance were calculated. E, F) AD cybrid cells showed significant decreases in anterograde and retrograde mitochondrial movement compared to non-AD controls. Data were collected from 25 processes of each non-AD or AD cybrid cell line. G) Kymographs generated from the live imaging movies showing non-AD and AD cybrid cells, respectively. In the kymographs, the X axis is mitochondrial position and the Y axis represents the time lapse (0–120 s). Vertical white lines represent stationary mitochondria and diagonal lines represent moving mitochondria. Anterograde movements are from left to right and retrograde movements are from right to left. Scale bars = 10 μm.

Fig. 4

Effects of antioxidant treatment on differentiation status and mitochondrial density in differentiated AD cybrid cells. A) Representative morphological images from differentiated AD cybrid cells with the addition of antioxidant (10 μM probucol in AII, 200 μM ascorbic acid/AA in AIII or vehicle in AI) or vehicle treatment. Scale bar = 50 μm. B) Quantification of neuronal process length of cybrid cells using the image J program. C) Representative morphological images of mitochondria in the processes of above differentiated AD cybrid cells with MitoRed staining (I, III, and V) and the corresponding enlarged images of processes (II, IV, and VI). I and II: AD cells under differentiation with the addition of antioxidants probucol (III and IV) or AA (V and VI), respectively. Mitochondrial density (D, E) was quantified in the processes of differentiated non-AD and AD cybrid cells. Total numbers of mitochondria were counted within processes per cybrid cell (D) and numbers of mitochondria were counted per 100 μm process length (E).

Fig. 5

Effects of antioxidant on mitochondrial function, membrane potential and ROS levels in differentiated AD cybrid cells. Enzymatic activity of complex I (A), IV (B), and cellular ATP levels (C) were determined in cell lysates from differentiated AD cybrid cells with or without the addition of antioxidants. Data were expressed as fold increase relative to AD cybrid cells without the addition of antioxidant (vehicle treatment). Generation of ROS was detected by electron paramagnetic resonance (EPR) spectra (D, E) and mitochondrial ROS levels were measured by MitoSox staining intensity (F, H). Mitochondrial membrane potential was measured by TMRM staining intensity (G, I). Quantifications were determined of the signal intensity of EPR (D), immunofluorescent intensity for MitoSox (F) and TMRM (G) in mitochondria of the indicated cybrid cells. The representative EPR and staining images were shown for EPR (E), and red fluorescence for Mitosox (H) and TMRM (I). Data were collected from 20–25 processes of each AD cybrid cell line with different treatments.

Fig. 6

Effect of antioxidant treatment on mitochondrial movement parameters in the neuronal processes from differentiated AD cybrid cells. A, B) The percentage of mobile (A) and stationary mitochondria (B) in the neuronal processes of cybrid cells. C) Average mitochondrial travel velocity and (D) mitochondrial travel distance were calculated. Antioxidant treatment significantly rescues anterograde and retrograde mitochondrial movement in AD cybrid cells (E, F). G) Kymographs generated from live imaging movies represent differentiated AD cybrid cells with or without antioxidant treatment. In the kymographs, the X axis is mitochondrial position and the Y axis represents time lapse (0–120 s). Vertical white lines represent stationary mitochondria and diagonal lines represent mobile mitochondria. Anterograde movements are from left to right and retrograde movements are from right to left. Scale bars = 10 μm.

Fig. 7

Schematic diagram depicting the role of antioxidant treatment on differentiation ability and axonal mitochondrial transport in AD cybrid cells. Compared to non-AD derived mitochondria (red), AD-derived mitochondria reveal higher levels of ROS, which arrests differentiation and mitochondria movement resulting in less movable mitochondria and more stationary mitochondria (blue) in AD cybrids. Antioxidants suppress ROS accumulation, which in turn rescue mitochondrial defects including axonal mitochondrial transport and neuronal differentiation.