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, 7 (8), e42983

HDAC6 Inhibitor Blocks Amyloid Beta-Induced Impairment of Mitochondrial Transport in Hippocampal Neurons

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HDAC6 Inhibitor Blocks Amyloid Beta-Induced Impairment of Mitochondrial Transport in Hippocampal Neurons

Chaeyoung Kim et al. PLoS One.

Abstract

Even though the disruption of axonal transport is an important pathophysiological factor in neurodegenerative diseases including Alzheimer's disease (AD), the relationship between disruption of axonal transport and pathogenesis of AD is poorly understood. Considering that α-tubulin acetylation is an important factor in axonal transport and that Aβ impairs mitochondrial axonal transport, we manipulated the level of α-tubulin acetylation in hippocampal neurons with Aβ cultured in a microfluidic system and examined its effect on mitochondrial axonal transport. We found that inhibiting histone deacetylase 6 (HDAC6), which deacetylates α-tubulin, significantly restored the velocity and motility of the mitochondria in both anterograde and retrograde axonal transports, which would be otherwise compromised by Aβ. The inhibition of HDAC6 also recovered the length of the mitochondria that had been shortened by Aβ to a normal level. These results suggest that the inhibition of HDAC6 significantly rescues hippocampal neurons from Aβ-induced impairment of mitochondrial axonal transport as well as mitochondrial length. The results presented in this paper identify HDAC6 as an important regulator of mitochondrial transport as well as elongation and, thus, a potential target whose pharmacological inhibition contributes to improving mitochondrial dynamics in Aβ treated neurons.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Reduction of acetylated α-tubulin in 5XFAD.
(A) Western blot of acetylated α-tubulin in the brains of both wild type (WT) and 5XFAD mice. Brain extracts were prepared from frontal cortex of 13-month-old mice. Actin is a loading control. (B) Quantitation of the acetylated α-tubulin normalized by total α-tubulin is shown as means ± SEM (WT n = 4, 5XFAD n = 3, *P<0.05).
Figure 2
Figure 2. Modulation of acetylated α-tubulin by Aβ and TBA.
(A) Western blot of acetylated α-tubulin in the rat hippocampal neurons. After being pretreated with Aβ (2 µM) for 24 hrs, cells were treated with TBA (5 µM) for 3 hrs and lysed with RIPA buffer. Actin is a loading control. (B) Quantitation of the acetylated α-tubulin normalized by total α-tubulin is shown as means ± SEM. Data were acquired from 4 independent experiments (*P<0.05, ***P<0.001). (C) Immunocytochemistry of acetylated α-tubulin in hippocampal neurons. Anti-acetylated α-tubulin antibody detects α-tubulin only when acetylated at Lys 40 (Scale bar = 100 µm).
Figure 3
Figure 3. Regulation of mitochondrial transport by Aβ and TBA.
(A) Representative kymographs of mitochondrial movement. Hippocampal neurons from rat (E18) were plated with densities of 6×104 cells in the somal side of the microfluidic chamber. Cells were transfected with pDsRed2-Mito after 7 days of culture. After being pretreated with Aβ (2 µM) for 24 hrs, cells were treated with TBA (5 µM) for 3 hrs. Images were acquired every 1 sec for 2 min at microgrooves. X axis of kymograph is axonal length (152.7 µm). Proximal to distal indicates the soma to axon terminal direction. Mitochondria which move from proximal to distal region show anterograde movement. Y axis is the time that mitochondria have moved (2 min). (B) Pictures of motile mitochondria for each group were shown every 5 sec (Scale bars = 10 µm). Arrows indicate motile mitochondria. (C) Average velocity of motile mitochondria. Anterograde and retrograde velocity were analyzed separately. (D) Motility of mitochondria. Motility stands for percentage of motile mitochondria over total mitochondria. Anterograde and retrograde motility were analyzed separately. Data were acquired from 4 independent experiments (Veh n = 41, TBA n = 44, Aβ n = 42, Aβ+TBA n = 43, *P<0.05, **P<0.01, ***P<0.001).
Figure 4
Figure 4. Alteration of mitochondrial length by Aβ and TBA.
(A) Average length of total mitochondria, including both motile (anterograde, retrograde transported) and stationary mitochondria. (B) Average length of motile and stationary mitochondria. ***P<0.001 significance of stationary mitochondria vs. motile mitochondria; ++P<0.01, +++P<0.001 among motile mitochondria; ###P<0.001 among stationary mitochondria. (C) The average number of mitochondria per 100 µm of axon. Data were obtained from 4 independent experiments (Veh n = 35, TBA n = 42, Aβ n = 38, Aβ & TBA n = 38, *P<0.05, **P<0.01, ***P<0.001).

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