Additional file 1: Figure S1. of Statins induce insulin-degrading enzyme secretion from astrocytes via an autophagy-based unconventional secretory pathway

Statins regulate cholesterol levels in astrocytes. (A) Cellular cholesterol levels were measured by filipin staining. MβCD is a positive control. (B) Quantitative analysis of Figure S1A using the Image J program (N = 3 experiments). ** p < 0.01, *** p < 0.001 vs. vehicle-treated cells. (PDF 217 kb

Additional file 4: Figure S4. of Statins induce insulin-degrading enzyme secretion from astrocytes via an autophagy-based unconventional secretory pathway

Cell death was not induced in this study. (A) MTS assay was used for checking cell viability under simvastatin, 3MA and/or bafilomycin treated condition. N = 5 experiments. (PDF 57 kb

Additional file 2: Figure S2. of Statins induce insulin-degrading enzyme secretion from astrocytes via an autophagy-based unconventional secretory pathway

Fluvastatin induces IDE secretion from astrocytes. (A) Increased IDE levels secreted from the primary astrocytes by fluvastatin in a concentration-dependent manner. Blots are representative of at least 3 independent experiments (N = 3 experiments). (B) Quantitative analysis of Figure S2A. ** p < 0.01 vs. vehicle-treated cells. (PDF 71 kb

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

<div><p>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.</p> </div

Insulin-degrading enzyme secretion from astrocytes is mediated by an autophagy-based unconventional secretory pathway in Alzheimer disease

<p>The secretion of proteins that lack a signal sequence to the extracellular milieu is regulated by their transition through the unconventional secretory pathway. IDE (insulin-degrading enzyme) is one of the major proteases of amyloid beta peptide (Aβ), a presumed causative molecule in Alzheimer disease (AD) pathogenesis. IDE acts in the extracellular space despite having no signal sequence, but the underlying mechanism of IDE secretion extracellularly is still unknown. In this study, we found that IDE levels were reduced in the cerebrospinal fluid (CSF) of patients with AD and in pathology-bearing AD-model mice. Since astrocytes are the main cell types for IDE secretion, astrocytes were treated with Aβ. Aβ increased the IDE levels in a time- and concentration-dependent manner. Moreover, IDE secretion was associated with an autophagy-based unconventional secretory pathway, and depended on the activity of RAB8A and GORASP (Golgi reassembly stacking protein). Finally, mice with global haploinsufficiency of an essential autophagy gene, showed decreased IDE levels in the CSF in response to an intracerebroventricular (i.c.v.) injection of Aβ. These results indicate that IDE is secreted from astrocytes through an autophagy-based unconventional secretory pathway in AD conditions, and that the regulation of autophagy is a potential therapeutic target in addressing Aβ pathology.</p

Modulation of acetylated α-tubulin by Aβ and TBA.

<p>(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).</p

Regulation of mitochondrial transport by Aβ and TBA.

<p>(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).</p

Alteration of mitochondrial length by Aβ and TBA.

<p>(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).</p

Reduction of acetylated α-tubulin in 5XFAD.

<p>(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).</p

Additional file 3: of Increased acetylation of Peroxiredoxin1 by HDAC6 inhibition leads to recovery of Aβ-induced impaired axonal transport

Pretreatment of TBA also decreases Aβ-induced ROS and Ca2+. HT22 cells were pretreated with TBA (0.5 μM) for 1 h before incubation with Aβ (2 μM, 24 h). a ROS level was measured by DCFDA assay in HT22 cells. Upper panel is representative images of DCFDA signals (top row) and bright field images (bottom row) and lower panel is quantification of fluorescent intensity (n = 6, independent experiments). b Ca2+ level was measured by Fluo-4 assay in HT22 cells. Left panel is representative images and right panel is quantification of fluorescent intensity (n = 4, independent experiments). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA, Bonferroni post-hoc test). Scale bar: 100 μm. (PDF 3678 kb