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, 2019, 7404815
eCollection

Resumption of Autophagy by Ubisol-Q 10 in Presenilin-1 Mutated Fibroblasts and Transgenic AD Mice: Implications for Inhibition of Senescence and Neuroprotection

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Resumption of Autophagy by Ubisol-Q 10 in Presenilin-1 Mutated Fibroblasts and Transgenic AD Mice: Implications for Inhibition of Senescence and Neuroprotection

Caleb Vegh et al. Oxid Med Cell Longev.

Abstract

Alzheimer's disease (AD) is the most prevalent form of dementia and is associated with loss of memory, amyloid-beta plaque buildup, and neurofibrillary tangles. These features might be a result of neuronal cell death in the cerebral cortex and hippocampal regions of the brain. AD pathologies can be attributed to a variety of biochemical consequences including mitochondrial dysfunction, increased oxidative stress, and autophagy inhibition. Unfortunately, current therapeutics are limited only to symptomatic relief and do not halt the progression of neurodegeneration. Previous in vitro experiments have shown that a water-soluble formulation of coenzyme-Q10, Ubisol-Q10, can stabilize the mitochondria, prevent oxidative stress, and inhibit premature senescence in fibroblasts of AD patients. Since autophagy plays a critical role in maintenance and survival of neurons, we hypothesized that Ubisol-Q10 treatment could result in resumption of autophagy. Indeed, we observed induction of autophagy by Ubisol-Q10 treatment in AD fibroblasts as well as in the brains of transgenic AD mice. We found increased expression of autophagy-related genes beclin-1 and JNK1 following Ubisol-Q10 treatment of AD fibroblasts. These results were confirmed at the protein level by immunofluorescence and Western blotting. Interestingly, despite reduction of oxidative stress in cells due to Ubisol-Q10 treatment, autophagy inhibition leads to resumption of premature senescence in these PS-1 mutated fibroblasts indicating that autophagy is critical to prevent the senescence phenotype. Withdrawal of Ubisol-Q10 treatment also leads to the return of the senescence phenotype in AD fibroblasts indicating that constant supplementation of Ubisol-Q10 is required. Additionally, Ubisol-Q10 supplementation in the drinking water of double transgenic AD mice leads to increased expression of beclin-1 and JNK1 in the cortical region. Thus, the activation of autophagy by Ubisol-Q10 could be the mechanism for its ability to halt the progression of AD pathology in transgenic AD mice shown previously.

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
(a) Autophagy-related gene expression profile for Normal Human Fibroblasts (NHF), untreated PSAF, PTS-treated PSAF, and Ubisol-Q10-treated PSAF. NHF cells served as a positive control whereas untreated and PTS-treated AD cells served as negative controls. Notably, autophagy-related genes beclin-1, MAPK8/JNK1, and CTSB were upregulated in AD cells treated with Ubisol-Q10 bringing expression levels similar to or higher than NHF. (b) Beclin-1 probing of whole cell lysates via Western blot from NHF and PSAF. Supporting gene analysis, Beclin-1 was upregulated in Ubisol-Q10-treated PSAF and not untreated or PTS-treated AD cells. (c, d) Immunofluorescence staining of NHF and PSAF probing for beclin-1 (green) and JNK1 (green) and quantification of fluorescence, respectively. Treatment of PSAF cells with Ubisol-Q10 led to increased staining for beclin-1 and JNK1 compared to the untreated and PTS-treated groups indicating upregulation of autophagic proteins. These Ubisol-Q10-treated PSAF were stained in a comparable manner to NHF cells. Nuclei were counterstained with Hoechst for visualization. Micrographs were taken at 200x magnification. Scale bar = 50 μm.
Figure 2
Figure 2
Ubisol-Q10-treated PSAF incubated with SP600125 JNK1 inhibitor showed a resumption of premature senescence bringing fibroblasts back to the original AD morphology. Cells were incubated in senescence-associated beta-galactosidase to identify senescent fibroblasts. Micrographs were taken at 200x. Scale bar = 25 μm.
Figure 3
Figure 3
Treatment with SP600125 leads to resumption of senescence phenotype and reduced autophagosome formation in Ubisol-Q10-treated cells. (a, b) Immunofluorescence staining of PSAF probing for p21 (green) and LC3 (red) and quantification of fluorescence, respectively. Treatment of PSAF with Ubisol-Q10 leads to increased staining for LC3 puncta an autophagosome indicator. Ubisol-Q10 also lead to reduced staining for p21, an indicator of senescence. Ubisol-Q10-treated PSAF incubated in the presence of SP600125 resulted in increased staining for p21 and reduced staining for LC3 puncta similar to that of untreated PSAF, PSAF treated with SP600125, and PSAF starved of Ubisol-Q10 for 48 hours. Nuclei were counterstained with Hoechst for visualization. Micrographs were taken at 400x magnification. Scale bar = 50 μm.
Figure 4
Figure 4
(a, c) Inhibition of autophagy via SP600125 leads to reduced autophagosome formation (blue) and an increase in oxidative stress (green) in PSAF following treatment with Ubisol-Q10. PSAF starved of Ubisol-Q10 returned to AD morphology. Cells were incubated with monodansylcadaverine (MDC) to visualize autophagic vacuoles. Cells were also incubated with 2′,7′-dichlorofluorescein diacetate which is oxidized to fluorescent 2′,7′-dichlorofluorescein (DCF) for visualization of reactive oxidative species (ROS) production. (b, d) Similarly to DCF, immunofluorescence staining for 4-hydroxynonenal (4-HNE) a peroxidized lipid and an oxidative stress indicator was reduced in Ubisol-Q10-treated PSAF. Ubisol-Q10-treated PSAF incubated in SP600125 and PSAF starved of Ubisol-Q10 for 48 hours showed increased staining for 4-HNE similar to untreated PSAF. Micrographs were taken at 200x. Scale bar = 100 μm.
Figure 5
Figure 5
Immunohistochemical staining of the cerebral cortex from double transgenic mice probing for beclin-1 and JNK1. Oral supplementation of Ubisol-Q10 results in the upregulation of beclin-1 and JNK1 in transgenic mice similar to wild-type mice as indicated by the increased immunoactivity in the Ubisol-Q10-treated AD group compared to the untreated and PTS-treated groups. Micrographs were taken at 100x. Scale bar = 25 μm.

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