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. 2018 Oct 19;430(21):3942-3953.
doi: 10.1016/j.jmb.2018.08.019. Epub 2018 Aug 29.

Ursodeoxycholic Acid Improves Mitochondrial Function and Redistributes Drp1 in Fibroblasts From Patients With Either Sporadic or Familial Alzheimer's Disease

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Free PMC article

Ursodeoxycholic Acid Improves Mitochondrial Function and Redistributes Drp1 in Fibroblasts From Patients With Either Sporadic or Familial Alzheimer's Disease

Simon M Bell et al. J Mol Biol. .
Free PMC article

Abstract

Alzheimer's disease (AD) is the leading cause of dementia worldwide. Mitochondrial abnormalities have been identified in many cell types in AD, with deficits preceding the development of the classical pathological aggregations. Ursodeoxycholic acid (UDCA), a treatment for primary biliary cirrhosis, improves mitochondrial function in fibroblasts derived from Parkinson's disease patients as well as several animal models of AD and Parkinson's disease. In this paper, we investigated both mitochondrial function and morphology in fibroblasts from patients with both sporadic and familial AD. We show that both sporadic AD (sAD) and PSEN1 fibroblasts share the same impairment of mitochondrial membrane potential and alterations in mitochondrial morphology. Mitochondrial respiration, however, was decreased in sAD fibroblasts and increased in PSEN1 fibroblasts. Morphological changes seen in AD fibroblasts include reduced mitochondrial number and increased mitochondrial clustering around the cell nucleus as well as an increased number of long mitochondria. We show here for the first time in AD patient tissue that treatment with UDCA increases mitochondrial membrane potential and respiration as well as reducing the amount of long mitochondria in AD fibroblasts. In addition, we show reductions in dynamin-related protein 1 (Drp1) level, particularly the amount localized to mitochondria in both sAD and familial patient fibroblasts. Drp1 protein amount and localization were increased after UDCA treatment. The restorative effects of UDCA are abolished when Drp1 is knocked down. This paper highlights the potential use of UDCA as a treatment for neurodegenerative disease.

Keywords: UDCA; mitochondrial morphology; neurodegeneration; presenilin; treatment.

Figures

Unlabelled Image
Fig. 1
Fig. 1
(A) Mitochondrial membrane potential in each of the seven sporadic and three familial AD lines. When compared to controls (n = 7), all sporadic lines and all familial lines showed a significant reduction in mitochondrial membrane potential (*p < 0.05, **p < 0.01, ***p < 0.005). (B) The total mitochondrial count was significantly reduced in both sporadic (*p < 0.05) and familial groups (*p < 0.05). (C) Perinuclear mitochondrial count was significantly increased in both sporadic (*p < 0.05) and familial (***p < 0.005) groups. (D) The percentage of cell area occupied by long mitochondria is increased in both sAD and PSEN1 patient fibroblasts (*p < 0.05). (E) Correlation of mitochondrial membrane potential and % long mitochondria shows negative correlation (p = 0.0002, R2 = 0.7). The circles represent controls; triangles, sAD; and squares, PSEN1. All measurements were carried out on three separate passages of each fibroblast line.
Fig. 2
Fig. 2
OCRs. Panels A–F show an OCR trace for controls (black circle solid line, n = 6), sporadic [gray squares and solid line, sAD1 (A), sAD2 (B), sAD3 (C), sAD4 (D), sAD5 (E)] and PSEN1 [gray squares and solid line, PSEN1 (F)] cell lines in the untreated condition. Sporadic patient fibroblasts have a reduction in OCR compared to controls at baseline and when measuring maximal capacity, but PSEN1 cell lines have an increase at both points. Panels A–F show the reduction seen in spare capacity in sporadic cell lines (*p < 0.05) and the increase seen in PSEN1 cell lines compared to controls (*p < 0.05). Fibroblasts after treatment with 100 nM UDCA for 24 h show increased mitochondrial respiration in both sAD (*p < 0.05) and PSEN1 (*p < 0.05) fibroblasts (A–F). Each measurement was repeated on three separate passages of each cell line. (G) Mitochondrial membrane potential shows correlation with spare respiratory capacity (p = 0.003, R2 = 0.65). The circles represent controls; squares, sAD; and triangles, PSEN1. The PSEN1 fibroblasts were excluded from linear regression calculations.
Fig. 3
Fig. 3
Recovery with UDCA treatment. (A) Fibroblasts are treated for 24 h with 100 nM UDCA. UDCA treatment, however, increases mitochondrial membrane potential. The increase from the untreated state for controls, sporadics and familial cell lines varies between 0 and 5% for controls, 12% and 28% for sAD and 19% and 21% for PSEN1 (*p < 0.05). (B) The increase in mitochondrial membrane potential when fibroblasts are treated with 100 nM of UDCA. The relative increase from the untreated state for controls, sporadics and familial cell lines is 0%, 11% and 25%, respectively (*p < 0.05). Each measurement was repeated on three separate passages of each cell line.
Fig. 4
Fig. 4
Drp1 protein expression. (A) Total Drp1 protein expression levels are reduced in both sAD (light gray bars) and PSEN1 (dark gray bars) fibroblasts (**p < 0.01). After treatment with 100 nM UDCA, total Drp1 levels are increased in the sAD fibroblasts (*p < 0.05). A(ii) Representative western blot showing control untreated (lane 1), control DMSO treated (lane 2), control 100 nM UDCA (lane 3), control 10 μM UDCA (lane 4), sAD untreated (lane 5), sAD DMSO treated (lane 6), sAD 100 nM UDCA treated (lane 7) and sAD 10 μM UDCA treated (lane 8) for Drp1 and loading control actin. (B) Drp1 cellular localization is altered in sAD and PSEN1 fibroblasts. Significantly less Drp1 colocalizes with the mitochondrial marker (TOMM20) in the sAD (light gray bars) and PSEN1 (dark gray bars) fibroblasts (***p < 0.005); however, after treatment with 100 nM UDCA for 24 h, the amount of Drp1 which colocalizes with the mitochondria is increased (*p < 0.05; **p < 0.01). B(ii) Representative images of control fibroblasts vehicle treated (a) and treated with UDCA (b) and sAD fibroblasts vehicle treated (c) and treated with UDCA (d) and PSEN1 vehicle treated (e) and treated with UDCA (f). Blue staining is Hoechst for the nucleus, green staining is Drp1 and red staining is TOMM20 for the mitochondria. Each measurement was repeated on three separate passages of each cell line; for immunocytochemistry measurements, at least 150 cells were imaged per fibroblast line per experiment.
Fig. 5
Fig. 5
Drp1 knockdown data. (A) Drp1 protein expression knockdown of 48% from scramble siRNA levels (*p < 0.05). (B) Mitochondrial form factor is increased in sAD and PSEN1 fibroblasts in the scramble siRNA condition, which is reduced again after UDCA treatment. Drp1 knockdown increases form factor in all fibroblasts. UDCA does not have an effect on mitochondrial form factor in the Drp1 knockdown condition. (C) Image showing Drp1 staining in green, mitochondria in red and nuclei in blue. A representative image is shown for each condition. Control scramble siRNA (i), control scramble siRNA + UDCA (ii), control Drp1 siRNA (iii), control Drp1 siRNA + UDCA (iv); sAD scramble siRNA (v), sAD scramble siRNA + UDCA (vi), sAD Drp1 siRNA (vii), sAD Drp1 siRNA + UDCA (viii); PSEN1 scramble siRNA (ix), PSEN1 scramble siRNA + UDCA (x), PSEN1 Drp1 siRNA (xi), PSEN1 Drp1 siRNA + UDCA (xii). (D) Mitochondrial membrane potential is reduced in sAD and PSEN1 fibroblasts in the scramble siRNA condition, which is increased with UDCA treatment. In the Drp1 siRNA condition, there is no further decrease in mitochondrial membrane potential; however, UDCA treatment does not increase mitochondrial membrane potential.
Supplementary Fig. 1
Supplementary Fig. 1
A-D show qPCR measurements of mRNA expression of Opa1 (A), Mfn1 (B), Mfn2 (C) and Drp1 (D). Black bars show controls, light grey bars show sporadic AD patient fibroblasts and dark grey bars show PSEN1 patient fibroblasts. Samples from each fibroblast line were run in triplicate and data presented is all controls, sAD and PSEN1 fibroblasts grouped. No significant differences are present in mRNA expression of any transcripts measured. Panels E and F show western blot data for Drp1, Opa1, Mfn1 and Mfn2. E shows individual western blot from each sAD fibroblast line sAD1-5. Showing a reduction in Drp1 protein levels in all sAD patient fibroblasts measured and an increase after UDCA treatment. Protein levels of Opa1, Mfn1 and Mfn2 were not changed in the sAD fibroblasts as quantified in F.

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