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, 9 (5), 6128-6143
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Drp1/Fis1 Interaction Mediates Mitochondrial Dysfunction, Bioenergetic Failure and Cognitive Decline in Alzheimer's Disease

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Drp1/Fis1 Interaction Mediates Mitochondrial Dysfunction, Bioenergetic Failure and Cognitive Decline in Alzheimer's Disease

Amit U Joshi et al. Oncotarget.

Abstract

Mitochondrial dynamics, involving a balance between fusion and fission, regulates mitochondrial quality and number. Increasing evidence suggests that dysfunctional mitochondria play a role in Alzheimer's disease (AD). We observed that Drp1 interaction with one of the adaptors, Fis1, is significantly increased in Aβ-treated neurons and AD patient-derived fibroblasts. P110, a seven-amino acid peptide, which specifically inhibits Drp1/Fis1 interaction without affecting the interaction of Drp1 with its other adaptors, attenuated Aβ42-induced mitochondrial recruitment of Drp1 and prevented mitochondrial structural and functional dysfunction in cultured neurons, in cells expressing mutant amyloid precursor protein (KM670/671NL), and in five different AD patient-derived fibroblasts. Importantly, sustained P110 treatment significantly improved behavioral deficits, and reduced Aβ accumulation, energetic failure and oxidative stress in the brain of the AD mouse model, 5XFAD. This suggests that Drp1/Fis1 interaction and excessive mitochondrial fission greatly contribute to Aβ-mediated and AD-related neuropathology and cognitive decline. Therefore, inhibiting excessive Drp1/Fis1-mediated mitochondrial fission may benefit AD patients.

Keywords: Alzheimer’s disease; Drp1; P110; mitochondrial dysfunction; patient-derived fibroblasts.

Conflict of interest statement

CONFLICTS OF INTEREST None.

Figures

Figure 1
Figure 1. P110 reduces Aβ42 injury and subsequent mitochondrial defects in SH-SY5Y cells
(A) Western blot analysis of immunoprecipitated Drp1 with its adaptors. n = 3. (B) Western blot showing Drp1 association with mitochondria-enriched fractions. VDAC, a mitochondrial membrane protein, was used as a loading control. Protein levels were quantified and represented as fold-change of vehicle treated control n = 5. (C) Western blot showing cytochrome c release into the cytosolic fraction; β-actin was used as loading controls. Protein levels were quantified and represented as fold-change of vehicle treated control n = 3. (D) Western blot of Drp1 in the total lysate; β-actin was used as loading controls. Protein levels were quantified and represented as fold-change of vehicle treated control n = 5. (E) Microscopy of stained SH-SY5Y cells with anti-TOM20 (a marker of mitochondria, 1:500 dilution) following incubation with Aβ42 for 24 hours. Scale bar: 5 µM. Inserts show enlarged areas of the white boxes. Below: Quantification of mitochondrial interconnectivity and mitochondrial elongation in SH-SY5Y cells following incubation with Aβ42. At least 150 cells per condition were counted. n = 5. (F) Cell death was measured using ApoTox-Glo™ after 24 hours of treatment as indicated. GSH/GSSG ratio was measured using GSH/GSSG-Glo™ 2 hours after adding oAβ1-42 (5 µM). Caspase 3/7 activity was measured using ApoTox-Glo™ after 24 hours of treatment. ATP levels were measured from using Mitochondrial ToxGlo™ after 24 hours of treatment. Mitochondrial superoxide production was determined using the mitochondrial superoxide indicator MitoSOX™ Red. Nuclei were stained with Hoechst (blue). Mitochondrial membrane potential was determined using JC-1 dye. n = 5. (G) Western blot of TOM20 and TIM17 as markers of mitochondrial mass in the total lysate; β-actin was used as loading controls. Protein levels were quantified and represented as fold-change of vehicle treated control n = 3. The activity of citrate synthase was also assayed using total cell lysates to determine the mitochondrial mass. Data information: Mean, standard deviation, and P-values are shown. #P < 0.05 versus controls. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. One-way ANOVA and Tukey’s post hoc test. C, control.
Figure 2
Figure 2. Treatment with P110 blocked increased Drp1 association with the mitochondria induced by Swe10 mutation and the associated mitochondrial damage in mouse N2a neuroblastoma cells
(A) Western blot showing Drp1 association with mitochondria-enriched fractions, VDAC was used as loading control. Protein levels were quantified and represented as fold-change of vehicle treated control. n = 5. (B) Western blot showing cytosolic cytochrome c levels, β-actin was used as loading control. Protein levels were quantified and represented as fold-change of vehicle treated control. n = 4. (C) Western blot showing Drp1 levels in total lysate; β-actin was used as loading control. Protein levels were quantified and represented as fold-change of vehicle treated control. n = 4. (D) Drp1 oligomerization in total lysates was measured under native conditions. Protein levels were quantified and represented as ratio. n = 4. (E) Drp1 phosphorylation levels were examined in total lysate by immunoblotting using anti-phosphorylated-S616-Drp1 or -S637-Drp1 antibodies in the presence or absence of P110 (1 µM/48 hours); β-actin was used as a loading control. Protein levels were quantified and represented as fold-change of N2a-WT. n = 3. (F) Microscopy of stained N2a WT and N2a Swe10 cells with anti-TOM20 (a marker of mitochondria, 1:500 dilution) incubated in the presence or absence of P110 (1 µM) for 12 hours in serum free medium. Scale bar: 0.5 µm. n = 3. (G) Quantification of mitochondrial interconnectivity and elongation scores in N2a WT and N2a Swe10 cells. At least 200 cells per condition were counted. n = 3. (H) Chymotrypsin-like activity was measured using fluorogenic substrate; Suc-LLVY-AMC to measure proteasome activity in homogenates of N2a cells. Activity levels were quantified and represented as %-change of N2a WT n = 3. (I) Amplex red assay was used to quantitate H2O2 generation and represented represented as %-change of N2a WT n = 3. (J) Cytotoxicity levels were measured using ApoTox-Glo™ after 48 hours of treatment. ATP levels were measured using ApoTox-Glo™ after 48 hours of treatment. Mitochondrial superoxide production was determined using the mitochondrial superoxide indicator MitoSOX™ Red. Mitochondrial membrane potential was determined using JC-1 dye. (K) Levels of p62, Beclin-1, ATG5, ATG3, LC3BII, LAMP-1, CathB, CathD (measures of Autophagic flux), Cleaved caspase -3, phosho JNK (measures of cell stress) and FL-APP, CTF α/β, BACE-1 (measures of APP processing) in total fractions were measured by immunoblotting in N2a Swe10 cells in the presence or absence of P110 (1 µM/24 hours); β-actin were used as a loading controls. Protein levels were quantified and represented as fold-change of vehicle. n = 3. Data information: Mean, standard deviation, and P-values are shown. #P < 0.05 versus controls. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. One-way ANOVA and Tukey’s post hoc test. C, control.
Figure 3
Figure 3. Drp1/Fis1 interaction leads to mitochondrial defects and bioenergetic failure in AD patient derived fibroblasts
(A) Western blot showing Drp1 association with mitochondria-enriched fractions after 72 hours incubation in 1% FBS medium. VDAC was used as loading control. Protein levels were quantified and represented as ratio. n = 3. (B) Western blot showing Drp1 levels in mitochondria-enriched fractions after 72 hours incubation in the presence or absence of P110 in 1% FBS medium. VDAC was used as loading control. Protein levels were quantified and represented as ratio. n = 3. (C) AD patient-derived and healthy subject-derived fibroblasts were incubated in serum free medium in the presence or absence of P110 for 72 hours, pooled total cell lysates were subjected to immunoprecipitation (IP) with anti-Fis1 or anti-Drp1 and the immunoprecipitates were analyzed by immunoblotting (IB); n = 3. (D) Mitochondrial membrane potential was determined using JC-1 dye after 48 hours. Mitochondrial superoxide production was determined using the mitochondrial superoxide indicator MitoSOX after 48 hours. Cell death was measured using ApoTox-Glo™ after 72 hours. ATP levels were measured from using Mitochondrial ToxGlo after 72 hours; n = 3. (E) OCR of healthy controls and AD-patient derived fibroblasts was measured at the basal level and then after sequential treatment with DMEM/200 mM Eto followed by 1 mM Oligo, 1.5 mM FCCP, 0.1 mM Rot and 1 mM Ant A using the XF-96 Seahorse system. The assay is representative of two independent experiments, run in triplicates. Bioenergetic parameters were quantified and are represented below. (F) Microscopy of stained healthy control fibroblasts H1: AG07123 from 62 year old male, H2:AG04146 from 57 year-old male and H3: HDFA and AD-patient derived fibroblasts A1: AG04402 from 47 year old male; Familial-APOE, A2:AG07377 from 60-year-old male; Sporadic AD; A3:AG06840 from 56 year-old male; Familial- PSEN1; A4:AG09908 from 81 year old male; Familial-PSEN2; A5:AG05810 from 79 year-old female; Familial-APOE with anti-TOM20 (a marker of mitochondria, 1:500 dilution) incubated in the presence or absence of P110 (1 µM) for 72 hours in serum free medium. Panels show enlarged areas of the boxes. Scale bar: 0.5 µm. Below, quantification of mitochondrial interconnectivity and elongation score in healthy control fibroblasts and AD-patient derived fibroblasts. (G) The activity of citrate synthase was determined in Healthy controls and AD-patient derived fibroblasts using total cell lysates to indicate the mitochondrial mass. Data information: Mean, standard deviation, and P-values are shown. #P < 0.05 versus controls. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. One-way ANOVA and Tukey’s post hoc test. C, control.
Figure 4
Figure 4. Inhibition of Drp1 association with Fis1 using P110 in the symptomatic phase improves survival and slows disease progression
(A) Aβ40 and Aβ42 levels were examined via ELISA in 5xTg-AD mitochondrial fractions. n = 5. (B) The ATP colorimetric/fluorometric assay kit was used to measure ATP levels in freshly isolated whole brain mitochondria. n = 5. (C) Lipid peroxide levels were determined by the leucomethylene blue assay in whole brain mitochondria. n = 5. (D) Rates of hydrogen peroxide production were determined in state-4 respiration by Amplex-Red hydrogen peroxide assay from whole brain mitochondria. n = 5. (E) Western blot showing Drp1, Mfn2 and Opa1 mitochondria-enriched fractions from whole brains. VDAC was used as loading control. Protein levels were quantified and represented as fold change. n = 4–6. (F) Non-learnt behavior as assessed by nesting behavior in home change. n = 4–6 per group. (G) Hindlimb clasping scores, as assessed in the tail suspension test. n = 10 per group. (H) Latencies to fall in the context of a wire hang test. n = 10 per group. (I) Body weight measurements separated into sexes. n = 5 per group. (J) Distance travelled was measured in a 10 min open field test after 10 min of habituation. n = 10 per group. Data information: Mean, standard deviation, and P-values are shown. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. One-way ANOVA and Tukey’s post hoc test.

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