Modulation of OSCP mitigates mitochondrial and synaptic deficits in a mouse model of Alzheimer's pathology

Abstract

Synaptic failure underlies cognitive impairment in Alzheimer's disease (AD). Cumulative evidence suggests a strong link between mitochondrial dysfunction and synaptic deficits in AD. We previously found that oligomycin-sensitivity-conferring protein (OSCP) dysfunction produces pronounced neuronal mitochondrial defects in AD brains and a mouse model of AD pathology (5xFAD mice). Here, we prevented OSCP dysfunction by overexpressing OSCP in 5xFAD mouse neurons in vivo (Thy-1 OSCP/5xFAD mice). This approach protected OSCP expression and reduced interaction of amyloid-beta (Aβ) with membrane-bound OSCP. OSCP overexpression also alleviated F1Fo ATP synthase deregulation and preserved mitochondrial function. Moreover, OSCP modulation conferred resistance to Aβ-mediated defects in axonal mitochondrial dynamics and motility. Consistent with preserved neuronal mitochondrial function, OSCP overexpression ameliorated synaptic injury in 5xFAD mice as demonstrated by preserved synaptic density, reduced complement-dependent synapse elimination, and improved synaptic transmission, leading to preserved spatial learning and memory. Taken together, our findings show the consequences of OSCP dysfunction in the development of synaptic stress in AD-related conditions and implicate OSCP modulation as a potential therapeutic strategy.

Keywords: Alzheimer's disease; Amyloid beta; Mitochondrial F1Fo ATP synthase; Oligomycin-sensitivity-conferring protein; Synaptic injury.

Fig. 1. Alleviated OSCP aberrations in Thy-1 OSCP/5xFAD mice.

(A&B) Immunoblotting analysis of OSCP and other major subunits of F1FO ATP synthase including α, β, γ, a, b, and c in brain mitochondria isolated from 4–5 (A1) and 9–10-month-old (B1) nonTg, 5xFAD, Thy-1 OSCP, and Thy-1 OSCP/5xFAD mice. TOM40 was used as a loading control. Two-way ANOVA followed by Bonferroni post hoc analysis. * P < 0.05 5xFAD vs other groups; NS, not significant. n = 3–7 mice per group. (A2&B2) Representative bands of immunoblotting. (C) Duolink PLA positive dots for OSCP/Aβ complex in cortex from 4–5 and 9–10-month-old 5xFAD and Thy-1 OSCP/5xFAD mice. Unpaired student t-test. * P < 0.05. n = 4–5 mice per group. The right panel is the 3-dimensional (3D) representative images of OSCP/Aβ PLA positive dots in the cortex. Red dots represent OSCP/Aβ interaction, blue are DAPI-labeled cell nuclei. Scale bar = 20 μm. (D) Co-immunoprecipitation (Co-IP) of OSCP and Aβ in isolated mitochondria or their membrane fractions from 9–10-month-old nonTg, 5xFAD, Thy-1 OSCP, and Thy-1 OSCP/5xFAD mice. Right panel are the representative images of Co-IP. Unpaired student t-test. * P < 0.05. n = 4–5 mice per group.

Fig. 2. Attenuated mitochondrial dysfunction in Thy-1 OSCP/5xFAD mice.

(A) F1FO ATP synthase catalytic activity of brain mitochondria isolated from 4–5 (A1) and 9–10-month-old (A2) nonTg, 5xFAD, Thy-1 OSCP, and Thy-1 OSCP/5xFAD mice. Unpaired student t-test. * P < 0.05, *** P < 0.001; NS, not significant. n = 4–7 mice per group. (B) Oligomycin sensitivity of isolated brain mitochondria from 4–5 (B1) and 9–10-month-old (B2) nonTg, 5xFAD, Thy-1 OSCP, and Thy-1 OSCP/5xFAD mice. Unpaired student t-test. * P < 0.05 5xFAD vs Thy1-OSCP/5xFAD, *** P < 0.001 5xFAD vs Thy1-OSCP/5xFAD. n = 3–7 mice per group. (C) Mitochondrial respiration control ratio in 4–5 (C1) and 9–10-month-old (C2) old nonTg, 5xFAD, Thy-1 OSCP, and Thy-1 OSCP/5xFAD mice. Unpaired student t-test. * P < 0.05, *** P < 0.001. n = 5–7 mice per group. (D) ATP production in isolated brain mitochondria from 4–5 (D1) and 9–10-month (D2) nonTg, 5xFAD, Thy-1 OSCP, and Thy-1 OSCP/5xFAD mice. Unpaired student t-test. ** P < 0.01, *** P < 0.001. n = 4–6 mice per group. (E) Mitochondrial ATP:O ratio in 4–5 (E1) and 9–10-month-old (E2) nonTg, 5xFAD, Thy-1 OSCP and Thy-1 OSCP/5xFAD mice. Unpaired student t-test. * P < 0.05 vs other groups, *** P < 0.001. n = 4–6 mice per group. (F) Mitochondrial swelling for 4–5 (F1) and 9–10-month-old (F2) nonTg, 5xFAD, Thy-1 OSCP, and Thy-1 OSCP/5xFAD mice. Unpaired student t-test. * P < 0.05 5xFAD vs Thy1-OSCP/5xFAD, *** P < 0.001 5xFAD vs Thy1-OSCP/5xFAD. n = 5–9 mice per group.

Fig. 3. Rescued axonal mitochondrial dynamic and motility by OSCP overexpression in Aβ-treated neurons.

(A1) The average length of axonal mitochondria was measured in primary nonTg and Thy1-OSCP neurons expressing mitoDsred. The neurons were treated with vehicle, 1 μM Aβ1–42, or 1 μM scramble Aβ for 24 hours. Two-way ANOVA followed by Bonferroni post hoc analysis. *** P < 0.001. n = 227–256 mitochondria per group. (A2) Representative images of axonal mitochondria in primary cultured neurons. Scale bar = 5 μm. (B) Cumulative distribution data of axonal mitochondrial length. n = 227–256 mitochondria per group. (C1) The average volume of axonal mitochondria was analyzed from nonTg and Thy1-OSCP neurons treated with vehicle, 1 μM Aβ1–42 or 1 μM scramble Aβ for 24 hours. Two-way ANOVA followed by Bonferroni post hoc analysis. *** P < 0.001. n = 223–256 mitochondria per group. (C2) Representative 3D images of axonal mitochondria in primary cultured neurons. Scale bar = 5 μm. (D) Cumulative distribution data of axonal mitochondrial volume. n = 223–256 mitochondria per group. (E1) Immunoblotting analysis of mitochondrial dynamic protein in isolated brain mitochondria from 9–10-month-old nonTg, 5xFAD, Thy-1 OSCP, and Thy-1 OSCP/5xFAD mice. p-Dlp1^Ser616, T-Dlp1, Mfn2, and OPA1 including short form (OPA1-S) and long form (OPA1-L) were detected. Two-way ANOVA followed by Bonferroni post hoc analysis. * P < 0.05 vs other groups. ** P < 0.01 vs other groups. n = 3–4 mice per group. (E2) Representative bands of immunoblotting in E1. (F-J) Axonal mitochondrial trafficking including percentage of stationary axonal mitochondria (F), percentages of movable (G), anterograding (H), and retrograding (I) mitochondria for vehicle and 24 hours 1 μM Aβ1–42 or 1 μM scramble Aβ-treated nonTg and Thy1-OSCP neurons. Unpaired student t-test. * P < 0.05 vs other groups. (J) Representative kymographs of axonal mitochondrial movement. Scale bar = 10 μm. Data were collected from 3 independent experiments.

Fig. 4. Preserved synaptic plasticity and transmission in Thy-1 OSCP/5xFAD mice.

(A1) Synaptic density of 4–5-and 9–10-month-old nonTg, 5xFAD, Thy-1 OSCP, and Thy-1 OSCP/5xFAD mice. Two-way ANOVA followed by Bonferroni post hoc analysis. *** P < 0.001 vs other groups. n = 5 mice per group. (A2) Representative 3D-reconstructed images of synapse staining. vGLUT1 (blue) and PSD95 (red) were used to visualize pre- and post-synaptic components, respectively. The overlaid staining of vGLUT1 and PSD95 indicates synapses. Scale bar = 20 μm. (B) Time course of long-term-potential (LTP) and representative fEPSP responses during the baseline period (black trace) and 30 seconds after theta burst stimulation (red trace) in four groups of mice at 4–5 (B1) and 9–10 (B2) months old. Two-way ANOVA followed by Bonferroni post hoc analysis. * P < 0.05 5xFAD vs other groups. ** P < 0.01 5xFAD vs other groups. n = 6–9 mice per group.

Fig. 5. Mitigated synapse trimming by microglia in Thy-1 OSCP/5xFAD mice.

(A1) Analysis of C1q-tagged synapses in cortex from 4–5 and 9–10-month-old nonTg, 5xFAD, Thy-1 OSCP, and Thy-1 OSCP/5xFAD mice. Two-way ANOVA followed by Bonferroni post hoc analysis. *** P < 0.001. n = 5 mice per group. (A2) Representative 3D-reconstructed images of C1q-tagged synapses in cortex. Synaptophysin (red) represents synapses. The overlaid staining of C1q and synaptophysin indicates C1q-tagged synapses. Scale bar = 10 μm. (B1) Synaptic pruning was examined through co-staining of microglia (Iba1, red) and synapses (synaptophysin, green). Two-way ANOVA followed by Bonferroni post hoc analysis. * P < 0.05, ** P < 0.01. n = 5 mice per group. (B2) 3D representative images of synaptic pruning by microglia. The overlaid staining of Iba1 and synaptophysin indicates microglia-engulfed synapses. Scale bar = 5 μm.

Figure 6.. Improved cognitive function in Thy-1 OSCP/5xFAD mice.

Spatial learning (A1&A2) and reference memory (B1&B2) of 4–5 and 9–10-month-old nonTg, 5xFAD, Thy-1 OSCP, and Thy-1 OSCP/5xFAD mice. One-way ANOVA followed by Bonferroni post hoc analysis. * P < 0.05 vs other groups, ** P < 0.01 vs other groups. n = 6–9 mice per group.