Synaptosomal bioenergetic defects are associated with cognitive impairment in a transgenic rat model of early Alzheimer's disease

Abstract

Synaptic bioenergetic deficiencies may be associated with early Alzheimer's disease (AD). To explore this concept, we assessed pre-synaptic mitochondrial function in hemizygous (+/-)TgMcGill-R-Thy1-APP rats. The low burden of Aβ and the wide array of behavioral and cognitive impairments described in 6-month-old hemizygous TgMcGill-R-Thy1-APP rats (Tg(+/-)) support their use to investigate synaptic bioenergetics deficiencies described in subjects with early Alzheimer's disease (AD). In this report, we show that pre-synaptic mitochondria from Tg(+/-) rats evidence a decreased respiratory control ratio and spare respiratory capacity associated with deficits in complex I enzymatic activity. Cognitive impairments were prevented and bioenergetic deficits partially reversed when Tg(+/-) rats were fed a nutritionally complete diet from weaning to 6-month-old supplemented with pyrroloquinoline quinone, a mitochondrial biogenesis stimulator with antioxidant and neuroprotective effects. These results provide evidence that, as described in AD brain and not proven in Tg mice models with AD-like phenotype, the mitochondrial bioenergetic capacity of synaptosomes is not conserved in the Tg(+/-) rats. This animal model may be suitable for understanding the basic biochemical mechanisms involved in early AD.

Keywords: Amyloid β; early-Alzheimer; hippocampal bioenergetics status; neurodegeneration; synaptosomes.

Figure 1.

Biochemical and metabolic characterization of synaptosomes in 6-month-old WT rats. (a) Representative Western blotting of mitochondrial (Mit.) and synaptosomal (Synap.) enriched fractions immunoreacted with the mitochondrial ATP synthase β-subunit (ATP syn-β) and synaptosomal synaptophysin (Synaptoph.) protein markers antibodies (upper panel); frozen-thawed plate-attached synaptosomes were imaged using an Olympus CKX41 microscope (lower panel). Synaptosome with the characteristic ‘doughnut’ shape is indicated by arrowhead. Inset, typical autofluoresence of synaptosomes aggregates imaged using an Olympus BX50 microscope. Scale bars, 10 µm; inset, 50 µm. (b), Representative profile of the oxygen consumption rate (OCR) for hippocampal synaptosomes from 6-month-old WT rats. OCR was determined in the presence of 15 mM glucose (Glc) or 15 mM glucose plus 10 mM pyruvate (Pyr) as substrates. Graph shows basal OCR, OCR after ATP synthase inhibition by 5 µM oligomycin and maximum OCR after the addition of 1 µM FCCP. Data are means ± SEM. (c) Graph shows the extracellular acidification rate (ECAR) determined in parallel with respiration shown in (b). (d) Bars show ATP synthesis rate in synaptosomes of 6-month-old WT rats assessed with luciferin-luciferase assay in digitonin-permeabilized samples in the presence of malate and glutamate (M + G) and after the addition of 1 µM oligomycin (O) or 5 µM antimycin (a). *p < 0.05 vs. M+G.

Figure 2.

Mitochondrial function of hippocampal synaptosomes from 6-month-old Tg(+/−) rats. (a) Representative Western blotting (upper panel) of pools corresponding to WT and Tg(+/−) synaptosomes showing Aβ oligomers (≈ 24 kDa) in Tg(+/−) in contrast to WT samples. Protein content of each lane is shown by anti-synaptophysin immunoreactivity (≈ 38 kDa) (lower panel). (b) Bars show the oxygen consumption rate (OCR) of synaptosomes of WT and Tg(+/−) rats expressed as % of WT basal value. *p < 0.05 vs. WT. (c) Representative Western blotting of synaptosomes of WT and Tg(+/−) rats immunoreacted with the synaptosomal (PSD-95) and mitochondrial ATP synthase β-subunit (ATP syn-β) protein markers (upper panel). Bars (lower panel) show the semi-quantification of 6–7 biological replicates of mitochondrial proteins normalized by synaptosomal content expressed in arbitrary units (A.U.) Data are means ± SEM. p = 0.9. (d) Bars show the citrate synthase activity of WT and Tg(+/−) synaptosomes expressed as nmol/min/mg protein. Data are means ± SEM. p = 0.6. (e) Bars show the specific activity of respiratory complex I, complex II and α-KGDHC of WT and Tg(+/−) synaptosomes expressed as nmol/min/mg protein. Data are means ± SEM. *p < 0.05 vs. WT. (f) The endogenous PGC-1α, NRF-1, and IDE-Met1 transcript levels were determined by RT-qPCR from hippocampal homogenates of WT and Tg(+/−) rats. Each point represents the mean value of at least three independent experiments performed by triplicate for each sample normalized by TATA-binding protein (TBP). The mean ± SEM in Tg(+/−) relative to WT are shown. Values between dashed lines (+1.5 and −1.5) were considered not different from WT=1. (g) Electron micrograph of a portion of the CA1 hippocampal region from WT and Tg(+/−) rats. Arrows, synapses; arrowheads, mitochondria. Scale bar = 2 µm. Inset, high magnification showing a representative mitochondria (M) and synapse (S). Scale bar = 500 nm. Bars (left panel) show the number of synapses/volume. Data are means ± SEM. p = 0.07.

Figure 3.

Effect of BioPQQ on anxiety, spatial working memory and recognition memory. Bars show in (a), entries into the center (left axis) and time spent in the center (right axis) of the OF; (b) total arm entries (left axis) and percentage of alternation (right axis) in the Y-maze; (c) exploration times for the two identical objects (A1 and A2) during the sample trial in the NORT; (d) exploration times for the familiar (A3, identical to A1 and A2) and novel (N) object. Data are means ± SEM of 8–10 rats per group. *p < 0.05, **p < 0.01.

Figure 4.

Treatment with BioPQQ prevented the spatial reference memory deficit exhibited by Tg(+/−) rats. (a) Graphs show latencies (upper-left panel) and paths length (upper-right panel) during the first and second day of the cued learning and latencies (lower-left panel) and paths length (lower-right panel) throughout the five days of spatial learning in the MWM test. (b) Bars show the percentage of time spent in quadrants (L, left; T, target; R, right; O, opposite). Data are means ± SEM of 8–10 per group. *p < 0.05 vs. percentage of time expected by chance (dashed line), ^#p < 0.05, ^##p < 0.01.

Figure 5.

Biochemical impact of BioPQQ on synaptic mitochondrial functionality and brain oxidative stress in 6-month-old Tg(+/−) rats. (a) Bars show the spare respiratory capacity of synaptosomes from Tg(+/−) rats under control (black) or BioPQQ (dashed) supplemented diet expressed as % of WT. *p < 0.05 vs. Tg(+/−) + BioPQQ; (b) The gene copy number of endogenous ND1 was determined by qPCR from CA1 iAβ positive neurons microdissected by LCM from Tg(+/−) and Tg(+/−)+BioPQQ rats. Each point represents the mean value of at least three independent experiments performed by triplicate for each sample normalized by cystic fibrosis transmembrane conductance regulator (CFTR) (left) and ND4 (right) gene, respectively. The mean ± SEM in Tg(+/−)+BioPQQ relative to Tg(+/−) is shown. Values between dashed lines (1.5 and −1.5) were considered not different from Tg(+/−)=1. (c) Representative Western blotting of synaptosomes of WT, Tg(+/−) and Tg(+/−)+BioPQQ rats immunoreacted with the synaptosomal (PSD-95) and mitochondrial ATP synthase β-subunit (ATP syn-β) protein markers (upper panel). Bars show the semi-quantification of 6–7 biological replicates of mitochondrial protein ATP syn-β normalized by PSD-95 content expressed in arbitrary units (A.U.). F(_2,13), p = 0.35. (d) Bars show TBARS accumulation expressed as nmol/mg protein in hippocampal mitochondria of Tg(+/−) rats exposed to control (black) or BioPQQ (dashed) diet. Data are means ± SEM. *p < 0.05.