The Protective Effect of Icariin on Mitochondrial Transport and Distribution in Primary Hippocampal Neurons from 3× Tg-AD Mice

Abstract

Icariin, a pharmacologically active component isolated from the Chinese herb Epimedium, has been shown to improve spatial learning and memory abilities in Alzheimer's disease (AD) rats through inhibition of Aβ production and tau protein hyperphosphorylation. However, the potential mechanism of icariin-induced protective effects against mitochondrial dysfunctions in AD still remains unclear. In the present study, we investigated the effect of icariin on the modulation of mitochondrial transport and distribution in primary hippocampal cultures from triple-transgenic (3× Tg) AD mice. The results showed that icariin enhanced mitochondrial motility and increased mitochondrial index and mitochondrial length and size in the diseased neurons. Additionally, the expression of the key mitochondrial enzyme, pyruvate dehydrogenase-E1α (PDHE1α), and the post synaptic density protein 95 (PSD95), was preserved in AD neurons after icariin treatment, accompanied by a downregulation of Aβ and phosphorylated tau expression in the corresponding areas. Further study showed that icariin treatment resulted in a decrease in mitochondrial fission protein dynamin-related protein 1 (Drp1) and an increase in fusion protein Mitofusin 2 (Mfn2). These data indicate that icariin can promote mitochondrial transport, protect mitochondria against fragmentation and preserve the expression of mitochondrial and synaptic functional proteins in AD neurons. Thus, icariin may be a potential therapeutic complement for AD and other mitochondrial malfunction-related neuronal degenerative diseases.

Keywords: Alzheimer’s disease; icariin; mitochondrial dysfunction; mitochondrial transport.

Figure 1

The overexpression of amyloid-β peptide (Aβ), tau and phosphorylated tau in 3× transgenic (Tg)-Alzheimer’s disease (AD) neurons. (a) Double-labeling analysis of neuronal marker microtubule-associated protein 2 (MAP2) with Aβ in neurons from nontransgenic (NTg) and Tg neurons. Tg cells showed an overexpression of Aβ in the soma and neurites as compared to NTg controls (white arrows); (b) Quantification revealed a significant difference in Aβ staining between Tg and NTg neurons. The enzyme linked immunosorbent assay (ELISA) demonstrated that the levels of extracellular and intracellular Aβ were higher in Tg neurons; (c) Total tau immunoreactivity in NTg and Tg neurons as measured with the tau 46 antibody. The immunoreactivity of tau 46 was more intense in the cell bodies and neuritic fibres of Tg neurons as compared to NTg controls (white arrows); (d) Quantification revealed a significant difference in tau 46 staining between Tg and NTg neurons. Western blot assay demonstrated that the level of total tau was significantly higher in Tg neurons; (e) Phosphorylated tau immunoreactivity in NTg and Tg neurons as measured with the phospho-tau (Ptau) antibody pS396. Tg neurons showed intense pS396 staining in the cytoplasm and neurites as compared to NTg controls (white arrows); (f) Quantification revealed a significant difference in pS396 staining between Tg and NTg neurons. Western blot assay demonstrated that the level of Ptau-396 was higher in Tg neurons. Bars graph (means ± SD) represented three independent experiments. * p < 0.05, ** p < 0.01. Scale bars in (a,c,e) = 20 μm.

Figure 2

Altered expressions of synaptic and mitochondrial proteins in 3× Tg-AD neurons. Areas boxed in a and c are shown at higher magnification in the right panels, respectively. (a) Double-labeling analysis of neuronal marker MAP2 with post synaptic density protein 95 (PSD95) in neurons from NTg and Tg neurons. PSD95 expression was weaker in the neurites of Tg cells as compared to NTg group and restricted to the soma (white arrows); (b) Western blot assay demonstrated that the level of PSD95 was lower in Tg neurons; (c) Double-labeling analysis of MAP2 with pyruvate dehydrogenase-E1α (PDHE1α) in neurons from NTg and Tg neurons. PDHE1α expression was more evenly distributed in NTg cells whereas Tg cells showed fragmented expression of PDHE1α along the neuronal processes (white arrows); (d) Western blot assay demonstrated that the level of PDHE1α was lower in Tg neurons. Voltage-dependent anion-selective channel protein (VDAC) was used as a mitochondrial loading control. Bars graph (means ± SD) represented three independent experiments. * p < 0.05. Scale bars in (a,c) = 20 μm.

Figure 3

Effect of icariin on mitochondrial trafficking and distribution within neurites of 3× Tg-AD neurons. (a) DsRed-mito transfected hippocampal neurons from NTg, Tg and Tg + ICA groups were imaged. Representative images are shown in upper panels. Areas boxed in a are shown at higher magnification in the lower panels, respectively; (b) Representative kymograph images of the three experimental groups; (c) Percentages of anterograde-transported, retrograde-transported, and total movable mitochondria were calculated. Calculations were based on analysis of kymographs; (d) The average transport speed of movable mitochondria was calculated; (e) Axonal mitochondrial index (the proportion of neuritic length occupied by mitochondria) in neurons from NTg, Tg and Tg + ICA groups; (f) Mitochondrial length was increased in Tg neurons after icariin treatment; (g) Axonal mitochondrial density was evaluated as number of mitochondria per micron axon. Bar graphs (means ± SD) represented three independent experiments. * p < 0.05, ** p < 0.01. Scale bar in (a) = 20 μm.

Figure 4

Effects of icariin on the modulation of mitochondrial dynamics and synaptic protein expression in 3× Tg-AD primary neuronal cells. (a) Western blot assay showed that icariin reduced the level of mitochondrial fission protein Drp1 and increased the level of mitochondrial fusion protein Mfn2 in Tg neurons; (b) Double-labeling analysis of PSD95 with PDHE1α in neurons from NTg, Tg and Tg + ICA groups. NTg neurons showed intense staining of PSD95 and PDHE1α in the cytoplasm and neurites. PSD95 was colocalized with mitochondrial PDHE1α in merged image (white arrows). In Tg cells, PSD95 and PDHE1α expression was reduced in the neurites and mainly restricted to the soma, which was partially recovered by icariin treatment (white arrows); (c) Quantification of PSD95 and PDHE1α immunoreactivity showed a significant increase of PSD95 and PDHE1α expression in Tg + ICA neurons relative to Tg cells. Bar graphs (means ± SD) represented three independent experiments. * p < 0.05 vs. PSD95 level in NTg neurons; ^# p < 0.05 vs. PDHE1α level in NTg neurons; ^$ p < 0.05 vs. PSD95 level in Tg neurons; ^@ p < 0.05, vs. PDHE1α level in Tg neurons. Scale bars in (b) = 20 μm.

Figure 5

Double-labeling analysis of Aβ and PDHE1α in neurons treated with icariin. (a) NTg neurons exhibited a moderate labelling of Aβ with an abundant expression of PDHE1α in the cytoplasm and neurites (white arrows). In Tg cells the overexpression of Aβ in the soma and neurites was correlated with a weaker signal in PDHE1α staining (white arrows). Icariin-treated Tg cells showed increased PDHE1α expression in the neurites, accompanied with a downregulation of Aβ (white arrows); (b) Quantification of Aβ and PDHE1α immunoreactivity showed a higher expression of Aβ and a lower expression of PDHE1α in Tg neurons relative to NTg cells, which was reversed by treatment with icariin. Bar graphs (means ± SD) represented three independent experiments. ** p < 0.01 vs. Aβ level in NTg neurons; ^# p < 0.05 vs. PDHE1α level in NTg neurons; ^$ p < 0.05 vs. Aβ level in Tg neurons; ^@ p < 0.05, vs. PDHE1α level in Tg neurons . Scale bars in (a) = 20 μm.

Figure 6

Double-labeling analysis of phosphorylated tau pS396 and PDHE1α in neurons treated with icariin. (a) NTg neurons showed a faint staining of pS396 in the neurites where PDHE1α expression was abundant (white arrows). In Tg cells, the strong pS396 staining was correlated with a weaker signal of PDHE1α immunoreactivity (white arrows). In icariin-treated Tg cells, the expression of PDHE1α was increased in the neurites, accompanied with a downregulation of pS396 (white arrows); (b) Quantification of pS396 and PDHE1α immunoreactivity showed a higher expression of pS396 and a lower expression of PDHE1α in Tg neurons relative to NTg cells, which was reversed by treatment with icariin. Bar graphs (means ± SD) represented three independent experiments. ** p < 0.01 vs. pS396 level in NTg neurons; ^# p < 0.05 vs. PDHE1α level in NTg neurons; ^$ p < 0.05 vs. pS396 level in Tg neurons; ^@ p < 0.05, vs. PDHE1α level in Tg neurons. Scale bars in (a) = 30 μm.