Furin promotes dendritic morphogenesis and learning and memory in transgenic mice

Abstract

Furin is a proprotein convertase implicated in a variety of pathological processes including neurodegenerative diseases. However, the role of furin in neuronal plasticity and learning and memory remains to be elucidated. Here, we report that in brain-specific furin transgenic (Furin-Tg) mice, the dendritic spine density and proliferation of neural progenitor cells were significantly increased. These mice exhibited enhanced long-term potentiation (LTP) and spatial learning and memory performance, without alterations of miniature excitatory/inhibitory postsynaptic currents. In the cortex and hippocampus of Furin-Tg mice, the ratio of mature brain-derived neurotrophic factor (mBDNF) to pro-BDNF, and the activities of extracellular signal-related kinase (ERK) and cAMP response element-binding protein (CREB) were significantly elevated. We also found that hippocampal knockdown of CREB diminished the facilitation of LTP and cognitive function in Furin-Tg mice. Together, our results demonstrate that furin enhances dendritic morphogenesis and learning and memory in transgenic mice, which may be associated with BDNF-ERK-CREB signaling pathway.

Keywords: BDNF; CREB; Furin; Learning and memory; Long-term potentiation (LTP).

Fig. 1

Furin expression is significantly increased in transgenic mice. A Schematic of furin construct. Full-length cDNA for mouse furin was cloned into MoPrP vector. B Germline transmission of the transgene was checked by PCR analysis of tail DNA using transgenic primers. 1–6, tail snip genomic DNA; 7, positive control vector DNA; M, DNA molecular weight standards. Yellow arrow indicated MoPrP-furin/+. Representative Western blots (C) and quantification (D) of furin protein levels in the hippocampus (hippo) and cortex in wild-type (WT) and furin transgenic (Tg) mice, respectively (data were analyzed by Student’s t test; n = 6. Error bars are SEM; *P < 0.05). The molecular weight of protein marker in kDa is shown to the right of each Western blot panel. E mRNA level of furin was determined by real-time PCR in WT and Tg mice (data were analyzed by Student’s t test; n = 6. Error bars are SEM; *P < 0.05). F Representative immunohistochemistry images from WT and Tg mice (a–f). Expression of furin is in the cytoplasm of neurons in the hippocampus (a, d) and cortex (c, f). Magnification of the marked area is shown (b, e). Scale bar: 100 µm. G A bar graph of the mean integral optical density (IOD) of furin shows a significant increase in the hippocampus (hippo) and cortex in Tg mice compared to WT mice. Data represent the mean ± SEM of four slices per genotype. **P < 0.01, ***P < 0.001 using Student’s t test. H Representative Western blot and quantification of furin protein level normalized to GAPDH in the cerebellum, olfactory bulb (OB) and midbrain from WT and Tg mice (data were analyzed by Student’s t test; n = 6. Error bars are SEM; *P < 0.05). I Representative Western blot and quantification of furin protein level normalized to GAPDH in the spleen, liver and kidney from WT and Tg mice (data were analyzed by Student’s t test; n = 6. Error bars are SEM)

Fig. 2

Furin increases dendritic spine density and neuronal proliferation. A Representative photomicrographs of dendritic spines in CA1 area of WT and Tg mice. Scale bar: 5 μm. B–F Quantification of dendritic spine density (total number per 10 μm) (B, n = 8 mice in each), size of soma (C, n = 40 in WT/42 in Tg), perimeter of soma (D, n = 40 in WT/42 in Tg), maximum distance from dendritic terminal to soma (E, n = 40 in WT/42 in Tg) and cross number with soma (F, n = 40 in WT/42 in Tg) in CA1 region of WT and Tg mice (data were analyzed by Student’s t test; error bars are SEM; *P < 0.05). G Representative confocal microscopic images showing the immunostaining of BrdU (red) and DCX (green) and merged images (yellow) in hippocampal slices from WT and Tg mice, respectively. Scale bar: 100 μm. Quantification of the total number of BrdU⁺ (H) and DCX⁺ neurons (I) (data were analyzed by Student’s t test; n = 5. Error bars are SEM; *P < 0.05, **P < 0.01). J The ratio of BrdU⁺–DCX⁺/total BrdU⁺ cells is not significantly different between WT and Tg mice (data were analyzed by Student’s t test; n = 5. Error bars are SEM)

Fig. 3

Tg mice show enhanced LTP but not excitatory/inhibitory neurotransmission. A Time-course of fEPSP in CA1 neurons of WT (n = 6) and Tg mice (n = 6). LTP is induced after high-frequency stimulation (HFS; 100 Hz for 1 s) at the indicated time by the arrow. Representative traces of the fEPSP before (1) and 1 h after (2) HFS are shown on the top. Scale bar: 0.5 mV/10 ms. B Bar graphs comparing LTP observed during the last 10 min in WT and Tg mice (data were analyzed by Student’s t test; n = 6/6 mice. Error bars are SEM; *P < 0.05). C–H Representative trace of mEPSC (C) or mIPSC (F) of WT and Tg mice in CA1 pyramidal cells, scale bar: 10 pA/5 s. Cumulative fractions and bar plots (insets) of mEPSC (D, E) or mIPSC (G, H) amplitude and frequency (inter-event intervals) in WT and Tg mice (data were analyzed by Student’s t test; n = 6/6 mice. Error bars are SEM)

Fig. 4

Spatial learning and memory performance are improved in Tg mice. Morris water maze test showing the mean escape latency from day 1 to 6 (A), the number of crossing (B) and the time spent in target quadrant (TQ) (C) (data were analyzed by Student’s t test; n = 14 in each group. Error bars are SEM; *P < 0.05, ***P < 0.001). D Typical pathlengths showing the movement trajectory of mice in hidden platform experiment on the fifth day. The circle represents the previous location of the platform

Fig. 5

Furin-associated proteins are altered in Tg mice. Representative Western blots (A) and quantitative analysis (B) of furin-related proteins in the hippocampus of WT and Tg mice, respectively (data were analyzed by Student’s t test; n = 8 in each group. Error bars are SEM; *P < 0.05). The molecular weight of protein marker in kDa is shown to the right of each Western blot panel. Representative Western blots (C) and quantitative analysis (D) of furin-related proteins in the cortex of WT and Tg mice, respectively (data were analyzed by Student’s t test; n = 8 in each group. Error bars are SEM; *P < 0.05, **P < 0.01). The molecular weight of protein marker in kDa is shown to the right of each Western blot panel

Fig. 6

CREB knockdown and anti-BDNF antibody inhibit LTP in Tg mice. A CREB protein levels in the hippocampus of WT and Tg mice injected with lentivirus bearing scrambled sequence (WT-lenti-scr, Tg-lenti-scr mice, n = 8) or shCREB (WT-lenti-shCREB, Tg-lenti-shCREB, n = 9) (data were analyzed by Student’s t test; error bars are SEM; **P < 0.01). The molecular weight of protein marker in kDa is shown to the right of each Western blot panel. B Time-course of fEPSP in CA1 neurons of WT and Tg mice treated with lenti-scr and lenti-shCREB. LTP is induced after high-frequency stimulation (HFS; 100 Hz for 1 s). Representative traces of the fEPSP before (1) and 1 h after (2) HFS are shown on the top. Scale bar: 0.5 mV/10 ms. C Bar graphs comparing LTP during the last 10 min in Tg-lenti-shCREB (n = 9), WT-lenti-shCREB (n = 9), Tg-lenti-scr (n = 8) and WT-lenti-scr mice (n = 8) (data were analyzed by Student’s t test; error bars are SEM; **P < 0.01). D Time-course of fEPSP in CA1 neurons of Tg mice injection with control rabbit IgG (anti-IgG, n = 6) and BDNF antibody (anti-BDNF, n = 6). LTP is induced after high-frequency stimulation (HFS; 100 Hz for 1 s). Representative traces of the fEPSP before (1) and 1 h after (2) HFS are shown on the top. Scale bar: 0.5 mV/10 ms. E Bar graphs comparing LTP during the last 10 min in anti-IgG and anti-BDNF mice. Data were analyzed by Student’s t test; n = 6/6 mice. Error bars are SEM; **P < 0.01. F Representative Western blots and quantitative analysis of p-CREB protein in the hippocampus of Tg mice injected with either IgG (n = 6) or antibody to BDNF (n = 6) (data were analyzed by Student’s t test; error bars are SEM; **P < 0.01). The molecular weight of protein marker in kDa is shown to the right of each Western blot panel

Fig. 7

CREB knockdown inhibits cognitive function in Tg mice. Morris water maze test showing the mean escape latency from day 1 to 6 (A), time spent in the target quadrant (B) and the number of crossing (C) (data were analyzed by Student’s t test; n = 14 in each group. Error bars are SEM; *P < 0.05). D Typical pathlengths showing the movement trajectory of mice in hidden platform experiment on the fifth day. The circle represents the previous location of the platform