Effects of NK-4 on Aβ_25–35-induced cognitive impairments in ICR Mice.

<p>Cognitive impairment was induced in ICR mice by icv injection of Aβ_25–35 solution as described in Material and Method. A low (50 µg/kg) or high (500 µg/kg) dose of NK-4 was administered intraperitoneally to mice twelve consecutive days starting from the next day of Aβ injection (day1). Mice were tested for object recognition (a), followed by passive avoidance (b). Values are means ± SEM (n = 10). Sham: sham-operated group, saline: saline-treated Aβ_25–35–icv group, NK-4l: low-dose NK-4-treated Aβ_25–35–icv group, NK-4h; high-dose NK-4-treated Aβ_25–35–icv group. *P<0.05, **P<0.01 vs. saline-treated group.</p

Effects of NK-4 on motor coordination in <i>hm</i>PCDs.

<p>(A) Effect of NK-4 on motor performance in the rota-rod test. Animals were tested weekly for the ability to remain on the rotating rod, and the time spent on the rod is shown. (B) Effect of NK-4 on frequency of falling in <i>hm</i>PCDs. Spontaneous falling of each animal was counted for 60 s and is presented as the falling frequency. Values are the mean ± SEM of 5 animals per group (2 males and 3 females). *P<0.05, **P<0.01 vs. vehicle-treated <i>hm</i>PCD.</p

Cytoprotective effects of NK-4 on Aβ_25–35-induced cytotoxicity in PC12 cells.

<p>PC12 cells were treated with 50 µM Aβ_25–35 for 72 hr in the absence (open bar) or presence of the indicated concentrations of NK-4 (closed bars). Control cells were incubated under the same conditions, but without Aβ_25–35. Cell viability was assessed by alamarBlue assay. Results are shown as means ± SD (n = 3). **P<0.01 vs. no NK-4.</p

Induction of Akt phosphorylation by NK-4 in PC12 cells.

<p>PC12 cells were treated with 250 nM NK-4 for the indicated times. Whole cell lysates were analyzed by Western blotting using anti-phospho-Akt antibody (upper panel) or anti-Akt antibody (lower panel). The graph shows the ratio of phosphorylated Akt to total Akt at each time point.</p

Effect of NK-4 on the plasma Aβ concentrations in Tg2576 mice.

<p>NK-4 did not interfere the optical density of both hAβ_1–40 (a) and hAβ_1–42 (b) ELISA systems, which using the antibody combinations of BAN50/BA27 and BAN50/BC05, respectively. Since these ELISA systems cannot distinguish N-terminal heterogeneity of Aβ peptides derived from biological samples, we represented hAβ species as hAβ_x-40 and hAβ_x-42, respectively. Plasma from mice aged 12 months was used for measurements of hAβ_x-40 (c) and hAβ_x-42 (d). Values are the mean ± SEM for saline-treated Tg2576 mice (control group, n = 10), a low dose NK-4 (100 µg/kg)-treated group (n = 9), and a high dose NK-4 (500 µg/kg)-treated group (n = 8). **P<0.01 vs. saline-treated Tg2576 group.</p

Effect of NK-4 on cognitive function of Tg2576 mice in a passive avoidance test.

<p>Mice were tested for learning at 9 months of age. The latency in the retention session performed 24 hr after the training session is shown. Values are means ± SEM (n = 10). **P<0.01 vs. saline-treated Tg2576 group.</p

Neurotrophic effects of NK-4 in PC12 cells.

<p>(<b>A</b>) Effect of NK-4 on PC12 cell growth. PC12 cells were raised in D-MEM containing 10% FBS for 72 hr in the presence or absence of NK-4. The number of cells was assessed by alamarBlue assay. (<b>B</b>) Effect of NK-4 on NGF-primed neurite-outgrowth in PC12 cells. PC12 cells were treated with NGF (5 ng/ml) plus NK-4 (indicated concentrations) for 72 hr. The percentage of PC12 cells with neurites longer than twice the diameter of the cell body was calculated. Results are shown as a mean ± SD (n = 3). **P<0.01 vs. the respective control. (<b>C</b>) Phase contrast micrographs of PC12 cells. PC12 cells treated with NK-4 (1000 nM) plus NGF (5 ng/ml) (c) had longer neurites than those treated with NK-4 (1000 nM) (a) or NGF (5 ng/ml) (b) for 72 hr. Bar: 50 µm.</p

Effect of NK-4 on brain soluble or insoluble Aβ_x-40 and Aβ_x-42 in Tg2576 mice.

<p>Brains from mice aged 12 months were homogenized and separated into soluble (c, d) and insoluble (a, b) fractions of Aβ, and assayed for hAβ_x-40 (a, c) and hAβ_x-42 (b, d), respectively. FA: SDS insoluble and formic acid soluble fractions of Aβ. SDS: SDS soluble fractions of Aβ. Correlations are shown between the plasma and brain levels of hAβ_x-40 (e) and hAβ_x-42 (f) in Tg2576 mice. Data are the mean ± SEM of hAβ_x-40 or hAβ_x-42 in each mouse in saline-treated Tg2576 controls (n = 10), a low dose NK-4 (100 µg/kg)-treated group (n = 9), and a high dose NK-4 (500 µg/kg)-treated group (n = 8). *P<0.05, **P<0.01 vs. saline-treated Tg2576 group.</p

Effect of NK-4 on learning ability of Tg2576 mice in a water maze test.

<p>Mice were tested for learning ability at 6 and 12 months of age. The mean latency represents the time spent finding a hidden platform placed in a fixed location in the pool. Two trials per day were conducted for 4 consecutive days. Data are expressed as means ± SEM of the averaged time on each day (n = 10). *P<0.05, **P<0.01 vs. saline-treated Tg2576 group.</p

Effects of NK-4 on cognitive function of Tg2576 mice in a novel object recognition test.

<p>A low (100 µg/kg) or high (500 µg/kg) dose of NK-4 was administered intraperitoneally to female Tg2576 mice from 3 to 12 months of age. Mice were tested for object recognition at 6 and 12 months of age. Values are means ± SEM (n = 10). **P<0.01 vs. saline-treated Tg2576 group. Wild: non-transgenic female 129S6 mice. Tg2576 (saline): saline-treated Tg2576 mice. Tg2576 (NK-4l): low-dose NK-4-treated Tg2576 mice. Tg2576 (NK-4h); high-dose NK-4-treated Tg2576 mice.</p