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

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

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

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 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

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

Effects of NK-4 on cerebellar atrophy and histopathology in <i>hm</i>PCDs.

<p>(A) Effect of NK-4 on cerebellar size in <i>hm</i>PCDs. Brains from 10-week-old animals were photographed from a constant distance and the cerebellar volume was calculated. (B) Effect of NK-4 on the Purkinje cell frequency in <i>hm</i>PCDs. Purkinje cells in the Purkinje cell layer were counted in the mid-sagittal section of the cerebellum from <i>hm</i>PCDs. (C) Immunohistochemistry using an anti-calbindin antibody on cerebellum sections for wild type (a), saline-treated <i>hm</i>PCD (b), and high dose NK-4 (100 µg/kg)-treated <i>hm</i>PCD (c). Bar: 30 µm. (D) Granule cell density in the cerebellum from <i>hm</i>PCDs and wild type controls. H&E stained sections of cerebellum cortex from <i>hm</i>PCDs and wild type controls were photographed and counted for granule cells in an area of 20000 µm² (a). Representative images for wild type (b), saline-treated <i>hm</i>PCD (c), and high dose NK-4-treated <i>hm</i>PCD (d). Bar: 10 µm. Graphs show the mean ± SEM of 6 hamsters at 10 weeks of age (3 males and 3 females). *P<0.05, **P<0.01 vs. <i>hm</i>PCD control. (E) Attenuation of cerebellar demyelination in the white matter. Representative microphotographs of cerebellar white matter for wild type (a), saline-treated <i>hm</i>PCD (b), and high dose NK-4-treated <i>hm</i>PCD (c). Bar: 50 µm.</p

Aβ deposition in brains of Tg2576 mice at 12 months of age.

<p>Coronal brain sections (cortex region) were stained by Congo Red (a) or anti-Aβ polyclonal antibody (b, c). The number of CR-positive plaques on the section was counted in cortex region (a). Typical images of Aβ-immunoreactive tangles of saline-treated Tg2576 mouse (b) and high-dose NK-4(500 µg/kg)-treated Tg2576 mouse (c). Bar represents 50 µm.</p