Effects of NK-4 in a Transgenic Mouse Model of Alzheimer's Disease

Beta-amyloid (Aβ) peptides are considered to play a major role in the pathogenesis of Alzheimer's disease (AD) and molecules that can prevent pathways of Aβ toxicity may be potential therapeutic agents for treatment of AD. We have previously reported that NK-4, a cyanine photosensitizing dye, displays neurotrophic and antioxidant activities. In this study, we report the effects of NK-4 on the toxicity of Aβ and on cognitive function and Aβ concentration in a transgenic mouse model of AD (Tg2576). In vitro, NK-4 effectively protected neuronal cells from toxicity induced by Aβ. In addition, it displayed profound inhibitory activities on Aβ fibril formation. In vivo, Tg2576 mice received an intraperitoneal injection at 100 or 500 µg/kg of NK-4 once a day, five times a week for 9 months. Administration of NK-4 to the mice attenuated impairment of recognition memory, associative memory, and learning ability, as assessed by a novel object recognition test, a passive avoidance test, and a water maze test, respectively. NK-4 decreased the brain Aβ concentration while increasing the plasma amyloid level in a dose-dependent manner. NK-4 also improved memory impairments of ICR mice induced by direct intracerebroventricular administration of Aβ. These lines of evidence suggest that NK-4 may affect multiple pathways of amyloid pathogenesis and could be useful for treatment of AD

Neurotrophic Effects of a Cyanine Dye via the PI3K-Akt Pathway: Attenuation of Motor Discoordination and Neurodegeneration in an Ataxic Animal Model

BACKGROUND: Neurotrophic factors may be future therapeutic agents for neurodegenerative disease. In the screening of biologically active molecules for neurotrophic potency, we found that a photosensitizing cyanine dye, NK-4, had remarkable neurotrophic activities and was a potent radical scavenger. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we evaluated the effect of NK-4 on the protection of neurons against oxidative damage and investigated the associated intracellular signaling pathways. Subsequently, we evaluated the effect of NK-4 in an animal model of neurodegeneration. In vitro, NK-4 showed dose-dependent protection of PC12 cells from toxicity induced by oxidative stress caused by hydrogen peroxide (H(2)O(2)) or 6-hydroxydopamine (6-OHDA). Comparison of extracellular signal-regulated kinase signaling pathways between treatment with NK-4 and nerve growth factor (NGF) using K252a, an inhibitor of the NGF receptor TrkA, revealed that NK-4 activity occurs independently of NGF receptors. LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, blocked the protective effect of NK-4, and NK-4 caused activation of Akt/protein kinase B, a downstream effector of PI3K. These results suggest that the neuroprotective effects of NK-4 are mediated by the PI3K-Akt signaling pathway. NK-4 treatment also attenuated stress-induced activation of SAPK/JNK, which suggests that NK-4 activates a survival signaling pathway and inhibits stress-activated apoptotic pathways independently of the TrkA receptor in neuronal cells. In vivo, administration of NK-4 improved motor coordination in genetic ataxic hamsters, as assessed by rota-rod testing. Histological analysis showed that cerebellar atrophy was significantly attenuated by NK-4 treatment. Notably, the Purkinje cell count in the treated group was threefold higher than that in the vehicle group. CONCLUSIONS/SIGNIFICANCE: These results suggest that NK-4 is a potential agent for therapy for neurodegenerative disorders based on the activation of survival signaling pathways

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

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 K252a, a Trk inhibitor, on PC12 cell growth (A) and neurite-outgrowth (B) induced by NK-4 (bold line) and/or NGF (broken line).

<p>PC12 cells were preincubated in serum-free D-MEM with the indicated concentrations of K252a for 15 min. NK-4 (250 nM) or NGF (50 ng/ml) was added and the cells were further incubated for 72 hr. In the neurite-outgrowth assay, 250 nM NK-4 with 5 ng/ml NGF, or 50 ng/ml of NGF alone was used. The number of cells was assessed by alamarBlue assay. The percentage of cells with neurites was quantified by phase contrast microscopy. Values are means ± SD in triplicate. **P<0.01 vs. control.</p

Promotion of PC12 cell growth and neurite-outgrowth through activation of PI3K by NK-4.

<p>Effects of LY294002, a PI3K inhibitor on cell growth (A) and neurite-outgrowth (B) induced by NK-4 (bold line) and/or NGF (broken line). PC12 cells were preincubated in serum-free D-MEM containing the indicated concentrations of LY294002 for 15 min. NK-4 (250 nM) or NGF (50 ng/ml) was added and the cells were further incubated for 72 hr. The number of cells was assessed by alamarBlue assay. In the neurite-outgrowth assay, 250 nM NK-4 with 5 ng/ml NGF, or 50 ng/ml of NGF alone was used. The percentage of cells with neurites was quantified by phase contrast microscopy. Values are means ± SD in triplicate. **P<0.01 vs. control.</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

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