**Effects of NK-4, a Cyanine Dye with Antioxidant Activities: Attenuation of Neuronal Deficits in Animal Models of Oxidative Stress-Mediated Brain Ischemia and Neurodegenerative Diseases**

Hitomi Ohta, Kenji Akita and Tsunetaka Ohta *R&D Center, HAYASHIBARA Co., Ltd., Japan* 

#### **1. Introduction**

368 Oxidative Stress – Environmental Induction and Dietary Antioxidants

Wolfram, S., Raederstorff, D., Wang, Y., Teixera, S.R. et al. TEAVIGO (Epigallocatechin

Xu, F.P., Chen, M.S., Wang, Y.Z., Yi, Q., et al. Leptin induces hypertrophy via endothelin-1-

Yamauchi, T., Kamon, J., Waki, H., Terauchi, Y., et al. The fat derived hormone adiponectin

Yang, B., Wang, J., Zhao, M., Liu, Y., Wang, W., Jiang, Y. Identification of polysaccharides

Youn, S.K., Kim, Y.J., Ahn, D.H. Antioxidative effects of chitosan in meat sausage. *J. Korean* 

Young, J.F., Nielsen, S.E., Haraldsdottir, J., Daneshvar, B., Lauridsen, S. T., Knuthsen, P., et

mass. *Ann. Nutr. Metab*. 2005, 49, 54-63.

antioxidant activities. *Carbohyd. Res.* 2006, 341, 634-8.

antioxidative status. *Am. J. Clin. Nutr*. 1999, 69, 87-94.

*Circulation* 2004, 110, 1269-75.

*Soc. Food Sci. Nutr*. 2001, 30, 477-81.

2001, 7, 941-46.

Gallate) supplementation prevents obesity in rodents by reducing adipose tissue

reactive oxygen species pathway in cultured neonatal rat cardiomyocytes.

reverses insulin resistance associated with both lipoatrophy and obesity. *Nat. Med*.

from pericarp tissues of litchi (Litchi chinensis Sonn.) fruit in relation to their

al. Effect of fruit juice intake on urinary quercetin excretion and biomarkers of

Cyanine photosensitizing dyes have been studied for over 150 years and continue to be of interest in their biology and medicine. They have been shown to possess various biological activities, including antimicrobial, antioxidant, macrophage activating, and oxidative phosphorylation uncoupling activities (Hayami, 1984; Ushio et al., 2009; Ishihara & Fujisawa, 2007; Kunikata et al., 2002; Takeguchi et al., 1985). Some dyes have been used as immunomodulators to treat allergy and rheumatoid arthritis, cancer, and to promote wound healing (Trauner et al., 1998; Motoyoshi et al., 1991). Since cumulative evidence suggests an involvement of oxidative stress and neuroinflammation as the common feature in the pathogenesis of neurodegenerative disorders (Amor et al., 2010; Wolozin & Behl, 2000a, 2000b), it would be reasonable to expect that cyanine dyes with anti-oxidative and antiinflammatory properties could protect the central nervous system from neuroinflammatoryrelated brain insults.

Recently, we screened more than 250 cyanine dyes for their neurotrophin-like activity and found that NK-4 and some other related compounds are potent neurotrophic agents for the promotion of growth and differentiation of neuronal rat adrenal pheochromocytoma cell line PC12. NK-4 is a divalent cationic pentamethine trinuclear cyanine dye that contains three quinolinium rings, N-alkyl side chains, and two iodine anions (Fig. 1A). Addition of NK-4 into the culture at nanomolar concentrations significantly augmented cell growth of PC12 cells in 3-day cultures (Fig. 1B). NK-4 also promoted nerve growth factor (NGF) primed neurite-outgrowth at micromolar and submicromolar concentrations (Fig. 1C). Since the intervention using neurotrophic or neuroprotective small molecules is thought to have potential for treating neurodegenerative disorders, we investigated the neuroprotective effects of NK-4 against neurotoxic insults *in vitro* and further evaluated its pharmacological effects using animal models of neurodegeneration, including ischemic stroke, cerebellar ataxia, and Alzheimer's disease (AD).

Effects of NK-4, a Cyanine Dye With Antioxidant Activities... 371

et al., 2002). Accordingly, the antioxidants that effectively scavenge hydroxyl radicals and other free radicals should be able to eliminate oxidative injury (Barinaga, 1996), and NK-4

NK-4 7.6 ± 0.6 5.2 ± 2.5 107 ± 2.2 Ascorbate 47.6 ± 0.2 30.5 ± 1.7 130 ± 24 Edaravone 779 ± 18 181 ± 19 > 3,800 Free radical-scavenging efficacy was evaluated in a cell-free *in vitro* system using ESR. Superoxides, hydroxyl radicals, and peroxy radicals were produced and stabilized in DMPO-hypoxanthine/XOD, DMPO-DTPA/H2O2/FeSO4 and DMPO-AAPH systems, respectively. The signal intensity of each radical was recorded in the presence or absence of test samples. The scavenging activity of the samples was expressed as IC50 against each radical. Means ± SD (n=3). Reproduced in part with permission from Biological & Pharmaceutical Bulletin Vol.33 No.11. Copyright [2010] Pharmaceutical Society of Japan.

Neurotrophins, such as NGF or brain-derived neurotrophic factor (BDNF) regulate the growth, survival, and differentiation of central neurons (Blum & Konnerth, 2005). For example, NGF protects PC12 cells against 6-hydroxydopamine (6-OHDA)- and hydrogen peroxide (H2O2)-induced oxidative stress (Salinas et al., 2003; Wang et al., 2001). Since NK-4 displayed a remarkable neurotrophin-like activity, namely the promotion of cell growth and NGF-primed neurite-outgrowth in PC12 cells (Fig. 1B, 1C), we next examined whether NK-4 protects neuronal cells from oxidative or starvation stress (Ohta et al., 2011). An acute oxidative stress challenge was induced by 2-hr treatment with H2O2 or 24 hr treatment with 6-OHDA. These treatments cause a significant decrease in metabolic capacity, which reflects a decline in cell viability, and this is accompanied by an enhancement of apoptotic markers (Franco et al., 2010). Particularly, 6-OHDA has been widely used in experimental models of Parkinsion's disease, and its neurotoxicity involves oxidative damage to catecholaminergic neurons via the generation of hydroxyl radicals, monoamine oxidase-mediated formation of H2O2, and mitochondrial generation

Treatment of PC12 cells with H2O2 decreased cell viability to approximately 50% of control. In contrast, NK-4 at concentrations of 6 µM or above significantly protected the cells from the acute oxidative damage (Fig. 2A). NK-4 also attenuated 6-OHDA-induced oxidative stress in PC12 cells at nanomolar concentrations in a dose-dependent manner (Fig. 2B). Next we tested the effect of NK-4 on trophic factor depletion-induced cell death. Extended serum depletion in PC12 cells triggers ATP shortage, which results in mitochondrial reactive oxygen species (ROS) generation and activation of apoptotic pathways (Troy et al., 2001). NK-4 at submicromolar concentrations significantly increased cellular viability of PC12 cells over 3 days of serum starvation (Fig. 2C). These results suggest that the antioxidative effects of NK-4, together with its neurotrophic

Hydroxyl Radical Peroxy Radical Superoxide

Compound IC50 (µM)

Table 1. Free radical-scavenging effects of NK-4 and other antioxidants.

**2.2 Neuroprotective effects against various cytotoxic stresses** 

could be one of such molecules.

of superoxide (Blum et al., 2001).

properties, engender strong survival signals in PC12 cells.

In this chapter, we focus on the neuroprotective effects of NK-4 against oxidative damage *in vitro* and *in vivo*. We introduce the multipotent properties of NK-4, which may act in concert to attenuate the common pathological pathways of neurodegeneration, and discuss the potential use of NK-4 for neurodegenerative disease therapy.

(A) Chemical structure of NK-4. It was synthesized in HAYASHIBARA Co., Ltd. (B) The proliferative effect of NK-4 on PC12 cells. Cell growth was assessed by AlamarBlue® assay in 3-day cultures. (C) The promotive effect of NK-4 on NGF (5 ng/ml)-primed neurite-outgrowth in PC12 cells. Neuriteoutgrowth was evaluated by counting neurite-outgrowth positive cells in 3-day cultures. The cells were defined as positive when the length of the longest neurite was >2-fold longer than that of the cell body. Data are means ± SD (n=3). \*P<0.05 and \*\* P<0.01 vs. control (no NK-4).

Fig. 1. Chemical structure and neurotrophin-like effects of NK-4.

#### **2.** *In Vitro* **Properties of NK-4**

#### **2.1 Free radical-scavenging activity**

As it has been known that some cyanine dyes show significant antioxidative property due to the extended π-electron conjugated system contained within the structure (Ishihara & Fujisawa, 2007), the free radical-scavenging capacity of NK-4 for superoxides, hydroxyl, and peroxy radicals was evaluated (Koya-Miyata et al., 2010). These radicals are produced *in vivo* in many experimental models of ischemia and reperfusion, and they are therefore generally regarded as the primary free radicals involved in oxidative stress-mediated injury. Half-maximal inhibitory concentrations (IC50) of NK-4 against these radicals are shown in Table 1.

Ascorbate, a well-characterized free radical-scavenging agent, and edaravone were used as positive controls. Edaravone, also known as MCI-186, is a hydroxy radical scavenger, and has been used for acute phase stroke therapy in Japan (Abe & Kogure, 1988). NK-4 displayed a direct and powerful hydroxyl radical-scavenging activity, significantly greater than those of ascorbate and edaravone. The scavenging activity for peroxy radicals was also potent in NK-4 and to a lesser extent in ascorbate. NK-4 and ascorbate acted on superoxides to a comparable extent; however, edaravone did not act on superoxides as previously reported (Tanaka, 2002). The highly reactive hydroxyl radical oxidizes cellular lipids, proteins, and DNA, leading to cell death, and is therefore highly detrimental (Gilgun-Sherki

In this chapter, we focus on the neuroprotective effects of NK-4 against oxidative damage *in vitro* and *in vivo*. We introduce the multipotent properties of NK-4, which may act in concert to attenuate the common pathological pathways of neurodegeneration, and discuss the

> **0 4 16 63 253 NK-4 conc. (nM)**

\*\* \*\*

\*\*

**0**

**0 253 507 1,013 2,027 NK-4 conc. (nM)**

\*\* \*\* \*\*

\*\*

**20**

**40**

**Neurite-outgrowth (%)**

**60**

**80**

\*

(A) Chemical structure of NK-4. It was synthesized in HAYASHIBARA Co., Ltd. (B) The proliferative effect of NK-4 on PC12 cells. Cell growth was assessed by AlamarBlue® assay in 3-day cultures. (C) The

outgrowth was evaluated by counting neurite-outgrowth positive cells in 3-day cultures. The cells were defined as positive when the length of the longest neurite was >2-fold longer than that of the cell body.

As it has been known that some cyanine dyes show significant antioxidative property due to the extended π-electron conjugated system contained within the structure (Ishihara & Fujisawa, 2007), the free radical-scavenging capacity of NK-4 for superoxides, hydroxyl, and peroxy radicals was evaluated (Koya-Miyata et al., 2010). These radicals are produced *in vivo* in many experimental models of ischemia and reperfusion, and they are therefore generally regarded as the primary free radicals involved in oxidative stress-mediated injury. Half-maximal inhibitory concentrations (IC50) of NK-4 against these radicals are shown in

Ascorbate, a well-characterized free radical-scavenging agent, and edaravone were used as positive controls. Edaravone, also known as MCI-186, is a hydroxy radical scavenger, and has been used for acute phase stroke therapy in Japan (Abe & Kogure, 1988). NK-4 displayed a direct and powerful hydroxyl radical-scavenging activity, significantly greater than those of ascorbate and edaravone. The scavenging activity for peroxy radicals was also potent in NK-4 and to a lesser extent in ascorbate. NK-4 and ascorbate acted on superoxides to a comparable extent; however, edaravone did not act on superoxides as previously reported (Tanaka, 2002). The highly reactive hydroxyl radical oxidizes cellular lipids, proteins, and DNA, leading to cell death, and is therefore highly detrimental (Gilgun-Sherki

promotive effect of NK-4 on NGF (5 ng/ml)-primed neurite-outgrowth in PC12 cells. Neurite-

potential use of NK-4 for neurodegenerative disease therapy.

**ABC**

**0**

**100**

**200**

**Cell Growth (%)**

Data are means ± SD (n=3). \*P<0.05 and \*\* P<0.01 vs. control (no NK-4). Fig. 1. Chemical structure and neurotrophin-like effects of NK-4.

**300**

**400**

N

N+

C2H5 I -

4,4'-[3-{2-(1-ethyl-4-(1-H)quinolidene)ethylidene}] propylene{bis(1-ethyl-quinolinium iodide)}

**2.** *In Vitro* **Properties of NK-4 2.1 Free radical-scavenging activity** 

<sup>N</sup> H5C2 <sup>+</sup> C2H5

I -

Table 1.


et al., 2002). Accordingly, the antioxidants that effectively scavenge hydroxyl radicals and other free radicals should be able to eliminate oxidative injury (Barinaga, 1996), and NK-4 could be one of such molecules.

Free radical-scavenging efficacy was evaluated in a cell-free *in vitro* system using ESR. Superoxides, hydroxyl radicals, and peroxy radicals were produced and stabilized in DMPO-hypoxanthine/XOD, DMPO-DTPA/H2O2/FeSO4 and DMPO-AAPH systems, respectively. The signal intensity of each radical was recorded in the presence or absence of test samples. The scavenging activity of the samples was expressed as IC50 against each radical. Means ± SD (n=3). Reproduced in part with permission from Biological & Pharmaceutical Bulletin Vol.33 No.11. Copyright [2010] Pharmaceutical Society of Japan.

Table 1. Free radical-scavenging effects of NK-4 and other antioxidants.

#### **2.2 Neuroprotective effects against various cytotoxic stresses**

Neurotrophins, such as NGF or brain-derived neurotrophic factor (BDNF) regulate the growth, survival, and differentiation of central neurons (Blum & Konnerth, 2005). For example, NGF protects PC12 cells against 6-hydroxydopamine (6-OHDA)- and hydrogen peroxide (H2O2)-induced oxidative stress (Salinas et al., 2003; Wang et al., 2001). Since NK-4 displayed a remarkable neurotrophin-like activity, namely the promotion of cell growth and NGF-primed neurite-outgrowth in PC12 cells (Fig. 1B, 1C), we next examined whether NK-4 protects neuronal cells from oxidative or starvation stress (Ohta et al., 2011). An acute oxidative stress challenge was induced by 2-hr treatment with H2O2 or 24 hr treatment with 6-OHDA. These treatments cause a significant decrease in metabolic capacity, which reflects a decline in cell viability, and this is accompanied by an enhancement of apoptotic markers (Franco et al., 2010). Particularly, 6-OHDA has been widely used in experimental models of Parkinsion's disease, and its neurotoxicity involves oxidative damage to catecholaminergic neurons via the generation of hydroxyl radicals, monoamine oxidase-mediated formation of H2O2, and mitochondrial generation of superoxide (Blum et al., 2001).

Treatment of PC12 cells with H2O2 decreased cell viability to approximately 50% of control. In contrast, NK-4 at concentrations of 6 µM or above significantly protected the cells from the acute oxidative damage (Fig. 2A). NK-4 also attenuated 6-OHDA-induced oxidative stress in PC12 cells at nanomolar concentrations in a dose-dependent manner (Fig. 2B). Next we tested the effect of NK-4 on trophic factor depletion-induced cell death. Extended serum depletion in PC12 cells triggers ATP shortage, which results in mitochondrial reactive oxygen species (ROS) generation and activation of apoptotic pathways (Troy et al., 2001). NK-4 at submicromolar concentrations significantly increased cellular viability of PC12 cells over 3 days of serum starvation (Fig. 2C). These results suggest that the antioxidative effects of NK-4, together with its neurotrophic properties, engender strong survival signals in PC12 cells.

Effects of NK-4, a Cyanine Dye With Antioxidant Activities... 373

**Cont**

**Aβ only**

**0 50**

> **0 300 0 300 NK-4 conc. (nM)**

**50 μM Aβ25-35**

\*\*

**100 150**

**ROS (% of control)**

**200 250**

**Aβ+NK-4(253nM)**

(A) Aβ25-35 was dissolved in saline and incubated at 37 °C for 4 days, which is termed an "aging" process. PC12 cells were treated with 50 µM aged Aβ25-35 for 72-hr in the absence or presence of the indicated concentrations of NK-4. Cell viability was assessed by AlamarBlue® assay. Results are shown as means ± SD (n=3). \*\*P<0.01 vs. no NK-4. (B) PC12 cells were treated as (A) and stained with Hoechst 33342 dye. (C) PC12 cells were treated as (A), but for 48 hr and the amount of intracellularly produced ROS was assessed by H2DCF-DA dye fluorescence (ex.492nm/em.527nm). Results are shown as means

Fig. 3. Cytoprotective effects of NK-4 on Aβ25-35-induced cytotoxicity in PC12 cells.

also found in the neurite-outgrowth of PC12 cells (data not shown).

induced neurotrophic effects in PC12 cells.

Neurotrophins act by binding to two kinds of plasma membrane receptors, the Trk receptor tyrosine kinases (Trks) and the p75 pan-neurotrophin receptor (p75NTR). There are several subtypes of Trk receptor kinases characterized by their specific affinities for different neurotrophins. NGF binds preferentially to TrkA, whereas BDNF and neutrophin-4/5 show a high affinity for TrkB (Berg et al., 1992). To address whether the effects of NK-4 are mediated by Trk activity, K252a, a non-specific inhibitor of Trks, was applied in growth assays of PC12 cells. Pretreatment with K252a dose-dependently inhibited NGF-induced cell growth, but did not inhibit growth induced by NK-4 (Fig. 4A). This suggests that NK-4 acts independently of TrkA activation in PC12 cells. A similar inhibitory profile of K252a was

We next examined whether NK-4 activates phosphatidylinositol 3-kinase (PI3K) and its downstream signaling effector Akt. This cascade is implicated in survival signaling mediated by NGF in serum-deprived PC12 cells, and in neuritogenesis in PC12 cells (Martin et al., 2004; Kim et al., 2004). LY294002, a specific PI3K inhibitor (Vlahos et al., 1994), blocked the cell growth-promoting activity of both NK-4 and NGF (Fig. 4B). Also, as shown in figure 4C, NK-4 strongly induced phosphorylation of Akt at Ser 473 in a time-dependent manner. The dose of NK-4 required for the induction of Akt phosphorylation was consistent with that required for promotion of PC12 cell growth. These results suggest that sequential activation of PI3K and its downstream signaling effector Akt are important for NK-4-

**A BC**

\*\*

**0**

**Cont 0 16 63 253 NK-4 conc. (nM)**

± SD (n=3). \*\*P<0.01 vs. no NK-4.

**2.4 Intracellular signaling** 

**50 μM Aβ25-35**

\*\*

**50**

**100**

**Viability (%)**

**150**

**200**

PC12 cells were seeded at a density of 2 х 104 cells/well and cultured in serum supplemented D-MEM for 24-hr at 37ºC in collagen-coated microplates. (A) H2O2 (200 µM) or (B) 6-OHDA (100 µM) was applied in the presence or absence of indicated concentrations of NK-4 and incubated for 2-hr or 24-hr, respectively. (C) After the pre-culture, serum-containing D-MEM was replaced with serum-free medium and the cells were cultured for additional 3 days. Cell viability was assessed by the AlamarBlue® method. Results are shown as means ± SD (n=3). \*P<0.05 and \*\*P<0.01 vs. no NK-4.

Fig. 2. Protective effect of NK-4 from cytotoxic stresses in PC12 cells.

#### **2.3 Neuroprotective effect against β-amyloid (Aβ) toxicity**

Next, we determined whether NK-4 was effective against β-amyloid (Aβ)-induced neurotoxicity *in vitro* using PC12 cells (Ohta et al., 2010a). PC12 cells are reported to be highly sensitive to Aβ peptide or the aggregated Aβ25-35 fragment (Shearman et al., 1994). The viability of PC12 cells treated with 50 µM of aged Aβ25-35 for 72-hr was about 25% compared with controls without Aβ25-35 (Fig. 3A). NK-4 dose-dependently attenuated the cytotoxic effect of Aβ25-35 and the effects were significant at doses over 60 nM. Aβ peptides induce morphological changes associated with apoptotic cell death, such as somal shrinkage, plasma membrane blebbing, chromatin condensation, and nuclear fragmentation (Ivins et al., 1999). Nuclear staining by Hoechst 333442 dye demonstrated the typical nuclear fragmentation in Aβ25-35–treated PC12 cells and this was clearly inhibited by the addition of NK-4 (Fig. 3B).

Since PC12 cells undergoing Aβ-mediated apoptosis produce large amounts of ROS due to deficits in mitochondrial function (Kadowaki et al., 2005), we next examined ROS generation in Aβ25-35–treated PC12 cells (Fig. 3C). Compared with the control, the amounts of intracellular ROS significantly increased after addition of 50 µM Aβ25-35 in 48-hr cultures, and 300 nM of NK-4 almost totally suppressed the ROS induction. Under normal culture conditions, NK-4 also decreased basal ROS levels in normal PC12 cells; however, the effect was not statistically significant. These data suggest that NK-4 attenuates Aβ-mediated cytotoxicity by reducing the unfavorable ROS accumulation in PC12 cells, probably through the restoration of mitochondrial function.

**A BC H2O2 6-OHDA serum starvation**

**0 9.8 352 12,670 NK-4 conc. (nM)**

PC12 cells were seeded at a density of 2 х 104 cells/well and cultured in serum supplemented D-MEM for 24-hr at 37ºC in collagen-coated microplates. (A) H2O2 (200 µM) or (B) 6-OHDA (100 µM) was applied in the presence or absence of indicated concentrations of NK-4 and incubated for 2-hr or 24-hr, respectively. (C) After the pre-culture, serum-containing D-MEM was replaced with serum-free medium and the cells were cultured for additional 3 days. Cell viability was assessed by the AlamarBlue® method. Results are shown as means ± SD (n=3). \*P<0.05 and \*\*P<0.01 vs. no NK-4.

Next, we determined whether NK-4 was effective against β-amyloid (Aβ)-induced neurotoxicity *in vitro* using PC12 cells (Ohta et al., 2010a). PC12 cells are reported to be highly sensitive to Aβ peptide or the aggregated Aβ25-35 fragment (Shearman et al., 1994). The viability of PC12 cells treated with 50 µM of aged Aβ25-35 for 72-hr was about 25% compared with controls without Aβ25-35 (Fig. 3A). NK-4 dose-dependently attenuated the cytotoxic effect of Aβ25-35 and the effects were significant at doses over 60 nM. Aβ peptides induce morphological changes associated with apoptotic cell death, such as somal shrinkage, plasma membrane blebbing, chromatin condensation, and nuclear fragmentation (Ivins et al., 1999). Nuclear staining by Hoechst 333442 dye demonstrated the typical nuclear fragmentation in Aβ25-35–treated PC12 cells and this was clearly inhibited by the addition of

Since PC12 cells undergoing Aβ-mediated apoptosis produce large amounts of ROS due to deficits in mitochondrial function (Kadowaki et al., 2005), we next examined ROS generation in Aβ25-35–treated PC12 cells (Fig. 3C). Compared with the control, the amounts of intracellular ROS significantly increased after addition of 50 µM Aβ25-35 in 48-hr cultures, and 300 nM of NK-4 almost totally suppressed the ROS induction. Under normal culture conditions, NK-4 also decreased basal ROS levels in normal PC12 cells; however, the effect was not statistically significant. These data suggest that NK-4 attenuates Aβ-mediated cytotoxicity by reducing the unfavorable ROS accumulation in PC12 cells, probably through

\*

\*\*

**Viability (%)**

**0 63 633 6,330 NK-4 conc. (nM)**

\*\* \*\*

**40**

**60**

**80**

**Viability (%)**

Fig. 2. Protective effect of NK-4 from cytotoxic stresses in PC12 cells.

**2.3 Neuroprotective effect against β-amyloid (Aβ) toxicity** 

\*\*

NK-4 (Fig. 3B).

the restoration of mitochondrial function.

**Viability (%)**

**0 0.63 6.3 63 NK-4 conc. (μM)**

\*\*

**100**

(A) Aβ25-35 was dissolved in saline and incubated at 37 °C for 4 days, which is termed an "aging" process. PC12 cells were treated with 50 µM aged Aβ25-35 for 72-hr in the absence or presence of the indicated concentrations of NK-4. Cell viability was assessed by AlamarBlue® assay. Results are shown as means ± SD (n=3). \*\*P<0.01 vs. no NK-4. (B) PC12 cells were treated as (A) and stained with Hoechst 33342 dye. (C) PC12 cells were treated as (A), but for 48 hr and the amount of intracellularly produced ROS was assessed by H2DCF-DA dye fluorescence (ex.492nm/em.527nm). Results are shown as means ± SD (n=3). \*\*P<0.01 vs. no NK-4.

Fig. 3. Cytoprotective effects of NK-4 on Aβ25-35-induced cytotoxicity in PC12 cells.

#### **2.4 Intracellular signaling**

Neurotrophins act by binding to two kinds of plasma membrane receptors, the Trk receptor tyrosine kinases (Trks) and the p75 pan-neurotrophin receptor (p75NTR). There are several subtypes of Trk receptor kinases characterized by their specific affinities for different neurotrophins. NGF binds preferentially to TrkA, whereas BDNF and neutrophin-4/5 show a high affinity for TrkB (Berg et al., 1992). To address whether the effects of NK-4 are mediated by Trk activity, K252a, a non-specific inhibitor of Trks, was applied in growth assays of PC12 cells. Pretreatment with K252a dose-dependently inhibited NGF-induced cell growth, but did not inhibit growth induced by NK-4 (Fig. 4A). This suggests that NK-4 acts independently of TrkA activation in PC12 cells. A similar inhibitory profile of K252a was also found in the neurite-outgrowth of PC12 cells (data not shown).

We next examined whether NK-4 activates phosphatidylinositol 3-kinase (PI3K) and its downstream signaling effector Akt. This cascade is implicated in survival signaling mediated by NGF in serum-deprived PC12 cells, and in neuritogenesis in PC12 cells (Martin et al., 2004; Kim et al., 2004). LY294002, a specific PI3K inhibitor (Vlahos et al., 1994), blocked the cell growth-promoting activity of both NK-4 and NGF (Fig. 4B). Also, as shown in figure 4C, NK-4 strongly induced phosphorylation of Akt at Ser 473 in a time-dependent manner. The dose of NK-4 required for the induction of Akt phosphorylation was consistent with that required for promotion of PC12 cell growth. These results suggest that sequential activation of PI3K and its downstream signaling effector Akt are important for NK-4 induced neurotrophic effects in PC12 cells.

Effects of NK-4, a Cyanine Dye With Antioxidant Activities... 375

In addition to Aβ deposition, intraneuronal tau aggregates in the brain is a hallmark of AD. Since the mutated tau transgenic mouse P301L exhibited a clear correlation between tau aggregates and neurodegeneration (Lewis et al., 2000), small molecule inhibition of tau aggregation is considered a potential therapeutic strategy, alongside Aβ anti-aggregative strategy (Pickhardt et al., 2007). Self-aggregation of tau protein occurs in the presence of polyanions such as heparin or poly(Glu), and can be quantitatively assessed by thioflavin S (ThS) fluorescence (Friedhoff et al., 1998). Therefore, we evaluated the inhibitory effect of NK-4 on tau aggregation using recombinant human tau-352 protein (0N3R variant, 15 µM) and heparin (2.5 µM) (Fig. 5C). As a result, NK-4 reduced ThS fluorescence in a dosedependent manner and 10 µM of NK-4 substantially inhibited ThS fluorescence of tau aggregation. This result, together with the inhibition of Aβ aggregation, strongly suggests that NK-4 is an effective inhibitor of protein aggregation *in vitro*. Probably, NK-4 associates at the interface of the β-sheet domains of proteins by its planar structure and would thus act

**Aβ<sup>42</sup>**

(A) Effects of NK-4 on Aβ fibril formation. Solutions of Aβ1-40 and Aβ1-42 (100 µM each) were incubated at 37°C for 3 days in the absence or presence of NK-4 at the indicated concentrations. Thioflavin T fluorescence was measured. Values are means ± SD (n=3). \*\*P<0.01 vs. control. (B) Electron microscopy of Aβ1-42 fibrils. The solution of 100 µM Aβ1-42 was incubated for 3 days alone (upper) or with 10 µM of NK-4 (lower). Incubated Aβ was adsorbed on copper grid and negatively stained by uranyl acetate, then observed with EM at 80 keV. (C) Effects of NK-4 on tau-352 aggregation. Solutions of tau-352 (15 µM) were incubated with 2.5 µM of heparin at 37°C in the absence or presence of NK-4 (2.5 or 10 µM)

As mentioned above, NK-4 showed remarkable neurotrophic and neuroprotective activities *in vitro*, and it might have potential to modulate Aβ and tau pathologies, which is implicated in AD. In relation to a possible application of NK-4 for AD, we examined whether NK-4 has an inhibitory effect on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) since the progressive deterioration of cholinergic innervation in the cerebral cortex leads to cognitive deficits in AD, and AChE inhibitors are still the primary choice for treatment of

**0**

**0 50 100 150 200 Incubation (hr)**

**tau-352 (0N3R variant)**

**vehicle NK-4 (2.5μM) NK-4 (10μM)**

**25**

**50**

**% ThS Fluorecsence**

**75**

**100**

as a "β-sheet breaker" (Suh & Checler, 2002).

**0**

AD (Ellis, 2005).

**% ThT Fluorescence**

**A BC**

**Aβ<sup>42</sup> + NK-4 <sup>0</sup>**

Fig. 5. NK-4 inhibits Aβ fibril formation as well as tau aggregation.

**\*\* \*\***

**Aβ1-40 Aβ1-42**

**0.01 0.1 1 10 100 1000 NK-4 conc. (μM)**

and thioflavin S fluorescence was measured.

**2.6 Cholinesterase (ChE) inhibitory activity** 

**\*\***

**\*\***

**\*\***

(A, B) Effects of K252a or LY294002 on PC12 cell growth induced by NK-4 (bold line) or NGF (broken line). PC12 cells were preincubated in serum-free D-MEM with the indicated concentrations of K252a (A) or LY294002 (B) for 15 min. NK-4 (250 nM) or NGF (50 ng/ml) was added and the cells were further incubated for 72-hr. Cell viabilities were assessed by AlamarBlue® assay. Values are means ± SD (n=3). \*P<0.05, \*\*P<0.01 vs. control. (C) PC12 cells were treated with 250 nM NK-4 for the indicated times. Whole cell lysates were analyzed by Western blotting using an anti-phospho-Akt (Ser 473) mAb (upper) or an anti-Akt mAb (lower). (D) PC12 cells were treated with 400 µM H2O2 for 2-hr in the presence of the indicated concentrations of NK-4. Phospho-SAPK/JNK (upper) and SAPK/JNK (lower) were analyzed by Western blotting using whole cell lysates.

Fig. 4. NK-4 alters phosphorylation of Akt and SAPK/JNK in a Trk-independent manner.

On the other hand, we also found that the SAPK/JNK stress-induced signaling pathway was altered by NK-4. SAPK/JNK is a major cellular stress-responsive protein activated by oxidative stress. Activated SAPK/JNK translocates from the cytosol to the nucleus and regulates the activity of transcription factors such as c-Jun, ATF-2, p53, SMAD4, and Elk-1 (Kyriakis & Avruch, 2001). PC12 cells treated with cytotoxic levels of H2O2 displayed augmented phosphorylation of SAPK/JNK, and this was attenuated by NK-4 in a concentration-dependent manner (Fig. 4D). This observation suggests that NK-4 might attenuate H2O2-induced cellular stress upstream of SAPK/JNK.

#### **2.5 Anti-aggregative effect on Aβ and tau**

To examine the mechanisms of NK-4-mediated protection from amyloid toxicity in neuronal cells, we evaluated the effect of NK-4 on fibrillization of Aβ (Fig. 5), since oligomerized or aggregated Aβ seems more toxic to neuronal cells (Lesne et al., 2006, Shearman et al., 1994). ThioflavinT (ThT) fluorescence is enhanced upon binding to Aβ fibrils in proportion to the amount of fibrils in solution (Lashuel et al., 2002). Therefore, the effect of NK-4 on Aβ fibril formation was evaluated using ThT (Fig. 5A). In the absence of NK-4, both Aβ1-40 and Aβ1-42 solutions displayed greatly enhanced emission at 482 nm, which is characteristic for ThT bound to amyloid fibrils. Significant decreases in ThT fluorescence were detected in the presence of NK-4 at concentrations of 10 µM or higher against 100 µM of Aβ. An equimolar concentration of NK-4 almost totally inhibited the increase in ThT fluorescence. Under electron microscopic observation, Aβ1-42 formed long and dense fibrils after 3 days of incubation (Fig. 5B, upper). In contrast, Aβ1-42 incubated with 10 µM of NK-4 produced fewer and shorter filaments in soluble assemblies (Fig. 5B, lower).

(A, B) Effects of K252a or LY294002 on PC12 cell growth induced by NK-4 (bold line) or NGF (broken line). PC12 cells were preincubated in serum-free D-MEM with the indicated concentrations of K252a (A) or LY294002 (B) for 15 min. NK-4 (250 nM) or NGF (50 ng/ml) was added and the cells were further incubated for 72-hr. Cell viabilities were assessed by AlamarBlue® assay. Values are means ± SD (n=3). \*P<0.05, \*\*P<0.01 vs. control. (C) PC12 cells were treated with 250 nM NK-4 for the indicated times. Whole cell lysates were analyzed by Western blotting using an anti-phospho-Akt (Ser 473) mAb (upper) or an anti-Akt mAb (lower). (D) PC12 cells were treated with 400 µM H2O2 for 2-hr in the presence of the indicated concentrations of NK-4. Phospho-SAPK/JNK (upper) and SAPK/JNK (lower) were

Fig. 4. NK-4 alters phosphorylation of Akt and SAPK/JNK in a Trk-independent manner.

On the other hand, we also found that the SAPK/JNK stress-induced signaling pathway was altered by NK-4. SAPK/JNK is a major cellular stress-responsive protein activated by oxidative stress. Activated SAPK/JNK translocates from the cytosol to the nucleus and regulates the activity of transcription factors such as c-Jun, ATF-2, p53, SMAD4, and Elk-1 (Kyriakis & Avruch, 2001). PC12 cells treated with cytotoxic levels of H2O2 displayed augmented phosphorylation of SAPK/JNK, and this was attenuated by NK-4 in a concentration-dependent manner (Fig. 4D). This observation suggests that NK-4 might

To examine the mechanisms of NK-4-mediated protection from amyloid toxicity in neuronal cells, we evaluated the effect of NK-4 on fibrillization of Aβ (Fig. 5), since oligomerized or aggregated Aβ seems more toxic to neuronal cells (Lesne et al., 2006, Shearman et al., 1994). ThioflavinT (ThT) fluorescence is enhanced upon binding to Aβ fibrils in proportion to the amount of fibrils in solution (Lashuel et al., 2002). Therefore, the effect of NK-4 on Aβ fibril formation was evaluated using ThT (Fig. 5A). In the absence of NK-4, both Aβ1-40 and Aβ1-42 solutions displayed greatly enhanced emission at 482 nm, which is characteristic for ThT bound to amyloid fibrils. Significant decreases in ThT fluorescence were detected in the presence of NK-4 at concentrations of 10 µM or higher against 100 µM of Aβ. An equimolar concentration of NK-4 almost totally inhibited the increase in ThT fluorescence. Under electron microscopic observation, Aβ1-42 formed long and dense fibrils after 3 days of incubation (Fig. 5B, upper). In contrast, Aβ1-42 incubated with 10 µM of NK-4 produced

**D**

**AB C**

analyzed by Western blotting using whole cell lysates.

**2.5 Anti-aggregative effect on Aβ and tau** 

attenuate H2O2-induced cellular stress upstream of SAPK/JNK.

fewer and shorter filaments in soluble assemblies (Fig. 5B, lower).

In addition to Aβ deposition, intraneuronal tau aggregates in the brain is a hallmark of AD. Since the mutated tau transgenic mouse P301L exhibited a clear correlation between tau aggregates and neurodegeneration (Lewis et al., 2000), small molecule inhibition of tau aggregation is considered a potential therapeutic strategy, alongside Aβ anti-aggregative strategy (Pickhardt et al., 2007). Self-aggregation of tau protein occurs in the presence of polyanions such as heparin or poly(Glu), and can be quantitatively assessed by thioflavin S (ThS) fluorescence (Friedhoff et al., 1998). Therefore, we evaluated the inhibitory effect of NK-4 on tau aggregation using recombinant human tau-352 protein (0N3R variant, 15 µM) and heparin (2.5 µM) (Fig. 5C). As a result, NK-4 reduced ThS fluorescence in a dosedependent manner and 10 µM of NK-4 substantially inhibited ThS fluorescence of tau aggregation. This result, together with the inhibition of Aβ aggregation, strongly suggests that NK-4 is an effective inhibitor of protein aggregation *in vitro*. Probably, NK-4 associates at the interface of the β-sheet domains of proteins by its planar structure and would thus act as a "β-sheet breaker" (Suh & Checler, 2002).

(A) Effects of NK-4 on Aβ fibril formation. Solutions of Aβ1-40 and Aβ1-42 (100 µM each) were incubated at 37°C for 3 days in the absence or presence of NK-4 at the indicated concentrations. Thioflavin T fluorescence was measured. Values are means ± SD (n=3). \*\*P<0.01 vs. control. (B) Electron microscopy of Aβ1-42 fibrils. The solution of 100 µM Aβ1-42 was incubated for 3 days alone (upper) or with 10 µM of NK-4 (lower). Incubated Aβ was adsorbed on copper grid and negatively stained by uranyl acetate, then observed with EM at 80 keV. (C) Effects of NK-4 on tau-352 aggregation. Solutions of tau-352 (15 µM) were incubated with 2.5 µM of heparin at 37°C in the absence or presence of NK-4 (2.5 or 10 µM) and thioflavin S fluorescence was measured.

Fig. 5. NK-4 inhibits Aβ fibril formation as well as tau aggregation.

#### **2.6 Cholinesterase (ChE) inhibitory activity**

As mentioned above, NK-4 showed remarkable neurotrophic and neuroprotective activities *in vitro*, and it might have potential to modulate Aβ and tau pathologies, which is implicated in AD. In relation to a possible application of NK-4 for AD, we examined whether NK-4 has an inhibitory effect on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) since the progressive deterioration of cholinergic innervation in the cerebral cortex leads to cognitive deficits in AD, and AChE inhibitors are still the primary choice for treatment of AD (Ellis, 2005).

Effects of NK-4, a Cyanine Dye With Antioxidant Activities... 377

**B**

**Edema ratio (%)**

The rat right middle cerebral artery was occluded by a silicon embolizer for 2-hr and then reperfused for 24-hr. Drugs were administered intravenously twice at the indicated doses 1-hr after the occlusion and at the start of reperfusion. (A) After the 24-hr reperfusion, neurological impairment scores of rats were rated with a maximum impairment score of 6.0. (B) Representative serial brain sections (2 mm thickness) stained with 2,3,5-triphenyltetrazolium chloride (TTC) after 2-hr MCAO and 24-hr reperfusion. (C) Quantification of hemispheric lesion volumes. Infarct volumes were calculated from serial brain sections by Scion Image software. (D) Cerebral edema ratios were calculated based on the values from volumetric analysis. NK-4 groups, n=5 each; edaravone group, n=8; control group, n=8. Data are means ± S.E.M. \*P<0.05, \*\*P<0.01 vs. vehicle control group. Reproduced in part with permission from Biological & Pharmaceutical Bulletin Vol.33 No.11. Copyright [2010] Pharmaceutical

Fig. 6. Suppressive effect of NK-4 on MCAO-induced infarct formation in rats.

We demonstrated that the efficacy of NK-4 was superior to that of an existing drug (edaravone) in scavenging free radicals *in vitro* (Table 1) and in protecting MCAO model rats from brain injury *in vivo* (Fig. 6). Thus the antioxidative function of NK-4 may foster an overall protective environment against neuronal damage after focal brain ischemia. Since NK-4 elicits neurotrophic activity *in vitro* in PC12 cells aside from its antioxidant function, it may also enhance endogenous neurotrophic signals *in vivo* and thereby play an important

In the ischemic stroke model, intravenously administered NK-4 showed a significant neuroprotective effect (Fig. 6). This model reflects an acute oxidative insult at MCA-affected areas in the brain. We next tested the effects of NK-4 on progressive chronic neurodegeneration using a genetic ataxia model in the Syrian hamster characterized by Purkinje cell degeneration (hmPCD model). This animal model was established in HAYASHIBARA Co., Ltd. and is thought to be homologous to the well-characterized *pcd* mutant mice. Both animal models display a suppressed expression of the brain *nna1* gene,

**Infarct volume (mm3**

Society of Japan.

**)**

**0.0 1.0 2.0 3.0 4.0 5.0 6.0**

**Neurological impairment score**

**A**

**1 mg/kg 100 μg/kg 50 μg/kg 25 μg/kg**

role in the protection of neuronal cells from ischemic damage.

**3.2 Ataxia model (PCD hamsters)** 

**\*\* \*\***

**1 mg/kg 100 μg/kg 50 μg/kg 25 μg/kg**

**C D**

**\*\* \* \***

**Control Edv NK-4**

**Control Edv NK-4**

**Control 100 µg/kg 50 µg/kg 25 µg/kg**

**1 mg/kg Edv**

**\*\***

**NK-4**

**\***

**1 mg/kg 100 μg/kg 50 μg/kg 25 μg/kg**

**\*\* \*\***

**Control Edv NK-4**

A comparison of IC50 values between NK-4 and other ChE inhibitors suggests that NK-4 is a potent and selective inhibitor of AChE (Table 2). The inhibitory effect of NK-4 against AChE was weaker than that of donepezil, the most popular drug worldwide for the treatment of dementia, but almost comparable to tacrine, and stronger than galantamine (Ohta et al., 2010a). These results suggest that NK-4 might have potential for the amelioration of disturbed cholinergic transmission in AD patients.


Inhibitory activities toward AChE and BChE by NK-4 and other ChE inhibitors are shown. IC50 values of NK-4 for AChE and BChE were determined based on the Ellman method using purified enzymes of human origin. Other data are cited from a review article (Sugimoto, 2004). Values are means ± SD.

Table 2. ChE inhibitory activities and specificity of NK-4 and other ChE inhibitors.

### **3. Anti-neurodegenerative effects of NK-4**

NK-4 exhibits a neurotrophin-like activity and a potent free radical-scavenging capacity *in vitro*. Moreover, it confers significant stress tolerance to neuronal cells via activation of survival signaling pathways. Therefore, we examined whether NK-4 produces effects in animal models of neurodegenerative disorders, including models of ischemic stroke, cerebellar ataxia, and AD.

#### **3.1 Stroke model (MCAO Rats)**

Stroke is the second most common cause of death and a major cause of long-lasting disability worldwide. In relation to the massive socio-economic impact of ischemic stroke, neuroprotective agents with different modes of action and/or extended application time windows are therefore urgently needed. We evaluated the effect of NK-4 on ischemic stroke using a focal and transient ischemia model in rats*.* The middle cerebral artery occlusion (MCAO) method in rats is an animal model of focal brain ischemia (Bederson et al., 1986) frequently used to evaluate drug efficacy.

In the brains of MCAO rats, infarct volume increased mainly in the ipsilateral cerebral cortex and stratum (sites affected by MCAO; Fig. 6B). Administration of NK-4 substantially decreased infarct size (Fig. 6C), especially in the cortex penumbra region, a zone of incomplete cerebral ischemia. Edaravone also reduced infarct volume by approximately 25% compared to the vehicle control, but the difference was not statistically significant (Fig. 6C). Regarding the edema ratio, both NK-4 and edaravone significantly attenuated brain swelling compared to the vehicle control (Fig. 6D). Although edaravone (1,000 µg/kg) and a lower dose of NK-4 (25 µg/kg) failed to significantly reduce infarct size, they still significantly decreased brain edema.

A comparison of IC50 values between NK-4 and other ChE inhibitors suggests that NK-4 is a potent and selective inhibitor of AChE (Table 2). The inhibitory effect of NK-4 against AChE was weaker than that of donepezil, the most popular drug worldwide for the treatment of dementia, but almost comparable to tacrine, and stronger than galantamine (Ohta et al., 2010a). These results suggest that NK-4 might have potential for the amelioration of

NK-4 88 ± 2.5 > 6,333 > 70 Donepezil 6.7 ± 0.35 7,400 ± 130 1100 Tacrine 77 ± 1.4 69 ± 1.4 0.90 Galantamine 1,200 ± 33 18,000 ± 333 15 Inhibitory activities toward AChE and BChE by NK-4 and other ChE inhibitors are shown. IC50 values of NK-4 for AChE and BChE were determined based on the Ellman method using purified enzymes of human origin. Other data are cited from a review article (Sugimoto, 2004). Values are means ± SD. Table 2. ChE inhibitory activities and specificity of NK-4 and other ChE inhibitors.

NK-4 exhibits a neurotrophin-like activity and a potent free radical-scavenging capacity *in vitro*. Moreover, it confers significant stress tolerance to neuronal cells via activation of survival signaling pathways. Therefore, we examined whether NK-4 produces effects in animal models of neurodegenerative disorders, including models of ischemic stroke,

Stroke is the second most common cause of death and a major cause of long-lasting disability worldwide. In relation to the massive socio-economic impact of ischemic stroke, neuroprotective agents with different modes of action and/or extended application time windows are therefore urgently needed. We evaluated the effect of NK-4 on ischemic stroke using a focal and transient ischemia model in rats*.* The middle cerebral artery occlusion (MCAO) method in rats is an animal model of focal brain ischemia (Bederson et al., 1986)

In the brains of MCAO rats, infarct volume increased mainly in the ipsilateral cerebral cortex and stratum (sites affected by MCAO; Fig. 6B). Administration of NK-4 substantially decreased infarct size (Fig. 6C), especially in the cortex penumbra region, a zone of incomplete cerebral ischemia. Edaravone also reduced infarct volume by approximately 25% compared to the vehicle control, but the difference was not statistically significant (Fig. 6C). Regarding the edema ratio, both NK-4 and edaravone significantly attenuated brain swelling compared to the vehicle control (Fig. 6D). Although edaravone (1,000 µg/kg) and a lower dose of NK-4 (25 µg/kg) failed to significantly reduce infarct size, they still

AChE BChE Specificity (B/A)

disturbed cholinergic transmission in AD patients.

**3. Anti-neurodegenerative effects of NK-4** 

cerebellar ataxia, and AD.

**3.1 Stroke model (MCAO Rats)** 

frequently used to evaluate drug efficacy.

significantly decreased brain edema.

Compound IC50 (nM)

The rat right middle cerebral artery was occluded by a silicon embolizer for 2-hr and then reperfused for 24-hr. Drugs were administered intravenously twice at the indicated doses 1-hr after the occlusion and at the start of reperfusion. (A) After the 24-hr reperfusion, neurological impairment scores of rats were rated with a maximum impairment score of 6.0. (B) Representative serial brain sections (2 mm thickness) stained with 2,3,5-triphenyltetrazolium chloride (TTC) after 2-hr MCAO and 24-hr reperfusion. (C) Quantification of hemispheric lesion volumes. Infarct volumes were calculated from serial brain sections by Scion Image software. (D) Cerebral edema ratios were calculated based on the values from volumetric analysis. NK-4 groups, n=5 each; edaravone group, n=8; control group, n=8. Data are means ± S.E.M. \*P<0.05, \*\*P<0.01 vs. vehicle control group. Reproduced in part with permission from Biological & Pharmaceutical Bulletin Vol.33 No.11. Copyright [2010] Pharmaceutical Society of Japan.

Fig. 6. Suppressive effect of NK-4 on MCAO-induced infarct formation in rats.

We demonstrated that the efficacy of NK-4 was superior to that of an existing drug (edaravone) in scavenging free radicals *in vitro* (Table 1) and in protecting MCAO model rats from brain injury *in vivo* (Fig. 6). Thus the antioxidative function of NK-4 may foster an overall protective environment against neuronal damage after focal brain ischemia. Since NK-4 elicits neurotrophic activity *in vitro* in PC12 cells aside from its antioxidant function, it may also enhance endogenous neurotrophic signals *in vivo* and thereby play an important role in the protection of neuronal cells from ischemic damage.

#### **3.2 Ataxia model (PCD hamsters)**

In the ischemic stroke model, intravenously administered NK-4 showed a significant neuroprotective effect (Fig. 6). This model reflects an acute oxidative insult at MCA-affected areas in the brain. We next tested the effects of NK-4 on progressive chronic neurodegeneration using a genetic ataxia model in the Syrian hamster characterized by Purkinje cell degeneration (hmPCD model). This animal model was established in HAYASHIBARA Co., Ltd. and is thought to be homologous to the well-characterized *pcd* mutant mice. Both animal models display a suppressed expression of the brain *nna1* gene,

Effects of NK-4, a Cyanine Dye With Antioxidant Activities... 379

from around 7 weeks of age and the frequency of falling increased with age. Animals treated with the low and high doses of NK-4 showed a significant reduction in falling frequency compared to saline-treated controls (Fig. 7B). These observations demonstrate that NK-4 is effective in treating motor discoordination associated with cerebellar ataxia in

The mutants also showed severe cerebellar atrophy and volumetric reduction at 10 weeks of age (Fig. 7C). Animals treated with low or high doses of NK-4 had a significantly larger cerebellum volume compared with saline-treated controls. H&E staining and calbindin immunohistochemistry of cerebellar cortical sections revealed a large reduction in the number of cerebellar PCs in the brain of hmPCDs at 10 weeks of age (Fig. 7D). The PC dendrites in high dose NK-4-treated hmPCDs were significantly longer and thicker than those in surviving PCs in saline-treated hmPCDs (data not shown). In the brain of hmPCDs, the cerebellar GC density was moderately reduced compared to wild type controls. NK-4 dose-dependently attenuated cellular atrophy and prevented the reduction in GCs (Fig. 7E). In this model, daily intraperitoneal injection of NK-4 at a dose of 20 or 100 µg/kg for 8 consecutive weeks was effective in attenuating motor discoordination and degenerative loss of both PCs and GCs with no detectable adverse events. This suggests that NK-4 can attenuate neurodegeneration in the central nervous system via a peripheral route of administration. PCs are susceptible to ischemic damage because of their reduced capacity to isolate glutamate and reduced ability to generate energy during anoxia (Welsh et al., 2002). GCs are also vulnerable to a variety of toxins that decrease glutathione levels and this makes the cells more vulnerable to cellular damage from ROS (Fonnum & Lock, 2004). Direct scavenging of free radicals by NK-4 may protect these cells. In addition, NK-4 appears to

activate survival-signaling pathways in degenerating cerebellar neurons.

AD, the most common form of dementia, is characterized clinically by ongoing declines in cognitive and functional ability and emergence of behavioral and psychological symptoms. More than 35 million people living with dementia worldwide in 2010, increasing to 65.7 million by 2030 and 115.4 million by 2050 (Wimo & Prince, 2010). However, no effective disease-modifying therapy is available. A report from Alzheimer's association predicts the total costs of care for AD patients will increase five-fold per year, and the treatments which delay the onset of Alzheimer's disease or slow the progression of this condition dramatically reduce the costs of Medicare or Medicaid (Alzheimer's association, 2010). Presently, the only approved therapies for AD are ChE inhibitors and an N-methyl-D-aspartate (NMDA) receptor antagonist, and the beneficial effects of these symptomatic treatments appear limited and are not long lasting (Lanctôt et al., 2009). Thus, more evidence-based effective therapies, whether

they are symptomatic treatment or disease-modifying strategies, are urgently needed.

NK-4 shows potent neuroprotective and neurotrophin-like activities *in vitro* (Fig. 1~3) and also *in vivo* (Fig. 6, 7). Furthermore, it remarkably and selectively inhibits AChE *in vitro* (Table 2). These multiple properties of NK-4 raised the possibility that it could halt or slow the progression of AD. To date, several mouse models for AD based on the amyloid hypothesis or the tau hypothesis have been developed (Bloom et al., 2005). Therefore, we tested the potential effects of NK-4 on AD using *in vivo* studies incorporating the amyloid

**3.3 Alzheimer's Disease (AD) mouse models** 

the hmPCD model.

which encodes a putative zinc carboxypeptidase originally identified by its induction in spinal motor neurons during axonal regeneration (Akita & Arai, 2009; Mullen et al., 1997; Fernandez-Gonzalez et al., 2002; Harris et al., 2000). Homozygous mutants of hmPCD show a moderate ataxia beginning at 7 weeks of age and exhibit an adult-onset degenerative loss of cerebellar Purkinje cells (PCs) followed by a slow, mild reduction in granule cell (GC) density (Akita et al., 2007).

To examine the effect of NK-4 on cerebellar ataxia in the hmPCD model, animals were administered 20 or 100 µg/kg/day of NK-4 (intraperitoneally) for 8 weeks, starting at 3 weeks of age. Motor coordination in ataxic and non-ataxic animals was evaluated weekly with a rota-rod test. As shown in figure 7A, a low dose of NK-4 (20 µg/kg) elicited a moderate, but significant, effect in attenuating the deterioration of motor function. A high dose of NK-4 (100 µg/kg) produced a considerable improvement in their rota-rod performance that lasted for the entire test period. NK-4 could not halt or reverse the disease symptoms in hmPCD, however; it profoundly delayed the progress of disease. At the end of the study (10 weeks of age), the motor ability of NK-4-treated animals was evaluated by counting the frequency of falling. The hmPCD animals began to fall frequently in their cage

(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 at a constant speed (6 rpm), and the time spent on the rod was recorded. (B) Effect of NK-4 on frequency of falling in hmPCDs (10 weeks of age). Spontaneous falling of each animal was counted for 60 s. (C) Effect of NK-4 on cerebellar size in hmPCDs. The volume of the hamster cerebella at 10 weeks of age was calculated as an approximate oval sphere. (D) Effect of NK-4 on the number of PCs in hmPCDs. PCs were counted in the mid-sagittal section of the cerebellum from hmPCDs. (E) GC density in the cerebellum from hmPCDs and wild type controls. H&E stained sections of cerebellum cortex from hmPCDs and wild type controls were counted for GCs in an area of 20,000 µm2. Graphs show the mean ± S.E.M. of 6 hamsters at 10 weeks of age. \*P<0.05, \*\*P<0.01 vs. hmPCD control. Adapted from Ohta H *et al*, *PLoS ONE* 6(2):e17137 (Ohta et al., 2011).

Fig. 7. Effects of NK-4 on motor coordination and cerebellar degeneration in hmPCDs.

which encodes a putative zinc carboxypeptidase originally identified by its induction in spinal motor neurons during axonal regeneration (Akita & Arai, 2009; Mullen et al., 1997; Fernandez-Gonzalez et al., 2002; Harris et al., 2000). Homozygous mutants of hmPCD show a moderate ataxia beginning at 7 weeks of age and exhibit an adult-onset degenerative loss of cerebellar Purkinje cells (PCs) followed by a slow, mild reduction in granule cell (GC)

To examine the effect of NK-4 on cerebellar ataxia in the hmPCD model, animals were administered 20 or 100 µg/kg/day of NK-4 (intraperitoneally) for 8 weeks, starting at 3 weeks of age. Motor coordination in ataxic and non-ataxic animals was evaluated weekly with a rota-rod test. As shown in figure 7A, a low dose of NK-4 (20 µg/kg) elicited a moderate, but significant, effect in attenuating the deterioration of motor function. A high dose of NK-4 (100 µg/kg) produced a considerable improvement in their rota-rod performance that lasted for the entire test period. NK-4 could not halt or reverse the disease symptoms in hmPCD, however; it profoundly delayed the progress of disease. At the end of the study (10 weeks of age), the motor ability of NK-4-treated animals was evaluated by counting the frequency of falling. The hmPCD animals began to fall frequently in their cage

**B**

**GC Density (/20,000 μm2**

**)**

**Frequency of falling (/min)**

**Normal**

**PCD adult**

**Saline**

**20**

**\*\* \*\***

**100**

**NK-4 (μg/ml)**

**NK-4 (µg/kg)**

**Wild Saline 20 100**

*hm* **PCD**

**NK-4 (μg/kg)**

density (Akita et al., 2007).

**Cerebellar Vol. (mm3**

**)**

**Time to fall (sec)**

**A**

**Wild Saline 20 100**

*hm* **PCD**

3 4 5 6 7 8 9 10 11 **Weeks of Age**

**Normal Saline NK-4 (100μg/kg) NK-4 (20μg/kg)**

**CD E**

**PCs (cells/section)**

control. Adapted from Ohta H *et al*, *PLoS ONE* 6(2):e17137 (Ohta et al., 2011).

**Wild Saline 20 100**

(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 at a constant speed (6 rpm), and the time spent on the rod was recorded. (B) Effect of NK-4 on frequency of falling in hmPCDs (10 weeks of age). Spontaneous falling of each animal was counted for 60 s. (C) Effect of NK-4 on cerebellar size in hmPCDs. The volume of the hamster cerebella at 10 weeks of age was calculated as an approximate oval sphere. (D) Effect of NK-4 on the number of PCs in hmPCDs. PCs were counted in the mid-sagittal section of the cerebellum from hmPCDs. (E) GC density in the cerebellum from hmPCDs and wild type controls. H&E stained sections of cerebellum cortex from hmPCDs and wild type controls were counted for GCs in an area of 20,000 µm2. Graphs show the mean ± S.E.M. of 6 hamsters at 10 weeks of age. \*P<0.05, \*\*P<0.01 vs. hmPCD

Fig. 7. Effects of NK-4 on motor coordination and cerebellar degeneration in hmPCDs.

*hm* **PCD**

**NK-4 (μg/kg)**

**NK-4 (μg/kg)**

from around 7 weeks of age and the frequency of falling increased with age. Animals treated with the low and high doses of NK-4 showed a significant reduction in falling frequency compared to saline-treated controls (Fig. 7B). These observations demonstrate that NK-4 is effective in treating motor discoordination associated with cerebellar ataxia in the hmPCD model.

The mutants also showed severe cerebellar atrophy and volumetric reduction at 10 weeks of age (Fig. 7C). Animals treated with low or high doses of NK-4 had a significantly larger cerebellum volume compared with saline-treated controls. H&E staining and calbindin immunohistochemistry of cerebellar cortical sections revealed a large reduction in the number of cerebellar PCs in the brain of hmPCDs at 10 weeks of age (Fig. 7D). The PC dendrites in high dose NK-4-treated hmPCDs were significantly longer and thicker than those in surviving PCs in saline-treated hmPCDs (data not shown). In the brain of hmPCDs, the cerebellar GC density was moderately reduced compared to wild type controls. NK-4 dose-dependently attenuated cellular atrophy and prevented the reduction in GCs (Fig. 7E).

In this model, daily intraperitoneal injection of NK-4 at a dose of 20 or 100 µg/kg for 8 consecutive weeks was effective in attenuating motor discoordination and degenerative loss of both PCs and GCs with no detectable adverse events. This suggests that NK-4 can attenuate neurodegeneration in the central nervous system via a peripheral route of administration. PCs are susceptible to ischemic damage because of their reduced capacity to isolate glutamate and reduced ability to generate energy during anoxia (Welsh et al., 2002). GCs are also vulnerable to a variety of toxins that decrease glutathione levels and this makes the cells more vulnerable to cellular damage from ROS (Fonnum & Lock, 2004). Direct scavenging of free radicals by NK-4 may protect these cells. In addition, NK-4 appears to activate survival-signaling pathways in degenerating cerebellar neurons.

#### **3.3 Alzheimer's Disease (AD) mouse models**

AD, the most common form of dementia, is characterized clinically by ongoing declines in cognitive and functional ability and emergence of behavioral and psychological symptoms. More than 35 million people living with dementia worldwide in 2010, increasing to 65.7 million by 2030 and 115.4 million by 2050 (Wimo & Prince, 2010). However, no effective disease-modifying therapy is available. A report from Alzheimer's association predicts the total costs of care for AD patients will increase five-fold per year, and the treatments which delay the onset of Alzheimer's disease or slow the progression of this condition dramatically reduce the costs of Medicare or Medicaid (Alzheimer's association, 2010). Presently, the only approved therapies for AD are ChE inhibitors and an N-methyl-D-aspartate (NMDA) receptor antagonist, and the beneficial effects of these symptomatic treatments appear limited and are not long lasting (Lanctôt et al., 2009). Thus, more evidence-based effective therapies, whether they are symptomatic treatment or disease-modifying strategies, are urgently needed.

NK-4 shows potent neuroprotective and neurotrophin-like activities *in vitro* (Fig. 1~3) and also *in vivo* (Fig. 6, 7). Furthermore, it remarkably and selectively inhibits AChE *in vitro* (Table 2). These multiple properties of NK-4 raised the possibility that it could halt or slow the progression of AD. To date, several mouse models for AD based on the amyloid hypothesis or the tau hypothesis have been developed (Bloom et al., 2005). Therefore, we tested the potential effects of NK-4 on AD using *in vivo* studies incorporating the amyloid

Effects of NK-4, a Cyanine Dye With Antioxidant Activities... 381

was lower compared to NK-4 treatments. Brain levels of Aβ were estimated separately in detergent-insoluble and -soluble fractions. Both detergent-insoluble and soluble Aβ1-40 levels were significantly decreased by NK-4 treatment (Fig. 9B, upper graphs). Donepezil did not affect Aβ concentrations in brain. Because the levels of Aβ1-42 were very low in all groups compared to those of Aβ1-40 (Fig. 9B, lower graphs), the levels of Aβ1-40 seem to reflect the

**B**

**Exploration preference (%)**

\* \*

**6 months 9 months 12 months**

Wild Tg (saline) Tg (DPZ) Tg (NK-4:100) Tg (NK-4:500)

\*

\*\* \*\*

**12 months**

**Day1 Day2 Day3 Day4**

NK-4 was injected intraperitoneally to Tg2576 mice at a dose of 100 or 500 µg/kg once a day, five times a week for 9 months, beginning at 3 months of age (n=10 each). Donepezil (DPZ) was administered at a dose of 200 µg/kg (n=5). Control mice received 200 µl of saline (wild type, n=10; Tg2576, n=10). (A) Morris water maze test at 6 and 12 months of age. The escape latency represents the average time to find a hidden platform placed in a fixed location in a circular pool (ø130 cm). Two trials per day were conducted for 4 consecutive days with an upper cutoff time of 120 sec. (B) Novel object recognition test at 6, 9 and 12 months of age. Two objects X and Y were placed in the test box and the exploration behavior of mice was recorded. A retention session was performed 24 hr after the training session. One of the familiar objects Y was replaced by a novel object Z. Mice were allowed to explore freely for 10 min, with the time spent in exploring each of the two objects recorded. The exploratory preference was expressed as a ratio of the time spent exploring the novel object (TZ) over that spent on the two objects (TX+TZ). Data are expressed as means ± S.E.M. \*P<0.05, \*\*P<0.01 vs. saline-treated Tg2576 group. Wild: non-transgenic mice. Tg(saline), saline-treated Tg2576 mice; Tg(DPZ), donepezil-treated Tg2576 mice; Tg(NK-4: 100), low-dose NK-4-treated Tg2576

We next surveyed brain Aβ deposition in Tg2576 mice by Aβ immunohistochemistry (Fig. 9C). Aβ-immunoreactive small diffuse plaques were abundantly present in the cortex of saline-treated Tg2576 mice (upper panel), and they were visibly reduced by high dose NK-4 treatment both in size and in quantity (bottom panel). The donepezil-treated group showed

Nine months of NK-4 administration to Tg2576 mice significantly attenuated the cognitive decline as assessed by behavioral tests (Fig. 8), and decreased the levels of Aβ in the brain (Fig. 9B, C) while augmenting levels in the plasma (Fig. 9A). These results imply that NK-4 induced cognitive improvement was attributable to decreased Aβ brain levels. Although there is still no consensus as to the mechanism by which some drugs, including anti-Aβ antibodies, alter amyloid deposition in the brain (Levites et al., 2006), Aβ may be cleared

Wild Tg (saline) Tg (DPZ) Tg (NK-4:100) Tg (NK-4:500)

total amount of Aβ in the brain.

**6 months**

**Day1 Day2 Day3 Day4**

**0**

mice; Tg2576 (NK-4: 500), high-dose NK-4-treated Tg2576 mice. Fig. 8. Effect of NK-4 on recognition memory in Tg2576 mice.

a slight decrease in Aβ-immunoreactivity (middle panel).

across the blood-brain barrier to the blood in NK-4-treated Tg2576 mice.

**30**

**60**

**90**

**120**

Wild Tg (saline) Tg (DPZ) Tg (NK-4:100) Tg (NK-4:500)

**A**

**0**

**30**

**60**

**Escape latency (s)**

**90**

**120**

precursor protein (APP) transgenic mice Tg2576 (Hsiao et al., 1996) and the Aβ-induced amnesia model (Maurice et al., 1996).

#### **3.3.1 Effects of NK-4 in APP-Tg (Tg2576) mice**

The Tg2576 mouse is the most thoroughly characterized AD mouse model and is considered to reflect human amyloid pathology most closely among mouse models. This mouse develops a considerable amount of Aβ deposits in the brain with age and this is accompanied by gradual declines in cognitive function (Hsiao, 2001). We examined whether chronic administration of NK-4 produced an effect on the cognitive deficits in Tg2576 mice (Ohta et al., 2010b). Tg2576 mice were administered NK-4 as doses of 100 or 500 µg/kg/day once a day, 5 times a week for 9 months, starting at 3 months of age. Donepezil, an existing drug for dementia based on its AChE inhibitory properties (Sugimoto, 2004) was used as a control drug and was administered at a dose of 200 µg/kg/day.

Wild type and Tg2576 mice were tested for spatial learning ability in a Morris water maze test (Morris, 1984) at ages 6 and 12 months. There were significant differences in escape latency between the wild type controls and saline-treated Tg2576 mice at 6 and 12 months of age (Fig. 8A). For the saline-treated Tg2576 group, the latency to reach the hidden platform did not shorten during the whole test period at 12 months of age, suggesting that the spatial learning ability of saline-treated Tg2576 mice became impaired with age. We found significant differences in spatial learning ability between the NK-4-treated Tg2576 mice (at both low and high doses) and saline-treated Tg2576 mice. Saline-treated Tg2576 mice consistently exhibited longer escape latencies compared to the NK-4-treated Tg2576 group at 6 and 12 months of age. Donepezil-treated mice showed a partial improvement in spatial memory deficits at 6 months of age, although the drug became less effective over time. At 12 months of age, the escape latencies of donepezil-treated and saline-treated Tg2576 mice showed no significant difference. These results suggest a long-lasting ameliorative effect of NK-4 on Aβ-mediated spatial memory impairment.

We also evaluated the object recognition memory of Tg2576 mice in a novel object recognition test (Nagai et al., 2003). Long-term recognition memory was tested at ages of 6, 9, and 12 months (Fig. 8B). Saline-treated Tg2576 mice displayed significantly decreased object recognition memory compared to wild type mice at the same ages. Tg2576 mice treated with low and high doses of NK-4 or donepezil spent a longer time exploring the novel object than did the saline-treated controls at 6 months of age. However, at 9 and 12 month of age, the exploration preference values of low dose NK-4 and donepezil-treated groups decreased, and the differences between these two groups and saline-treated group became insignificant. In contrast, the high dose of NK-4 group spent a significantly longer time exploring the novel object versus the saline-treated mice throughout the test period, and the exploratory preference was comparable to that of wild type controls at 12 months of age. This suggests that NK-4 administration for a longer period and at high dosing might be more effective for the improvement of object recognition memory.

At the end of experiment (12 months of age; after 9-month treatment), the effects of NK-4 on plasma and brain Aβ levels in Tg2576 mice were evaluated (Fig. 9). Dose-dependent increases in Aβ1-40 and Aβ1-42 were observed in plasma from the NK-4-treated mice (Fig. 9A). Although donepezil treatment also increased both plasma Aβ1-40 and Aβ1-42 levels, the effect

precursor protein (APP) transgenic mice Tg2576 (Hsiao et al., 1996) and the Aβ-induced

The Tg2576 mouse is the most thoroughly characterized AD mouse model and is considered to reflect human amyloid pathology most closely among mouse models. This mouse develops a considerable amount of Aβ deposits in the brain with age and this is accompanied by gradual declines in cognitive function (Hsiao, 2001). We examined whether chronic administration of NK-4 produced an effect on the cognitive deficits in Tg2576 mice (Ohta et al., 2010b). Tg2576 mice were administered NK-4 as doses of 100 or 500 µg/kg/day once a day, 5 times a week for 9 months, starting at 3 months of age. Donepezil, an existing drug for dementia based on its AChE inhibitory properties (Sugimoto, 2004) was used as a

Wild type and Tg2576 mice were tested for spatial learning ability in a Morris water maze test (Morris, 1984) at ages 6 and 12 months. There were significant differences in escape latency between the wild type controls and saline-treated Tg2576 mice at 6 and 12 months of age (Fig. 8A). For the saline-treated Tg2576 group, the latency to reach the hidden platform did not shorten during the whole test period at 12 months of age, suggesting that the spatial learning ability of saline-treated Tg2576 mice became impaired with age. We found significant differences in spatial learning ability between the NK-4-treated Tg2576 mice (at both low and high doses) and saline-treated Tg2576 mice. Saline-treated Tg2576 mice consistently exhibited longer escape latencies compared to the NK-4-treated Tg2576 group at 6 and 12 months of age. Donepezil-treated mice showed a partial improvement in spatial memory deficits at 6 months of age, although the drug became less effective over time. At 12 months of age, the escape latencies of donepezil-treated and saline-treated Tg2576 mice showed no significant difference. These results suggest a long-lasting ameliorative effect of

We also evaluated the object recognition memory of Tg2576 mice in a novel object recognition test (Nagai et al., 2003). Long-term recognition memory was tested at ages of 6, 9, and 12 months (Fig. 8B). Saline-treated Tg2576 mice displayed significantly decreased object recognition memory compared to wild type mice at the same ages. Tg2576 mice treated with low and high doses of NK-4 or donepezil spent a longer time exploring the novel object than did the saline-treated controls at 6 months of age. However, at 9 and 12 month of age, the exploration preference values of low dose NK-4 and donepezil-treated groups decreased, and the differences between these two groups and saline-treated group became insignificant. In contrast, the high dose of NK-4 group spent a significantly longer time exploring the novel object versus the saline-treated mice throughout the test period, and the exploratory preference was comparable to that of wild type controls at 12 months of age. This suggests that NK-4 administration for a longer period and at high dosing might be

At the end of experiment (12 months of age; after 9-month treatment), the effects of NK-4 on plasma and brain Aβ levels in Tg2576 mice were evaluated (Fig. 9). Dose-dependent increases in Aβ1-40 and Aβ1-42 were observed in plasma from the NK-4-treated mice (Fig. 9A). Although donepezil treatment also increased both plasma Aβ1-40 and Aβ1-42 levels, the effect

amnesia model (Maurice et al., 1996).

**3.3.1 Effects of NK-4 in APP-Tg (Tg2576) mice** 

control drug and was administered at a dose of 200 µg/kg/day.

NK-4 on Aβ-mediated spatial memory impairment.

more effective for the improvement of object recognition memory.

was lower compared to NK-4 treatments. Brain levels of Aβ were estimated separately in detergent-insoluble and -soluble fractions. Both detergent-insoluble and soluble Aβ1-40 levels were significantly decreased by NK-4 treatment (Fig. 9B, upper graphs). Donepezil did not affect Aβ concentrations in brain. Because the levels of Aβ1-42 were very low in all groups compared to those of Aβ1-40 (Fig. 9B, lower graphs), the levels of Aβ1-40 seem to reflect the total amount of Aβ in the brain.

NK-4 was injected intraperitoneally to Tg2576 mice at a dose of 100 or 500 µg/kg once a day, five times a week for 9 months, beginning at 3 months of age (n=10 each). Donepezil (DPZ) was administered at a dose of 200 µg/kg (n=5). Control mice received 200 µl of saline (wild type, n=10; Tg2576, n=10). (A) Morris water maze test at 6 and 12 months of age. The escape latency represents the average time to find a hidden platform placed in a fixed location in a circular pool (ø130 cm). Two trials per day were conducted for 4 consecutive days with an upper cutoff time of 120 sec. (B) Novel object recognition test at 6, 9 and 12 months of age. Two objects X and Y were placed in the test box and the exploration behavior of mice was recorded. A retention session was performed 24 hr after the training session. One of the familiar objects Y was replaced by a novel object Z. Mice were allowed to explore freely for 10 min, with the time spent in exploring each of the two objects recorded. The exploratory preference was expressed as a ratio of the time spent exploring the novel object (TZ) over that spent on the two objects (TX+TZ). Data are expressed as means ± S.E.M. \*P<0.05, \*\*P<0.01 vs. saline-treated Tg2576 group. Wild: non-transgenic mice. Tg(saline), saline-treated Tg2576 mice; Tg(DPZ), donepezil-treated Tg2576 mice; Tg(NK-4: 100), low-dose NK-4-treated Tg2576 mice; Tg2576 (NK-4: 500), high-dose NK-4-treated Tg2576 mice.

Fig. 8. Effect of NK-4 on recognition memory in Tg2576 mice.

We next surveyed brain Aβ deposition in Tg2576 mice by Aβ immunohistochemistry (Fig. 9C). Aβ-immunoreactive small diffuse plaques were abundantly present in the cortex of saline-treated Tg2576 mice (upper panel), and they were visibly reduced by high dose NK-4 treatment both in size and in quantity (bottom panel). The donepezil-treated group showed a slight decrease in Aβ-immunoreactivity (middle panel).

Nine months of NK-4 administration to Tg2576 mice significantly attenuated the cognitive decline as assessed by behavioral tests (Fig. 8), and decreased the levels of Aβ in the brain (Fig. 9B, C) while augmenting levels in the plasma (Fig. 9A). These results imply that NK-4 induced cognitive improvement was attributable to decreased Aβ brain levels. Although there is still no consensus as to the mechanism by which some drugs, including anti-Aβ antibodies, alter amyloid deposition in the brain (Levites et al., 2006), Aβ may be cleared across the blood-brain barrier to the blood in NK-4-treated Tg2576 mice.

Effects of NK-4, a Cyanine Dye With Antioxidant Activities... 383

and Aβ25-35-injected ICR mice. Both low (50 µg/kg) and high (500 µg/kg) doses of NK-4 successfully prolonged the step-through latencies with statistically significant differences (p<0.05 for the low dose and p<0.01 for the high dose, respectively; Fig. 10A). Improved long-term memory retention following NK-4 administration was also confirmed using this

**Sham**

(A) Passive avoidance test of Aβ25-35–icv mice. Male ICR mice were injected aged Aβ25-35 (9 nmol/mouse) into the left lateral ventricle using the following coordinates from Bregma: 0.5 mm posterior, 1.0 mm lateral, and 2.0 mm ventral. A low (50 μg/kg) or high (500 μg/kg) dose of NK-4 was administered intraperitoneally to mice for twelve consecutive days starting from the next day of Aβ injection. Then, mice were tested for step-through passive avoidance during days 9-12. The step-through latency in the retention session (24-hr after the training session) was recorded. Values are means ± S.E.M. (n=10). Sham, sham-operated group; saline, saline-treated Aβ25-35–icv group; NK-4, NK-4-treated Aβ25-35–icv groups. \*P<0.05, \*\*P<0.01 vs. saline-treated group. (B) Histological damage scores in the hippocampal CA1 region. Hippocampal neuronal cell loss was assessed in coronal H&E sections. Scores are

These beneficial effects of NK-4 on recognition memory are in agreement with histological findings shown in figure 10B. A single icv injection of Aβ25-35 caused significant hippocampal neuronal loss mainly in the CA1 region. The difference in histological scores was not statistically significant between the saline-treated (1.6 ± 0.41) and the high dose NK-4–treated mice (0.9 ± 0.41, p = 0.067). However, NK-4 ameliorated the Aβ25-35-induced injury of pyramidal neurons in the hippocampus (Fig. 10B). Reportedly, associative learning in the passive avoidance test strongly depends on hippocampal function (Phillips & LeDoux, 1992). In this context, neuronal injury in the hippocampal CA1 region following Aβ25-35-icv injection provides a reasonable explanation for the impaired long-term memory. These results, in combination with the results from Tg2576 mice, and AChE inhibitory property of NK-4 suggest that NK-4 treatment effectively improves cognitive deficits in AD model mice by both attenuating Aβ pathology and enhancing cholinergic transmission in the brain.

In this chapter, we showed that NK-4, a type of cyanine dye, exerts a wide spectrum of biochemical and biological activities implicated in neuroprotection. NK-4 significantly ameliorated neurological and cognitive deficits, as well as neurodegeneration, in four

**Saline NK-4 (500)**

**Saline NK-4(500)**

**0.0 1.0 2.0 3.0**

**Histological score**

animal model in a novel object recognition test (data not shown).

**0**

**Sham**

**Saline**

**50**

\*

**A B**

**500**

represented as means ± S.E.M. (n=10). Arrows indicate the sites of degeneration.

Fig. 10. Effects of NK-4 on Aβ25-35-induced cognitive impairments in ICR mice.

\*\*

**NK-4 (μg/kg)**

**50**

**100**

**Latency (s)**

**4. Conclusion** 

**150**

**200**

(A) Plasma Aβ concentrations of Tg2576 mice at 12 months of age. Aβ levels were separately measured by ELISA for Aβ1-40 and Aβ1-42. (B) Brain Aβ levels of detergent-insoluble or soluble fractions at 12 months of age. Hemi-brains from Tg2576 mice were homogenized and separated into detergent-insoluble and soluble fractions. Aβ1-40 and Aβ1-42 in individual samples were measured and are expressed as picomoles/g wet brain weight. Values are the means ± S.E.M. Saline (Sal), n=10; Donepezil (DPZ), n=5; NK-4 (100), n=9; and NK-4 (500), n=8. \*P<0.05, \*\*P<0.01 vs. saline-treated group. (C) Aβ-immunohistochemistry of the cerebral cortex from Tg2576 mice at 12 months of age. Hemi-brains were fixed with 10% buffered formalin and embedded in paraffin. Coronal sections of cerebral cortex with 5-µm thickness were immunostained with rabbit anti-Aβ-peptide polyclonal antibody. Bar represents 50-µm.

Fig. 9. Effect of NK-4 on the plasma and brain concentrations of Aβ in Tg2576 mice.

Oxidative mechanisms are thought to be involved in cell loss and other neuropathologies associated with AD (Zhu et al., 2001; Cutler et al., 2004). During AD pathogenesis, ROS impair mitochondrial redox activity and further increase ROS generation (Shearman et al., 1994; Hensley et al., 1994). Aβ induces the production of ROS and leads to apoptotic neuronal cell death that can be inhibited by antioxidants (Behl et al., 1994; Mattson & Goodman, 1995). Pathological and biochemical studies suggest that ROS induced by fibrillar Aβ produce neurotoxic effects (Gevais et al., 1999). Since NK-4 is a potent scavenger of ROS (Table 1) and is an inhibitor of Aβ aggregation (Fig. 2), these features might work in concert to attenuate Aβ toxicity in AD.

#### **3.3.2 Effects of NK-4 in Aβ-icv mice**

To further determine whether the effect of NK-4 was directly attributable to mitigation of Aβ pathology, an Aβ-induced amnesia model was employed (Ohta et al., 2010a). In this model, ICR mice received intracerebroventricular (icv) administration of aggregated Aβ25-35 peptide to induce memory deficits due to cholinergic dysfunction (Maurice et al., 1996). Long-term recognition memory was evaluated by the step-through type passive avoidance test 12 days after Aβ25-35 injection. In this behavioral assay, daily NK-4 treatment significantly and dose-dependently improved memory deficits induced by Aβ25-35 (Fig. 10A). There was a significant difference in step-through latencies between sham operated mice

\*\*

**0 0.5 1 1.5 2 2.5**

**0 0.02 0.04 0.06** **Sal DPZ 100 500 NK-4 (μg/kg)**

**Sal DPZ 100 500 NK-4 (μg/kg)**

\* \*

**Saline**

**DPZ**

**NK-4 (500)**

**A C B**

**plasma insoluble soluble**

**Aβ1-40 Aβ1-40 Aβ1-40**

**Sal DPZ 100 500 NK-4 (μg/kg)**

**Aβ1-42 Aβ1-42 Aβ1-42**

**Sal DPZ 100 500**

(A) Plasma Aβ concentrations of Tg2576 mice at 12 months of age. Aβ levels were separately measured by ELISA for Aβ1-40 and Aβ1-42. (B) Brain Aβ levels of detergent-insoluble or soluble fractions at 12 months of age. Hemi-brains from Tg2576 mice were homogenized and separated into detergent-insoluble and soluble fractions. Aβ1-40 and Aβ1-42 in individual samples were measured and are expressed as picomoles/g wet brain weight. Values are the means ± S.E.M. Saline (Sal), n=10; Donepezil (DPZ), n=5; NK-4 (100), n=9; and NK-4 (500), n=8. \*P<0.05, \*\*P<0.01 vs. saline-treated group. (C) Aβ-immunohistochemistry of the cerebral cortex from Tg2576 mice at 12 months of age. Hemi-brains were fixed with 10% buffered formalin and embedded in paraffin. Coronal sections of cerebral cortex with 5-µm thickness were immunostained with rabbit anti-Aβ-peptide polyclonal

Fig. 9. Effect of NK-4 on the plasma and brain concentrations of Aβ in Tg2576 mice.

Oxidative mechanisms are thought to be involved in cell loss and other neuropathologies associated with AD (Zhu et al., 2001; Cutler et al., 2004). During AD pathogenesis, ROS impair mitochondrial redox activity and further increase ROS generation (Shearman et al., 1994; Hensley et al., 1994). Aβ induces the production of ROS and leads to apoptotic neuronal cell death that can be inhibited by antioxidants (Behl et al., 1994; Mattson & Goodman, 1995). Pathological and biochemical studies suggest that ROS induced by fibrillar Aβ produce neurotoxic effects (Gevais et al., 1999). Since NK-4 is a potent scavenger of ROS (Table 1) and is an inhibitor of Aβ aggregation (Fig. 2), these features might work in concert

To further determine whether the effect of NK-4 was directly attributable to mitigation of Aβ pathology, an Aβ-induced amnesia model was employed (Ohta et al., 2010a). In this model, ICR mice received intracerebroventricular (icv) administration of aggregated Aβ25-35 peptide to induce memory deficits due to cholinergic dysfunction (Maurice et al., 1996). Long-term recognition memory was evaluated by the step-through type passive avoidance test 12 days after Aβ25-35 injection. In this behavioral assay, daily NK-4 treatment significantly and dose-dependently improved memory deficits induced by Aβ25-35 (Fig. 10A). There was a significant difference in step-through latencies between sham operated mice

**NK-4 (μg/kg)**

**Aβ1-40 (pmol/g)**

**Aβ1-42 (pmol/g)**

\*\* \*\*

\*\*

**Aβ1-40 conc. (pM)**

**Aβ1-42 conc. (pM)** **Sal DPZ 100 500 NK-4 (μg/kg)**

\*\*

\*\*

\*\*

antibody. Bar represents 50-µm.

to attenuate Aβ toxicity in AD.

**3.3.2 Effects of NK-4 in Aβ-icv mice** 

**Sal DPZ 100 500 NK-4 (μg/kg)** and Aβ25-35-injected ICR mice. Both low (50 µg/kg) and high (500 µg/kg) doses of NK-4 successfully prolonged the step-through latencies with statistically significant differences (p<0.05 for the low dose and p<0.01 for the high dose, respectively; Fig. 10A). Improved long-term memory retention following NK-4 administration was also confirmed using this animal model in a novel object recognition test (data not shown).

(A) Passive avoidance test of Aβ25-35–icv mice. Male ICR mice were injected aged Aβ25-35 (9 nmol/mouse) into the left lateral ventricle using the following coordinates from Bregma: 0.5 mm posterior, 1.0 mm lateral, and 2.0 mm ventral. A low (50 μg/kg) or high (500 μg/kg) dose of NK-4 was administered intraperitoneally to mice for twelve consecutive days starting from the next day of Aβ injection. Then, mice were tested for step-through passive avoidance during days 9-12. The step-through latency in the retention session (24-hr after the training session) was recorded. Values are means ± S.E.M. (n=10). Sham, sham-operated group; saline, saline-treated Aβ25-35–icv group; NK-4, NK-4-treated Aβ25-35–icv groups. \*P<0.05, \*\*P<0.01 vs. saline-treated group. (B) Histological damage scores in the hippocampal CA1 region. Hippocampal neuronal cell loss was assessed in coronal H&E sections. Scores are represented as means ± S.E.M. (n=10). Arrows indicate the sites of degeneration.

Fig. 10. Effects of NK-4 on Aβ25-35-induced cognitive impairments in ICR mice.

These beneficial effects of NK-4 on recognition memory are in agreement with histological findings shown in figure 10B. A single icv injection of Aβ25-35 caused significant hippocampal neuronal loss mainly in the CA1 region. The difference in histological scores was not statistically significant between the saline-treated (1.6 ± 0.41) and the high dose NK-4–treated mice (0.9 ± 0.41, p = 0.067). However, NK-4 ameliorated the Aβ25-35-induced injury of pyramidal neurons in the hippocampus (Fig. 10B). Reportedly, associative learning in the passive avoidance test strongly depends on hippocampal function (Phillips & LeDoux, 1992). In this context, neuronal injury in the hippocampal CA1 region following Aβ25-35-icv injection provides a reasonable explanation for the impaired long-term memory. These results, in combination with the results from Tg2576 mice, and AChE inhibitory property of NK-4 suggest that NK-4 treatment effectively improves cognitive deficits in AD model mice by both attenuating Aβ pathology and enhancing cholinergic transmission in the brain.

#### **4. Conclusion**

In this chapter, we showed that NK-4, a type of cyanine dye, exerts a wide spectrum of biochemical and biological activities implicated in neuroprotection. NK-4 significantly ameliorated neurological and cognitive deficits, as well as neurodegeneration, in four

Effects of NK-4, a Cyanine Dye With Antioxidant Activities... 385

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distinct animal models (MCAO rats, ataxic hamsters, APP-transgenic mice, and Aβ-icv mice). Molecular mechanisms by which NK-4 acts against neurodegeneration remain unclear, although it was found that NK-4 activates the PI3K-Akt pathway independently of Trk receptors. Thus, activated Akt may be a key mediator of the beneficial effects on neural cell survival. In addition, since activated Akt is required for PI3K-mediated synaptic plasticity and memory consolidation via activation of the downstream regulator CREB (Brightwell et al., 2007), induction of Akt phosphorylation might play a critical role in NK-4 mediated memory improvement in animal models of dementia. In addition to the modification of intracellular signaling, a direct antioxidative property of NK-4 may also be involved in survival and functional maintenance of neurons.

Regarding the safety profile, there were no specific adverse events in mice that received intraperitoneal injections of NK-4 at doses of up to 500 µg/kg/day, 5 days a week, for 9 months (3.3.1. Tg2576 study). Furthermore, NK-4 was well tolerated in rats up to 4,000 mg/kg in an acute toxicity study and 100 mg/kg in a subacute toxicity study (both via the oral rout of administration) based on mortality, clinical observations, body weight, hematology, blood chemistry, organ weights, and histological examination of a complete tissue list (Ohmori et al., 1983). Furthermore, NK-4 has been used as an active ingredient of over the counter (OTC) medicine in Japan for treating allergy, and for promoting wound healing since the 1950s (Suzue, 1969). Additionally, NK-4 was not mutagenic in the standard Ames test (our unpublished data). These observations suggest that NK-4 is a safe compound that will not cause serious adverse reactions.

Application of small neurotrophic molecules that modulate neuronal survival and synaptic function is a promising and valid therapeutic approach for neurodegenerative disorders. Lines of evidence described here strongly suggest the potential utility of NK-4 as a treatment for neurodegenerative disease. The evaluations of pharmacokinetics, bioavailability, as well as the efficacy of orally administered NK-4 are ongoing. In addition, assessment of NK-4 in human neurodegenerative therapy will require further studies.

#### **5. Acknowledgment**

We would like to thank Dr. S. Arai, Ms. C. Ushio, and Dr. M. Takeuchi (HAYASHIBARA Co., Ltd.) for their helpful support.

#### **6. References**


http://www.alz.org/documents\_custom/trajectory.pdf

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distinct animal models (MCAO rats, ataxic hamsters, APP-transgenic mice, and Aβ-icv mice). Molecular mechanisms by which NK-4 acts against neurodegeneration remain unclear, although it was found that NK-4 activates the PI3K-Akt pathway independently of Trk receptors. Thus, activated Akt may be a key mediator of the beneficial effects on neural cell survival. In addition, since activated Akt is required for PI3K-mediated synaptic plasticity and memory consolidation via activation of the downstream regulator CREB (Brightwell et al., 2007), induction of Akt phosphorylation might play a critical role in NK-4 mediated memory improvement in animal models of dementia. In addition to the modification of intracellular signaling, a direct antioxidative property of NK-4 may also be

Regarding the safety profile, there were no specific adverse events in mice that received intraperitoneal injections of NK-4 at doses of up to 500 µg/kg/day, 5 days a week, for 9 months (3.3.1. Tg2576 study). Furthermore, NK-4 was well tolerated in rats up to 4,000 mg/kg in an acute toxicity study and 100 mg/kg in a subacute toxicity study (both via the oral rout of administration) based on mortality, clinical observations, body weight, hematology, blood chemistry, organ weights, and histological examination of a complete tissue list (Ohmori et al., 1983). Furthermore, NK-4 has been used as an active ingredient of over the counter (OTC) medicine in Japan for treating allergy, and for promoting wound healing since the 1950s (Suzue, 1969). Additionally, NK-4 was not mutagenic in the standard Ames test (our unpublished data). These observations suggest that NK-4 is a safe compound

Application of small neurotrophic molecules that modulate neuronal survival and synaptic function is a promising and valid therapeutic approach for neurodegenerative disorders. Lines of evidence described here strongly suggest the potential utility of NK-4 as a treatment for neurodegenerative disease. The evaluations of pharmacokinetics, bioavailability, as well as the efficacy of orally administered NK-4 are ongoing. In addition, assessment of NK-4 in human

We would like to thank Dr. S. Arai, Ms. C. Ushio, and Dr. M. Takeuchi (HAYASHIBARA

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**5. Acknowledgment** 

0167-7063

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neurodegenerative therapy will require further studies.

imperative. 16.06.2010, Available from:

http://www.alz.org/documents\_custom/trajectory.pdf


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## *Edited by Volodymyr I. Lushchak*

This book focuses on the numerous applications of oxidative stress theory in effects of environmental factors on biological systems. The topics reviewed cover induction of oxidative stress by physical, chemical, and biological factors in humans, animals, plants and fungi. The physical factors include temperature, light and exercise. Chemical induction is related to metal ions and pesticides, whereas the biological one highlights host-pathogen interaction and stress effects on secretory systems. Antioxidants, represented by a large range of individual compounds and their mixtures of natural origin and those chemically synthesized to prevent or fix negative effects of reactive species are also described in the book. This volume will be a useful source of information on induction and effects of oxidative stress on living organisms for graduate and postgraduate students, researchers, physicians, and environmentalists.

Oxidative Stress - Environmental Induction and Dietary Antioxidants

Oxidative Stress

Environmental Induction

and Dietary Antioxidants

*Edited by Volodymyr I. Lushchak*

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