Masanori Katakura

Green tea catechins prevent cognitive deficits caused by Aβ1–40 in rats

Amyloid beta peptide (Abeta)-induced oxidative stress is involved in the pathogenesis of Alzheimers disease (AD). In contrast, green tea catechins confer potent antioxidative defense to brain neurons. Therefore, we examined whether long-term administration of green tea catechins [Polyphenon E (PE): 63% of epigallocatechin-3-gallate, 11% of epicatechin, 6% of (-)-epigallocatechin and 6% of (-)-epicatechin-gallate] prevents cognitive impairment in an animal model of AD, rats infused with Abeta1-40 into the cerebral ventricle. Five-week-old male Wistar rats fed with an MF diet were randomly divided into two groups: 0.0% PE (rats administered with water only) and 0.5% PE (rats administered with 5 g/L of PE). Twenty weeks after the PE administration, the 0.0% PE group was divided into the Vehicle group (rats infused with the solvent used for dissolving Abeta) and the Abeta(1-40)-infused rat group (Abeta group), whereas the 0.5% PE group was divided into the PE+Vehicle group (PE-preadministered vehicle-infused rats) and the PE+Abeta group (PE-preadministered Abeta-infused rats). Abeta1-40 or vehicle was infused into the cerebral ventricle using a mini osmotic pump. Behavioral changes in the rats were assessed by an eight-arm radial maze. PE administration for 26 weeks significantly decreased the Abeta-induced increase in the number of reference and working memory errors, with a concomitant reduction of hippocampal lipid peroxide (LPO; 40%) and cortico-hippocampal reactive oxygen species (ROS; 42% and 50%, respectively). Significantly reduced levels of LPO in the plasma (24%) and hippocampus (25%) as well as those of ROS in the hippocampus (23%) and cortex (41%) were found in the PE+Vehicle group as compared with the Vehicle group. Furthermore, rats with preadministered PE had higher ferric-reducing antioxidation power of plasma as compared with the Vehicle group. Our results suggest that long-term administration of green tea catechins provides effective prophylactic benefits against Abeta-induced cognitive impairment by increasing antioxidative defenses.

DOCOSAHEXAENOIC ACID PROMOTES NEURONAL DIFFERENTIATION BY REGULATING BASIC HELIX-LOOP-HELIX TRANSCRIPTION FACTORS AND CELL CYCLE IN NEURAL STEM CELLS

Recent studies have suggested that docosahexaenoic acid (DHA) enhances neuronal differentiation of neural stem cells (NSCs) isolated from rat embryonic day 14.5. However the underlying mechanism remains largely unknown. One hypothesis supported by DHA controls the expression level of basic helix-loop-helix (bHLH) transcription factors, such as hairy and enhancer of split 1 (Hes1), Mash1, neurogenin1, and NeuroD; another is that previous studies in retinal progenitor cells DHA affects the cell cycle. In this study, we show that treatment with DHA under differentiation conditions without basic fibroblast growth factor, (1) increases Tuj-1 and MAP2 positive cells in NSCs, (2) that the expression level of Hes1 mRNA and protein decreased significantly from day 1 to day 4, on the other hand, the NeuroD mRNA expression level increased from day 1 to day 4 after treatment with DHA and (3) decreased the percentage of S-phase cells, which correlated with prolonged expression of cyclin-dependent kinase inhibitor p27(kip1), suggesting that DHA enhances neuronal differentiation of NSCs, in part, by controlling the bHLH transcription factors and promoting cell cycle exit. We therefore speculate that DHA is one of the essential key molecules for neuronal differentiation of NSCs.

Docosahexaenoic acid disrupts in vitro amyloid β1-40 fibrillation and concomitantly inhibits amyloid levels in cerebral cortex of Alzheimer’s disease model rats

We have previously reported that dietary docosahexaenoic acid (DHA) improves and/or protects against impairment of cognition ability in amyloid beta1‐40 (Aβ1‐40)‐infused Alzheimer’s disease (AD)‐model rats. Here, after the administration of DHA to AD model rats for 12 weeks, the levels of Aβ1‐40, cholesterol and the composition of fatty acids were investigated in the Triton X100‐insoluble membrane fractions of their cerebral cortex. The effects of DHA on the in vitro formation and kinetics of fibrillation of Aβ1‐40 were also investigated by thioflavin T fluorescence spectroscopy, transmission electron microscopy and fluorescence microscopy. Dietary DHA significantly decreased the levels of Aβ1‐40, cholesterol and saturated fatty acids in the detergent insoluble membrane fractions of AD rats. The formation of Aβ fibrils was also attenuated by their incubation with DHA, as demonstrated by the decreased intensity of thioflavin T‐derived fluorescence and by electron micrography. DHA treatment also decreased the intensity of thioflavin fluorescence in preformed‐fibril Aβ peptides, demonstrating the anti‐amyloidogenic effects of DHA. We then investigated the effects of DHA on the levels of oligomeric amyloid that is generated during its in vitro transformation from monomers to fibrils, by an anti‐oligomer‐specific antibody and non‐reducing Tris‐Glycine gradient (4–20%) gel electrophoresis. DHA concentration‐dependently reduced the levels of oligomeric amyloid species, suggesting that dietary DHA‐induced suppression of in vivo Aβ1‐40 aggregation occurs through the inhibitory effect of DHA on oligomeric amyloid species.

Mechanism of docosahexaenoic acid-induced inhibition of in vitro Aβ1–42 fibrillation and Aβ1–42-induced toxicity in SH-S5Y5 cells

The mechanism of the effect of docosahexaenoic acid (DHA; C22:6, n‐3), one of the essential brain nutrients, on in vitro fibrillation of amyloid β (Aβ1–42), Aβ1–42‐oligomers and its toxicity imparted to SH‐S5Y5 cells was studied with the use of thioflavin T fluorospectroscopy, laser confocal microfluorescence, and transmission electron microscopy. The results clearly indicated that DHA inhibited Aβ1–42‐fibrill formation with a concomitant reduction in the levels of soluble Aβ1–42 oligomers. The polymerization (into fibrils) of preformed oligomers treated with DHA was inhibited, indicating that DHA not only obstructs their formation but also inhibits their transformation into fibrils. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (12.5%), Tris–Tricine gradient(4–20%) gel electrophoresis and western blot analyses revealed that DHA inhibited at least 2 species of Aβ1–42 oligomers of 15–20 kDa, indicating that it hinders these on‐pathway tri/tetrameric intermediates during fibrillation. DHA also reduced the levels of dityrosine and tyrosine intrinsic fluorescence intensity, indicating DHA interrupts the microenvironment of tyrosine in the Aβ1–42 backbone. Furthermore, DHA protected the tyrosine from acrylamide collisional quenching, as indicated by decreases in Stern–Volmer constants. 3‐[4,5‐Dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide‐reduction efficiency and immunohistochemical examination suggested that DHA inhibits Aβ1–42‐induced toxicity in SH‐S5Y5 cells. Taken together, these data suggest that by restraining Aβ1–42 toxic tri/tetrameric oligomers, DHA may limit amyloidogenic neurodegenerative diseases, Alzheimer’s disease.

Omega-3 polyunsaturated fatty acids enhance neuronal differentiation in cultured rat neural stem cells

Polyunsaturated fatty acids (PUFAs) can induce neurogenesis and recovery from brain diseases. However, the exact mechanisms of the beneficial effects of PUFAs have not been conclusively described. We recently reported that docosahexaenoic acid (DHA) induced neuronal differentiation by decreasing Hes1 expression and increasing p27kip1 expression, which causes cell cycle arrest in neural stem cells (NSCs). In the present study, we examined the effect of eicosapentaenoic acid (EPA) and arachidonic acid (AA) on differentiation, expression of basic helix-loop-helix transcription factors (Hes1, Hes6, and NeuroD), and the cell cycle of cultured NSCs. EPA also increased mRNA levels of Hes1, an inhibitor of neuronal differentiation, Hes6, an inhibitor of Hes1, NeuroD, and Map2 mRNA and Tuj-1-positive cells (a neuronal marker), indicating that EPA induced neuronal differentiation. EPA increased the mRNA levels of p21cip1 and p27kip1, a cyclin-dependent kinase inhibitor, which indicated that EPA induced cell cycle arrest. Treatment with AA decreased Hes1 mRNA but did not affect NeuroD and Map2 mRNA levels. Furthermore, AA did not affect the number of Tuj-1-positive cells or cell cycle progression. These results indicated that EPA could be involved in neuronal differentiation by mechanisms alternative to those of DHA, whereas AA did not affect neuronal differentiation in NSCs.

n-3 fatty acids effectively improve the reference memory-related learning ability associated with increased brain docosahexaenoic acid-derived docosanoids in aged rats.

We investigated whether a highly purified eicosapentaenoic acid (EPA) and a concentrated n-3 fatty acid formulation (prescription TAK-085) containing EPA and docosahexaenoic acid (DHA) ethyl ester could improve the learning ability of aged rats and whether this specific outcome had any relation with the brain levels of EPA-derived eicosanoids and DHA-derived docosanoids. The rats were tested for reference memory errors (RMEs) and working memory errors (WMEs) in an eight-arm radial maze. Fatty acid compositions were analyzed by GC, whereas brain eicosanoid/docosanoids were measured by LC-ESI-MS-MS-based analysis. The levels of lipid peroxides (LPOs) were measured by thiobarbituric acid reactive substances. The administration of TAK-085 at 300 mg·kg⁻¹day⁻¹ for 17 weeks reduced the number of RMEs in aged rats compared with that in the control rats. Both TAK-085 and EPA administration increased plasma EPA and DHA levels in aged rats, with concurrent increases in DHA and decreases in arachidonic acid in the corticohippocampal brain tissues. TAK-085 administration significantly increased the formation of EPA-derived 5-HETE and DHA-derived 7-, 10-, and 17-HDoHE, PD1, RvD1, and RvD2. ARA-derived PGE2, PGD2, and PGF2α significantly decreased in TAK-085-treated rats. DHA-derived mediators demonstrated a significantly negative correlation with the number of RMEs, whereas EPA-derived mediators did not exhibit any relationship. Furthermore, compared with the control rats, the levels of LPO in the plasma, cerebral cortex, and hippocampus were significantly reduced in TAK-085-treated rats. The findings of the present study suggest that long-term EPA+DHA administration may be a possible preventative strategy against age-related cognitive decline.

Effects of docosahexaenoic acid on in vitro amyloid beta peptide 25–35 fibrillation

Amyloid beta peptide(25-35) (Abeta(25-35)) encompasses one of the neurotoxic domains of full length Abeta(1-40/42), the major proteinaceous component of amyloid deposits in Alzheimers disease (AD). We investigated the effect of docosahexaenoic acid (DHA, 22:6, n-3), an essential brain polyunsaturated fatty acid, on the in vitro fibrillation of Abeta(25-35) and found that it significantly reduced the degree of fibrillation, as shown by a decrease in the intensity of both the thioflavin T and green fluorescence in confocal microscopy. Transmission electron microscopy revealed that DHA-incubated samples were virtually devoid of structured fibrils but had an amorphous-like consistency, whereas the controls contained structured fibers of various widths and lengths. The in vitro fibrillation of Abeta(25-35) appeared to be pH-dependent, with the strongest effect seen at pH 5.0. DHA inhibited fibrillation at all pHs, with the strongest effect at pH 7.4. It also significantly decreased the levels of Abeta(25-35) oligomers. Nonreductive gradient gel electrophoresis revealed that the molecular size of the oligomers of Abeta(25-35) was 10 kDa (equivalent to decamers of Abeta(25-35)) and that DHA dose-dependently reduced these decamers. These results suggest that DHA decreases the in vitro fibrillation of Abeta(25-35) by inhibiting the oligomeric amyloid species and, therefore, Abeta(25-35)-related neurotoxicity or behavioral impairment could be restrained by DHA.

Docosahexaenoic acid withstands the Aβ25-35-induced neurotoxicity in SH-SY5Y cells

BACKGROUND Docosahexaenoic acid (DHA, C22:6, n-3) ameliorates the memory-related learning deficits of Alzheimers disease (AD), which is characterized by fibrillar amyloid deposits in the affected brains. Here, we have investigated whether DHA-induced inhibition of Amyloid β-peptide(25-35) (Aβ(25-35)) fibrillation limits or deteriorates the toxicity of the human neuroblastoma cells (SH-SY5Y). EXPERIMENTAL METHODS In vitro fibrillation of Aβ(25-35) was performed in the absence or presence of DHA. Afterwards, SH-SY5Y cells were incubated with Aβ(25-35) in absence or presence 20 μM DHA to evaluate its effect on the Aβ(25-35)-induced neurotoxicity by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)]-redox and TUNEL (TdT-mediated dUTP-biotin nick end-labeling) assay and immunohistochemistry. The level of Aβ(25-35)-induced lipid peroxide (LPO) was determined in the absence or presence of oligomer-specific antibody. Fatty acid profile was estimated by gas chromatography. RESULTS DHA significantly reduced the Aβ(25-35) in vitro fibrillation, as indicated by fluorospectroscopy and transmission electron microscopy. Aβ(25-35) decreased the MTT-redox activity and increased the apoptotic damage and levels of LPO when compared with those of the controls. However, when the SH-SY5Y cells were treated with Aβ(25-35) in the presence of DHA, MTT redox potential significantly increased and the levels LPO decreased, suggesting an inhibition of the Aβ(25-35)-induced neurotoxicity. DHA improved the Aβ(25-35) induced DNA damage and axodendritic loss, with a concomitant increase in the cellular level of DHA, suggesting DHA protects the cell from neurotoxic degeneration. CONCLUSION DHA not only inhibits the in vitro fibrillation but also resists the Aβ(25-35)-induced toxicity in the neuronal cells. This might be the basis of the DHA-induced amelioration of Aβ-induced neurodegeneration and related cognitive deficits.

Omega-3 Fatty Acids Protect Renal Functions by Increasing Docosahexaenoic Acid-Derived Metabolite Levels in SHR.Cg-Leprcp/NDmcr Rats, a Metabolic Syndrome Model

The omega-3 polyunsaturated fatty acids (ω-3 PUFAs) docosahexaenoic acid (DHA) and/or eicosapentaenoic acid (EPA) protect against diabetic nephropathy by inhibiting inflammation. The aim of this study was to assess the effects of highly purified DHA and EPA or EPA only administration on renal function and renal eicosanoid and docosanoid levels in an animal model of metabolic syndrome, SHR.Cg-Leprcp/NDmcr (SHRcp) rats. Male SHRcp rats were divided into 3 groups. Control (5% arabic gum), TAK-085 (300 mg/kg/day, containing 467 mg/g EPA and 365 mg/g DHA), or EPA (300 mg/kg/day) was orally administered for 20 weeks. The urinary albumin to creatinine ratio in the TAK-085-administered group was significantly lower than that in other groups. The glomerular sclerosis score in the TAK-085-administered group was significantly lower than that in the other groups. Although DHA levels were increased in total kidney fatty acids, the levels of nonesterified DHA were not significantly different among the 3 groups, whereas the levels of protectin D1, resolvin D1, and resolvin D2 were significantly increased in the TAK-085-administered group. The results show that the use of combination therapy with DHA and EPA in SHRcp rats improved or prevented renal failure associate with metabolic syndrome with decreasing triglyceride levels and increasing ω-3 PUFA lipid mediators.

Hydrogen-rich water inhibits glucose and α,β -dicarbonyl compound-induced reactive oxygen species production in the SHR.Cg-Leprcp/NDmcr rat kidney

BackgroundReactive oxygen species (ROS) production induced by α,β-dicarbonyl compounds and advanced glycation end products causes renal dysfunction in patients with type 2 diabetes and metabolic syndrome. Hydrogen-rich water (HRW) increases the H2 level in blood and tissues, thus reducing oxidative stress in animals as well as humans. In this study, we investigated the effects of HRW on glucose- and α,β-dicarbonyl compound-induced ROS generation in vitro and in vivo.MethodsKidney homogenates from Wistar rats were incubated in vitro with glucose and α,β-dicarbonyl compounds containing HRW, following which ROS levels were measured. In vivo animal models of metabolic syndrome, SHR.Cg-Leprcp/NDmcr rats, were treated with HRW for 16 weeks, following which renal ROS production and plasma and renal α,β-dicarbonyl compound levels were measured by liquid chromatograph mass spectrometer.ResultsHRW inhibited glucose- and α,β-dicarbonyl compound-induced ROS production in kidney homogenates from Wistar rats in vitro. Furthermore, SHR.Cg-Leprcp/NDmcr rats treated with HRW showed a 34% decrease in ROS production. Moreover, their renal glyoxal, methylglyoxal, and 3-deoxyglucosone levels decreased by 81%, 77%, and 60%, respectively. Positive correlations were found between renal ROS levels and renal glyoxal (r = 0.659, p = 0.008) and methylglyoxal (r = 0.782, p = 0.001) levels.ConclusionThese results indicate that HRW inhibits the production of α,β-dicarbonyl compounds and ROS in the kidneys of SHR.Cg-Leprcp/NDmcr rats. Therefore, it has therapeutic potential for renal dysfunction in patient with type 2 diabetes and metabolic syndrome.