Kentaro Matsuzaki

Nobiletin, a Citrus Flavonoid, Improves Memory Impairment and Aβ Pathology in a Transgenic Mouse Model of Alzheimer's Disease

Increasing evidence suggests that the elevation of β-amyloid (Aβ) peptides in the brain is central to the pathogenesis of Alzheimers disease (AD). Our recent studies have demonstrated that nobiletin, a polymethoxylated flavone from citrus peels, enhances cAMP/protein kinase A/extracellular signal-regulated kinase/cAMP response element-binding protein signaling in cultured hippocampal neurons and ameliorates Aβ-induced memory impairment in AD model rats. For the first time, we report that this natural compound improves memory deficits in amyloid precursor protein (APP) transgenic mice that overexpress human APP695 harboring the double Swedish and London mutations [APP-SL 7-5 transgenic (Tg) mice]. Our enzyme-linked immunosorbent assay (ELISA) also showed that administration of nobiletin to the transgenic mice for 4 months markedly reduced quantity of guanidine-soluble Aβ1–40 and Aβ1–42 in the brain. Furthermore, consistent with the results of ELISA, by immunohistochemistry with anti-Aβ antibody, it was evidently shown that the administration of nobiletin decreased the Aβ burden and plaques in the hippocampus of APP-SL 7-5 Tg mice. These findings suggest that this natural compound has potential to become a novel drug for fundamental treatment of AD.

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.

Nobiletin, a Citrus Flavonoid, Reverses Learning Impairment Associated with N-Methyl-D-aspartate Receptor Antagonism by Activation of Extracellular Signal-Regulated Kinase Signaling

Recent studies have indicated that learning-induced activation of extracellular signal-regulated kinase (ERK) signaling via N-methyl-d-aspartate (NMDA) receptors is required for consolidation of the resultant learning. These findings raise an idea that control of ERK signaling may be a potential target for treatment of cognitive dysfunction. Our recent studies have demonstrated that nobiletin, a polymethoxylated flavone from Citrus depressa, enhances cAMP/protein kinase A/ERK signaling in cultured rat hippocampal neurons and PC12D cells. Here, we, for the first time, present the evidence that this natural compound reverses learning impairment associated with NMDA receptor antagonism by activation of ERK in the hippocampus. Treatment with 50 mg/kg nobiletin reversed the NMDA receptor antagonist MK-801 (dizocilpine maleate)-induced learning impairment in mice. Western blot analysis also showed that nobiletin reversed MK-801-induced inhibition of learning-associated ERK activation in the hippocampus of the animals. Furthermore, consistent with these results, in cultured rat hippocampal neurons, nobiletin restored MK-801-induced impairment of NMDA-stimulated phosphorylation of ERK in a concentration-dependent manner. Taken together, the present study suggests that compounds that activate ERK signaling improve cognitive deficits associated with NMDA receptor hypofunction and that nobiletin may give us a new insight into therapeutic drug development for neurological disorders exhibiting cognitive impairment accompanied by a hypofunction of NMDA receptor-ERK signaling.

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.

Docosahexaenoic acid: one molecule diverse functions.

Abstract Docosahexaenoic acid (DHA, C22:6, ω-3) is a highly polyunsaturated omega-3 fatty acid. It is concentrated in neuronal brain membranes, for which reason it is also referred to as a “brain food”. DHA is essential for brain development and function. It plays an important role in improving antioxidant and cognitive activities of the brain. DHA deficiency occurs during aging and dementia, impairs memory and learning, and promotes age-related neurodegenerative diseases, including Alzheimer’s disease (AD). For about two decades, we have reported that oral administration of DHA increases spatial memory acquisition, stimulates neurogenesis, and protects against and reverses memory impairment in amyloid β peptide-infused AD rat models by decreasing amyloidogenesis and protects against age-related cognitive decline in the elderly. These results demonstrate a robust link between DHA and cognitive health. Rodents that were fed a diet low in ω-3 polyunsaturated fatty acids, particularly those that were DHA-deficient, frequently suffered from anxiety, depression and memory impairment. Although the exact mechanisms of action of DHA in brain functions are still elusive, a host of mechanisms have been proposed. For example, DHA, which inherently has a characteristic three-dimensional structure, increases membrane fluidity, strengthens antioxidant activity and enhances the expression of several proteins that act as substrates for improving memory functions. It reduces the brain amyloid burden and inhibits in vitro fibrillation and amyloid-induced neurotoxicity in cell-culture model. In this review, we discuss how DHA acts as a molecule with diverse functions.

Upregulation of aquaporin expression in the salivary glands of heat-acclimated rats

It is known that aquaporin (AQP) 5 expression in the apical membrane of acinar cells in salivary glands is important for the secretion of saliva in rodents and humans. Although heat acclimation enhances saliva secretion in rodents, the molecular mechanism of how heat induces saliva secretion has not been determined. Here, we found that heat acclimation enhanced the expression of AQP5 and AQP1 in rat submandibular glands concomitant with the promotion of the HIF-1α pathway, leading to VEGF induction and CD31-positive angiogenesis. The apical membrane distribution of AQP5 in serous acinar cells enhanced after heat acclimation, while AQP1 expression was restricted to the endothelial cells in the submandibular glands. A network of AQPs may be involved in heat-acclimated regulation in saliva secretion. Because AQPs probably plays a crucial role in saliva secretion in humans, these findings may lead to a novel strategy for treating saliva hyposecretion.

Cellular heat acclimation regulates cell growth, cell morphology, mitogen-activated protein kinase activation, and expression of aquaporins in mouse fibroblast cells.

The heat shock response has been extensively studied by a number of investigators to understand the molecular mechanism underlying the cellular response to severe heat stress (higher than 42°C). But, body or tissue temperature increases by only a few degrees Celsius during physiological events. Therefore, the physiological cellular response to mild heat stress rather than severe heat stress is likely to be more important. Repeated exposure to hyperthermia for consecutive 5 days induces heat acclimation which is an adaptive physiological process in humans and animals. However, thus far, the effect of continuous exposure to heat stress on cells has not been fully evaluated. In this study, we investigated an adaptive physiological process that is induced in culture cells by continuous exposure to mild heat stress for 5 days. Exposure to heat activated p38-mitogen-activated protein kinase; inhibited cell growth without apoptosis; and increased the levels of HSPs and HSF-1 in mouse fibroblast cells. Interestingly, exposure to heat regulated the expression of aquaporins and induced morphological change. In a physiological sense, these results suggested that continuous exposure to mild heat stress for 5 days, in which heat acclimation is attained in humans and animals, might induce molecular adaptation to heat in cells.

Aging attenuates acquired heat tolerance and hypothalamic neurogenesis in rats

This study investigated age‐dependent changes in heat exposure‐induced hypothalamic neurogenesis and acquired heat tolerance in rats. We previously reported that neuronal progenitor cell proliferation and neural differentiation are enhanced in the hypothalamus of long‐term heat‐acclimated (HA) rats. Male Wistar rats, 5 weeks (Young), 10–11 months (Adult), or 22–25 months (Old) old, were subjected to an ambient temperature of 32°C for 40–50 days (HA rats). Rats underwent a heat tolerance test. In HA rats, increases in abdominal temperature (Tab) in the the Young, Adult, and Old groups were significantly smaller than those in their respective controls. However, the increase in Tab of HA rats became greater with advancing age. The number of hypothalamic bromodeoxyuridine (BrdU)‐immunopositive cells double stained with a mature neuron marker, neuronal nuclei (NeuN), of HA rats was significantly higher in the Young group than that in the control group. In Young HA, BrdU/NeuN‐immunopositive cells of the preoptic area/anterior hypothalamus appeared to be the highest among regions examined. Large numbers of newborn neurons were also located in the ventromedial and dorsomedial nuclei, as well as the posterior hypothalamic area, whereas heat exposure did not increase such numbers in the Adult and Old groups. Aging may interfere with heat exposure‐induced hypothalamic neurogenesis and acquired heat tolerance in rats. J. Comp. Neurol. 523:1190–1201, 2015.

β-amyloid infusion into lateral ventricle alters behavioral thermoregulation and attenuates acquired heat tolerance in rats

We investigated behavioral thermoregulatory function and acquired heat tolerance of β-amyloid (Aβ)-infused rats. Male Wistar rats were anesthetized and implanted in the intraperitoneal cavity with a temperature transmitter. Aβ peptide (4.9–5.5 nmol) was dissolved in a solvent of 35% acetonitrile and 0.1% trifluoroacetic acid (pH 2.0). The solvent was used as the vehicle. An osmotic pump contained 234 ± 13.9 μl of Aβ solution was subcutaneously implanted in the back and was cannulated into the left cerebral ventricle. Moreover, 0.5 µg of AlCl3 was injected into the right cerebral ventricle with a micro syringe pump (Aβ-infused rats). The solvent-infused rats were used as control rats (CN rats). After 2 weeks, rats were placed in a thermal gradient and their intra-abdominal temperature (Tab) and their ambient temperatures (Ta) selected (Ts) were measured for 3 consecutive days. In an additional study, rats were kept at a Ta of 32°C for 4 weeks to attain heat acclimation. Then, rats were subjected to a heat tolerance test, i.e. they were exposed to a Ta of 36°C for 160 min. Although there were clear day-night variations of Ts and Tab in CN rats, patterns were significantly abolished in Aβ-infused rats. Moreover, heat tolerance obtained by heat acclimation was attenuated in Aβ-infused rats. These results suggest that Aβ-infusion in the lateral ventricle modifies behavioral thermoregulation and lowers an ability to acclimate to heat in rats.

Theobromine up-regulates cerebral brain-derived neurotrophic factor and facilitates motor learning in mice.

Theobromine, which is a caffeine derivative, is the primary methylxanthine produced by Theobroma cacao. Theobromine works as a phosphodiesterase (PDE) inhibitor to increase intracellular cyclic adenosine monophosphate (cAMP). cAMP activates the cAMP-response element-binding protein (CREB), which is involved in a large variety of brain processes, including the induction of the brain-derived neurotrophic factor (BDNF). BDNF supports cell survival and neuronal functions, including learning and memory. Thus, cAMP/CREB/BDNF pathways play an important role in learning and memory. Here, we investigated whether orally administered theobromine could act as a PDE inhibitor centrally and affect cAMP/CREB/BDNF pathways and learning behavior in mice. The mice were divided into two groups. The control group (CN) was fed a normal diet, whereas the theobromine group (TB) was fed a diet supplemented with 0.05% theobromine for 30 days. We measured the levels of theobromine, phosphorylated vasodilator-stimulated phosphoprotein (p-VASP), phosphorylated CREB (p-CREB), and BDNF in the brain. p-VASP was used as an index of cAMP increases. Moreover, we analyzed the performance of the mice on a three-lever motor learning task. Theobromine was detectable in the brains of TB mice. The brain levels of p-VASP, p-CREB, and BDNF were higher in the TB mice compared with those in the CN mice. In addition, the TB mice performed better on the three-lever task than the CN mice did. These results strongly suggested that orally administered theobromine acted as a PDE inhibitor in the brain, and it augmented the cAMP/CREB/BDNF pathways and motor learning in mice.