DHA-enriched fish oil ameliorates deficits in cognition associated with menopause and the APOE4 genotype in rodents

Female APOE4 carriers have a greater predisposition to developing Alzheimer’s disease (AD) compared to their male counterparts, which may partly be attributed to menopause. We previously reported that a combination of menopause and APOE4 led to an exacerbation of cognitive and neurological deficits, which were associated with reduced brain DHA and DHA:AA ratio. Here, we explored whether DHA-enriched fish oil (FO) supplementation mitigated the detrimental impact of these risk factors. Whilst DHA-enriched fish oil improved recognition memory (NOR) in APOE4 VCD (4-vinylcyclohexene diepoxide)-treated mice (p < 0.05), no change in spatial working memory (Y-maze) was observed. FO supplementation increased brain DHA and nervonic acid and the DHA:AA ratio. The response of key bioenergetic and blood–brain barrier related genes and proteins provided mechanistic insights into these behavioural findings, with increased BDNF protein concentration as well as mitigation of aberrant Erβ, Cldn1 and Glut-5 expression in APOE4 mice receiving fish oil supplementation (p < 0.05). In conclusion, supplementation with a physiologically relevant dose of DHA-enriched fish oil appears to offer protection against the detrimental effects of menopause, particularly in “at-risk” APOE4 female carriers

Altered SPMs and age-associated decrease in brain DHA in APOE4 female mice: Brain SPMs and DHA, sex, ageing and APOE genotype

An Apolipoprotein E4 (APOE4) genotype is the most important, common genetic determinant for Alzheimer’s disease (AD), and female APOE4 carriers present with an increased risk compared to males. The study quantified cortical and hippocampal fatty acid and phospholipid profiles along with select eicosapentaenoic acid (EPA)- and docosahexaenoic acid (DHA)-derived specialised proresolving mediators (SPMs) in 2-, 9- and 18-month-old APOE3 and APOE4 male and female mice. A 10% lower cortical DHA was evident in APOE4 females at 18 months compared to 2 months, with no significant decrease in APOE3 or APOE4 males. This decrease was associated with a reduction in DHA-phosphatidylethanolamine. Older APOE4 females had a 15% higher oleic acid content compared to young mice. Although no sex*APOE genotype interactions were observed for SPMs expressed as a ratio of their parent compound, higher cortical (±) 18 HEPE, resolvin D3, protectin D1, 10S,17S-diHDHA, maresin 1, 17S-HDHA and 14S-HDHA were evident in females, and lower cortical 17R-resolvin D1, 10S,17S-diHDHA and (±) 18 HEPE in APOE4. Our findings show a strong association between age, female sex and an APOE4 genotype, with decreased cortical DHA and a number of SPMs, that together may contribute to the development of cognitive decline and AD patholog

APOE4 genotype exacerbates the impact of menopause on cognition and synaptic plasticity in APOE‐TR mice

The impact of sex and menopausal status in Alzheimer’s disease remains understudied despite increasing evidence of greater female risk, particularly in APOE4 carriers. Utilizing female APOE‐TR mice maintained on a high‐fat diet background we induced ovarian failure through repeated VCD injections, to mimic human menopause. At 12 months of age, recognition memory and spatial memory were assessed using object recognition, Y‐maze spontaneous alternation, and Barnes maze. A VCD*genotype interaction reduced the recognition memory (P < .05), with APOE4 VCD‐treated animals unable to distinguish between novel and familiar objects. APOE4 mice displayed an additional 37% and 12% reduction in Barnes (P < .01) and Y‐maze (P < .01) performance, indicative of genotype‐specific spatial memory impairment. Molecular analysis indicated both VCD and genotype‐related deficits in synaptic plasticity with BDNF, Akt, mTOR, and ERK signaling compromised. Subsequent reductions in the transcription factors Creb1 and Atf4 were also evident. Furthermore, the VCD*genotype interaction specifically diminished Ephb2 expression, while Fos, and Cnr1 expression reduced as a consequence of APOE4 genotype. Brain DHA levels were 13% lower in VCD‐treated animals independent of genotype. Consistent with this, we detected alterations in the expression of the DHA transporters Acsl6 and Fatp4. Our results indicate that the combination of ovarian failure and APOE4 leads to an exacerbation of cognitive and neurological deficits

The Influence of APOE Genotype, DHA, and Flavanol Intervention on Brain DHA and Lipidomics Profile in Aged Transgenic Mice

The apolipoprotein E4 (APOE4) genotype is predictive of Alzheimer’s disease (AD). The brain is highly enriched with the omega-3 polyunsaturated fatty acid (n3-PUFA), docosahexaenoic acid (DHA). DHA’s metabolism is defective in APOE4 carriers. Flavanol intake can play a role in modulating DHA levels. However, the impact of flavanol co-supplementation with fish oil on brain DHA uptake, status and partitioning, and according to APOE genotype is currently unknown. Here, using a humanised APOE3 and APOE4 targeted replacement transgenic mouse model, the interactive influence of cocoa flavanols (FLAV) and APOE genotype on the blood and subcortical brain PUFA status following the supplementation of a high fat (HF) enriched with DHA from fish oil (FO) was investigated. DHA levels increased in the blood (p < 0.001) and brain (p = 0.001) following supplementation. Compared to APOE3, a higher red blood cell (RBC) DHA (p < 0.001) was evident in APOE4 mice following FO and FLAV supplementation. Although FO did not increase the percentage of brain DHA in APOE4, a 17.1% (p < 0.05) and 20.0% (p < 0.001) higher DHA level in the phosphatidylcholine (PC) fraction in the HF FO and HF FO FLAV groups, and a 14.5% (p < 0.05) higher DHA level in the phosphatidylethanolamine (PE) fraction in the HF FO FLAV group was evident in these animals relative to the HF controls. The addition of FLAV (+/− FO) did not significantly increase the percentage of brain DHA in the group as a whole. However, a higher brain: RBC DHA ratio was evident in APOE3 only (p < 0.05) for HF FLAV versus HF. In conclusion, our data shows only modest effects of FLAV on the brain DHA status, which is limited to APOE3

Neuroinflammatory processes in cognitive disorders:Is there a role for flavonoids and n-3 polyunsaturated fatty acids in counteracting their detrimental effects?

Neuroinflammatory processes are known to contribute to the cascade of events culminating in the neuronal damage that underpins neurodegenerative disorders such as Parkinson's and Alzheimer's disease. With the ageing population and increased cases of neurodegenerative diseases, there is a crucial need for the development of new strategies capable to prevent, delay the onset or treat brain dysfunction and associated cognitive decline. Growing evidence sheds light on the use of dietary polyphenols and n-3 long chain polyunsaturated fatty acids to improve cognitive performances and reduce the neuroinflammatory and oxidative stress responses occurring with age and neurodegenerative pathologies. This review will summarise the most recent information related to the impact and mechanisms underlying the neuroinflammatory processes in neurodegenerative disorders. We will also detail the current evidence indicating that flavonoids and n-3 polyunsaturated fatty acids are strong candidate in preventing neuroinflammation and modulating age-related memory decline, and will describe the potential mechanisms of action underlying their neuroprotective effects. As such, these dietary bioactives represent important precursor molecules in the quest to develop of a new generation of drugs capable of counteracting neuroinflammation and neurodegenerative diseases

Vascular smooth muscle phenotypic diversity and calcium signaling

Vascular smooth muscle cells (VSMCs) exhibit different physiological properties when they are localized in the wall of large conductance artery like the aorta, or small resistance arteries as resistance mesenteric artery (RMA). Cytosolic Ca2+ increase is crucial for controlling vascular tone. Contractile tension of arteries is mainly regulated by agonist acting on G protein-coupled receptors. In addition, in small RMA, cytosolic Ca2+ increase triggers a myogenic contraction in response to increased intravascular pressure. Cytosolic Ca2+ increase occurs through voltage-operated Ca2+ channels (VOCC) or receptor- or store-operated Ca2+ channels. Mechanisms of cytosolic Ca2+ increase are still not completely elucidated. It has been previously shown that Rho kinase (ROCK) contributes to non-voltage-dependent and non-capacitative Ca2+ entry in rat aorta, and that the Ca2+-dependent kinase of myosin light chain (MLCK) is involved in Ca2+ channels regulation in HEK293 cells. Furthermore, in pathological conditions such as hypertension or atherosclerosis, VSMCs change from a contractile to a non-contractile synthetic proliferative phenotype that can be mimicked by culturing VSMCs. Ca2+ signaling pathways in cultured VSMCs differ from the situation in whole artery. The aim of the present work was to investigate and compare the role of ROCK and MLCK in cytosolic Ca2+ increase regulatory mechanisms between rat large and small arteries, and between whole rat aorta and cultured rat aortic smooth muscle cells. In addition, particular attention was paid to the role of ROCK in Ca2+ signaling and myogenic contraction in response to increased intravascular pressure in RMA. We showed that in fura-2 loaded small RMA, ROCK is not involved in the regulation of cytosolic Ca2+ increase in response to noradrenaline. Conversely, the myogenic response and Ca2+ entry following pressure elevation in pressurized-RMA is dependent on ROCK activity. We also demonstrated that MLCK participates in the VOCC-mediated Ca2+ entry in response to noradrenaline in RMA. In aorta, we further characterized the Ca2+ entry induced by noradrenaline and concluded that actin cytoskeleton is not involved in the ROCK-dependent Ca2+ entry. However, myosin cytoskeleton is a potential candidate for Ca2+ influx regulation as the inhibition of MLCK by ML-7 depresses the VOCC-insensitive Ca2+ entry. MLCK could be therefore responsible for Ca2+ channels trafficking to the plasma membrane in arteries. Additionally, although cultured cells are usually used to investigate cellular functions, we highlighted that in cultured VSMCs, neither ROCK nor MLCK contribute to the vasopressin-induced Ca2+ signaling pathways. The lack of sensitivity to ROCK and MLCK inhibition could result from lower MLCK expression in cultured VSMCs. Eventually, we observed that MLCK regulates differentiation markers as well as Ca2+ channels mRNA expression in the rat aortic cell line A7r5, where MLCK could be involved in the regulation of transcription. Together, our results stress important differences in Ca2+ signaling between conductance and resistance arteries and between whole artery and cultured VSMCs.(SBIM 3) -- UCL, 201

Rho kinase regulation of vasopressin-induced calcium entry in vascular smooth muscle cell: comparison between rat isolated aorta and cultured aortic cells

In addition to its role in artery contraction, Rho kinase (ROCK) is reported to be involved in the Ca(2+) response to vasoconstrictor agonist in rat aorta. However the signaling pathway mediated by ROCK had not been investigated so far and it was not known whether ROCK also contributed to Ca(2+) signaling in cultured vascular smooth muscle cells (VSMC), which undergo profound phenotypic changes. Our results showed that in VSMC, ROCK inhibition by Y-27632 or H-1152 had no effect on the Ca(2+) response to vasopressin, while in aorta the vasopressin-induced Ca(2+) entry was significantly decreased. The inhibition of myosin light chain kinase (MLCK) by ML-7 depressed the vasopressin-induced Ca(2+) signal in aorta but not in VSMC. The difference in ROCK sensitivity of vasopressin-induced Ca(2+) entry between aorta and VSMC was not related to an alteration of the RhoA/ROCK pathway. However, MLCK expression and activity were depressed in cultured cells compared to aorta. We concluded that the regulation of vasopressin-induced Ca(2+) entry by ROCK in aorta could involve the myosin cytoskeleton and could be prevented by the downregulation of MLCK in VSMC. These results underline the important differences in Ca(2+) regulation between whole tissue and cultured cells

Vasorelaxant activity of essential oils from Croton zambesicus and some of their constituents.

In this study, we determined the vasorelaxant activity of essential oils of different samples of CROTON ZAMBESICUS collected in the same area in Benin at different periods and analysed their compositions by GC-FID and GC-MS. 68 compounds were identified among which 20 have not been described previously in this plant's essential oils. We observed quantitative differences among essential oils but all possess significant vasorelaxant activity on intact rat aortae contracted by KCl (IC (50) 5.6-11.8 µg/mL). This activity may, at least in part, be explained by the presence of vasorelaxant diterpenes such as ENT-18-hydroxy-trachyloban-3-one, isopimara-7,15-dien-3β-ol, and ENT-18-hydroxy-isopimar-7,15-dien-3β-ol, previously isolated from the dichloromethane extract of the leaves, but also to linalool (IC (50) 43.4 µg/mL) and particularly to caryophyllene oxide (IC (50) 2.5 µg/mL)

Vascular activity of a natural diterpene isolated from Croton zambesicus and of a structurally similar synthetic trachylobane.

The aim of this study was to determine the vasorelaxant activity of a natural diterpene extracted from Croton zambesicus, ent-18-hydroxy-trachyloban-3-one (DT6), and a synthetic diterpene of similar structure, ent-trachyloban-14,15-dione (DT10) in rat aorta. DT6 and DT10 inhibited aorta contraction in a concentration-dependent manner. Both were more potent inhibitors of KCl-evoked contraction than noradrenaline-evoked contraction. Nitric oxide (NO) synthase inhibition did not significantly affect DT6 effect whereas it significantly decreased DT10 inhibitory potency. In fura-2 loaded aorta rings, DT10 simultaneously inhibited KCl-evoked contraction and cytosolic calcium increase in a concentration-dependent manner. Furthermore, DT10 significantly inhibited calcium channel current recorded by the patch-clamp technique in human neuroblastoma cells SH-SY5Y. However, despite potentiation of 8-bromo-cGMP-response, DT6 and DT10 as verapamil depressed acetylcholine-evoked relaxation, DT6 being the most potent, while only DT6 and DT10 depressed SNAP-evoked relaxation. In conclusion, these data suggest that vasorelaxant activity of diterpenes (DT) is associated with the blockade of L-type voltage-operated calcium channels. Inhibition of NO-dependent relaxation by DT could be related to a decrease in NO availability

Different effect of Rho kinase inhibition on calcium signaling in rat isolated large and small arteries.

In addition to its role in the regulation of artery contraction, Rho kinase (ROCK) was reported to be involved in the cytosolic calcium response to vasoconstrictor agonists in rat aorta and superior mesenteric artery (SMA). However, it remains to be determined whether ROCK also contributes to calcium signaling in resistance arteries, which play a major role in blood pressure regulation. The investigation of the effect of ROCK inhibition on the calcium and contractile responses of rat resistance mesenteric artery (RMA), in comparison with aorta and SMA, indicated that the calcium response to noradrenaline was inhibited by the ROCK inhibitor Y-27632 in aorta and SMA but not in RMA. The effect of Y-27632 on the calcium signal was unaffected by cytochalasin-D. ROCK activation in noradrenaline-stimulated arteries was confirmed by the inhibition of myosin light chain phosphorylation by Y-27632. Moreover, noradrenaline-induced calcium signaling was similarly inhibited by nimodipine in aorta, SMA and RMA, but nimodipine sensitivity of the contraction increased from the aorta to the RMA, suggesting that the contraction was controlled by different sources of calcium. In pressurized RMA, Y-27632 and H-1152 depressed pressure-induced calcium responses and abolished myogenic contraction. These results stress the important differences in calcium signaling between conductance and resistance arteries