Targeted Suppression of Lipoprotein Receptor LSR in Astrocytes Leads to Olfactory and Memory Deficits in Mice.

Perturbations of cholesterol metabolism have been linked to neurodegenerative diseases. Glia-neuron crosstalk is essential to achieve a tight regulation of brain cholesterol trafficking. Adequate cholesterol supply from glia via apolipoprotein E-containing lipoproteins ensures neuronal development and function. The lipolysis-stimulated lipoprotein receptor (LSR), plays an important role in brain cholesterol homeostasis. Aged heterozygote Lsr+/- mice show altered brain cholesterol distribution and increased susceptibility to amyloid stress. Since LSR expression is higher in astroglia as compared to neurons, we sought to determine if astroglial LSR deficiency could lead to cognitive defects similar to those of Alzheimer's disease (AD). Cre recombinase was activated in adult Glast-CreERT/lsrfl/fl mice by tamoxifen to induce astroglial Lsr deletion. Behavioral phenotyping of young and old astroglial Lsr KO animals revealed hyperactivity during the nocturnal period, deficits in olfactory function affecting social memory and causing possible apathy, as well as visual memory and short-term working memory problems, and deficits similar to those reported in neurodegenerative diseases, such as AD. Furthermore, GFAP staining revealed astroglial activation in the olfactory bulb. Therefore, astroglial LSR is important for working, spatial, and social memory related to sensory input, and represents a novel pathway for the study of brain aging and neurodegeneration.

Use of Active Salmon-Lecithin Nanoliposomes to Increase Polyunsaturated Fatty Acid Bioavailability in Cortical Neurons and Mice.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) play an important role in the development, maintenance, and function of the brain. Dietary supplementation of n-3 PUFAs in neurological diseases has been a subject of particular interest in preventing cognitive deficits, and particularly in age-related neurodegeneration. Developing strategies for the efficient delivery of these lipids to the brain has presented a challenge in recent years. We recently reported the preparation of n-3 PUFA-rich nanoliposomes (NLs) from salmon lecithin, and demonstrated their neurotrophic effects in rat embryo cortical neurons. The objective of this study was to assess the ability of these NLs to deliver PUFAs in cellulo and in vivo (in mice). NLs were prepared using salmon lecithin rich in n-3 PUFAs (29.13%), and characterized with an average size of 107.90 ± 0.35 nm, a polydispersity index of 0.25 ± 0.01, and a negative particle-surface electrical charge (-50.4 ± 0.2 mV). Incubation of rat embryo cortical neurons with NLs led to a significant increase in docosahexaenoic acid (DHA) (51.5%, p < 0.01), as well as palmitic acid, and a small decrease in oleic acid after 72 h (12.2%, p < 0.05). Twenty mice on a standard diet received oral administration of NLs (12 mg/mouse/day; 5 days per week) for 8 weeks. Fatty acid profiles obtained via gas chromatography revealed significant increases in cortical levels of saturated, monounsaturated, and n-3 (docosahexaenoic acid,) and n-6 (docosapentaenoic acid and arachidonic acid) PUFAs. This was not the case for the hippocampus or in the liver. There were no effects on plasma lipid levels, and daily monitoring confirmed NL biocompatibility. These results demonstrate that NLs can be used for delivery of PUFAs to the brain. This study opens new research possibilities in the development of preventive as well as therapeutic strategies for age-related neurodegeneration.

Nanoliposomes and Nanoemulsions Based on Chia Seed Lipids: Preparation and Characterization.

Polyunsaturated fatty acids (PUFA) are important in reducing the risk for cardiovascular, metabolic and neurodegenerative diseases. Chia (Salvia hispanica L.) seeds contain high levels of omega-3 PUFA, α-linolenic acid (ALA) in particular, and are a potential source for development of omega-3 PUFA-based products. Our objective was to obtain and characterize chia seed lipids, focusing on phospholipid fraction, and to investigate their use in the formulation of nanoemulsions (NE) and nanoliposomes (NL). Solvent-based lipid extraction was performed on the ORURO variety of chia seeds, followed by lipid composition analysis using GC and LC-MS and physico-chemical characterization of chia NL and NE. Folch extraction led to a slightly higher yield of ALA as compared to Soxhlet extraction. Lipid, phospholipid, and fatty acid composition analysis of the oil and residue revealed that the residue was rich in phospholipids; these were used to prepare NE and NL. Physico-chemical characterization showed that NE and NL were generally spherical (transmission electron microscopy), with a size of <120 nm under hydrated conditions that remained stable over 5 days. In conclusion, chia oil and phospholipid-rich residue can be used to obtain stable NL or NE using a simple method that involves spontaneous emulsification during lipid hydration, which potentially may be useful in cosmetics, pharmaceutical, and other health applications.

Anxiolytic-like effect of a salmon phospholipopeptidic complex composed of polyunsaturated fatty acids and bioactive peptides.

A phospholipopeptidic complex obtained by the enzymatic hydrolysis of salmon heads in green conditions; exert anxiolytic-like effects in a time and dose-dependent manner, with no affection of locomotor activity. This study focused on the physico-chemical properties of the lipidic and peptidic fractions from this natural product. The characterization of mineral composition, amino acid and fatty acids was carried out. Stability of nanoemulsions allowed us to realize a behavioral study conducted with four different tests on 80 mice. This work highlighted the dose dependent effects of the natural complex and its various fractions over a period of 14 days compared to a conventional anxiolytic. The intracellular redox status of neural cells was evaluated in order to determine the free radicals scavenging potential of these products in the central nervous system (CNS), after mice sacrifice. The complex peptidic fraction showed a strong scavenging property and similar results were found for the complex as well as its lipidic fraction. For the first time, the results of this study showed the anxiolytic-like and neuroprotective properties of a phospholipopeptidic complex extracted from salmon head. The applications on anxiety disorders might be relevant, depending on the doses, the fraction used and the chronicity of the supplementation.

Effects of lactational exposure to benzo[alpha]pyrene (B[alpha]P) on postnatal neurodevelopment, neuronal receptor gene expression and behaviour in mice.

The harmful effects of exposure to benzo[alpha]pyrene (B[alpha]P), which is a neurotoxic pollutant, on mammalian neurodevelopment and/or behaviour as yet remain widely unclear. In the present investigation, we evaluated the impact of the lactational exposure to B[alpha]P on postnatal development of pups and behaviour of young mice. The neurobiological effects of B[alpha]P during lactation were also evaluated on pups' brain. Here, we found that lactational exposure to B[alpha]P at 2 and 20mg/kg affects the neuromaturation of pups by significantly decreasing their reflex as highlighted in surface righting reflex and negative geotaxis tests. However, we noted a significant increase in muscular strength of lactationally B[alpha]P mg/kg-exposed pups, which was probably due to the impact of the exposure to this toxic compound on body weight gain. At the pup stage, lactational exposure to B[alpha]P also provoked a neurobiological change, which was assessed by determination of neuronal receptor gene expression. Indeed, a significant reduction in gene expression of 5HT(1A) receptors in pups exposed to B[alpha]P through lactation was found in comparison to controls. Additionally, attenuation in the expression of MOR(1) mRNA was observed, but statistically significant only in animals receiving the higher dose. Neither the expression levels of ADRA(1D) nor GABA(A) mRNA were altered. Interestingly, the harmful effects of lactational exposure to B[alpha]P on behaviour and cognitive function were still found despite a long post-weaning period. Young mice whose mothers were exposed to B[alpha]P displayed a disinhibition behaviour towards the aversive spaces of the elevated plus maze. Furthermore, a significant increase of spontaneous alternation in the Y-maze was observed, but only in young mice whose mothers were orally exposed to the lower dose of B[alpha]P. Our results suggest a close link between the neurobiological change highlighted in pups' brain and the different behavioural disturbances observed during postnatal development period until young adult stage.

Lactational exposure of mice to low levels of non-dioxin-like polychlorinated biphenyls increases susceptibility to neuronal stress at a mature age.

Lactational exposure to low levels of the sum of the six indicator polychlorinated biphenyls (Σ6 NDL-PCBs, 10ng/kg/day) is known to lead to persistent anxious behavior in young and adult offspring mice at postnatal days 40 and 160, respectively. At more advanced life stages, we evaluated the effects on the mouse brain of neuronal stress induced by the synaptotoxic amyloid-beta (Aß) peptide. Perinatal exposure of lactating mice to Σ6 NDL-PCBs did not affect short-term memory performances of their offspring male mice aged 14 months as compared to control PCB-naive mice. However, following intracerebroventricular injection of soluble Aß oligomers, significant impairments in long-term memory were detected in the mice that had been lactationally treated with Σ6 NDL-PCBs. In addition, immunoblot analyses of the synaptosomal fraction of hippocampal tissues from treated mice revealed a lower expression of the synaptic proteins synaptophysin and PSD-95. Though preliminary, our findings suggest for the first time that early exposure to low levels of NDL-PCBs induce late neuronal vulnerability to amyloid stress. Additional experiments are needed to confirm whether early environmental influences are involved in the etiology of brain aging and cognitive decline.

No evidence for oxidative stress in the cerebellar tissues or cells of juvenile male mice exposed via lactation to the 6 non-dioxin-like PCBs at levels below the regulatory safe limits for humans.

The developing central nervous system is particularly vulnerable to environmental contaminants such as non-dioxin-like polychlorinated biphenyls (NDL-PCBs). This study investigated the potential oxidative effects in mice pups exposed via lactation to the sum of the six indicator NDL-PCBs (∑6 NDL-PCBs) at 0, 1, 10 and 100 ng/kg per 14 days, constituting levels below the guidance values fixed by French food safety agencies for humans at 10 ng/kg body weight per day. For this purpose, the oxidative status was assessed by flow cytometry via dichloro-dihydro-fluorescein diacetate in the cerebellum of juvenile male offspring mice during brain growth spurt [postnatal day (PND) 14]. No significant differences were found in the levels of reactive oxygen species in the cerebellar neurons or glial cells (astrocytes, oligodendrocytes and microglia) of lactationally exposed male mice at PND 14 (p>0.05). Concordantly, oxidative-stress related gene expression was measured by qPCR for catalase, copper zinc superoxide dismutase 1, glyoxalase 1, glutathione peroxidase 1, and glutathione reductase 1, in the cerebellum at PND 14 appeared unaffected, as also verified at the protein level by immunoblots. Moreover, transcriptomic data from our previous work have not shown differences in the mRNA expressions of genes belonging to GO terms involved in oxidative stress in neurons of male mice exposed to ∑6 NDL-PCBs compared to controls; except for glyoxalase 1 which was downregulated in neurons isolated from exposed group compared to controls. Our findings suggest that lactational exposure to NDL-PCBs at environmental relevant concentrations may not cause significant oxidative effect on juvenile cerebellum.

Lactational exposure to low levels of the six indicator non-dioxin-like polychlorinated biphenyls induces DNA damage and repression of neuronal activity, in juvenile male mice.

Previously, we evaluated the effects of lactational exposure to a representative mixture of the six indicator non-dioxin-like polychlorinated biphenyls (∑6 NDL-PCBs) at low levels on the neurobiological changes and developmental/behavioral performances in mice. In this study, we analyzed the global gene expression profile in cerebellar neurons isolated from male mice presenting the most significant induction of anxiety-like behavior in our previous study (10 ng/kg ∑6 NDL-PCBs). Our results revealed changes in the expression of 16658 genes in the neurons of the exposed mice. Among these, 693 upregulated [fold change (FC)>2; p<0.05] and 665 downregulated (FC<2; p<0.05) genes were statistically linked to gene ontology terms (GO). Overexpressed genes belonged to GO terms involved with the cell cycle, DNA replication, cell cycle checkpoint, response to DNA damage stimulus, regulation of RNA biosynthetic processes, and microtubule cytoskeleton organization. Downregulated genes belonged to terms involved with the transmission of nerve impulses, projection neurons, synapse hands, cell junctions, and regulation of RNA biosynthetic processes. Using qPCR, we quantified gene expression related to DNA damage and validated the transcriptomic study, as a significant overexpression of Atm-Atr Bard1, Brca2, Fancd2, Figf, Mycn, p53 and Rad51 was observed between groups (p<0.001). Finally, using immunoblots we determined the expression level of six selected proteins. We found that changes in the protein expression of Atm Brca1, p53, Kcnma1, Npy4r and Scn1a was significant between exposed and control groups (p<0.05), indicating that the expression pattern of these proteins agreed with the expression pattern of their genes by qPCR, further validating our transcriptomic findings. In conclusion, our study showed that early life exposure of male mice to a low level of ∑6 NDL-PCBs induced p53-dependent responses to cellular stress and a decrease in the expression of proteins involved in the generation, conduction, and transmission of electrical signals in neurons.

Neurodevelopmental and behavioral toxicity via lactational exposure to the sum of six indicator non-dioxin-like-polychlorinated biphenyls (∑6 NDL-PCBs) in mice.

In this study, the neurobehavioral toxicity of lactational exposure to a representative mixture of the six indicator non-dioxin-like-polychlorinated biphenyls (∑6 NDL-PCBs 28, 52, 101, 138, 153 and 180) found in contaminated fish matrices were assessed in neonatal (postnatal day 0) to adult (postnatal day 275) mice. Thus, a battery of developmental, behavioral and cognitive tests was performed. The performance of mice whose mothers were orally exposed to ∑6 NDL-PCBs at environmental doses of 1 ng/kg, 10 ng/kg or 100 ng/kg was compared to that of mice whose mothers were orally exposed to vehicle. Our results showed that neonatal offspring mice exposed to ∑6 NDL-PCBs through lactation exhibited significantly longer turning reflexes on postnatal days 7 and 9 (p=0.001, p=0.002, respectively) at 100 ng/kg and showed a reduction in their general activity at 1 ng/kg (p=0.002) and 10 ng/kg (p=0.001) compared to controls. However, these developmental alterations were sex-dependent; only the female reflexes and male locomotor activity were affected. These disturbances were transient, and they disappeared with age. In addition, the males' visuomotor integration was also altered at the doses of 1 ng/kg (p=0.02) and 100 ng/kg (p=0.004), as revealed by the WESPOC test. Nevertheless, lactational exposure to ∑6 NDL-PCBs (1-100 ng/kg) resulted in persistent disturbances despite a long post-weaning period; the exposed mice exhibited anxious behavior that was detected at more progressive life stages, i.e., at postnatal days 40 and 160, using an elevated plus maze and the light/dark choice test, respectively. This persistent anxious behavior could be related to the overexpression of RyR3 in the cerebellum via the disruption of calcium signaling in the neurons. We found no differences in the offspring mice with regard to their cognitive function and mood or mRNA neurotransmitter receptor gene expression in several brain areas, including 5-HT(1A), MOR1 and GABA(Aα1), suggesting the absence of adverse effects of postnatal exposure to ∑6 NDL-PCBs under these conditions. Therefore, our results suggest that regular consumption of contaminated fish matrices by lactating women could be detrimental to the neurodevelopment of their newborns.

Subacute oral exposure to benzo[alpha]pyrene (B[alpha]P) increases aggressiveness and affects consummatory aspects of sexual behaviour in male mice.

Benzo[alpha]pyrene (B[alpha]P) is a neurotoxic pollutant which is also able to affect some behaviour and cognitive function. Here we report that a subacute oral exposure to B[alpha]P increases aggressiveness and affects copulatory behaviour in male mice. Indeed, after 3 weeks of exposure to B[alpha]P at 0.02 and 0.2mg/kg, we have observed that B[alpha]P 0.02 mg/kg-treated male mice are more aggressive than control mice in resident-intruder test because a significant decrease in the latency time of the first attack and a significant increase in the number of attacks in B[alpha]P 0.02 mg/kg-treated mice were found. On the other hand, we have found that subacute exposure (4 weeks) to B[alpha]P, does not affect the appetitive aspects and sexual motivation in copulatory behaviour because the latency to the first mount between control and B[alpha]P-treated male mice was not significantly different. We have nevertheless, surprisingly found that B[alpha]P (0.02-0.2)mg/kg-treated mice have performed significantly more sexual behavioural acts including mounting, intromission latency and intromission frequency than control mice. Although these last results suggest that B[alpha]P improves the consummatory aspects of sexual behaviour, we cannot conclude that this neurotoxic pollutant has advantage of sexual function because B[alpha]P has been shown to alter the monoaminergic neurotransmitter system and causes endocrine dysregulation via toxic effect.