Neuroprotective effects of DHA in Alzheimer’s disease models

Lipidomix (JE2482), ENSAIA Institut National Polytechnique de Lorraine, 15, rue du Bois de la Champelle, 54505 Vandœuvre-lès-Nancy, France <Thierry.Pillot@mtm.nancy.inserm.fr> Abstract: Alzheimer’s disease (AD) is a major public health concern in all developped countries. Although the precise cause of AD is still unknown, a growing body of evidence supports the notion that soluble oligomers of amyloid b-peptide (Ab) may be the proximate effectors of synaptic injuries and neuronal death in the early stages of AD. AD patients display lower levels of docosahexaenoic acid (DHA, C22:6; n-3) in plasma and brain tissues as compared to control subjects of same age. Furthermore, epidemiological studies suggest that high DHA intake might have protective properties against neurodegenerative diseases. These observations are supported by in vivo studies showing that DHA-rich diets limit the synaptic loss and cognitive defects induced by Ab peptide. Although the molecular basis underlying these neuroprotective effects remains unknown, several mechanisms have been proposed such as (i) regulation of the expression of potentially protective genes, (ii) activation of antiinflammatory pathways, (iii) modulation of functional properties of the synaptic membranes along with changes in their physicochemical and structural features. We recently demonstrated that DHA protects neurons from soluble Ab oligomer-induced apoptosis. Indeed, DHA pretreatment was observed to significantly increase neuronal survival upon Ab treatment by preventing cytoskeleton perturbations, caspase activation and apoptosis, as well as by promoting ERK-related survival pathways. These data suggest that DHA enrichment most likely induces changes in neuronal membrane properties with functional outcomes, thereby increasing protection from soluble Ab oligomers. Such neuroprotective effects could be of major interest in the prevention of AD and other neurodegenerative diseases.


Introduction
Alzheimer's disease (AD) is a progressive dementia that manifests in early stages primarily as a profound inability to form new memories.Mounting evidence suggests that this syndrome begins with subtle alterations of hippocampal synaptic dysfunction associated with neuronal cell death involving apoptosis [1].The molecular basis for this specificity is unknown, but evidence favors the involvement of neurotoxins derived from the amyloid-b peptide (Ab), a normal product of intracellular proteolysis of a precursor protein (bAPP) [2].Indeed, studies from our group have demonstrated that exposure of cells to soluble Ab could lead to neuronal apoptosis following oxidative stress, pro-inflammatory signals and cytoskeleton perturbation [3,4].Such neurotoxicity strongly suggests that soluble Ab oligomers could be the proximate effectors of the neuronal injury and death occurring in the precocious stages of AD [2].Due to their amphiphilic properties, soluble Ab oligomers may directly interact with the neuronal plasma membrane and affect its functioning [5], thereby initiating intracellular pro-apoptotic signaling pathways.It is thus essential to identify the biological factors that could modulate these early interactions and their fatal consequences.Also, considerable attention has been focused in the past several years on the possible influence of lipid status, especially that of n-3 polyunsaturated fatty acids (PUFA), in the central nervous system on the development of AD.Docosahexaenoic acid (DHA, C22:6 D4, 7,10,13,16,19 ; n-3) represents the longest and the most unsaturated fatty acid (FA) commonly found in biological systems and is mainly present in fish and algae.It is the major n-3 PUFA constituent of the neuronal membranes in the grey matter of the cerebral cortex and in retinal photoreceptor cells [6].Because it is highly unsaturated, DHA is expected to increase the fluidity of neuronal membranes, thereby playing a role in various neurochemical processes in brain [7].Given the enormous economical and societal burdens, there is an enormous medical need for the development of novel therapeutic strategies that target or even better prevent the molecular mechanisms leading to AD dementia.In this review, we wished to provide an overview on the beneficial effect of DHA on brain tissue in general and in the particular context of AD, leading to the idea that dietary DHA supplementation could be an efficient preventive strategy for delaying or preventing AD and other neurodegenerative diseases.

DHA, an essential fatty acid for the central nervous system
DHA is the major n-3 PUFA constituent in the neuronal membranes, present in approximately 30-40% of the phospholipids of the gray matter of cerebral cortex and photoreceptor cells in the retina [6].In the last trimester of fetal life and the first two years of childhood, the brain undergoes a period of rapid growth termed the "brain growth spurt".During this period, the need in this PUFA is dramatically elevated because of the increase in brain size and in relative DHA contents.Animal studies have demonstrated that reductions in perinatal brain DHA accrual are associated with deficits in neuronal arborisation, multiple indices of synaptic pathology including deficits in serotonin and mesocorticolimbic dopamine neurotransmission, neurocognitive deficits, elevated behavioral indices of anxiety, aggression and depression and decreased visual acuity [8].In primates and humans, preterm delivery has been shown to be associated with the same troubles which can be reverted by n-3 PUFA supplementation [9].After the perinatal brain development, DHA intake remains essential for the normal maintenance of brain functions including synaptic plasticity, neurotransmission and vision [10].Because neurons lack the enzymes necessary for de novo DHA and arachidonic acid (AA, C20:4 D5, 8,11,14 ; n-6) synthesis, these FA are derived either directly from the diet or are mainly synthesized from the dietary precursors, a-linolenic acid (ALA, C18:3 D9, 12,15 ; n-3) and linoleic acid (LA, C18:2 D9,12 ; n-6) in liver and in a minor way in cerebral endothelium or in astrocytes from where they are exported to neuronal cells [7] (figure 1).

DHA and Alzheimer's disease
Recent findings suggest a possible role of diet in age-related cognitive decline and impairments.Among the nutritional factors influencing AD occurrence, moderate fish consumption as a proxi of n-3 PUFA intake was related to a reduced risk of impaired cognitive functions [11].In the same way, a recent populationbased study among middle aged women suggests that dietary cholesterol and to a lesser extent saturated FA intake was associated with an increased risk of AD, while consumption of n-3 PUFA such as eicosapentaenoic acid (EPA ; C20:5 D5,8,11,14,17 ) and DHA was associated with a decreased risk of cognitive impairment, independently of differences in age, gender, education, smoking, total energy uptake and cardiovascular risk factor [12].Importantly, lower contents of n-3 PUFA have also been measured in the plasma [13] as well as in the brain [14] of AD patients.Recent in vivo studies have reported that reduction of dietary n-3 PUFA in Tg2576 AD mouse model resulted in a loss of postsynaptic proteins and behavioral deficits, while a DHA-enriched diet could prevent these effects [15].Furthermore, dietary DHA was not only shown to be protective against Ab production, accumulation and toxicity in Tg2576 mice [16] and AD model rats [17], but it could also ameliorate cognitive impairments in Ab-infused rats [18].This there-fore provides a link between neuronal DHA homeostasis, AD pathogenesis and Ab effects.

Neuroprotective effects of DHA
For one simple molecule to affect so many seemingly unrelated processes, DHA must function at a fundamental level, common to most cells such as transcription events, membrane structure and functions and/or signal transduction (figure 2) [19].A nutrigenomic approach with high-density microarrays revealed changes in the expression of brain genes in response to different PUFA-enriched diets.It emphasised significant changes in the expression of several genes, as demonstrated by altered transcription of various genes, including that encoding the Ab-scavenger transthyretin, in hippocampus of aged rats fed with fish oil [20].Some reports have concluded that DHA or fish oil supplementation resulted in antioxidant effects in hippocampus and cortex of an AD model rat [17] as well as in rat hippocampal cultures exposed to glutamate [21].It could then be suggested that the preventive effect described in epidemiological studies could be due to antioxidant properties of this FA.We recently demonstrated that DHA strongly protects rat cortical neurons from soluble Ab oligomer-induced neurodegeneration and apoptosis (figure 3) [22].It is noteworthy that DHA prevents soluble Ab oligomer-mediated cytosketelon perturbation [23], as well as activation of both neutral and acidic sphingomyelinases [24].We also reported that DHA pretreatment preserves the capacity of neurons to phosphorylate ERK1/2 upon exposure to soluble Ab (figure 4).The enrichment of membrane in DHA has thus proven its crucial interest in maintaining a sufficient rate of these phosphorylated/active proteins, whose associated survival pathways are thereby promoted in neurons.This suggests that not only DHA is required as an essential membrane constituent, but it also likely acts as a sensitive switch of major importance for modulating most signaling pathways including cell apoptosis and survival.Further experiments are required to iden- tify the proteins and domains in the plasma membrane that could act as protective sensors able to induce the antiapoptotic response triggered by DHA enrichment in neurons.Focusing on architectural changes PUFA enrichment could induce in neuronal plasma membrane, the most interesting hypothesis to explain neuroprotective effects of DHA might concern its impact on lipid rafts, defined as compositionally distinct platforms for compartmentalizing dynamically regulated signaling assemblies at the plasma membrane.DHA and PUFA enrichment is known to be accompanied by lateral phase separation and local lipid redistribution, leading to membrane remodeling [25].In our experimental model, protection of rat cortical neurons from Ab-induced apoptosis was obtained by supplementing the medium with nanomolar DHA concentrations, likely resulting in DHA enrichment of specific phospholipids species or membrane microdomains.We thus hypothesized that subtle changes could have occurred in rafts, affecting their lipid as well as protein components.Accordingly, we studied the raft-specific ganglioside M1 and flotillin by immunocytochemistry and showed that exposition of cortical neurons to soluble Ab peptide leads to a membrane disorganisation, whereas neurons pretreated with DHA still exhibit intense fluorescence labeling (figure 4).
This could suggest that the apoptosis induced by Ab oligomers involves structural and qualitative changes in lipid rafts, which are prevented after DHA pretreatment.These changes are expected to have functional outcomes in terms of regulating neuronal signaling cascades.

Conclusion
Diet strongly influences the incidence and outcome in major age-related disease including cardiovascular diseases, cancer and dementia such as AD.Several epidemiological and experimental data suggest that DHA intake and enrichment in neuronal membranes could provide a substantial protective effect against these devastating pathologies, which undoubtedly represents one of the most promising preventive approaches to develop with the aim to prevent or to delay the onset of AD.Different ways of action could contribute to the neuroprotective as well as neurotrophic properties of DHA.Further studies are still necessary to identify the preferential mechanism(s) with the view to optimize this approach and to improve its interest on rational and scientifically established bases instead of nutritional or nutraceutical treatments that are still often proposed on empirical considerations.Also, it is likely that the neuroprotective effects of DHA might be further enhanced by coupling the FA  [22] but preliminary results suggest that DHA might not only prevent the initiation of apoptotic cascade but also maintain survival signalling through fine membrane remodelling.The maintenance of the ERK1/2 signalling pathway is here presented as an example of these neuroprotective effects.
to anti-inflammatory and/or antioxidant molecules such as polyphenols, given that many papers have also associated the consumption of vegetables and fruits with a lower AD risk [26].Original DHA-based formulations might therefore provide essential health benefits in preventing AD for which no disease-modifying therapies are currently available.

Figure 2 .Figure 1 .
Figure 2. Implication of DHA in neuronal integrity and survival.

Figure 3 .Figure 4 .
Figure 3. Docosahexaenoic acid prevents neuronal apoptosis induced by soluble Ab oligomers.DHA prevents both cytoskeleton perturbations and apoptosis induced by Ab soluble oligomers as demonstrated by a cytoskeleton integrity analysis after b-tubulin labelling and by a visualization after 4,6-diamidino-2-phenylindole staining respectively[22].