Carriers of an apolipoprotein E epsilon 4 allele are more vulnerable to a

dietary deficiency in omega-3 fatty acids and cognitive decline

Tanya Gwendolyn Nock, Raphaël Chouinard-Watkins, Mélanie Plourde

PII: S1388-1981(17)30135-X
DOI: doi:10.1016/j.bbalip.2017.07.004
Reference: BBAMCB 58177

To appear in: BBA - Molecular and Cell Biology of Lipids

Received date: 26 March 2017

Revised date: 5 July 2017
Accepted date: 15 July 2017

Please cite this article as: Tanya Gwendolyn Nock, Raphaël Chouinard-Watkins, Mélanie
Plourde, Carriers of an apolipoprotein E epsilon 4 allele are more vulnerable to a dietary
deficiency in omega-3 fatty acids and cognitive decline, BBA - Molecular and Cell Biology
of Lipids (2017), doi:10.1016/j.bbalip.2017.07.004

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 ACCEPTED MANUSCRIPT
Carriers of an apolipoprotein E epsilon 4 allele are more vulnerable to a dietary deficiency in omega-3
fatty acids and cognitive decline.

Tanya Gwendolyn Nock, Raphaël Chouinard-Watkins and Mélanie Plourde.

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Research Center on Aging, Centre Intégré Universitaire de Santé et Services Sociaux de l’Estrie-Centre

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Hospitalier Universitaire de Sherbrooke, Canada
Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Canada

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Institute of Nutrition and Functional Foods, Quebec City, Canada

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To whom correspondence should be addressed:
Mélanie Plourde, Ph.D. MA
Research Center on Aging
1036 Belvédère Sud
Sherbrooke, Canada, J1H 4C4
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Tel : 819-780-2220 extension 45664

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Fax : 819-829-7141
Mail : Melanie.plourde2@usherbrooke.ca
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Running title:
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Abbreviations used:
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AA: arachidonic acid

ALA: alpha-linolenic acid
APOE: apolipoprotein E
Aβ : β-amyloid
BBB: blood-brain-barrier
CNS: central nervous system
DHA: docosahexaenoic acid, 22:6 n-3
DHAEE: DHA ethyl ester
E4: epsilon 4 allele of apolipoprotein E
EPA: eicosapentaenoic acid
FA: fatty acid
F2: second generation
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GLUT: glucose transporter
LA: linoleic acid
MRI: magnetic resonance imaging
n-3: omega-3
PBD: peroxisome digenesis disorder

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PEX: peroxisomal biogenesis factor

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Abstract (170 words):

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Carriers of an epsilon 4 allele (E4) of apolipoprotein E (APOE) develop Alzheimer’s disease (AD)
earlier than carriers of other APOE alleles. The metabolism of plasma docosahexaenoic acid (DHA,

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22:6n-3), an omega-3 fatty acid (n-3 FA), taken up by the brain and concentrated in neurons, is
disrupted in E4 carriers, resulting in lower levels of brain DHA. Behavioural and cognitive
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impairments have been observed in animals with lower brain DHA levels, with emphasis on loss of
spatial memory and increased anxiety. E4 mice provided a diet deficient in n-3 FA had a greater
depletion of n-3 FA levels in organs and tissues than mice carrying other APOE alleles. However,
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providing n-3 FA can restore levels of brain DHA in E4 animals and in other models of n-3 FA
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deficiency. In E4 carriers, supplementation with DHA as early as possible might help to prevent the
onset of AD and could halt the progression of, and reverse some of the neurological and behavioural
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consequences of their higher vulnerability to n-3 FA deficiency.

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Introduction
Today, there are around 44 million men and women worldwide with Alzheimer’s disease (AD).
The onset of AD in carriers of an epsilon 4 allele of apolipoprotein E (E4) is 8-15 years earlier than in
carriers of other apolipoprotein E (APOE) alleles [1]. It is unclear as to how E4 modulates AD
pathology but neuropathological changes associated with AD such as β-amyloid (Aβ) plaque

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deposition occur as early as 30 years of age in E4 carriers [2]. However, one intriguing aspect is that

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not all E4 carriers will develop AD suggesting that there is a window of action for environmental risk
factors to modulate the clinical manifestation of AD.

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To be healthy and function well, the brain needs a constant supply of blood to deliver oxygen
and nutrients. The blood-brain-barrier (BBB) is an obligatory interface between the cardiovascular

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system and the brain. Recent evidence suggests that in humans, there is an age-dependent loss of BBB
integrity in the hippocampus [3]. In 2014, two papers published in Nature reported a surprising dual
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role for the BBB transporter Mfsd2a both in establishing BBB integrity and the uptake of
docosahexaenoic acid (DHA) [4, 5], a long chain omega-3 fatty acid (n-3 FA) concentrated in brain
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membranes. Mutations in conserved residues of the MFSD2A gene lead to lethal microcephaly
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syndrome linked to inadequate brain uptake of FAs such as DHA [6]. Mice knockdown for MFSD2A
have significantly lower DHA in their brains, neuronal cell loss in the hippocampus and the
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cerebellum, neurological and behavioural deficits and reduced brain size [5]. The brain has a limited
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capacity to produce DHA de novo and obtains DHA from the plasma. The level of DHA in the plasma
reflects a balance between uptake and release of DHA from organs and tissues. For the past 7 years, we
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have shown that plasma DHA metabolism is disrupted in E4 carriers. Hence, the following sections
will describe evidence supporting the role of n-3 FA deficiency in neurological and behavioural
consequences and explain the rationale behind our hypothesis of a connection between the dysregulated
DHA metabolism occurring in E4 carriers, the neurological consequences observed thereafter, and the
increase in risk of developing AD in E4 carriers.

N-3 fatty acid deficiency and neurological consequences

Consumption of n-3 FA and n-6 FA in the western diet has changed markedly over the 20th
century largely due to the increased production of soybean oil and new food production methodologies.
One study attempted to reconstruct consumption patterns and the availability of essential fatty acids in
traditional methods (1909) and in current practices (1999) to predict the net effects of altered intakes on
the concentration of FA in tissues. A discussion and comparison of reconstructed estimates to available
data from other studies in the prediction of tissue content have been discussed [7]. The study found the
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availability of linoleic acid (LA), an n-6 FA most commonly found in soybean oil, increased from
2.79% to 7.21% of energy and found substantial declines in the dietary availability of n-6 arachidonic
acid (AA), and n-3 FA eicosapentaenoic acid (EPA) and DHA. The dietary increase in n-6 FA and
decrease in n-3 FA results in n-6 FA replacing n-3 FA in the tissue membrane prevents DHA from
carrying out its multiple roles in the neuronal membrane. Predicted net effects of these dietary changes

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included declines in tissue n-3 highly unsaturated fatty acid status by one-third and declines in the

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estimated omega-3 index, a direct measure of erythrocyte EPA + DHA as a percentage of total fatty
acids by half. The apparent increased consumption of LA, has likely decreased tissue concentrations of

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EPA and DHA during the 20th century and provides evidence that the western diet is deficient in DHA
[8]. This has negative consequences because an excess of n-6 FA and a deficient amount of n-3 FA is

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stimulating thrombogenesis, lowering immune response, increasing inflammation and decreasing
neuronal membrane fluidity and function [9-11]. In fact, many experimental protocols in animals now
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commonly use a diet with a composition of 0% DHA as a typical western diet in their methodologies
without citing a rationale for its composition [12, 13]. The ratio of n-6:n-3 FA in the US western diet is
around 15:1. This ratio is lower in most European countries however, and in Nordic countries the ratio
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is below 5:1. Ratios less than 5:1 were associated with decreased mortality and a reduced risk of
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chronic diseases such as colorectal cancer and rheumatoid arthritis by reducing rectal cell proliferation
and suppressing inflammation, respectively [14]. Further to this point, association studies have linked
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the dietary n-6:n-3 ratio to cognitive decline and the incidence of dementia [15]. Since there is no clear
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definition of a deficient diet in n-3 FA in humans, the Western diet is therefore categorized as a diet
low in n-3 FA because of its n-6:n-3 FA ratio of around 15:1.
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Cumulative evidence in animal models beginning in the late 1970’s supports that diets devoid
of n-3 FA produce observable pathologies in the brain which will be discussed herein [16]. Studies of
n-3 fatty acid deficiency span a wide range of animal models including rat, monkey, mouse, guinea pig,
frog, honey bee as well as humans with certain genetic disorders predisposing them to endogenous n-3
FA deficiency [17-20]. Deficiency in n-3 FAs, namely DHA or α-linolenic acid (ALA), modifies the
FA composition of several organs in the body with the greatest changes in FA composition found in
the highly metabolically active tissues of the brain, heart, muscle, retina and liver [21]. Modifications
to FA in n-3 deficiency have been studied in the neuronal membranes throughout the brain with the
greatest loss of n-3 FA at 90% of DHA from control found in the hippocampus of the n-3 deficient
rat brain [22] and are unique to the region of the brain studied [23-26]. Furthermore, an alteration in
fatty acid composition was observed in the synaptosomes and microsomes generated from the
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neuronal membrane [27] as well as a 3-fold reduction in the capillaries of the adult rat brain deficient
in n-3 [28]. In addition to the change in lipid composition of neuronal membranes, a couple of groups
found the neurons of the DHA-deficient rat smaller in size (with no change in density, cell number, or
cell volume) in the hypothalamus and parietal cortex in weaning, in the piriform cortex in maturity, and
in the substantia nigra with corresponding decreases in cell number and total protein quantity [22, 29-

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31]. In addition to characterizing the neuron, a couple of groups looked at neuronal plasticity in the n-3

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FA deficient developing frog and rat. In the frog, a decrease in dendritic branching with a smaller
dendritic arbor was observed and in the rat, an increase in axonal sprouting and a delay in their pruning

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was observed in one study and decreased growth cone formation in another [20, 32, 33]. Altered
development of the neuron may be the result of a concomitant decrease in nerve growth factor, known

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to maintain survival and stimulate differentiation of cells [34].
The modifications in lipid composition from n-3 FA deficiency are also dependent on diet
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[35-37], stage of development [38-40], age [41], sex [42], generational n-3 FA status and any
combination of these factors. Generational n-3 FA status indicates whether the diet of the previous
generation(s) was also deficient in n-3 FA, for example, mice pups fed an n-3 FA–deficient diet,
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whose mothers were also fed the same diet, had a 21% reduction in DHA brain content [43] in
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comparison to studies using a multigenerational model or chronic studies of n-3 FA deficiency which
generate a larger decrease of ~75% in brain DHA content [44]. In general, the reduction in n-3 FA of
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the lipid membrane is met by a compensatory increase in docosapentaenoic acid (22:5 n-6) to maintain
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22-C levels and consistent brain volume in white and gray matter during aging [31].
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Overall, levels of DHA in the brain are lower in dietary n-3 FA deficient animals compared to
controls in the frontal cortex, striatum and cerebellum.

Human syndromes with inherent n-3 FA deficiency

In humans, certain genetic disorders result in a deficiency of n-3 FA in afflicted individuals.
Most notably, peroxisome biogenesis disorders (PBD) are characterized by biochemical abnormalities,
including low DHA in the brain, retina and other tissues [45]. Peroxisomes perform the biogenesis of
DHA and in Zellweger syndrome, the most severe PBD initially referred to as cerebro-hepato-renal
syndrome [46], there is an absence of all peroxisomes from all organs. Children with Zellweger
syndrome have a profound deficiency in DHA and the syndrome is usually fatal within the first year of
life. Neuropathology of the disease has been studied using mouse models of Zellweger syndrome with
brain-restricted deficiency of the peroxisome biogenesis factor (PEX) 13, a cytosolic docking factor in
the peroxisome translocation machinery [47]. Pex13(-/-) mice reproduce many of the features of
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Zellweger syndrome and PEX13 deficiency in humans [48]. The brains of these animals showed
disordered lamination in the cerebral cortex, consistent with an observed developmental delay in
neuronal migration leading to neuronal dysfunction. Mice homozygous for the targeted deletion of the
peroxisome integral membrane protein, PEX2, developed the same delay in migration resulting in the
development of mice with such severe cerebellar defects as abnormal Purkinje cell development and an

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altered folial pattern. Inbred mice with a PEX2 mutation show significant embryonic lethality and

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widespread neuronal lipidosis throughout the brain. Biochemical analysis of PEX2 mutant mice shows
the characteristic accumulation of very long chain fatty acids as well as a deficiency in plasmalogen, a

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phospholipid essential for proper brain function. DHA levels were reduced in the brain of mutant mice
[49]. Attempts to explain the neuropathogenesis have implicated peroxisomal metabolic dysfunction,

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and more specifically the loss of peroxisomal products, such as plasmalogens and DHA, and the
accumulation of peroxisomal substrates, such as very-long-chain-fatty acids [50]. In a particular case of
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human, LA deficiency of a 6 year old, similar neurological abnormalities were observed [51, 52].
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Behavioural Consequences of n-3 FA deficiency

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One of the most important outcomes of n-3 deficiency is that it negatively affects certain animal
behaviours presumably the result of structural and functional changes in the brain that manifest in
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learning and locomotor defects [37, 53-55]. These behaviours can be measured using established tests
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with measurable outcomes. Impaired learning performance has been observed in several experimental
paradigms of n-3 deficiency using a number of behavioural measures including the Morris water maze,
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the Barnes Maze, elevated plus maze and the learning of olfactory or visual discrimination tasks [26,
37, 44, 56-58]. Impairments in learning were supported by the expression pattern of genes involved in
cerebral activity. The expression of c-fos mRNA was induced in the olfactory bulb, piriform and
neocortex, the areas of the brain depleted of n-3 fatty acids, whereas glucocorticoid-induced receptor
and GLUT1 mRNA were absent in the piriform cortex, indicating a lack of cerebral activity in the area
processing olfactory cues not detecting the smells [26]. The second generation (F2), n-3-deficient adult
male rats had 82% decrease in DHA and showed impaired learning- and memory-related behaviors.
This was observed by significantly more total errors in a 7-problem, 2-odor discrimination task
compared to the n-3-adequate group [44].

F2 generation, n-3 deficient young adult mice had 50% decrease in DHA and had learning impairments
via passive avoidance test [17] supporting evidence for learning impairment on habituation [54, 55] and
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the elevated plus maze learning protocol [53]. Despite a 76% lower brain DHA, n-3-deficient rats were
able to acquire most simple 2-odor discrimination tasks, but were deficient in the acquisition of a 20-
problem olfactory learning set. This deficit could not be attributed to changes in sensory capacity and
rather instead, appeared to represent a deficit in higher order learning [59].

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A neuroimaging study found that resting-state functional connectivity among prefrontal cortical
networks was impaired in monkeys raised on an n-3 FA deficient diet compared with monkeys raised

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on fish oil-fortified diet [60]. Specifically, n-3 FA deficiency during perinatal development was
associated with reduced resting-state connectivity between multiple regions in the brain. Altogether,

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impairment of learning discrimination tasks as well as MRI studies support the hypothesis that DHA is
involved in the communication with structures involved in higher order learning involving multiple

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brain regions.
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One study also reported that ALA deficiency reduced the appetite, which can thereafter lead to other
nutrient deficiencies [61]. Mice pups from n-3 FA deficient mothers fed an n-3 FA-deficient diet had
reduced sensitivity to sucrose and a reduced ability to feel pleasure which can also lead to reduced
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appetite and further nutrient deficiencies [61, 62].

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Due to the fact that multigenerational studies of n-3 FA deficiency have shown a larger decrease in n-3
FA accrual in the brain, a number of groups looked at the resultant behavioural outcomes. For instance,
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in a second generation n-3 FA deficient rat model, a model made to replicate the dietary deficiency of
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current adolescents today, cognitive and motivated behaviour impairments distinct from the deficits
observed in adults were observed and these include behavioural measures that reflected hyperactivity,
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increased anxiety, increased goal-irrelevant and decreased goal-directed activity, and reduced
behavioural flexibility [41]. These behavioral deficits in animals fed a diet deficient in n-3 FA could be
because DHA deficiency impairs communication between neurons and glia of the brain and between
the brain and the vascular system [63, 64]. Indeed, cortical DHA deficiency during development is also
associated with neuroinflammation [65]. It was previously shown that n-3 FA-deficient rats have
impaired communication between neurons and glia during the reduction of electrical brain activity
following stimulation in addition to cortical spreading depression, something that is more pronounced
in second generation n-3 FA deficiency in rats [66]. Deficiency in n-3 FA was also shown to affect
brain energy metabolism by modulating glucose through the expression of brain glucose transporters,
Glut1 and Glut3 [67-69] as well as mRNA of GLUT1 [26]. The level of Glut1 may be responsible for
learning and memory deficits since neuronal activation requires an enhanced glucose demand [70].
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Glucose transport was decreased by 30% and utilization via cytochrome oxidase activity was decreased
by 20-40% in the three brain regions that share a high rate of energy metabolism [69]. Therefore, there
are a number of studies supporting that n-3 FA deficiency leads to deficits in behavior and function of
the brain.

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Effect of dietary n-3 FA deficiency on anxiety-like and depressive-like behaviours

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Nutritional n-3 FA deficiency produces anxiety and depressive-like symptoms [71-74]. Behavioural

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studies suggest that development deficits in brain DHA accrual are associated with elevated
behavioural indices of depression that emerge after puberty [39, 75]. In contrast, dietary fish oil

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fortification significantly decreases depression-like behavior similar to antidepressant medications [76,
77]. Perinatal n-3 FA deficiency is associated with increased home cage stereotypy and locomotion
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bouts [78] which is consistent with dysregulated mesolimbic dopamine activity.

Recent neuroimaging findings suggest that low n-3 FA intake may impair cortical structural and
functional maturation in corticolimbic regions repeatedly implicated in psychopathology [79]. Indeed,
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rats subjected to n-3 FA deficiency during 15 weeks from weaning display higher depressive-like
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symptoms in the forced swimming test [75, 80].

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A few groups have reported that n-3 FA deficiency appears to abolish the role of endocannabinoid
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signaling in long-term potentiation in different areas of the brain required for the consolidation of
memories [81-83] and behaviourally, these mice display increased anxiety-like behaviour [71].
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Organisms such as the honey bee use spatial and time-dependent cues for disseminating information of
food location to the hive. A diet deficient in n-3 FA delayed proper learning by conventional
behavioural tests. In addition, the hypopharyngeal glands, used for collecting pollen, were smaller in
the n-3 FA deficient bees which further hampers proper nutrient intake in this model [19]. Altogether,
there is evidence in animal models suggesting that dietary n-3 FA deficiency contributes to anxiety-
like- and depressive-like behaviours.

N-3 deficiency and dysrupted Dopaminergic Pathway

There are some studies that looked at dietary deficiency in n-3 FA and the dopaminergic system.
Dopamine is a major neurotransmitter in the central nervous system (CNS) with three major projections
arising from the dopamine perikarya in the substantia nigra and ventral tegmental area. The substantia
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nigra plays an important role in reward and movement and the ventral tegmental area is critical to an
organism's capacity to detect rewards and novelty and to enlist appropriate behavioural responses. A
decrease in dopamine availability was observed in the frontal cortex of n-3 FA deficient rats [62, 84] as
well as other areas of the brain [85] and is accompanied by a prolonged compensatory increase in
dopaminergic receptor levels [86]. The increase in receptor levels continues to rise up to 4 weeks

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suggesting that the dopaminergic system is adaptive to n-3 FA deficiency although inadequate in the

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absence of supplementation. There is also evidence supporting the idea that n-3 FA deficiency in rats
leads to a 40–75% lower level of endogenous dopamine in the frontal cortex according to age and a

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10% reduction in density of dopaminergic D2 receptors [62]. Reduced central dopaminergic function
could be the result of a structural change at the synapse. One group showed lower levels of dopamine

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release in the frontal cortex and nucleus accumbens because of a depletion of the dopamine vesicular
storage pool in animals fed a deficient diet in n-3 FA [87]. It is noteworthy that not all areas of the
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brain, such as the corpus striatum and the striatum, have a dopaminergic vulnerability to n-3 FA
deficiency [23, 88]. Impaired rat spatial learning in the Barnes maze [57] may be the behavioural
manifestation of altered dopamine release as well as impaired neurogenesis in the dentate gyrus of the
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hippocampus [89]. The mechanism leading to this modification is yet unknown and could involve
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vesicle turnover, membrane fluidity, or vesicular monoamine transporter. Another group conducted
studies in a second generation n-3 FA deficiency rat model due to its similarities in the trajectory of n-3
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FA deficiency to that of the adolescents of today. The interest in adolescents stems from the reality that
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they are the second generation of humans that would have been born and first been exposed to n-3 FA
deficiencies in the 1960s and 1970s (4-5 decades ago) when dietary trends toward decreased
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consumption of these fats began. The group found an elevation of dopamine synthesis specific to the
dorsal striatum and unique to the adolescent stage. Surprisingly, in this model, a dietary n-3 FA
deficiency across consecutive generations increased dopamine availability [41]. These findings are in
agreement with recent reports of enhanced dopamine availability in young individuals at high risk of
developing schizophrenia [90]. Hence, it is not clear if today’s increased rate of neurological disorders
such as attention deficit hyperactivity disorder, autism and schizophrenia are linked to the current
multigenerational n-3 FA deficiency or to better diagnosis at earlier stages.

E4 allele and DHA deficiency: E4 carriers might be particularly vulnerable to a dietary deficiency in
n-3 FAs as demonstrated by an experiment we conducted on 14C-DHA uptake in the brain in which E4
mice were fed a diet deficient in n-3 FAs. The plasma DHA pool is critical to bring DHA to the brain
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and it is dynamic, constantly exchanging FA with organs and tissues. Under conditions of chronic low
dietary n-3 FA, the liver usually upregulates its ability to synthesize DHA and presumably receives
ALA from the adipose tissue. Hence, it is suggested that there is an adipose-liver-brain FA axis [91].
We have shown that under an n-3 FA deficient diet, E4 mice had similar plasma DHA levels to that of
E3 mice, but the liver and the adipose tissue DHA levels were ~46% lower in E4 mice than in E3 mice

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fed the same diet, suggesting that to maintain plasma DHA levels, E4 mice had to pull DHA from, or

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prevent DHA getting into, the liver and the adipose tissue (Figure 1) [92]. In terms of lipid
metabolism, there is evidence for both decreased tissue uptake as well as a global metabolic shift

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towards lipid oxidation and enhanced thermogenesis in E4 mice. Specifically, one study showed E4
mice displayed elevated markers of insulin resistance, a reduced respiratory quotient during the

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postprandial period (0.95±0.03 versus 1.06±0.03, P<0.001), indicating increased usage of lipids as
opposed to carbohydrates as a fuel source. Finally, E4 mice showed increased body temperature
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(37.30±0.68°C versus 36.9±0.58°C, P=0.039), augmented cold tolerance and more metabolically active
brown adipose tissue compared with E3 mice [93]. Furthermore, our group showed that E4 mice had
higher levels of liver carnitine palmitoyltransferase 1, a protein needed for the entrance of FA in the
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mitochondria and their oxidation thereafter [94].

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In support of decreased uptake, another group found that a difference in a single amino acid between
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apoE4 and apoE3 changes the organization and stability of both the N-terminal helix bundle domain
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and separately folded C-terminal domain conferring an enhanced lipid binding ability of apoE4 over
apoE3 to the surface of very low density lipoprotein particles. This impairs the lipolytic processing in
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the circulation so that apoE4 is associated with a more pro-atherogenic lipoprotein-cholesterol

distribution (higher very low density lipoprotein-cholesterol/high density lipoprotein-cholesterol ratio)
[95]. Therefore, in the long term, E4 carriers will have a flat reservoir of ALA and DHA, hence
accentuating their vulnerability to n-3 FA deficiency. We have data in 8-12 months E4 mice showing
that ALA levels in the liver and the adipose tissue were >80% and >40% lower, respectively, compared
to the levels in E3 mice when fed a deficient diet in n-3 FA (Figure 1). Moreover, E4 mice fed the n-3
FA-deficient diet had ~80% lower liver ALA/LA ratio, than in E3 mice (control). This ratio gives an
indication of Δ6 desaturase activity which is an indicator of the capacity to convert ALA in DHA.
Hence, carrying the E4 allele tended to amplifies the DHA deficiency in the liver, adipose tissue and
brain, and this process might be accentuated during aging because there is a loss of Δ6 desaturase
activity [96].
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Brain function in E4 carriers in cognitive decline: In E4 carriers, brain atrophy is more pronounced
compared to that of the non-carriers [97]. In E4 mice, it was shown that vascular defects precede
neuronal dysfunctions and can initiate neurodegeneration [1, 98]. In E4 mice, cerebral vascularization
is reduced and vascular walls are thinner compare to mice carrying other APOE alleles [99]. This result
is consistent with human post-mortem brain observations where the basement membrane surface area

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was lower in E4 carriers compared to the non-carriers [100]. One recent study conducted on post-

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mortem human brains reported that pericyte degradation was accelerated in E4 carriers with AD > non
carriers and with AD > non-AD control [101]. Pericytes maintain the integrity of the BBB and

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degenerate in AD. To our knowledge, it is not clear when disruption of BBB permeability and cerebral
vascularization including the number, size and length of veins and arteries are initiated in humans and

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mice with E4 allele. Previous studies reported lower integrity of the BBB in E4 mice compared to E3
mice [99]. MA
Brain function in cognitively intact E4 carriers: Data from MRI studies seems to provide strong
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evidence of lower brain mass in AD-afflicted areas of healthy E4 carriers (not known to have Aβ
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plaques or phosphorylated tau tangles) compared to non-carriers. These brain-based studies have been
done with similar findings in neonates [102], infants (2-25 months), [103], and children and
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adolescents (aged 8– 20) [104]. The evidence on cognitive effects of the E4 allele in midlife however,
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has been mixed [105]. One study demonstrated that the E4 allele disrupts the resting state connectivity
in MRI in the healthy older individuals (mean age = 62) [106]. In another study, there were no
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structural differences in hippocampal or frontal volumes with respect to E4 carriers in healthy

professionals without any presenting memory problems and without selection for a family history of
AD. The data does show that late-middle age and older E4 carriers have lower memory recall
performance than non-carriers and that memory recall was correlated positively with hippocampal
volume. Their findings point to basic memory testing as a sensitive tool for detecting E4-related
influences on memory in aging workers [107]. However, most of those studies used standardized
neuropsychological tests, which may not be sensitive to subtle cognitive change (e.g., Kozauer et al.,
[108] used the Mini-Mental State Exam) since deficits in working memory and semantic memory have
been shown to be the earliest functions to undergo decline during the AD prodrome [109]. Greenwood
et al., [110], have also previously validated an information-processing working memory task sensitive
to APOE genotype in middle-age [111, 112].
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Using an advanced dynamic contrast-enhanced magnetic resonance imaging protocol with high spatial
and temporal resolution to quantify regional BBB permeability in humans, the team of Zlokovic
reported age-dependent BBB permeability damage in the hippocampus and its CA1 and dentate gyrus
subdivisions [3]. Cerebrospinal fluid markers of BBB permeability damages such as albumin quotient,
fibrinogen and CypA seems to be greater in late-onset AD patients and E4 carriers compared with

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neurologically healthy human [113]. More importantly, young cognitively normal E4 carriers have

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impaired cerebrovascular reactivity in response to memory task and CO2 inhalation [114]. However,
the age at which BBB permeability is damaged in E4 carriers remains elusive but having this

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information could help us to design intervention to limit progression of these damages. One potential
intervention is to provide DHA in the diet of E4 carriers but this may be conditional to DHA efficiency.

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Providing DHA in the diet has recently been shown to improve cerebrovascular parameters in several
different scenarios. In a rat model of transient focal cerebral ischemia, the integrity of the BBB was
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improved by a reduction in the expression of the intercellular adhesion molecule-1, an increase in the
expression of collagen IV and a decrease in the extravasation of fluid from the vessels into the tissue in
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DHA-treated rats [115]. Another group, used transgenic Mfsd2a-/- mice [4] and showed that genetic
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deletion of Mfsd2a leads to BBB defects in the developing CNS. Furthermore, Nguyen et al. [116]
showed that Mfsd2a-/- mice have significantly reduced levels of DHA in brain accompanied with
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neuronal cell loss in the hippocampus and cerebellum, neurological and behavioral deficits, and
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reduced brain size. Mfsd2a is responsible for establishing a unique lipid environment that inhibits
caveolae vesicle formation in CNS endothelial cells to suppress transcytosis and ensure BBB integrity
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[117]. Addullah et al., [118], observed an E4 dependent difference in mfsd2a expression and DHA-
containing phospholipids. Among AD patients, E4 homozygotes had lower expression of mfsd2a than
E4 heterozygotes and non-carriers and lower of expression of mfsd2a correlated to lower quantity of
DHA and a higher quantity of AA in the phospholipids of the membrane [119].

We observed a shorter latency in the Barnes maze test in E4 mice fed a DHA diet as compared to E4
carriers fed a control diet [120]. One could hypothesize the improvement in cognitive decline may be
the result of an interaction with DHA and the BBB but this has yet to be investigated. One of the
primary events in cognitive decline is a breakdown of the BBB and neuroinflammation so potentially,
providing DHA in the diet to E4 carriers could restore the integrity of the BBB if provided prior to the
irreversible stage of decline [121].
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E4 allele modifies brain DHA levels? APOE regulates lipid transport and metabolism [122]. The
brain has its own pool of apoE [123] that plays critical roles in lipid transport to neurons. Using in situ
intracerebral perfusion, we showed that brain uptake of 14C-DHA was 24% lower in 4- and 13-month-
old E4 mice compared to uptake in E2 mice but that cortex DHA was significantly lower only in 13-
month-old E4 mice. This suggests that in E4 mice, lower cortex DHA levels are age-dependent but

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plasma DHA relative % was higher in E4 mice [124]. This pattern i.e., lower brain DHA uptake, was
14

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observed in mice knocked-down for the Mfsd2a transporter [6]. In the study using C-DHA brain
uptake, mice were fed a diet deficient in n-3 FA. Therefore, DHA imbalance in E4 mice might

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contribute to the loss of BBB integrity in E4 carriers. Yassine et al also proposes two other mechanisms
of how the E4 allele can be involved in brain DHA levels. One of these mechanisms is an increased

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level of liver catabolism of DHA in E4 carriers compared to non-carriers as it was outlined by
Chouinard-Watkins et al [125]. The other mechanism suggested by this group is a hypolipidated, or
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decreased number of, apoE particles leading to less efficient transport of DHA in the brain [126]. There
is also an association between DHA and A deposition in pre-dementia [127] but not during or after
the disease has expressed suggesting that timing is important to intervene to delay onset of the disease.
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Is it possible to rebalance n-3 FA deficiencies and to reverse neurological consequences?

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There is ample evidence in the literature that n-3 FA supplementation following deficiency allows for
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the rebalancing of the fatty acid composition in the cell membranes and capillaries of the brain in
animal models and human syndromes of n-3 deficiency when administered in the therapeutic window.
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Studies of DHA repletion include but are not limited to, the monkey [128, 129], maternal rats [130], F2
generation rats of DHA deficiency [131] and Zellweger syndrome [45, 132].

The rebalancing of DHA in the brain after a period of n-3 FA deficiency occurs over time in the young
adult F2 generation rats studied with partial recovery of 20% of the n-3 adequate group at 1 week rising
to 35% at 2 weeks and completely resolving at 8 weeks after implementation of the DHA repletion diet
[131]. The slow reversal of DHA accrual in the brain is in contrast to the repletion levels of DHA in the
serum and liver were approximately 90% and 100% replaced, respectively, within 2 weeks of diet
repletion. Similar results were obtained in intrauterine n-3 FA deficient monkeys, depleted of 70-90%
of n-3 FA at birth, requiring 15 weeks for total recovery of DHA in the cerebral cortex in contrast to
the required 4 weeks and 8 weeks for plasma and erythrocytes, respectively [128]. The difference in the
amount and time course of DHA accrual in the nervous system compared to the liver and circulation
 ACCEPTED MANUSCRIPT
suggests that transport-related processes may limit the rate of DHA repletion in the brain. There was a
recovery of n-3 FA in the capillaries of an adult rat after substitution with a non-deficient diet however,
the recovery was slow and resolved over 2.5 months in contrast to a relatively immediate recovery in
the choroid plexus [28]. Switching to a fish oil-supplemented diet induced a recovery in DHA content
in the frog embryos within 20 weeks and diminished the deprivation effects observed on neurons

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involved in vision [20]. In maternal rats, fish oil supplementation on an n-3 FA deficient diet during

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pregnancy and lactation, prevents depletion of maternal brain regional DHA levels, and reduces anxiety
as observed in the elevated plus maze test [133]. In aged mice, there were no significant differences

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between those fed an n-3 FA deficient diet throughout their lifespan and those supplemented with ALA
after an n-3 FA deficient diet in the open field test, used to assess capability and drive in exploratory

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activity [134]. However, the impaired learning performance observed in Morris water maze in mice fed
the n-3 deficient diet was completely restored in old n-3 FA deficient mice supplemented with DHA
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[56]. In human studies of patients afflicted with Zellweger Syndrome, normalization of DHA levels in
the brain was achieved by a quantity of 100-500 mg/day of DHA ethyl ester (DHAEE) for variable
periods of time. The daily doses varied between 100 mg and 500 mg of DHAEE, depending on age and
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degree of DHA deficiency, but never on a body-weight basis. This was done because the brain has the
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largest DHA requirements during early development; so that the youngest patients with a marked DHA
deficiency after regular plasma fatty acid analysis received the highest doses. Improvement was
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observed in myelination via MRI and this translated into a marked improvement in liver function,
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growth, muscle tone, vision, and social contact in those patients, most significantly when the treatment
was started before 6 months of age [45]. In contrast, DHA supplementation of 100 mg/kg/day did not
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improve the growth of treated individuals with PBD with an inherent n-3 deficiency during an average
of 1 year of follow-up in patients aged 1 to 144 months [135]. A similar result occurred in a patient
with classic Zellweger syndrome treated with DHAEE for three months during which the patient
showed no clear clinical improvement over the short period of treatment. Five other patients with
Zellweger variants (four of them under one-year-old and a five-year-old) were also treated with
DHAEE until normalization of the DHA levels in erythrocytes and in general, had accelerated
developmental curves with the infants becoming more alert and acquiring better social contact [136].
Follow-up to these patients, MRI has shown advances in brain myelination [137]. In the case of the 6
year old child with human ALA deficiency, when the diet was supplemented to an emulsion containing
linolenic acid, the neurological symptoms disappeared [51].
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At the molecular level, dysregulation of multiple neurotransmitter systems in cortical DHA deficiency
is reversible with early, but not later, postnatal n-3 FA supplementation [38, 138]. For example, n-3 FA
supplementation before weaning to n-3 deficient rats, is able to reverse changes in serotonergic
transmission however, increased basal serotonergic transmission and decreased stimulated release
persisted if supplementation was introduced after weaning [138]. Therefore, there is evidence

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supporting the idea that tissue n-3 FA deficiency can be rebalanced if n-3 FA are provided in the diet at

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early stages and doing so, may reverse some of the neurological consequences caused by the n-3
deficiency. In humans, there is evidence that an increase in consumption of DHA via supplementation

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could improve cognitive decline by decreasing the level of inflammation in the brain. One study
demonstrated the ability of DHA to transfer from the plasma to the CSF in AD patients and to modify

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the level of inflammation in the brain. The increase of DHA in CSF, and therefore uptake in the brain,
was inversely correlated with CSF levels of total and phosphorylated tau markers of AD and directly
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correlated with soluble interleukin-1 receptor type II, an anti-inflammatory marker. [139]. Thus, as
DHA increased in CSF, markers of AD decreased and anti-inflammatory markers increased [139].
Another group demonstrated the ability of EPA and DHA to reduce inflammation by increasing
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phagocytosis of Alzheimer’s disease related-Aβ42 by human microglia, shown by decreasing pro-

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inflammatory markers CD40 and CD36 and increasing production of brain-derived neurotrophic factor
[140]. However, the direct link between plasma DHA, brain DHA and risk of cognitive decline is not
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crystal-clear. For instance, vegetarians have 50% lower plasma levels of n-3 FA compared to that of
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omnivores but the overall mortality in vegetarians does not differ significantly from that of omnivores
[141]. Therefore, blood DHA levels may not be linearly related to tissue DHA levels. Moreover, one
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would have thought that the n-6:n-3 ratio of the vegetarian diet would be lower than that of the Western
diet, which would reduce competition for Δ6 desaturase and improve conversion of ALA to EPA and
DHA. However, Kornsteiner et al [142] reported that in Austria, the vegetarian and vegan diet have an
n-6:n-3 ratio of 10:1. Therefore, the n-6:n-3 ratio is not tremendously lower in vegetarians and vegans
compared to those eating the Western diet hence suggesting that there is probably something else than
dietary fatty acids in the diet somehow protecting vegetarians and vegans from chronic diseases and
risk of cognitive decline. Also important, long-term dietary restriction of n-3 FA such as following a
vegetarian diet, could upregulate the expression and activity of the enzymes elongase and desaturase
increasing its sensitivity, as found in studies of cats and rodents in order for the liver to make DHA and
transport it to the brain thereby maintaining cerebral levels [143, 144]. The relation of plasma DHA to
brain DHA concentration and metabolism in humans has been previously investigated. There was a
slight correlation between plasma and brain n-3 FA with DHA and one area of the brain but only in
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non-cognitively impaired indicating a tenuous link between plasma and tissue levels in the healthy
human brain [146]. Despite apparently low DHA intake in AD, brain DHA levels are frequently the
same as in controls, suggesting that low DHA intake results in low plasma DHA but does not
necessarily reduce brain DHA in humans [145, 146]. One study determined the change in whole-body
DHA synthesis rates from varied amounts of dietary ALA [147]. Rats fed a diet with adequate ALA

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had a 2-fold greater capacity to synthesize DHA than did rats fed either a diet rich, or deficient, in ALA

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and a DHA synthesis rate that was similar to that of rats fed the diet high in ALA. However, rats fed
the diet deficient in ALA had a 750% lower DHA synthesis rate than rats fed the diet adequate or rich

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in ALA. Therefore, increasing dietary ALA from 3% to 10% of FA did not increase DHA synthesis
rates, because of a decreased capacity to synthesize DHA in rats fed the diet high in ALA. Another

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study using intakes, food sources, and status of n−3 FAs according to dietary habit (fish-eaters and
non-fish-eating meat-eaters, vegetarians, or vegans), estimated the conversion between dietary ALA
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and circulating n−3 FAs [148]. They found n-3 FA intakes in non-fish-eaters were 57-80% of those in
fish-eaters, but tissue differences were considerably smaller potentially due to a higher conversion rate
of ALA with low intake rates.
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Is it possible to rebalance n-3 FA metabolic disturbances in E4 mice? In a recent experiment, we

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fed E4 mice a control diet containing ALA (0.14%) or a diet rich in DHA (0.7%). In mice fed the
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control diet, E4 mice had twice as much DHA in the liver compared to E3 mice (Figure 2). ALA levels
in the liver were lower, but Δ6 desaturase activity was also estimated to be lower in E4 mice than in E3
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mice, suggesting that a greater conversion of ALA to DHA did not explain the higher levels of DHA in
the liver of E4 mice fed the control diet. The only possible explanation we have to date is that the FA
binding protein 1 was upregulated in the livers of E4 mice fed the control diet. FA binding protein 1
has a critical role in FA uptake and intracellular transport together with regulating lipid metabolism and
cellular signaling pathways [149]. However, E4 mice fed the control diet had plasma and cortex DHA
levels similar to those of E3 mice fed the same diet suggesting that the E4-specific disruption in liver
DHA did not contribute to lower cortical DHA levels. When fed the DHA diet, E4 mice had or tended
to have lower plasma, liver and cortex DHA levels than did E3 mice (Figure 2). Despite the fact that E4
mice fed the DHA diet had more than twice the level of plasma DHA than when receiving the control
diet, cortex DHA levels barely increased. We propose that this was because E4 carriers have poor brain
protection, poor brain repair mechanisms, lower neurogenesis and lower vascular function as well as
lower limitations. Hence, providing DHA in mice vulnerable to DHA deficiency helps to minimize
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these detrimental processes. In support of this hypothesis, we tested the behaviour of 12-month old E3
(n = 32) and E4 (n = 38) fed a control or a DHA diet. Our results suggest that feeding DHA to E4 mice
prevented spatial memory deficits due to the observation that escape latency of E4 mice in the Barnes
maze was equal to E3 mice in the DHA supplemented group in contrast to a longer latency in E4 mice
compared to E3 mice fed a control diet [120]. These results were independent of anxiety, as tested by

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the light and dark test, but were accompanied by modifications of hippocampal and liver AA content.

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Indeed, we reported a significant genotype x diet interaction for the levels of AA in the liver and the
hippocampus where the DHA diet suppressed the E4-specific excess of AA in the liver and the

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hippocampus [120, 150]. The results of these studies suggest that AA metabolic disturbances might
play a crucial role in the cognitive deficits reported in E4 mice, which in turn, could contribute to

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disrupt DHA metabolism since both AA and DHA compete for the same enzymatic pathways [151]. In
another study, E4 mice, receiving a dietary treatment of Fortasyn (a supplementation including
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docosahexaenoic acid, eicosapentaenoic acid, uridine, choline, phospholipids, folic acid, vitamins B12,
B6, C, and E, and selenium) ameliorated the reduced cerebral blood flow, accelerated synaptic loss,
improved white matter integrity and functional connectivity in both aging E4 and wild type mice as
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well as illustrating enhanced protective mechanisms on vascular and synapse health [36]. The results
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were found to be independent of APOE genotype however, genotype effects could have been masked
by the inclusion of coconut oil into the control diet. Coconut oil contains medium chain triglycerides
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and 5% PUFA. Medium chain triglycerides produce ketone bodies which is an alternative fuel to
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glucose for the brain [152]. Therefore, we believe that the use of coconut oil has largely biased this
study design. Recently, our group also provided evidence that plasma and liver cholesterol levels are
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modified by the APOE genotype and diet and these findings should be considered when designing
animal trials with E4 mice [153].

E4 carriers and DHA kinetics: Our group has consistently reported that DHA metabolism is disrupted
in E4 carriers. In humans not supplemented with n-3 FA, E4 carriers have approximately twice as much
DHA in the plasma triglycerides than the non-carriers. In a trial in which DHA uniformly labelled with
carbon 13 (13C-DHA) and provided as a single oral dose of 40mg, the mean plasma 13C-DHA level in
E4 carriers was 31% lower than in the non-carriers but 13
C-DHA was 50% more degraded via -
13
oxidation in E4 carriers. Plasma C-DHA half-life was similar between E4 carriers and non-carriers,
but the whole-body half-life in E4 carriers was 32 days vs. 140 days in the non-carriers [125]. The
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discrepancy between plasma C-DHA half-life vs whole-body half-life suggests that, in E4 carriers
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and compared to the non-carriers, C-DHA might be cleared preferentially from non-plasma stores
such as the liver or the adipose, but this hypothesis needs to be confirmed. Then, 13C-DHA kinetics in
E4 carriers and non-carriers was evaluated again after they were supplemented with 3 g/d of n-3 FAs
for 5 months to assess if a dietary supplementation with n-3 FAs could prevent DHA metabolic
disturbances in E4 carrier. In that context, we showed that E4 carriers (n = 4) had a greater

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13
accumulation of C-DHA in the plasma than the non-carriers, suggesting a slower clearance of DHA

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to and/or use by tissues, and this was opposite to what we reported in E4 participants not supplemented
with n-3 FA [154]. Hence, when given a high dose of DHA, 13C-DHA kinetics were rebalanced in E4

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carriers. In a more recent trial, we outlined that disturbances in DHA metabolism in E4 carriers might
be dependent on body mass index [155]. In that study, E4 carriers were lower plasma responders to a

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DHA supplement compared to the non-carriers, but only if they were overweight.

The brain has a limited capacity to produce DHA de novo and obtains DHA from the plasma.
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Recently, the orphan receptor, Mfsd2a was identified as a transporter of DHA esterified to
lysophosphatidylcholine from the plasma and a receptor involved in BBB integrity. One group reported
lower Mfsd2a transporter levels in E4 homozygotes AD patients compared to control and
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heterozygotes E4 AD patients [119]. This group has also performed experiments in an in vitro BBB
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model in the presence of different apoE isoforms. They showed that the brain apical-to-basolateral
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transit of AA and DHA is lower when using human serum of homozygotes for E4 allele compared to
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homozygotes for E3 allele [118]. In addition, another group found Msfd2a-deficient mice exhibit a
substantial reduction in brain DHA levels due to deficiency in a membrane transporter involved in
endothelial cell uptake of esterified DHA [116]. Therefore, providing a DHA supplement rebalances
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plasma DHA and might contribute to the prevention in the loss of BBB integrity in aging E4 carriers.
However, the plasma pool into which DHA is incorporated might be key to this success since the non-
esterified FA and the lysophosphatidylcoline pool, the latter being the preferred pool for Mfsd2a
transporter, might be more important than the other plasma pools [156]. Recently, the team of Yassine
and collaborators suggested that a high dose of DHA supplementation in E4 carriers might be a
promising strategy to prevent development of AD but this treatment has to be provided before onset of
any clinical manifestation of AD [126]. Our efforts for designing better clinical trials and combining
our trials with state-of-the art technology will certainly help our team to understand the time-
specificities where we have more chances to succeed by providing DHA supplementation at optimal
doses for this at-risk population of developing late-onset AD.
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Conclusion

Altogether, this review provided evidence that dietary n-3 FA deficiencies lead to behavioural and
functional deficits. E4 carriers may be more vulnerable to these dietary n-3 FA deficits because they
catabolise more DHA and DHA is acquired mainly through the diet. It seems that to maintain a certain

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level of DHA in the plasma of E4 carriers, DHA and ALA are pulled from other organs and tissues
such as the liver and the adipose. However, providing exogenous DHA in the diet seems to rebalance

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some of the behavioural and functional deficits in E4 mice and improve clinical outcomes of patients
with inherent DHA deficiencies early in brain development when DHA accretion is maximal. DHA

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may have multiple roles beyond its incorporation into brain membranes and a need for understanding
these diverse roles and their involvement in the prevention of AD is critical. Since there are no efficient

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drugs for treating the symptoms of AD or limiting the progression of the disease, we believe that
preventative strategies are urgently needed in this area.
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Acknowledgments
The authors declare no conflict of interest. Supported by the Canadian Institutes of Health
Research (MOP119454) and the Natural Sciences and Engineering Research Council of Canada for the
project funding, a Fonds de la recherche du Québec-Santé for a Junior 2 salary award to MP. MP also
holds a New Investigator salary award from the Canadian Institutes for Health Research and a Chair on

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lipid metabolism during aging donated by Center of Medical Research of University of Sherbrooke.

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Figure legends
Figure 1: Plasma, liver, and adipose tissue concentrations of DHA (left panels) and ALA (right panels)
in APOE3 and APOE4 mice fed a diet deficient in n-3 FA. To maintain plasma DHA and ALA levels,
E4 mice had to pull DHA from or prevent DHA getting into the liver and the adipose tissue [92]

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Figure 2: Mean ± SEM of DHA levels in the plasma (left panel, n = 9-13 in each group), liver (middle,

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n = 9-13 in each group) and cortex (right, n = 4-5 in each group) in E3 and E4 mice. Mice consumed a
control or a diet enriched with DHA.

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Figure 2

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Highlights

 E4 carriers are more vulnerable to dietary n-3 FA deficits than other APOE alleles.
 DHA is pulled from the liver and adipose in E4 carriers to maintain plasma levels.
 Providing n-3 FA can restore levels of brain DHA in E4 mice.

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 DHA supplementation in E4 carriers may help to prevent or halt cognitive decline.

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