Alzheimer’s disease (AD) is a progressive, irreversible, neurodegenerative disorder and one of the most common causes of dementia in old age. AD currently affects about 35 million people in the world [1
]. The Global Deterioration Scale (GDS) provides an indication of the seven different stages of cognitive function for AD patients. Stages 1–3 are the pre-dementia period and stages 4–7 correspond to the dementia step. At the beginning of stage 5, the individual needs assistance [2
]. The neurodegeneration produced in AD might originate from the accumulation of amyloid β-peptide (Aβ) in the brain, and it is the primary influence driving AD pathogenesis. The rest of the disease process, including formation of neurofibrillary tangles containing tau protein, has been proposed as the result from an imbalance between Aβ production and clearance [3
]. In AD, the ability of synapses to transmit information is reduced, the number of synapses is decreased, and then death of the neurons occurs [4
]. High-fat Western diets seem to promote the progression of AD-like pathology through enhancement of cerebral myeloid angiopathy and oxidative stress [5
], but the leading mechanisms are still unclear.
Mediterranean lifestyle patterns could be beneficial for AD since dietary intake of n-3 fatty acids (FA) and weekly consumption of fish may reduce the risk of AD [6
]. The more abundant n-3 polyunsaturated fatty acids (PUFA) in fish oil, docosahexaenoic acid (DHA; 22:6n-3), and eicosapentaenoic acid (EPA; 20:5n-3), are proposed to be beneficial components in the prevention of AD [8
]. DHA could exert protective effects against β-amyloid production, accumulation, and potential downstream toxicity [6
]. DHA is implicated in a diversity of physiological processes, as well as memory formation, aging, synaptic membrane function, biogenesis, function of photoreceptors, and neuroprotection [9
]. A diet enriched with DHA reduces amyloid burden in an aged Alzheimer animal mouse model [10
]. Nevertheless, n-3 PUFA (polyunsaturated fatty acids) supplementation might not be useful in the advanced stages of AD [11
] in which significant neuronal failure has already occurred [8
]. The meta-analysis from de Wilde et al. reported significantly lower levels of DHA in blood and brain from AD patients [12
]. Thus, mechanisms to improve DHA in brain of AD are of major interest.
Given that DHA synthesis in brain is limited [13
], the uptake from serum PUFA plays a critical role in the maintenance of brain lipid composition in neurological diseases [14
]. The blood–brain barrier (BBB) is a specialized multicellular membrane of astrocytes, pericytes, and endothelial cells that control the selective uptake of molecules into the brain and keep away toxins and pathogens. BBB provides an optimal environment for brain function [15
]. Recently, a transmembrane protein named Major Facilitator Superfamily Domain containing 2A (MFSD2a) was described as the primary carrier for the DHA uptake, and other long-chain FA such as lyso-phospholipids (Lyso-PL) in BBB [16
] and in retinal pigment epithelium [17
]. MFSD2A is expressed by the endothelial cells and presents 12 transmembrane domains composed by two evolutionary duplicated six transmembrane units [18
]. MFSD2a is a carrier with a dual role in the brain, the uptake of unsaturated lyso-PL as DHA, and the establishment of BBB integrity by the inhibition of caveolae-mediated transcytosis [16
Humans with homozygous mutations that inactivate the MFSD2a gene present intellectual disability and severe microcephaly [20
]. Furthermore, MFSD2a knock-out mice showed reduced levels of DHA in brain, deficits in learning and memory, neuronal cell loss in hippocampus and cerebellum, and severe microcephaly [16
]. Additionally, in a mouse model it has been observed that MFSD2a is required at the BBB for normal postnatal brain growth and for the maintenance plasma membrane phospholipid composition [23
On the other hand, short-term fish oil treatment in AD mice did not affect the expression of MFSD2a in the brain, while it tended to be enhanced in the liver [24
]. Due to its important role in the central nervous system (CNS) and the BBB physiology, MFSD2a means an interesting issue to study in neurodegenerative disorders, such as AD. It is unknown whether MFSD2a is been altered somehow in AD or if its level in blood could be a potential indicator of the AD. Since it is so difficult to evaluate the expression of MFSD2a in the brain of AD patients in vivo, new biomarkers based on MFSD2a that could be analyzed in easily-to-obtain samples such as blood should be explored.
We aimed to study whether MFSD2a level is altered in the blood of AD patients, which are easy-to-obtain samples by noninvasive procedures, on different stages of the disease as a potential biomarker of AD. In addition, we studied in a small subset of samples the MFSD2a levels and fatty acid profiles in postmortem brain samples from AD and control subjects.
This is the first study that has analyzed MFSD2a levels in the blood of humans under different stages of AD. The participants of this study were non-supplemented with n-3 PUFA. We demonstrated for the first time that MFSD2a protein expression decreased significantly in blood samples obtained from AD patients compared to healthy subjects. Nevertheless, the reduction of MFSD2a protein expression observed in blood was not accompanied by the correspondent reduction in the brain, which could indicate that MFSD2a protein level in the brain could be differently regulated in AD. Low levels of MFSD2a in the whole blood could affect peripheral tissue functions in AD patients, which should be explored in future studies.
AD has been associated with metabolic disorders such as obesity, hypertension, hypercholesterolemia, and diabetes [26
]. In the present study, we found a significantly higher concentration of total lipids in AD (Table 2
) and more than 50% of AD patients suffered from hypertension, while nearly 26% were diabetics (Table 1
), which could explain the higher levels of total lipids in serum of AD patients, although this point should be further explored by comparison with more complete records of data from control subjects.
A significant decline in the percentage of n-3 LC-PUFA was observed in serum concomitantly to the progression and severity of AD (Table 2
). This result is related with the decrease of both DHA and EPA percentages in the serum of AD groups (Figure 2
). In agreement with previous studies [24
], the decrease of n-3 FA percentages in serum leads to a significant increase in the n-6/n-3 PUFA ratio (Table 2
). These data are consistent with previous studies performed in old people with dementia that reported also lower concentrations of plasma n-3 PUFA [12
]. A disturbed n-6/n-3 ratio is related with inflammation and some neurological diseases, and several studies have described an improvement of n-6/n-3 ratio after short-term fish oil supplementation [24
]. Many factors such as diet, nutrient absorption, metabolic disturbances, or the increased use of nutrients during the processes related to the AD pathology may alter nutrient levels in the plasma of these patients [26
]. Furthermore, subjects receiving DHA supplements were excluded in the present study. Although we did not evaluate dietary intake of patients, the clear decline of DHA levels already in GDS4 subjects point towards AD as the most probable cause of the lower serum n-3 FA levels in such patients, due to an impaired systemic availability of numerous nutrients [26
]. In addition, Wang et al., proposed that high oxidative stress in AD patients may decrease PUFA level, since they can suffer lipid peroxidation and break down into toxic compounds, like malonaldehyde. In fact, they postulated DHA as a potential biomarker of AD, because its alterations could reflect metabolic changes taking place during AD development [31
In the present study, we demonstrated a significant continuous decline of MFSD2a protein level in blood of patients with AD (Figure 1
). This suggests that blood cells, easily obtainable through peripheral blood sampling, express the MFSD2a carrier and that its expression could be related with AD. This result would be in line with the behavior of other FA carriers in blood of AD patients such as fatty acid translocase (FAT/CD36), whose expression levels also decreased in peripheral leukocytes of these patients [32
]. Apart from being a FA carrier, FAT/CD36 has an important role as a scavenger receptor that recognizes amyloid plaques, and has been reported to trigger oxidant production by macrophages and microglia [33
]. Our results suggest that altered MFSD2a levels in blood might inform us about metabolic disorders and/or the nutrient transport across other human tissues, which ought to be studied in future.
In this research, both MFSD2a protein level in blood and DHA percentage in serum decreased in advanced stages of AD, although the blood level of MFSD2a did not lineally correlate with the serum level of DHA observed. Despite the differences in age between Control and AD groups, patients from GDS4 and GDS6 had similar age but a gradient to lower MFSD2a and DHA levels in blood respect to controls. Thus, the pathology seems to have the highest effect on the parameters analyzed. MFSD2A plays different roles, as it is a carrier of lipid metabolism, but also participates in body growth and development, and motor function; and its codifying gene is nutritionally regulated in mice [34
]. MFSD2a is not an exclusive carrier of DHA, because can also transport other ligands [21
]; so, the decrease of MFSD2a levels in the advanced stages of AD might not be necessarily parallel the decline of DHA levels. While, other mechanisms apart from blood MFSD2a may be contributing as well to the decreased percentage of DHA observed in the serum of AD patients. In addition, it is important to highlight that the decline of MFSD2a levels in blood is more acute and occurs at earlier stages of AD than the reduction of DHA levels in serum. Therefore, MFSD2a could be postulated as a more sensitive biomarker to diagnose different stages of AD in easy-to-obtain blood samples, without the necessity of applying other more invasive methods.
We also studied MFSD2a expression in the hippocampus in another small set of postmortem subjects with GDS6, but no statistical differences were found compared to controls (Figure 3
). Thus, MFSD2a expression protein seems to be strongly regulated in the hippocampus of AD patients not supplemented with n-3 PUFA. Our results were similar to those obtained by Milanovic et al. [24
], who reported no changes in MFSD2a in both liver and brain in an AD mouse model after a fish oil supplementation period. However, in the liver, strong trends of enhanced MFSD2a protein expression was observed by supplementation with fish oil for a short term (3 weeks), while no significant increases of MFSD2a were found in the brain of AD models [24
]. Although our low number of brain samples might have limited the power of the analysis, it is also possible that the higher expression of MFSD2a observed in brain could try to act as a compensatory mechanism in an attempt to improve DHA accretion into this tissue. Berger et al., reported that the expression of MFSD2a in brain is ubiquitous and slowly induced [34
]. Thus, it appears to be difficult to modify MFSD2a levels in the brain. On the other hand, Sandoval et al. demonstrated opposite differences of MFSD2a expression between the cortical and subcortical regions in healthy animals after long term supplementation with different FA, probably due to the different cellular composition and nutrient transport requirements of each brain region [35
]. We analyzed MFSD2a level and FA profile only in the hippocampus, but not in the cortex or any other areas of the brain. Thus, the study of other brain regions could report additional information on the regulation of MFSD2a expression in the whole brain. Changes in brain components are very complex and would require many further studies. Regrettably, it was not possible to analyze MFSD2a levels in blood of such postmortem patients because the corresponding blood samples were not available in the biobank.
DHA has a significant role in the hippocampus, which is critical for learning [10
]. E-series resolvins (RvEs) derived from eicosapentaenoic acid (EPA), and D-series resolvins (RvDs), protectin/neuroprotectin (PD/NPD) and maresins (MaRs) derived from DHA have local potent pro-resolving actions [36
]. We did not find significant differences in the percentages of DHA or EPA in the hippocampus of the postmortem subjects between controls and GDS6 AD patients, probably due to the low number of samples available (Table 3
). A meta-analysis reported significantly lower levels of DHA in the brain of AD patients [12
], although six studies of such meta-analysis reported significantly lower levels of DHA in AD patients than in controls, while seven studies reported no significant differences between groups [12
]. In the OmegAD study in AD patients, 6 months of supplementation with 2.3 g of DHA enhanced both EPA and DHA in cerebrospinal fluid and plasma [39
]. However, changes of EPA and n-3 docosapentaenoic acid (22:5) in cerebrospinal fluid and plasma were strongly correlated. In contrast, DHA in cerebrospinal fluid and plasma were not correlated [39
]. Despite the existence of a disturbed uptake of DHA respect to EPA in BBB in AD patients, a different metabolization of these two FA within the brain could also occur. Regardless, the changes in DHA levels in cerebrospinal fluid were inversely related to phosphorylated tau protein and inflammatory biomarkers, which highlight the importance to have appropriate DHA levels in the brain of someone with AD [39
We could not find a correlation of MFSD2a level with DHA and other FA percentages in the hippocampus. This suggests that other mechanisms apart from MFSD2a are contributing to DHA percentage in the serum and the brain of AD patients. It was previously reported, in an AD animal model, that a short-term fish oil supplementation did not modify DHA levels in the brain of AD animals, while in wild type animals both EPA and DHA were increased. Instead, in the liver, fish oil increased DHA in both AD and wild type animals [24
]. Thus, the n-3 fatty acid profile is strongly protected in the brain compared with other tissues, especially in AD patients.
In conclusion, MFSD2a carrier is expressed in blood and its expression is reduced concomitantly with the progression of AD. Therefore, MFSD2a protein level in blood could be an additional potential biomarker of AD progression. The function of MFSD2a and localization in blood needs further clarification.