*4.3. Some Other Neuropathologies*

Beneficial effects of DHA or derivatives have been observed in other models of neuroinflammation. Even if these pathologies are not neurodegenerative diseases, they are of interest because of their neuroinflammation component accompanied by microglial activation and pro-inflammatory factor release.

For example, synaptamide could be used to treat chronic neuropathic pain. In vitro, the addition of synaptamide to the SIM-A9 murine microglia cell line prevented LPSinduced NO overproduction and ROS production. In vivo, rats with sciatic nerve chronic constriction injury (CCI) treated with synaptamide showed lower concentrations of hippocampal Iba-1, CD86, IL-6, and IL-1β than the CCI group without synaptamide treatment. Furthermore, more doublecortin-positive neurons and proliferating cell nuclear-positive cells have been counted in the dentate gyrus subgranular zone in the CCI synaptamide treated rats compared to the CCI rats. Behavioral improvements were also observed in the synaptamide-treated groups [45].

Others have shown anti-inflammatory effects of NPD-1 in old mice in a model of postoperative delirium, with reduction of IL-6, TNF-α, glial fibrillary acidic protein (GFAP), and Iba-1 compared with controls. IL-10 increase was also observed [46].

Taken together, all of these studies suggest strong benefits of DHA mediated by microglial cells on neuroinflammation in neurodegenerative disorders. However, the mechanisms of action of the anti-inflammatory in vivo are still not elucidated.

#### **5. DHA, Clinical Trials, and Neurodegenerative Diseases**

Various results of clinical trials have been published concerning the use of DHA in neurodegenerative diseases (summarized in Table 3). A majority of clinical trials focus on AD. In a first clinical trial on this pathology, a food supplementation composed of xanthophyll carotenoids and omega 3 fatty acids was tested [47]. Two conditions were tested: the first condition was lutein/meso-zeaxanthin/zeaxanthin at 10:10:2 mg/day and the second condition was the formulation used in the first condition plus 1 g/day of fish oil containing 430 mg DHA and 90 mg EPA. It turned out that the formulation containing DHA was the most effective in slowing down AD, showing that the consumption of xanthophyll carotenoids combined with DHA (fish oil) has a better protective effect than xanthophyll carotenoids used alone [47]. Disease progression is reduced with this formulation with improvement in memory, sight, and mood. An OmegAD clinical trial (NCT00211159), enrolling 204 participants, studied the effects of DHA-rich dietary supplementation on cognitive impairment in patients with AD. Preliminary results showed that DHA (capsule EPAX 1050TG; Pronova Biocare A/S, one capsule: 430 mg DHA and 150 mg EPA) supplementation for 6 months induces DNA hypomethylation in blood cells [48].These results provide a new possible mechanism of action for these compounds: they could modulate gene expression by hypomethylation. The authors postulate that it could then be interesting to treat AD with hypomethylating agents [48]. Another work from the OmegAD clinical trial (NCT00211159) studied the plasma levels of fatty acids following DHA intake. It was shown that the higher the plasma levels of omega-3 fatty acids, the better the cognitive functioning, regardless of gender [49]. However, body weight is important and DHA doses should be adjusted to it [49]. Another part of the clinical trial was to study immune function. Since it is difficult to work on microglia directly, the

authors used peripheral blood mononuclear cells (PBMCs), which can infiltrate the brains of Alzheimer's patients like T lymphocytes and monocytes and participate in the development of inflammation. PBMCs were recovered before and after supplementation with DHA and EPA and then treated with the Aβ40 peptide. DHA/EPA supplementation prevented the reduction of specialized proresolving mediators (SPM, lipoxin A4, and RvD1 released from PBMCs) [50]. Furthermore, inflammation resolution is disrupted in patients with AD; DHA/EPA supplementation (EPAX1050TG; Pronova Biocare A/S, Lysaker, Norway) could improve it [50]. As the authors point out, it remains to be determined whether the same effects can be observed in microglia and whether the use of SPM or their precursors could be effective in the treatment of AD [50]. Another component of the study was to evaluate the effects of oral dietary omega-3 supplementation on inflammatory biomarkers and oxidative stress. Patients were supplemented for 6 months with a DHA/EPA complex (four capsules of EPAX 1050TG, i.e., 1.7 g of DHA and 0.6 g of EPA); urine samples were collected before and after supplementation [51]. In these samples, the levels of major F2-isoprostane, 8-iso-prostaglandinF2α (biomarker oxidative stress), and 15-keto-dihydroprostaglandin F2α (biomarker of inflammation) were measured [51]. The results obtained indicate that DHA/EPA supplementation does not have a well-defined effect on oxidative stress as measured but may have a possible role in immunoregulation. Since AD affects the brain, it was interesting to know if fatty acids are able to cross the blood–brain barrier. Therefore, fatty acid profiling was performed in cerebrospinal fluid (CSF) to assess whether supplementation was able to alter this profile [52]. Patients received 6 months of DHA supplementation (four capsules of EPAX 1050TG, i.e., 1.7 g of DHA and 0.6 g of EPA). After 6 months, changes in the fatty acid profile were observed, with a significant increase in eicosapentaenoic acid (EPA), DHA, and total n-3 FA levels in CSF [52]. A correlation was also made with the markers of AD and, the more DHA levels increased in the CSF, the more changes there were in the biomarkers of the pathology (tau, phosphorylation of the tau protein, IL-1 receptor). Their results also showed that supplementation failed to stop disease progression and that DHA supplementation would likely need to be taken early to see an effect on disease progression [52]. In AD, deposits of the Aβ protein are present in the brain. Transthyretin (TTR) can bind to amyloid β and thus reduce its presence. Patients received DHA/EPA supplementation (four capsules of EPAX 1050TG, i.e., 1.7 g of DHA and 0.6 g of EPA) for 6 months; it was observed that this treatment could increase plasma levels of TTR, which could influence Aβ peptide deposits in the brain, results that need to be confirmed by further experiments [53]. DHA/EPA supplementation was also evaluated on gene expression in peripheral blood mononuclear cells [54]. Patients received a DHA/EPA complex (four capsules of EPAX 1050TG, i.e., 1.7 g of DHA and 0.6 g of EPA) for 6 months and the expression of 8000 genes was studied. Modulations of the expression of several genes (decrease (10) or increase (9)) were measured. The upregulated genes are *MS4A3* (role in signal transduction), *NAIP* (apoptosis inhibitory protein), *DRG1* (stress and hormone responses, cell growth, differentiation), *CD36* (cell adhesion, cell migration), *HSD17B11* (regulation of inflammation, modulation of intracellular glucocorticoid levels), *RAB27A* (signal transduction), *CASP4* (inflammatory caspase), *SUPT4H1* (RNA synthesis), and *UBE2V1* (ubiquitination). The negatively regulated genes are *RHOB* (inflammation), *VCP* (vesicle transport, fusion, and ubiquitin-dependent protein degradation), *LOC3999491*, *ZNF24* (transcription factor), *SORL1* (regulation of processing of amyloid precursor protein), *MAN2A1* (inflammation regulation), *PARP1* (differentiation, proliferation, tumor transformation, DNA damage reparation), *SSRP1* (action on transcription), *ARIH1* (ubiquitination process), and *ANAPC5* (cell cycle progression). Many of these genes are involved in inflammation regulation and neurodegeneration, and in ubiquitination processes [54]. The impact of DHA (four capsules of EPAX 1050TG, i.e., 1.7 g of DHA and 0.6 g of EPA) on inflammation was confirmed by another study performed in this clinical trial, which showed that DHA decreased the release of PGF2α from LPS-stimulated PBMCs and that it could be hypothesized that DHA could act via anti-inflammatory and neuroprotective lipid mediators on the resolution phase of inflammation [55]. Using the same protocol

(PBMCs treated with LPS following DHA/EPA supplementation, four capsules of EPAX 1050TG, i.e., 1.7 g of DHA and 0.6 g of EPA), the authors also showed that the increase in plasma DHA concentration was correlated with a reduction in the release of IL-1β, IL-6, and the granulocyte colony-stimulating factor of PBMCs [56].


**Table 3.** Clinical trials involving DHA.

In an independent study of the OmegAD study, omega-3 fatty acid supplementation (capsules containing a total of 625 mg of DHA and 600 mg of EPA) increased plasma DHA and EPA concentrations in people with cognitive impairment no dementia, and AD. However, no beneficial effect on cognition and mood was observed in these populations. As emphasised by the authors, the sample used to conduct this study was quite small (76 participants), and the duration of the study was short (4 months). One hypothesis raised by the authors was to adapt the dose according to the pathology studied and the level of progression of the pathology [57]. These same observations were made in a study carried out by Paul S. Aisen's group, where DHA supplementation did not slow cognitive decline in patients with mild or moderate AD [58]. This study involved 402 patients and lasted for 18 months. DHA supplementation was performed with an algal-derived DHA (Martek Biosciences, Columbia, Maryland) in capsule form. Twice a day, patients took 1 g, for a total daily dose of 2 g, knowing that these capsules contained approximately 45% to 55% DHA by weight and did not contain eicosapentaenoic acid. The authors suggest that DHA may have an effect if the patients do not have overt dementia [58]. A third study shows that omega-3 supplementation (EPAX1050TG™ from Pro-nova Biocare A/S, Lysaker, Norway, four capsules) in patients with mild to moderate AD did not induce effects on neuropsychiatric symptoms, but had possible positive effects on depressive symptoms in non-ApoEω4 carriers and on agitation symptoms in ApoEω4 carriers [59]. In contrast to these three studies, a supplementation study using Aravita capsules (Suntory Ltd., Osaka, Japan), containing 40 mg/capsule of arachidonic acid (ARA) and DHA and 0.16 mg/capsule of astaxanthin (antioxidant of PUFA), showed significant improvements in the memory of patients with organic brain damage or mild cognitive impairment [60]. The authors hypothesize that these changes may be due to neural circuit remodeling (possible upregulation of synaptogenesis and/or neurogenesis with ARA), as well as improvement of membrane function and regional cerebral blood flow by DHA [60].

A few clinical trials have focused on spinocerebellar ataxias, which are very heterogeneous neurodegenerative diseases, clinically and genetically. The main characteristic of these pathologies is cerebellar syndrome, associated with walking and balance disorders. Spinocerebellar ataxia 38 (SCA38) is caused by a mutation in the elongation of the very long chain fatty acid protein 5 (ELOVL5) gene and is associated with reduced serum DHA levels. The team of Borroni et al. studied the effect of short-term (16–40 weeks) and long-term (2 years) DHA supplementation [61,62]. The DHA was derived from algal oil (Sofedus, Milan, Italy) and administered as sachets dosed at 600 mg/day. Improvement in clinical symptoms and no degradation of neurophysiological parameters were observed following fish oil-derived DHA intake.

The published results of clinical studies (mainly on AD) underline the interest in DHA, particularly through its ability to cross the blood–brain barrier and to influence inflammation. It is not known to what extent DHA crosses this barrier and whether microglia, the orchestral leader of inflammatory reactions in the brain, is affected by DHA. Many authors also mention the need to adapt the dose according to the severity of the disease or the weight of the patients. With DHA, the problem is not that it does not cross the blood–brain barrier, but that the quantity of DHA that crosses the barrier is not sufficient and does not only target the microglia. It is therefore important to look at the contribution of nanotechnologies in the targeting of therapeutic molecules of interest, such as DHA, to the microglia.
