Summary
Alzheimer’s disease is the most prevalent form of dementia worldwide. It is characterized by progressive loss of brain functions such as memory, behavior regulation and general cognition. This loss eventually extends to basic functions such as swallowing, leading to death. Intriguingly, research has uncovered links between DHA and Alzheimer’s disease risk and progression. Studies show that DHA supplementation can play a positive role in certain forms of Alzheimer’s disease.

Introduction
Symptoms and disease progression
Alzheimer’s disease (AD) is perhaps the most well-known type of dementia. More than 50 million people are affected by AD worldwide, making it the most prevalent form of dementia. Classical symptoms of AD include cognitive decline, memory impairment and behavioral changes, which worsen as the disease progresses. In advanced stages of the disease, even basic functions of the brain such as swallowing and breathing can be lost, resulting in death. During the course of AD, the brain progressively loses neurons, which are the brain cells that store, send and receive information through electrical signalling. Therefore, AD is called a neurodegenerative disorder. On a biological level, AD is defined by pathological aggregation (clumping together) and spread of two protein types through the brain: Tau and amyloid-beta (Aβ). The spread of these protein aggregations occurs in an AD-specific pattern through the brain, which is thought to contribute to the dysfunction of neurons, loss of neurons, and clinical symptoms of AD. Though much is still unknown about the disease mechanism of AD, inflammation of neurons is increasingly recognized as an important contributor to AD pathology.

Diagnosing AD
A common method to assess (the rate of) cognitive decline in AD is the Mini-Mental State Examination (MMSE), which can be used to obtain a Clinical Dementia Rating (CDR). This can be combined with certain brain scans to diagnose AD. With an MRI-scan of the brain, changes in the volume of certain brain regions are determined, giving information about neurodegeneration. A PET-scan of the brain can give information about pathological aggregation of Tau and Aβ. Health care professionals may also elect to perform a lumbar punction. This procedure collects cerebrospinal fluid from the spinal cord; this liquid is in contact with our brain and contains markers that can give indications about the brain’s health and nutrient status. Finally, blood plasma can also contain biomarkers that indicate presence of AD pathology. A combination of clinical symptoms and biological indicators is often necessary to establish with certainty that a person has AD.

Sporadic Alzheimer’s disease and familial Alzheimer’s disease
Alzheimer’s disease can have a clear genetic contributing factor, in which case we refer to familial AD. It can also develop without clear cause, in which case we refer to it as sporadic AD.

In familial AD, persons have one or multiple ‘risk genes’, which are variants of genes that enhance the probability of developing Alzheimer’s disease. This depends on the type of gene, and whether persons receive a risk gene from one or both parents. For each gene that we have, we receive both a maternal and paternal copy. A multitude of gene variants have been identified that enhance the probability of developing AD, these are referred to as genetic risk factors. In addition, genetic variants exist that outright cause AD. A common risk factor is APOE ε4, which is a variant of the APOE gene. Multiple genetic variations of the APOE gene exist, such as APOE ε2, APOE ε3 & APOE ε4. Compared to people without an APOE ε4 gene copy (such as APOE ε3/ ε3), persons with one copy of this gene (such as APOE ε3/ε4) are 3-4 times as likely to develop AD. Persons with two copies of APOE ε4 (APOE ε4/ε4) are 12 times more likely to develop AD than those without this gene variant. Crucially, DHA transport to the brain is thought to be altered by the APOE ɛ4 genotype.

In sporadic AD, there is not one clear factor that causes development of AD. In addition to risk genes, many other factors have been identified that can contribute to the development of sporadic AD, and increase the probability of AD development in persons that carry risk genes. Examples of these are aging-related changes in the brain such as inflammation, but also lifestyle factors such as depression.

DHA & the (AD) brain 
DHA is one of the most prevalent fatty acids in the brain, making up to 40% of all brain fatty acids. One of its roles in the brain is supporting neuronal health functioning. A further crucial function of DHA in the context of AD is aiding in the production and breaking down of Aβ, one of the proteins that aggregates and accumulates in AD. Additionally, DHA can be converted into certain compounds with anti-inflammatory properties. These may aid against the neuroinflammation that is associated with AD.

Research findings
Lower levels of DHA in plasma AD patients
Chu and collaborators researched possible links between diet, concentrations of omega-3 fatty acids in blood and cognitive decline in AD patients receiving acetylcholinesterase inhibitors (Chu et al. 2022). Acetylcholinesterase inhibitors are commonly given to AD patients to provide a temporary improvement in cognitive symptoms. For a period of two years, 129 AD patients (men and women) were periodically tested to assess nutritional and cognitive status. At the beginning, halfway, and at the end of the study, Clinical Dementia Rating was established using the Mini-Mental State Examination. Additionally, blood samples were taken during these moments. Based on changes Clinical Dementia Rating, patients were divided into two groups: the cognitive decline group (worsening CDR scores) and the cognitively stable group (stable CDR scores). In the group comparison analysis, Chu and colleagues discovered that the cognitively stable group had significantly higher DHA and EPA blood levels than the cognitive decline group. They further reported that in AD patients receiving acetylcholinesterase inhibitors, a lower baseline DHA (but not EPA) blood concentration is associated with an increased risk of cognitive decline.

Discovering the importance of the APOE ε4 risk gene for effects of DHA in AD patients
In a study by Quinn and colleagues, DHA was given to patients with mild to moderate AD in an attempt to slow the deterioration of certain cognitive capacities and neurodegeneration (Quinn et al. 2010). Contrary to the authors’ expectations, the 18-month supplementation of 2 grams DHA per day period did not result in significant differences in the measured parameters compared to the placebo treatment. However, a sub-group analysis performed by the authors where participants with AD were divided into APOE ε4 carriers and APOE ε4 non-carriers revealed an effect of DHA supplementation. APOE ε4 non-carriers who took the DHA supplementation had a slower decline in cognitive performance in Alzheimer’s Disease Assessment Scale than those who received the placebo treatment. This effect was not seen for APOE ε4 carriers. The authors concluded that the APOE gene variant influences susceptibility to positive effects of DHA treatment in AD. The effect of the APOE ɛ4 gene in the context of DHA and AD has since been studied intensively, as can be read in the following sections.

APOE ε4 and DHA supplementation in non-demented individuals
Coughlan and colleagues researched associations between blood levels of DHA and volume of certain brain regions, comparing these between cognitively healthy APOE ɛ4 carriers and noncarriers (Coughlan et al. 2021). A group of 53 non-demented men and women with a mean age of 64 were tested for the APOE ɛ4 genotype. The age range was selected because it is roughly 10 years before the typical onset of AD. This way, the authors could observe clinical and biological changes during the period in which AD develops. The participants were divided into two groups for comparative analyses: APOE ɛ4 carriers (APOE ɛ3/ɛ4) and APOE ɛ4 non-carriers (APOE ɛ3/ɛ3). MRI brain scans were conducted to assess the volume of certain brain regions called the entorhinal cortex and hippocampus. Both these regions are crucial for memory function, and are affected early on in AD. DHA levels in blood serum were measured to test associations with the APOE genotype and volume of these specific brain regions.

The authors found a positive association between serum DHA levels and brain volume of the entorhinal cortex was found, though only in the APOE ɛ4 non-carriers. This means that APOE ɛ4 non-carriers had a larger entorhinal cortex volume if they had higher levels of serum DHA. In contrast, APOE ɛ4 carriers genotype showed a trend towards a negative association between serum DHA levels and entorhinal cortex volume. Similar results were seen for the hippocampus, albeit just shy from reaching statistical significance.

The authors then wanted to assess if the established associations between APOE ɛ4 and DHA in non-demented adults affect memory and navigational function. They recruited a new group of 46 participants with the same characteristics as the first group. Again, participants were screened for APOE genotype and divided into APOE ɛ4 carrier (APOE ɛ3/ɛ4) and APOE ɛ4 noncarrier (APOE ɛ3/ɛ3) groups. Participants’ blood serum DHA levels were determined, which was used to assess if this was associated with performance in a memory and navigation test all participants undertook. These cognitive skills are often affected early in the AD process, the authors performed their analysis to establish if DHA is associated with any AD-related cognitive deficits. Almost counterintuitively, it was found that higher levels of blood serum DHA were associated with poorer performance in the spatial navigation test for the APOE ɛ4 carrier group. No association was found between DHA and performance in the cognitive test in the APOE ɛ4 non-carrier group. The authors suggest that the inverse relation between the APOE ɛ3/ɛ4 genotype and spatial navigation test performance could come from impairment of DHA uptake by the brain in this group. This would mean that the elevated levels in their blood serum are a result of their brain’s inability to take up DHA, coupling with specific cognitive deficits.

Aiming to assess the relevance of APOE ε4 genotype on uptake ability of DHA supplementation in non-demented adults, Arellanes and fellow researchers set up a randomized clinical trial (Arellanes et al. 2020). A group of 33 cognitively and physically healthy participants aged 55 and over was tested for APOE ε4 genotype and divided into a treatment arm and placebo arm. The treatment arm consisted of 7 APOE ε4 carriers and 8 APOE ε4 non-carriers, the placebo arm consisted of 8 APOE ε4 carriers and 10 APOE ε4 non-carriers. For a period of 6 months, the treatment group took 2151 mg DHA per day; the placebo group received identical-looking corn/soy oil capsules. Most DHA supplementation clinical trials, including those investigating AD, use daily doses of up to 1000 mg. The rationale of this study was to test if higher doses of DHA can counter the suspected negative effect of the APOE ε4 genotype on DHA uptake in the brain. Lumbar punctures (to obtain cerebrospinal fluid), MRI scans, cognitive tests and blood plasma collections were conducted at the beginning and end of the supplementation period. After the supplementation period, no difference in cognitive test performance or volume of the entorhinal cortex and hippocampus was found between the two groups. In the cerebrospinal fluid analysis, the authors reported a 28% increase of DHA and a 43% increase of EPA in the DHA supplementation group, compared to the placebo group. EPA is normally a precursor of DHA, but can also be made by the body via retro-conversion of DHA; likely explaining the large increase of EPA in the cerebrospinal fluid of the supplementation group. A subgroup comparison between APOE ε4 carriers and non-carriers in the DHA supplementation group provided interesting results. After DHA supplementation, APOE ε4 carriers showed lower EPA levels and a trend towards lower DHA levels in the cerebrospinal fluid compared to non-carriers. Therefore, the authors concluded that the APOE ε4 genotype likely negatively influences the brain’s ability to take up DHA in non-demented adults.

APOE ε4 and DHA supplementation in AD patients
The studies above have provided evidence for the influence the APOE ɛ4 genotype has on the effects of DHA in non-demented individuals. Tomaszewski and colleagues set up a clinical trial to test what influence the APOE ɛ4 genotype has on the effects of DHA supplementation in persons with mild AD (Tomaszewski et al. 2020). They recruited 275 persons with mild AD and randomly divided them into two groups: one receiving DHA supplementation, the other a placebo. The supplementation period lasted 18 months, during which participants either took 2040mg of DHA (161 participants) or a corn/soy oil placebo (114 participants) every day. Baseline dietary intake levels of DHA was determined at the start of the study, which did not differ per APOE genotype group. An MRI-brain scan (to assess neurodegeneration), blood plasma and cerebrospinal fluid was also obtained for some of the participants at the start and end of the study. With this data, baseline ratio and changes in ratio of (anti-inflammatory) omega-3 fatty acids DHA and EPA to the (pro-inflammatory) omega-6 fatty acid AA were assessed.

One of the first findings of their study was that the APOE genotype affects the blood plasma ratio of DHA/AA. At the baseline measurements, APOE ɛ2/ɛ4 carriers and APOE ɛ3/ɛ3 carriers had a greater ratio of DHA/AA compared to APOE ɛ4/ɛ4 carriers.

After the 18-month supplementation period, all participants who received supplementation had a higher ratio of DHA/AA in their blood plasma compared to their counterparts who received the placebo. However, APOE ɛ4/ɛ4 carriers showed the smallest increase of all groups, which was significant when compared to APOE ɛ3/ɛ4, APOE ɛ3/ɛ3 and APOE ɛ2/ɛ3 carriers. The authors then plotted this data against measured changes in DHA/AA ratio in the cerebrospinal fluid. They found that increases in blood plasma DHA/AA ratio were more strongly associated with increase in cerebrospinal fluid DHA/AA ratio in APOE ɛ4 non-carriers compared to APOE ɛ4 carriers. This means that if APOE ɛ4 non-carriers showed higher ratios of blood plasma DHA/AA, they were more likely to have higher ratios of cerebrospinal fluid DHA/AA, compared with APOE ɛ4 carriers.

Finally, the authors looked at changes in volume of the right and left hippocampus following the supplementation period. They did not observe a significant change between the DHA supplementation and placebo group, nor did they find an association between increasing plasma DHA/AA ratio and hippocampal volume in the DHA group. However, when the authors looked at the EPA/AA blood plasma ratio, they found that a higher ratio correlated with a lower decline in right hippocampal volume in APOE ɛ4 non-carriers. This effect was not seen in APOE ɛ4 carriers. As DHA can be retro-converted to EPA in the brain, the authors propose that DHA supplementation can counter degeneration of the right hippocampus by providing a source for EPA. However, this effect seems to be limited to APOE ɛ4 non-carriers.

Conclusion
Although the mechanism behind AD remains to be fully understood, the scientific community has identified certain lifestyle and genetic risk factors that can increase the chances of AD development. Overall, non-demented individuals overall have higher levels of DHA and EPA in their blood plasma compared to persons with AD, and lower levels of DHA are associated with an increased risk of cognitive decline. However, the APOE ε4 risk gene influences DHA metabolism characteristics and associations with brain health and function.

One of the first indications regarding the importance of APOE ε4 in this context was found in a sub-group analysis in a study by Quinn and collaborators. These authors reported that APOE ε4 non-carriers had a slower cognitive decline following DHA supplementation compared to those who received placebo treatment. This effect was not observed in APOE ε4 carriers. Moreover, research shows that in non-demented persons, the positive association between serum DHA levels and volume of the brain region called the entorhinal cortex is exclusive to APOE ε4 non-carriers.

When given high doses of DHA supplementation, non-demented APOE ε4 carriers have lower increases in levels of blood plasma DHA compared to their APOE ε4 non-carrier counterparts. Moreover, compared to APOE ε4 non-carriers, APOE ε4 carriers show lower EPA levels and a trend towards lower DHA levels in their cerebrospinal fluid after DHA supplementation. The APOE ε4 genotype likely negatively influences the body and brain’s ability to take up DHA and retro-convert it to EPA in non-demented adults. Accordingly, when DHA supplementation is given to persons with mild AD, APOE ε4 carriers show a lower increase in plasma DHA/AA and EPA/AA ratios. This couples with levels of hippocampal volume loss following DHA supplementation as well. AD patients given DHA supplementation show an increase in plasma EPA/AA that correlates with the less decline in right hippocampal volume only in APOE4 noncarriers, but not in APOE4 carriers.

The effects of DHA in AD are likely influenced by medicinal treatment, the disease stage of AD, nutritional status and presence of genetic risk factors. The start of the pathological process of AD is thought to occur a decade or more before the onset of clinical symptoms, during which the irreversible process of neurodegeneration is already in motion. Therefore, the time window in which DHA supplementation is taken by an individual likely influences any effects it may have on AD development and progression. Based on the studies researching the importance of APOE ɛ4 in AD, this might be especially relevant for APOE ɛ4 carriers.

References
Arellanes, Isabella C., Nicholas Choe, Victoria Solomon, Xulei He, Brian Kavin, Ashley E. Martinez, Naoko Kono, David P. Buennagel, Nalini Hazra, Giselle Kim, Lina M. D’Orazio, Carol McCleary, Abhay Sagare, Berislav V. Zlokovic, Howard N. Hodis, Wendy J. Mack, Helena C. Chui, Michael G. Harrington, Meredith N. Braskie, Lon S. Schneider, and Hussein N. Yassine. 2020. ‘Brain Delivery of Supplemental Docosahexaenoic Acid (DHA): A Randomized Placebo-Controlled Clinical Trial’. eBioMedicine 59. doi: 10.1016/j.ebiom.2020.102883.

Chu, Che-Sheng, Chi-Fa Hung, Vinoth Kumar Ponnusamy, Kuan-Chieh Chen, and Nai-Ching Chen. 2022. ‘Higher Serum DHA and Slower Cognitive Decline in Patients with Alzheimer’s Disease: Two-Year Follow-Up’. Nutrients 14(6):1159. doi: 10.3390/nu14061159.

Coughlan, Gillian, Ryan Larsen, Min Kim, David White, Rachel Gillings, Michael Irvine, Andrew Scholey, Neal Cohen, Cristina Legido-Quigley, Michael Hornberger, and Anne-Marie Minihane. 2021. ‘APOE Ε4 Alters Associations between Docosahexaenoic Acid and Preclinical Markers of Alzheimer’s Disease’. Brain Communications 3(2):fcab085. doi: 10.1093/braincomms/fcab085.

Quinn, Joseph F., Rema Raman, Ronald G. Thomas, Karin Yurko-Mauro, Edward B. Nelson, Christopher Van Dyck, James E. Galvin, Jennifer Emond, Clifford R. Jack, Michael Weiner, Lynne Shinto, and Paul S. Aisen. 2010. ‘Docosahexaenoic Acid Supplementation and Cognitive Decline in Alzheimer Disease: A Randomized Trial’. JAMA 304(17):1903–11. doi: 10.1001/jama.2010.1510.

Tomaszewski, Natalie, Xulei He, Victoria Solomon, Mitchell Lee, Wendy J. Mack, Joseph F. Quinn, Meredith N. Braskie, and Hussein N. Yassine. 2020. ‘Effect of APOE Genotype on Plasma Docosahexaenoic Acid (DHA), Eicosapentaenoic Acid, Arachidonic Acid, and Hippocampal Volume in the Alzheimer’s Disease Cooperative Study-Sponsored DHA Clinical Trial’. Journal of Alzheimer’s Disease 74(3):975–90. doi: 10.3233/JAD-191017