The role of dietary DHA in addressing a leaky BBB in Parkinson's disease models

Background and wider context of proposed research

1. What is the background and wider context of your proposed research?

This study in neurodegenerative disorder sufferers is a natural sequel to recent ground-breaking research into pathological fleatures of Alzheimer’s disease [1]. It will explore the role of (low) fatty acid (bioavailability) imbalance on the permeability of the blood brain barrier (BBB), and interference with the development of Parkinson’s disease (PD).  

The importance of the n-3 fatty acid docosahexaenoic acid (DHA) in neurological disorders with a neuroinflammatory component has led to examining the role of diet in regulating its levels; however, mechanistic details regarding its uptake into the brain have been relatively scarce.

The nutrient exists in several forms in nature. One of them is phospholipid and the other is free atty acid esters. The former, found in whole fish, is allegedly, at least in Alzheimer’s cohorts, more efficiently utilised by the brain [1]. There exists a gap in the literature on the comparative, dose-response effect of different forms of the essential n-3 fatty acid, DHA.

I propose a mechanism explaining how increasing dietary DHA may present a plausible route to overcome a leaky BBB which allow xenobiotics (chemicals, toxins and LPS), involved in dopaminergic cell death, to perfuse into the brain. This n-3 appears to help maintain the integrity of the BBB by enhancing tight junction protein expression [6, 9,12].

The present n-3 DHA bioavailability trial will focus on fatty acid trafficking of two DHA dosage forms whilst possibly uncovering the role of phospholipid DHA in improving outcomes in PD.

The primary objective of the study is looking at the comparative bioavailability of a particular dosage form, concentration, and regimen effects of n-3 DHA (here in nonesterified and esterified form) and identifying a dose range that would likely provide adequate exposure to yield an important clinical effect. This effect will be considered clinically significant when there is an observable change in brain DHA accretion after randomisation to dietary DHA or supplemental DHA.

This is an area worthy of study as research suggests that DHA metabolism may be intricately linked to the following clinical outcomes:

• Preserving the integrity of the BBB [13]
• Protecting the brain by increasing glutathione reductase (GSH) whilst decreasing accumulation of oxidized proteins in the brain [15,16]
• Promoting adequate sleep-wake activity and lipid signalling, and prevention of REM Sleep Behaviour Disorder [11,14]
• Alleviating depression, PD’s main non-motor symptom [17]
• Increasing neurogenesis, helping to maintain cognitive function in Lewy Body Dementia [18].

All clinical trials to date that have evaluated the effect of DHA on cognitive function and brain morphology, separately or together, have used fish oil supplements, which do not contain DHA in phospholipid form. When DHA is consumed in phospholipid form (as from dietary fish or seafood) more DHA appears in the plasma as DHA-lysoPC, which may be better transported across the BBB. Dietary sources of DHA in phospholipid form may provide a means to increase plasma levels of DHA-lysoPC, thereby decreasing the risk of PD.

Research question and aims

2. What is your research question? What are the aims and objectives of your research?

“Does dietary fish lead to higher DHA accretion than supplemental DHA in Parkinson’s disease animal models?”

1. The wider aim of this research is the learning of ways, in which an essential nutrient may affect secondary pathologies linked to PD, that have potential to change the approach to disease. The improvement of n-3 DHA stores through aging constitutes an easily implementable target in those dealing with neurodegeneration.

2. Generating interest for further potential research looking at the impact of DHA metabolism on “primary” secondary symptoms of PD. That is, starting with the most impactful endpoint(s) and gradually moving down the list of less significant outcomes.

3. What is your research design?

My study is experimental, prospective and carried out in engineered mice models of PD. The study explores fatty acid trafficking in the brain of PD animal models in the short-term through, continuous, punctuated intervention with supplementation or dietary DHA.

In this 8-week dose-response bioavailability crossover trial, a total of n=80 mice (40 per group) will be randomized in this study in a 1:1 ratio to test and placebo. A group of mice will be split into equal fish oil consuming and isocaloric EFA-sufficient fish roe consuming groups or a mixture of both for comparison to a low-omega-3 diet for 8 weeks. A range of doses and dosing intervals will be investigated in a sequential manner, after which time blood will be sampled weekly and DHA accretion rates will be estimated. It may be the case that an average increase of a certain magnitude will be required for either treatment to be deemed effective.

4. Who or what will make up your samples? How many participants need to be recruited? What are your inclusion and exclusion criteria? Why those participants and not others? What are some potential limitations of your sample?

The sample will consist of two randomized groups of n=80 mice models of PD. Dietary and supplemental input will be isocaloric. Other confounders, such as nutrient-specific interactions involved in the metabolism of DHA [5], will, too, be accounted for in the final analysis. Genotyping will also help reduce variability in the control population.

In terms of inclusion and exclusion criteria, the benefits of treatment allocation should be obtained from a group of animals who are most representative of the real-world patients to whom the protocol would be given. Mice need to be diagnosed as having PD. This can be achieved through engineering mice to induce the onset of some of selected pathological features. The protocol will state the precise criteria that constitute relevant measurable clinical signs PD, such as elevations in certain inflammatory markers, dystonia, chronic illness.  

Research design

The main limitation of the sample is that the metabolism of animals and humans is closely related but also significantly distinct. Therefore, any outcome from this trial will have to be interpreted as the big picture model of the Parkinson’s brain.

5. How will you get access to your sample?

I would seek access to samples through a partnering research facility studying rodents’ brain physiology and metabolism. There is such a facility at the Rowett Institute, here in Aberdeen. They are researching slightly different questions, but have a high collective understanding of the brain, in addition to being a rich learning environment to evolve in a lab.

6. What methods of data collection will you use? Why is this method the most appropriate? Are there any potential limitations of this method?

I have identified valuable research methods for conducting scientific inquiry here, in line with the two leading methods in the field: the observation of transcription factors coding for proteins regulating DHA brain metabolism as well as plasma DHA pools [2,7,9].

The most common methods of data collection in use for the study of brain PUFA metabolism today include metabolite isotope tracers and surrogate endpoints of brain DHA accretion, detailed in the next section [3,12]. Tracer studies, in particular, suggest that phospholipid DHA (PL-DHA) more effectively targets the brain than triglyceride DHA (TAG-DHA). For this type of experiment, rats are supplemented for n weeks with TAG-DHA (fish oil), PL-DHA (roe PL) or a mixture of both for comparison to a low-omega-3 diet. Then, brain tissues are collected, and blood sampled weekly. DHA accretion rates are estimated using the balance method.

Both biological and methodologic confounders complicate the interpretation of brain DHA uptake data during aging [6]. Animal studies provide a higher level of control. But there are also many expected limitations. While one might think that dietary studies would be the gold standard for determining the effects of DHA in the brain, they are often confounded by other dietary factors and by concurrent decreases in peripheral tissue DHA levels [8,10].

7. What is the procedure for collecting your data?

The conductor of the study must first seek licence and access to samples. Measurements of pharmacokinetic data will be collected on several occasions, at various time points, before the start of the trial, at several times during administration/exposure, and at least once after the administration when its effect is likely to be minimal (for example, 24h later).

Considering that the difference in response (to different forms of n-3) may lie in the way the brain transports the different forms of DHA, comparative data from tracer studies to estimate DHA accretion rates would be useful. The brain relies on different transport systems for shuttling the varied forms of the metabolized DHA across the BBB [12]. The metabolized DHA across the BBB can be quantified in rodents by looking at various markers: a) Plasma non-esterified fatty acid (NEFA) pool and DHA-lysoPC, a biomarker for brain DHA levels, b) Tracer work using DHA labelled stable isotope, which is limited to blood samples, c) Genetic mapping of brain PUFA metabolism (e.g., ACSL, FATP), d) Recycled DHA in the Lands’ cycle (surrogate), e) Measures of stores of DHA in brain membrane phospholipids.

For the purposes of this study, at least two of those biomarkers will be chosen, or a combination of two, with a leaning towards a) and b).

8. How will you analyse your data? What method of analysis will you use? Why is this method appropriate? Are there any potential limitations of this method?