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Zooming the link between diet and brain health: how phenolic metabolites modulate brain inflammation

Periodic Reporting for period 2 - LIMBo (Zooming the link between diet and brain health: how phenolic metabolites modulate brain inflammation)

Reporting period: 2020-10-01 to 2022-03-31

A current big concern of our aging society is to efficiently delay the onset of neurodegenerative diseases, like Parkinson’s disease, which are progressively rising in incidence. The paradigm that a diet rich in phenolics, abundant compounds in e.g. fruits, is beneficial to brain health has reached the public. However, their mechanistic actions in brain functions remain to be disclosed, particularly since the nature of those acting in the brain remains overlooked.
LIMBO’s project addresses this gap by identifying candidate compounds that can support the development of effective strategies to delay neurodegeneration. In particular, we have been analyzing the potential of dietary phenolics in both prevention and treatment (i.e. delay) of neuroinflammation – a key process shared among neurodegenerative diseases. We are focused on the low molecular weight (LMW) (poly)phenol metabolites derived from dietary phenolics and investigate their brain permeability and their effects in both established and unknown responses of microglia cells - the innate immune cells of the central nervous system, either alone or when communicating with other brain cells. Ultimately, to attain an integrated view of their effects, we will establish nutrition trials in mice. LIMBo considers both pro- and anti- inflammatory processes to preliminary validate the action of any promising metabolite in prevention and/or therapeutics.
LIMBo can be divided into 3 major objectives as listed below:
1. Identify LMW (poly)phenol metabolites in the brain and screen their capability to attenuate microglia-mediated neuroinflammation
2. Elucidate the molecular mechanisms mediating the effects of LMW (poly)phenol metabolites in brain cells undergoing inflammation
3. Attain an integrated perception of dietary phenolics effects in a mice model of neuroinflammation and in a mice model of Parkinson’s disease.

The evaluation of all these datasets is essential to build an integrated vision of dietary phenolics effects in brain health, particularly how they influence brain dysfunction with inflammation and ultimately of their potential in the prevention and/or treatment of neurodegenerative diseases.
We listed all described LMW (poly)phenol metabolites and reviewed the molecular mechanisms underlying their effects on the brain and their specific role in neuroinflammation, identifying major gaps. Moreover, we also gathered the sparse data available regarding brain bioavailability of these human LMW (poly)phenol metabolites, including in silico, in vitro and in vivo evidence about blood-brain barrier transposition. Based on this, we decided to update our library with all described LMW (poly)phenol metabolites (around 130), either acquired commercially or by performing the chemical synthesis of the remaining.
To get a comprehensive overview of LMW (poly)phenol metabolites’ preventive neuroprotective effects towards neuroinflammation, we screened their anti-neuroinflammatory properties to protect microglia cells from a strong inflammatory insult, monitoring the release of pro-inflammatory cytokines and neurotoxic factors. Kinetics and dose-response of their protective effects using near-physiological conditions of both concentration and residence time for each metabolite were established.
The brain permeability of the top 5 LMW (poly)phenol metabolites was evaluated in silico and for the 3 top compounds, presenting higher anti-neuroinflammatory activity, their brain permeability was confirmed in vivo. Compounds were injected intravenously at levels comparable to those reported as physiological post-absorption after an intake of berry fruits serving - hence nutritionally relevant. Brain extracts are currently being analyzed for detection of the LMW (poly)phenol metabolites and their end-route metabolites by untargeted metabolomics.
LMW (poly)phenol metabolites’ ability to prevent LPS-mediated internalization of toll-like receptor 4 (TLR4), together with a multiplex cytokine analysis, reinforced their remarkable anti-inflammatory potential in N9 microglial cells. Parallelly, LMW (poly)phenol metabolites’ neuroprotective capacity against a dopaminergic insult in LUHMES cells, both in 2D and 3D models (i.e. cell viability, ATP levels, thiolome, apoptosis-related genes, and stress response-related genes) supports further investigations in more physiologically relevant model systems. Both human and rodent cell lines have been used to disclose relevant pathways modulation by the LMW (poly)phenol metabolites during a systemic inflammatory insult, in microglia-neuron crosstalk, as well as to unravel astrocytes’ participation in an inflammatory insult.
Human co-culture and mixed cultures set-up optimization with the different cell lines are currently being developed. Initial data points for a more physiological cell system to study cells crosstalk and implementation of microfluidics BBB on-a-chip model will be a more robust model for translational results. Such systems will be used to undoubtfully disclose (poly)phenol metabolites’ mode of action towards neuroinflammation.
Our final goal is to estimate their potential in vivo in two animal models – MPTP-induced Parkinsons’ disease-like pathology, and in a LPS model of neuroinflammation. Since the neuroinflammation is a hallmark of both models, our focus will be on estimating the potential of berry-enriched food to modulate the main hallmarks of neuroinflammation (i.e. pro-inflammatory cytokine production, microglial and astroglial activation, peripheral macrophage infiltration) but also to unravel the molecular mechanisms behind the observed effects of the LMW (poly)phenol metabolites. Moreover, the potential of the LMW (poly)phenol metabolites to affect the oxidative stress and neurodegeneration within the brain will be analysed. Since any nutritional paradigm represents systemic intervention, we decided on an integrative approach to create a comprehensive image of LMW (poly)phenol metabolites effect, not only in the brain but in the entire organism. To accomplish this integrated view, we benefited from the diverse expertise of our collaborators and a broad-based analysis of different organs/tissues was done. Currently, a pilot trial for both models was done to confirm that our protocols are adequate to evaluate the capacity of berries phenolics to mitigate neuroinflammation and to analyze the outcome of a nutritional intervention in mice symptomatic of Parkinson’s disease. Some of the analyses have already been performed while others are still in progress.
The comprehensive analysis of LMW (poly)phenol metabolites’ ability to reach the brain and capacity to attenuate microglial activation, therefore reducing neuroinflammation, fills the currently identified gaps in this area, constituting progress beyond the state of the art. Moreover, we predict to uncover the molecular targets of the most active metabolites and validate the effects and targets in vivo. In the end, LIMBo will provide invaluable scientific insights for future implementation of healthy brain diets. LIMBo will also create far-reaching opportunities by generating knowledge that impacts our fundamental understanding about the diversity of phenolic metabolites and their specific impacts in neuroinflammation and potential use as prodrugs.