Circulating (Poly)phenol Metabolites: Neuroprotection in a 3D Cell Model of Parkinson's Disease

Scope Diets rich in (poly)phenols have been associated with positive effects on neurodegenerative disorders, such as Parkinson's disease (PD). Several low‐molecular weight (poly)phenol metabolites (LMWPM) are found in the plasma after consumption of (poly)phenol‐rich food. It is expected that LMWPM, upon reaching the brain, may have beneficial effects against both oxidative stress and neuroinflammation, and possibly attenuate cell death mechanisms relate to the loss of dopaminergic neurons in PD. Methods and Results This study investigates the neuroprotective potential of two blood‐brain barrier permeant LMWPM, catechol‐O‐sulfate (cat‐sulf), and pyrogallol‐O‐sulfate (pyr‐sulf), in a human 3D cell model of PD. Neurospheroids were generated from LUHMES neuronal precursor cells and challenged by 1‐methyl‐4‐phenylpyridinium (MPP+) to induce neuronal stress. LMWPM pretreatments were differently neuroprotective towards MPP+ insult, presenting distinct effects on the neuronal transcriptome. Particularly, cat‐sulf pretreatment appeared to boost counter‐regulatory defense mechanisms (preconditioning). When MPP+ is applied, both LMWPM positively modulated glutathione metabolism and heat‐shock response, as also favorably shifting the balance of pro/anti‐apoptotic proteins. Conclusions Our findings point to the potential of LMWPM to trigger molecular mechanisms that help dopaminergic neurons to cope with a subsequent toxic insult. They are promising molecules to be further explored in the context of preventing and attenuating parkinsonian neurodegeneration.


Neurospheroids monitoring along time
At different time points along culture progression, viability of differentiated neurospheroids was assessed using a fluorescence microscopy-based method, the fluoresceine diacetate (FDA)/propidium iodide (PI) assay. [1] Moreover, cell proliferation was also assessed by fluorescence microscopy using Click-iT Plus EdU AlexaFluor TM 488 Assay kit (Life Technologies). Neurospheroids sampling was performed along differentiation and neurospheroids disrupted in 0.1 M citric acid with 1% Triton X-100, at 37 °C, overnight, and nuclei stained with 0.1 % crystal violet and counted in a Fuchs-Rosenthal haemocytometer chamber. [2] A sample size of at least 1 mL of culture was used to reliably determine cell concentration, neurospheroids counting and perform aggregate size measurements using ImageJ software.
Exocytosis was stimulated with 100 mM KCl buffer and samples were visualized live using a fluorescence microscope (Leica DMI6000) in order to monitor the decrease in fluorescence intensity over time. Fluorescence intensity was measured using ImageJ software.
Spontaneous synaptic activity of LUHMES neurospheroids was assessed by their response in calcium assay. [5] Neurospheroids with 7 days of differentiation were incubated with 1x Fluo-4 Direct calcium reagent (Invitrogen) for 30 min at 37 °C, 5% CO2 and for 15 min at RT. Samples were then imaged live using spinning disk microscopy (Nikon Eclipse Ti-E, confocal scanner: Yokogawa CSU-x1). Fluorescence change over time is defined as ΔF/F0 = (F-F0)/F0, where F is the fluorescence at any time point, and F0 the baseline fluorescence determined by baseline fitting across the whole movie for each cell using SparkMaster plugin of ImageJ software.

Immunofluorescence microscopy
Neurospheroids were collected at 7 days of differentiation and processed for immunofluorescence staining. [4] Briefly, neurospheroids were fixed in 4% (w/v) paraformaldehyde (Sigma-Aldrich) solution in PBS with 4% (w/v) sucrose and processed directly for immunostaining.
The primary antibodies used for cell characterization were anti-βIII-tubulin (βIII-tub, Millipore), antiglial fibrillary acidic protein (GFAP, DAKO) and anti-tyrosine hydroxylase (TH, Santa Cruz Biotechnology). The secondary antibodies used were goat anti-mouse IgG-AlexaFluor 488 and goat anti-rabbit IgG AlexaFluor 594 (Life Technologies). Cell nuclei were counterstained with 4',6diamidino-2-phenylindole (DAPI, Life Technologies). Preparations were visualized using a point-scan confocal (Leica SP5) microscope. Merge between channels, maximum z-projections, and orthogonal projections, as well as linear brightness and contrast adjustment of the images were created using ImageJ software.