Dihydroxy-Metabolites of Dihomo-γ-linolenic Acid Drive Ferroptosis-Mediated Neurodegeneration

Even after decades of research, the mechanism of neurodegeneration remains understudied, hindering the discovery of effective treatments for neurodegenerative diseases. Recent reports suggest that ferroptosis could be a novel therapeutic target for neurodegenerative diseases. While polyunsaturated fatty acid (PUFA) plays an important role in neurodegeneration and ferroptosis, how PUFAs may trigger these processes remains largely unknown. PUFA metabolites from cytochrome P450 and epoxide hydrolase metabolic pathways may modulate neurodegeneration. Here, we test the hypothesis that specific PUFAs regulate neurodegeneration through the action of their downstream metabolites by affecting ferroptosis. We find that the PUFA dihomo-γ-linolenic acid (DGLA) specifically induces ferroptosis-mediated neurodegeneration in dopaminergic neurons. Using synthetic chemical probes, targeted metabolomics, and genetic mutants, we show that DGLA triggers neurodegeneration upon conversion to dihydroxyeicosadienoic acid through the action of CYP-EH (CYP, cytochrome P450; EH, epoxide hydrolase), representing a new class of lipid metabolites that induce neurodegeneration via ferroptosis.


Supporting Figures and
): Figure S1: Dose response curve: the effect of different DGLA concentrations on degeneration of ADE neurons at Day 1 and Day 8 of adulthood. The slope for dose response curve on day 8 adulthood is significantly different compared to day1 adulthood, suggesting there may be different mechanism for neurodegeneration at these 2 timepoints.
S4 Figure S2: Ethanol does not alter ADE neuron phenotypes. Percentage (%) of worms with healthy ADE dopaminergic neurons in dat-1::gfp transgenic worms +/supplementation with 10 µl absolute ethanol. This test was done to determine whether ethanol in PUFA supplementation (which is 10 µl) affects the overall healthspan of dopaminergic neurons. N=3, and about 20 worms were tested in each replicate.. Two-way analysis of variance (ANOVA), Tukey's multiple comparison test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P < 0.0001, ns: not significant.  This result suggests that DGLA could trigger ferroptosis-mediated neurodegeneration independent of the GPX pathway, which is not without precedent. It has been reported that cytochrome P450 oxidoreductase mediates ferroptosis distinct from the GPX pathway(Zou et al., 2020). Another likely possibility is that there is redundancy, and we have only tested one of the seven isoforms of GPX. More testing on other GPX isoforms is underway. Figure S5: DGLA Supplementation significantly changes the EEDs and DHEDs levels in worm. Oxylipin profile representing the pmol/g of Epoxy-and dihydroxy-PUFA levels in worms treated with 100 µM of DGLA compared to control. The worms were supplemented at the L4 stage, and were tested at day 1 of adulthood. Black boxes represent the values are those that were inconsistent in different trials or were out of standard curve range.

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S8 Figure S6: AUDA changes the EEDs and DHEDs levels in worm supplemented by DGLA, through inhibition of epoxide hydrolase enzyme. Oxylipin profile representing the pmol/g of Epoxy-and dihydroxy-PUFA level in worms treated with 100 µM of DGLA ±100 µM AUDA compared to control. The worms were supplemented at the L4 stage and were tested at day 1 of adulthood. Black boxes represent the values are those that were inconsistent in different trials or were out of standard curve range.   Oxylipin profile representing the pmol/g of Epoxy, hydroxy, and dihydroxy-PUFA regioisomers, CYP/EH metabolites in worms treated with 100 µM of either DGLA or EEDs, or DHEDs compared to control. Worms were supplemented at the L4 stage and were tested at day 1 of adulthood. Black boxes represent the values those that were inconsistent in different trials or were out of standard curve range. S12

Experimental Sections:
Reagent and resource: Deuterated standards used for oxylipin analysis.

Age synchronized worms:
The age-synchronized population was prepared by transferring specific numbers Finally, L4 worms were resuspend in s-basal solution and transferred to the supplemented or control plates seeded with OP50.
During lifespan, every day the age synchronized population was filtered through a 40 μm cell strainer placed on top of a 50 mL centrifuge tube. The progeny was collected in the filtrate and removed. The age-synchronized adult worms were removed from the surface of the cell strainer and placed on a freshly seeded NGM/supplemented plate. The filtration process was repeated every day during early adulthood of the age synchronized population to avoid any S15 contamination from the progeny, and to provide fresh supplementation for worm during their lifespan.

Fatty acid supplementation:
In order to supplement worms with fatty acids and/or their downstream metabolites, 10 ul of each compound at desired concentration was spread on the NGM plate, and then immediately seeded with 250-400 µl E. coli OP50 (2.8 ×10 8 cell/ml). The seeded plates were sealed with parafilm? and kept for 2 days at room temperature (20-23°C) and then transferred to the refrigerator to be used later. In all experiments in this study, the NGM solution plates were made using standard methods 3 .

Supplementations for ferroptosis studies:
To study the possible role of DGLA and EEDs supplementation in ferroptosis, 10 µl of 100 µM of DGLA or EEDs was spread on NGM plates, followed by spreading 10 µl of 100 µM of liproxstatin-1 (Lip-1) solution in ethanol. Immediately after that, 250-400 µl E. coli OP50 was plated and allowed to dry for two days. The plates were then either used immediately or kept in the fridge (4°C) for later use. The same procedure was used for the 2,2-bipyridine (BP) (100 µM) and Trolox (500 µM), For the control experiments, 10 µl of ethanol solution was used.

Fluorescence microscopy imaging for tracking dopaminergic neurons.
In order to track neurodegeneration, age-synchronized worms with dat-1::gfp transcriptional fusions were used. The age-synchronized worms were analyzed based on a previously published protocol with some modifications 4 . First agarose gel pad were prepared as previously described. (For quantitative analyses of changes in DAergic neuron cell morphology, 20-25 worms were mounted on the layer of the agar pad and paralyzed with 5 mM NaN3 for 5 S16 minutes (Fig.15). Finally, a coverslip was placed onto an agar pad containing worms. A fluorescent microscope (Eclipse Ti2-E-Nikon) was used to image the worms and NIS-Elements software was used to analyze the data. All 8 DAergic neurons were analyzed in each worm. The ADE neurons were the ones with significant neurodegeneration with the treatment of DGLA and its downstream metabolites. Therefore, in all microscopic tests in this study, neurodegeneration refers to the absence of fluorescent signal in the ADE neurons. Worms with healthy ADE are those with both ADE cell bodies or processes that could be seen under fluorescent microscope. The same procedure was followed for dopaminergic neuron analyses using the cat-2::gfp (EM641).
Step 1: Collecting and freezing worm samples for oxylipin analysis Oxylipins are a class of bioactive oxidized lipid metabolites derived from PUFAs via cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) enzymatic pathways.
To investigate the oxylipin profile in C. elegans, we collected about 5 mg of worms per trial to ensure that the whole worm lysates contain a sufficient concentration of oxylipins for detection. A sufficiently sized population of worms was generated using a minimum of 7 P100 plates with a diameter of 100 mm?, per trial. To generate 5 mg of whole worm lysates, we prepared approximately 2000-3000 worms (300-400 worms per plate). The age-synchronized population of worms was generated and maintained using the filtration method illustrated and described above.
When a population of worms was ready for isolation and collection, the entire population of seven plates per trial was transferred and filtered using s-basal solution and a cell strainer with a pore size of 40 µm. The worms that collected on the surface of the cell strainer were transferred using a Pasteur pipet to an Eppendorf vial to avoid C. elegans from sticking on the wall of the pipet. The worms were rinsed with s-basal medium and centrifuged. The supernatant was collected and discarded. The worms were then washed four more times with s-basal medium to ensure that all bacteria and PUFA supplements were removed. After the bacteria and supplements were removed, the Eppendorf vials containing each worm sample were transferred to a benchtop centrifuge. The vials were centrifuged for 10 minutes at 10,000 rpm at 4°C. The supernatant was removed using 100 µL and 10 µL pipets. A 20 µL pipet with a long tip was pushed to the bottom of the vial to remove the liquid between the worms. Lastly, the standard filter paper was cut and inserted into the Eppendorf vials to remove any remaining liquid within the worm sample. After all liquid was S17 removed, the worm samples flash frozen using liquid nitrogen and stored in the -80°C freezer.
Step 3: Solid phase extraction to isolate the oxylipins from the whole worm lysate To isolate the oxylipins from the whole worm lysates solid phase extraction (SPE) (Waters Oasis-HLB cartridges, (Part No. WAT094226, Lot No. 176A30323A) was used. We used a polar stationary phase to trap the extremely polar biological material such as sugars. The oxylipins that S19 we are isolating are significantly less polar in comparison. The SPE column was prepared by sequential washing with 2 mL ethyl acetate, 2 mL methanol twice, and 2 mL of 95:5 (v/v) mixture of water and methanol containing 0.1% acetic acid. The column was kept moist during preparation.
The process of SPE column preparation is illustrated in the Figure S12.  After the column was loaded with the sample and completely dried, 0.5 mL of methanol was added to begin the elution step. Eluted compounds were collected to an Eppendorf vial containing 6 µL of 30% glycerol in methanol, which serves as a trap solution. The column was allowed to gravity elute until the column appeared dry. A 5 mL syringe was filled with air and placed on the top of S20 the SPE column to gently push the remaining solvent out of the column with air. Once the column was completely dry, 1 mL of ethyl acetate was added to the column. The solvent was allowed to gravity elute until the column appeared dry to the eye. The remaining solvent was again removed using a 5 mL syringe and gently pushing air through the column. The process of eluting is illustrated in the Figure S14.  detector. Electrospray was operated as ionization source for negative multiple reaction monitoring (MRM) mode. To generate the best selectivity and sensitivity, each analyte standards were infused into the mass spectrometer and multiple reaction monitoring was used to analyze the desired compound.  were then added, and the layers were separated. The aqueous layer was extracted with EtOAc (4 x 10 mL) and the organic phase was washed with saturated aqueous NaCl (40 mL), dried over Na2SO4, concentrated, and azeotroped with hexanes (3 x 20 mL) to remove residual formic acid. oxylipin analysis was used to confirm the mixture of regioisomer and the purity of the compound Figure S16 shows the different regioisomers ratio in EEDs and DHEDs supplementation used in this study. Confirmation of desired structure was done by examining ratio of integrations of alkene protons (~5 -6 ppm) to the methyl ester (~3.6 ppm) on 1 H NMR. HRMS was also done to ensure desired mass. Separations and characterization were performed to determine relative percentages of each isomer in mixture used for biological testing.

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The synthesis followed published procedure 5 . To a suspension of 12-aminododecanoic acid (1g,