Neurorestorative targets of dietary long-chain omega-3 fatty acids in neurological injury

Abstract

Long-chain omega-3 polyunsaturated fatty acids (LC-O3PUFAs) exhibit therapeutic potential for the treatment and prevention of the neurological deficits associated with spinal cord injury (SCI). However, the mechanisms implicated in these protective responses remain unclear. The objective of the present functional metabolomics study was to identify and define the dominant metabolic pathways targeted by dietary LC-O3PUFAs. Sprague-Dawley rats were fed rodent purified chows containing menhaden fish oil-derived LC-O3PUFAs for 8 weeks before being subjected to sham or spinal cord contusion surgeries. We show, through untargeted metabolomics, that dietary LC-O3PUFAs regulate important biochemical signatures associated with amino acid metabolism and free radical scavenging in both the injured and sham-operated spinal cord. Of particular significance, the spinal cord metabolome of animals fed with LC-O3PUFAs exhibited reduced glucose levels (-48 %) and polar uncharged/hydrophobic amino acids (less than -20 %) while showing significant increases in the levels of antioxidant/anti-inflammatory amino acids and peptides metabolites, including β-alanine (+24 %), carnosine (+33 %), homocarnosine (+27 %), kynurenine (+88 %), when compared to animals receiving control diets (p < 0.05). Further, we found that dietary LC-O3PUFAs impacted the levels of neurotransmitters and the mitochondrial metabolism, as evidenced by significant increases in the levels of N-acetylglutamate (+43 %) and acetyl CoA levels (+27 %), respectively. Interestingly, this dietary intervention resulted in a global correction of the pro-oxidant metabolic profile that characterized the SCI-mediated sensorimotor dysfunction. In summary, the significant benefits of metabolic homeostasis and increased antioxidant defenses unlock important neurorestorative pathways of dietary LC-O3PUFAs against SCI.

Figure 1. Timeline outlining experimental design and animal groups

Rats were fed control or LC-O3PUFA-enriched chows for 8 weeks before being subjected to sham or SCI operations. Rats were removed for terminal global metabolomics analyses during acute and chronic injury stages. Values for weights and food consumption are average grams (g) ± S.D; n = at least 16 rats per group.

Figure 2. Dietary LC-O3PUFAs significantly modulate the non-lipid spinal cord metabolome during acute injury stages

(A) Partial least square discriminant analysis (PLS-DA) distinguished subgroups based on operation and dietary intake at 1 week post-operation. This model was constructed using scaled intensity peaks of the global non-lipid detected features. Permutation provided statistically significant separations between subgroups (p < 0.05; not shown). (B) The variable influence on projection (VIP) reflects the importance of amino acids and antioxidant peptides in the generated PLS model.

Figure 3. Dietary LC-O3PUFAs significantly modulate the non-lipid spinal cord metabolome during chronic injury stages

(A) Partial least square discriminant analysis (PLS-DA) discriminated between groups based on dietary intake at 8 weeks post-operation. The model was generated using scaled intensity peaks of the global non-lipid detected features. Permutation provided statistically significant separations between subgroups (p < 0.05; data not shown). (B) The variable influence on projection (VIP) indicates the importance of carbohydrates, amino acids, and antioxidant peptides at 8 week-post operations.

Figure 4. Results of LC-MS/MS data analysis using Ingenuity Pathways Analysis (IPA) software

The data set was filtered for non-lipid small-molecules that met the 1.5 fold cut-off criteria. These metabolites were associated with biological functions in the IPA Knowledge Base. The p-value was calculated using right-tailed Fischer’s exact test and represents the probability that each biological function assigned to that data set is due to random chance alone. P-value < 0.05 were considered statistically significant.

Figure 5. Metabolic pathways targeted by dietary LC-O3PUFA in the sham rat spinal cord

Dietary LC-O3PUFAs target the metabolism of carnosine and homocarnosine, as evidenced by increased levels of alanine, carnosine, homocarnosine, and 4-guanidinobutanoate in the spinal cord of animals fed with LC-O3PUFAs. The diet rich in LC-O3PUFAs also altered the glutamine-glutamate cycling. A distinctive group of amino acid systems were affected by the diet, including threonine and tryptophan. In particular, animals fed with LC-O3PUFAs showed dramatic increases in the levels of kynurenines, which can regulate mitochondrial homeostasis and oxidative stress, inflammation, and glutamate excitotoxicity through NMDA receptor inhibition. Notably, the diet increased the levels of ornithine and urea, while decreasing glucose and glucose-6-P levels, showing selective alterations in the spinal cord cell bioenergetics. This support that LC-O3PUFAs fuel energy production largely by oxidative phosphorylation via the tricarboxylic acid (TCA) cycle and pentose phosphate pathway (PPP) rather than glycolysis, which are essential pathways for the synthesis of necessary macromolecules (i.e. amino acids, neurotransmitters, glutathione, nucleosides and lipids required for assembling new cells). These pathways may represent important mechanisms by which dietary LC-O3PUFAs confer prophylaxis against neurodegeneration and dysfunction in SCI. This reservoir of protective molecules and antioxidant bioavailability is expected to make neurons and glia more resistant against calcium overload, glutamate toxicity, and cell death following SCI. Metabolites in red increased with the dietary intervention. Features in green decreased with the LC-O3PUFA diet when compared to controls. Putative enzymatic/receptor targets are highlighted in red ovals. Abbreviations: AA, amino acid (polar); GABA, gamma-aminobutyrate; NADPH, nicotinamide adenine dinucleotide phosphate; NMDA, N-methyl-D-aspartate; PPP, pentose phosphate pathway; R5P, ribose 5-phosphate; Thr, threonine.

Figure 6. Metabolic pathways targeted by dietary LC-O3PUFA in the injured spinal cord

Although we did not characterize the specific sources of ROS in the present study, our metabolomics dataset supports the role of mitochondrial dysfunction as a major source during both acute and chronic injury stages. Interestingly, we identified glutathione (GSH) metabolism as a molecular target of dietary LC-O3PUFAs. For instance, the animals receiving the dietary intervention showed increased spinal cord levels of γ-glutamylglutamine, cystathione, hippurate, GSH, and GSSH, suggesting increased production and/or reduced depletion of antioxidant pools after SCI. Notably, the levels of heme were increased in the spinal cord of rats exposed to the LC-O3PUFA-rich diet, proposing a novel protective mechanism for LC-O3PUFAs. Similar to the findings observed in sham rats, LC-O3PUFAs altered the TCA and Urea cycle. The increased levels of purine nucleotides and acetyl-lysine suggests a mechanism for which chronic dietary supplementation with LC-O3PUFAs modulates plasticity, growth, and gene expression. Metabolites in red increased with the dietary intervention. Features in green decreased with the LC-O3PUFA diet when compared to controls. Putative enzymatic and protein targets are highlighted in red ovals. Abbreviations: 5-OPase, 5-oxoprolinase; Arg1, arginase; CYGB, cytoglobin; cys-gly, cysteine-glycine; GSH, glutathione, reduced; GSSH, glutathione disulfide, oxidized; GCL, γ-glutamylcysteine ligase; GS, glutathione synthase; GPx, glutathione peroxidase; GR, glutathione reductase; GST, glutathione-S-transferase; γGT, γ-glutamyltransferase; GCT, γ-glutamylcylotransferase; MTA, 5′-methylthioadenosine; Ngb, neuroglobin; TCA, tricarboxylic acid.