Fig 1.
Serum metabolomic response to aging.
(A) Metabolites (phosphatidylcholines, sphingomyelins, acylcarnitines) showing differential patterns in the serum of mice aged 2 (n = 2), 4 (n = 3), 14 (n = 2), and 21 months (n = 3). Results are shown as mean (solid line) ± standard error of the mean (SEM; dotted line). (B) Metabolites that decreased with aging. Statistical significance is indicated in the heatmap with asterisks. (C) Metabolites that increased with aging. Statistical significance is indicated in the heatmap with asterisks. In (A) to (C), PC and SM represent phosphatidylcholine and sphingomyelin, respectively. The terms aa, ae, and -OH represent diacyl, acyl-alkyl, and hydroxylated forms, respectively. In (B) and (C), data were analyzed with an ordinary two-way ANOVA with Tukey’s multiple comparison test; *p < 0.05, **p < 0.01.
Fig 2.
Palmitoyl-L-carnitine increases tau phosphorylation.
(A) shows changes in protein levels of pTau (T181), pTau (S262), PHF-1, and total tau after treatment with palmitoyl-L-carnitine in SH-SY5Y cells. Full blots are provided in S1 File. (B) Histogram illustrating protein levels of pTau (T181), pTau (S262), and PHF-1 in cells treated with palmitoyl-L-carnitine, shown as mean ± standard error of the mean (SEM; n = 3). Tau phosphorylation levels were normalized to the total tau protein. In (A) and (B), pTau (T181), pTau (S262), and PHF-1 represent tau phosphorylated at threonine 181, serine 262, and serine 396/404, respectively. BSA and BSA-PC represent bovine serum albumin and BSA-conjugated palmitoyl-L-carnitine, respectively. Statistical significance was determined using an unpaired two-tailed t-test with Welch’s correction; *p < 0.05, **p < 0.01.
Fig 3.
Palmitoyl-L-carnitine leads to mitochondrial fission.
(A) shows changes in protein levels of pDRP1 (S616), DRP1, pMFF (S146), and MFF after palmitoyl-L-carnitine treatment in SH-SY5Y cells. pDRP1 (S616) and pMFF (S146) reflect DRP1 phosphorylated at serine 616 and MFF phosphorylated at serine 146, respectively. Full blots are provided in S1 File. (B) Histogram illustrating changes in protein levels of pDRP1 (S616), DRP1, pMFF (S146), and MFF after palmitoyl-L-carnitine treatment, shown as mean ± standard error of the mean (SEM; n = 3). (C) shows changes in protein levels of OPA1, Mitofusin-1, and Mitofusin-2 after palmitoyl-L-carnitine treatment in SH-SY5Y cells. Full blots are provided in S1 File. (D) Histogram illustrating changes in protein levels of OPA1, Mitofusin-1, and Mitofusin-2 after palmitoyl-L-carnitine treatment, shown as mean ± standard error of the mean (SEM; n = 3). (E) shows changes in mitochondrial dynamics after palmitoyl-L-carnitine treatment in GFP-Mito transfected SH-SY5Y cells. The images represent findings from at least three independent experiments (n = 3), with more than four fields of view analyzed per replicate. The rectangular box with a white dotted line indicates a magnified field of view. In (A) to (E), BSA and BSA-PC represent bovine serum albumin and BSA-conjugated palmitoyl-L-carnitine, respectively. Statistical significance was determined using an unpaired two-tailed t-test with Welch’s correction; *p < 0.05, **p < 0.01.
Fig 4.
Palmitoyl-L-carnitine results in calcium overload in the cells.
(A) shows changes in calcium overload after palmitoyl-L-carnitine treatment in SH-SY5Y cells. (B) shows a histogram illustrating changes in fluorescence intensity of calcium staining induced by palmitoyl-L-carnitine. Data represent three independent experiments (n = 3) and are expressed as mean ± standard error of the mean (SEM). Fluorescence intensity of calcium staining (Fluo-4 AM) was quantified using Image J software. In (A) and (B), BSA and BSA-PC represent bovine serum albumin and BSA-conjugated palmitoyl-L-carnitine, respectively. Statistical significance was determined using an unpaired two-tailed t-test with Welch’s correction; **p < 0.01.
Fig 5.
Tau kinases are involved in palmitoyl-L-carnitine-mediated tau phosphorylation.
(A) shows changes in protein levels of pGSK-3β (S9), GSK-3β, CDK5, and p25 after treatment with palmitoyl-L-carnitine in SH-SY5Y cells. pGSK-3β (S9) indicates GSK-3β phosphorylated at serine 9. Full blots are provided in S1 File. (B) Histogram illustrating changes in protein levels of pGSK-3β (S9), CDK5, and p25 after treatment with palmitoyl-L-carnitine in SH-SY5Y cells, shown as mean ± standard error of the mean (SEM; n = 3). GSK-3β phosphorylation levels were normalized to the total GSK-3β protein. (C) shows changes in protein levels of pTau (T181), pTau (S262), PHF-1, total tau, GSK-3β, CDK5, and p25 after treatment with tau kinase inhibitors in palmitoyl-L-carnitine-treated SH-SY5Y cells. pTau (T181), pTau (S262), and PHF-1 represent tau phosphorylated at threonine 181, serine 262, and serine 396/404, respectively. SB216763, Roscovitine, and PD150606 are GSK-3β inhibitor, CDK5 inhibitor, and calpain inhibitor, respectively. Full blots are provided in S1 File. (D) Histogram illustrating changes in protein levels of pTau (T181), pTau (S262), PHF-1, total tau, GSK-3β, CDK5, and p25 after treatment with tau kinase inhibitors in palmitoyl-L-carnitine-treated SH-SY5Y cells, shown as mean ± standard error of the mean (SEM; n = 3). Tau phosphorylation levels were normalized to the total tau protein. In (A) and (D), BSA and BSA-PC represent bovine serum albumin and BSA-conjugated palmitoyl-L-carnitine, respectively. Statistical significance was determined using an unpaired two-tailed t-test with Welch’s correction and an ordinary two-way ANOVA with Tukey’s multiple comparison test; ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 6.
Palmitoyl-L-carnitine might play a key role in AD pathology with aging.
A schematic illustration showing the mechanism by which palmitoyl-L-carnitine induces tau phosphorylation in SH-SY5Y neurons. Palmitoyl-L-carnitine causes calcium overload by closely interacting with mitochondrial malfunction, including the fission process. This increased calcium overload activates tau kinases (GSK-3β and CDK5/p25), leading to significant tau phosphorylation. Therefore, elevated serum levels of palmitoyl-L-carnitine are likely to contribute significantly to the development of AD pathology with aging.