CMS121, a fatty acid synthase inhibitor, protects against excess lipid peroxidation and inflammation and alleviates cognitive loss in a transgenic mouse model of Alzheimer's disease

Abstract

The oxidative degradation of lipids has been shown to be implicated in the progression of several neurodegenerative diseases and modulating lipid peroxidation may be efficacious for treating Alzheimer's disease (AD). This hypothesis is strengthened by recent findings suggesting that oxytosis/ferroptosis, a cell death process characterized by increased lipid peroxidation, plays an important role in AD-related toxicities. CMS121 is a small molecule developed against these aspects of neurodegeneration. Here we show that CMS121 alleviates cognitive loss, modulates lipid metabolism and reduces inflammation and lipid peroxidation in the brains of transgenic AD mice. We identify fatty acid synthase (FASN) as a molecular target of CMS121 and demonstrate that modulating lipid metabolism through the inhibition of FASN protects against several AD-related toxicities. These results support the involvement of lipid peroxidation and perturbed lipid metabolism in AD pathophysiology and propose FASN as a target in AD-associated toxicities.

Keywords: Alzheimer’s disease; Cognition; Fatty acid synthase; Ferroptosis; Lipid peroxidation; Oxytosis.

Graphical abstract

Fig. 1

CMS121 alleviates cognitive dysfunction in APPswe/PS1ΔE9 transgenic AD mice. After 3 months of treatment with CMS121, the spatial memory (A), the anxiety response (B) and the contextual memory (C) in 12 months old APPswe/PS1ΔE9 transgenic AD mice were normalized to age-matched WT levels. A. While all mice were able to locate the visible platform after 4 trials (V4) in the Morris Water Maze (MWM), untreated AD mice spent significantly more time locating the hidden platform. In contrast, CMS121-treated AD mice could, identical to WT mice, locate the platform after only one trial (T1). B. The disinhibition response was assessed by measuring the time the mice spent in the open arms of the elevated plus maze (EPM) over a 5min trial. Untreated AD mice spent a significantly longer amount of time in the open arms, compared to the other test groups, confirming the disinhibition phenotype seen in AD. C. Similarly, the contextual memory of AD mice was impaired, as measured by the fear conditioning test. The first day, the mice received an aversive stimulus after receiving a tone. After 24h, the mice were placed back in the same chamber for 120s but without the tone. The time the untreated AD mice spent frozen was significantly lower than the other test groups, suggesting a loss of contextual memory. CMS121 treatment normalized contextual memory back to baseline WT levels. (*p<0.05, **p<0.01, ***p<0.001, one-way ANOVA, Tukey’s post-hoc test, n = 12).

Fig. 2

CMS121 decreases lipid peroxidation and has anti-inflammatory properties. Using Bodipy 581/591 reagent we found that CMS121 prevents the increase in lipid peroxidation induced by the GPX4 inhibitor RSL3 in HT22 neuronal cells (A) and by LPS in BV2 microglial cells (B). Results are expressed as mean fold changes ±SD; relative to untreated control cells: fold changes are thus calculated by using untreated control cells as baseline (***p<0.001, T-test, n = 3–4). C. Relative levels of 4-hydroxynonenal (4HNE) protein adducts as a measure of lipid peroxidation are increased in untreated AD compared to untreated WT mice. Treatment of AD mice with CMS121 reduced 4HNE adduct levels back to WT levels. To measure the level of 4HNE protein adducts, the optical density of the entire lane was determined (the whole lane was considered as a single band) and normalized against the respective actin levels. CMS121 decreases 15LOX2 (D) and GFAP (E), both markers of inflammation in the hippocampi of AD mice. Results are expressed as mean ± SEM (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. One-way ANOVA, n = 5–6).

Fig. 3

Intraneuronal Aβ increases eicosanoids and this is countered by CMS121. A. Representative blot of intracellular Aβ induction after Tet removal (Tet-) in MC65 cells. CMS121 prevents the accumulation of Aβ. B. CMS121 prevents cell death induced by intracellular Aβ in MC65 cells (n = 3). C. Eicosanoid analysis of MC65 cells shows a general increase in eicosanoid levels secreted into the culture medium by cells when Aβ toxicity is induced (Tet-). Treatment of cells with CMS121 prevents this increase (Tet- + CMS121). Results are expressed as fold changes normalized over Tet + baseline levels. Every dot represents the average fold change of a single eicosanoid (****p<0.0001, Wilcoxon matched-pairs signed rank test, n = 4). D,E. Individual eicosanoids derived from arachidonic acid (D), linoleic acid (E) and docosahexaenoic acid (E) organized by the main enzymatic system involved in the metabolism of the respective eicosanoid (COX: cyclooxygenase; CYP: cytochrome p450; LOX: lipoxygenase). Relative lipid levels, normalized against + Tet controls, are depicted (n = 4).

Fig. 3

Intraneuronal Aβ increases eicosanoids and this is countered by CMS121. A. Representative blot of intracellular Aβ induction after Tet removal (Tet-) in MC65 cells. CMS121 prevents the accumulation of Aβ. B. CMS121 prevents cell death induced by intracellular Aβ in MC65 cells (n = 3). C. Eicosanoid analysis of MC65 cells shows a general increase in eicosanoid levels secreted into the culture medium by cells when Aβ toxicity is induced (Tet-). Treatment of cells with CMS121 prevents this increase (Tet- + CMS121). Results are expressed as fold changes normalized over Tet + baseline levels. Every dot represents the average fold change of a single eicosanoid (****p<0.0001, Wilcoxon matched-pairs signed rank test, n = 4). D,E. Individual eicosanoids derived from arachidonic acid (D), linoleic acid (E) and docosahexaenoic acid (E) organized by the main enzymatic system involved in the metabolism of the respective eicosanoid (COX: cyclooxygenase; CYP: cytochrome p450; LOX: lipoxygenase). Relative lipid levels, normalized against + Tet controls, are depicted (n = 4).

Fig. 3

Intraneuronal Aβ increases eicosanoids and this is countered by CMS121. A. Representative blot of intracellular Aβ induction after Tet removal (Tet-) in MC65 cells. CMS121 prevents the accumulation of Aβ. B. CMS121 prevents cell death induced by intracellular Aβ in MC65 cells (n = 3). C. Eicosanoid analysis of MC65 cells shows a general increase in eicosanoid levels secreted into the culture medium by cells when Aβ toxicity is induced (Tet-). Treatment of cells with CMS121 prevents this increase (Tet- + CMS121). Results are expressed as fold changes normalized over Tet + baseline levels. Every dot represents the average fold change of a single eicosanoid (****p<0.0001, Wilcoxon matched-pairs signed rank test, n = 4). D,E. Individual eicosanoids derived from arachidonic acid (D), linoleic acid (E) and docosahexaenoic acid (E) organized by the main enzymatic system involved in the metabolism of the respective eicosanoid (COX: cyclooxygenase; CYP: cytochrome p450; LOX: lipoxygenase). Relative lipid levels, normalized against + Tet controls, are depicted (n = 4).

Fig. 4

Lipid metabolism is dysregulated in AD and CMS121 affects several lipid classes in mouse cortex. A. Random forest analysis of the top 30 metabolites indicates the importance of lipids in the separation of the different treatment groups. B. In general, metabolites classified as lipids are more elevated in untreated AD mice than CMS121-treated AD mice. C. Levels of endocannabinoids, fatty acids, PUFAs and ceramides are most clearly affected by CMS121. Eicosanoids, metabolites related to fatty acid (FA) metabolism, lysophospholipids and sphingolipids are not significantly altered in AD or affected by CMS121 treatment. Lipid levels are expressed as relative levels, normalized against untreated wildtype mice (WT). Statistical significance is determined by the Wilcoxon matched-pairs signed rank test. Cortices of 5 animals per treatment group were analyzed; each datapoint represents the average relative level of a specific metabolite. Only the most important lipid groups are presented. The entire metabolomics data set is available via https://data.mendeley.com/datasets/v6gjkzggfr/draft?a=587e71a7-f962-414f-869f-54f4ed7009d4https://doi.org/10.17632/v6gjkzggfr.1. D-E. Palmitate levels (D) as well as protein levels of FASN (see also blot below graph) (E), support a general increase in lipid levels in AD brains. Results are expressed as mean ± SEM for relative protein levels, and as box and min. to max. whisker plots for palmitate (*p<0.05, **p<0.01, one-way ANOVA, n = 5–6).

Fig. 5

FASN is a target of CMS121. A. CMS121 inhibits the enzymatic activity of FASN measured in HT22 cell lysates in a dose-dependent manner. The Vmax during the linear phase of the kinetic curve decreases with increasing concentrations of CMS121. Triclosan, a known FASN inhibitor was used as a positive control. Fold changes of treatment over vehicle controls are plotted. Results are expressed as mean ± SD. Significance is calculated using one-way ANOVA, the mean of each column was compared with the mean of the control column (N = 18, with 6 independent experiments). FASN knockdown (siFASN) of HT22 cells protects against oxytosis/ferroptosis induced by glutamate, erastin and RSL3 (B-D). (***q<0.001, ****q<0.0001, multiple T-tests, n = 9 per 3 independent transfections). Transfection efficiency was tested by immunoblotting for FASN (E). Exposure of control siRNA (siCTL) transfected HT22 cells to 2 mM glutamate (F), 250 nM erastin (G) or 25 nM RSL3 (H) for 24h leads to ≥50% cell death. 1 μM CMS121, as well as knockdown of FASN significantly increased survival rates in all cases. In siFASN-treated cells, 1 μM CMS121 offers additional protection against glutamate and erastin, but not against RSL3 toxicity. Significance is calculated with 2-way ANOVA and Sidak’s post-hoc multiple comparisons test (ns = not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). I,J. 4HNE adduct formation as a measure of lipid peroxidation is decreased in HT22 cells transfected with siRNA against FASN (siFASN), compared to control siRNA transfected cells (siCTL) (****p<0.0001, 2-way ANOVA, n = 3). 30min exposure of HT22 cells to RSL3 increases 4HNE adduct formation in siCTL cells (#p<0.05, 2-way ANOVA, Sidak’s multiple comparisons test), but not in siFASN transfected cells. In siFASN transfected cells, CMS121 does not have an added protective effect against 4HNE adduct formation. K. 4HNE adduct formation correlates significantly with FASN protein expression levels in HT22 cells (r = 0.86, p<0.0001, 2-tailed Pearson correlation, with H₀ stating the absence of association between 4HNE adduct formation and FASN protein levels, n = 6). Both CMS121 (L–O) and FASN knockdown (P–U), were able to decrease the levels of the inflammatory markers iNOS (L,P), COX2 (M,Q), and TNFα (N,R). Results are expressed as mean ± SD. Significance was calculated using the unpaired T-test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n = 3–4). Knockdown efficiency of FASN in the BV2 microglia was assessed by immunoblotting for FASN (S). V. In AD patients, protein levels of FASN are significantly increased. Results are expressed as relative protein expression, normalized against human control samples (**p<0.01, unpaired T-test, n = 8). W. Knocking down FASN protects MC65 cells against intracellular Aβ-induced cell death. Results are expressed as mean ± SD % viability against untreated, Tet + control cells (****p<0.0001, unpaired T-test, n = 3).