Long-term atorvastatin improves cognitive function by modulating SIRT2-mediated dynamic transition of NFL lysine 272 crotonylation to ubiquitination in naturally aging rats

Abstract

Proteins' reversible post-translational modifications (PTMs) are essential for cellular regulation, but their involvement in statin-mediated neuroprotection remains elusive. Our previous research demonstrated that long-term atorvastatin intervention ameliorates cognitive decline in naturally aged rats, although the underlying mechanisms were unknown. Here, we employed a proteomic and PTMomic approach using mass spectrometry-based quantitative proteomics and bioinformatics analyses to elucidate the molecular underpinnings. We also utilized isolated and cultured rat hippocampal neuronal cells to investigate the regulation of neurofilament light chain (NFL) modifications, including crotonylation and ubiquitination, using techniques such as immunoprecipitation, cell transfection, protein imprinting, and PCR. We identified several novel findings: (1) Long-term atorvastatin treatment significantly reduced NFL protein levels in both brain tissue and serum compared to controls; (2) This intervention decreased NFL crotonylation while enhancing ubiquitination via SIRT2 upregulation; (3) SIRT2 reversibly modulated NFL crotonylation and ubiquitination at lysine 272 (NFLK272); (4) Increased NFL ubiquitination promoted its degradation, reducing neurofibrillary tangle (NFT) formation, which colocalizes with NFL in the brain. These results suggest that long-term atorvastatin enhances NFL ubiquitination through SIRT2-mediated reversible regulation at NFLK272, leading to reduced NFT pathology and improved cognitive function. These findings not only redefine the pleiotropic neuroprotective actions of statins but also nominate SIRT2-mediated PTM interplay as a druggable node for mitigating neurofilamentopathy-driven cognitive decline.

Fig. 1. Analysis of differential protein expression and functional enrichment in response to high-dose treatment.

A Technical wiring diagrams. B Quantitative volcano plot of differentially expressed proteins in the high-dose and control groups. C Classification of differential protein subcellular structural localization. D Statistical distribution of differentially expressed proteins in gene ontology (GO) secondary classification. E–G GO enrichment analysis of differentially expressed proteins. Containing three main categories: biological process, cellular component, and molecular function.

Fig. 2. Crotonylomic analysis of high-dose treatment effects.

A Quantitative volcano plot of differentially expressed modification sites for crotonylation modification in the high-dose group and control group. B Classification of subcellular structural localization. C Statistical distribution of differentially expressed proteins with crotonylation modifications in GO secondary classification. D–F GO enrichment analysis of differentially expressed proteins modified by crotonylation. Containing three main categories: biological processes, cellular components, and molecular functions.

Fig. 3. Effects of atorvastatin on NFL in the aging rat brain and serum.

A, B Relative protein levels of NFL in the brain tissue of each group. Y: Youth group (6-month-old rats); M: Middle-aged group (12-month-old rats); C: Aged group (18-month-old rats); L: Low-dose atorvastatin intervention in the aged group; H: High-dose atorvastatin intervention in the aged group. Data are expressed as mean ± standard deviation (n = 3 per group) and were analyzed using one-way ANOVA and LSD-t test. *P < 0.05, **P < 0.01, ***P < 0.001. C Determination of NFL in the serum of each group by the method of Simoa. Data are expressed as mean ± standard deviation (n = 8 per group) using one-way ANOVA and LSD-t test. **P < 0.01. D Immunofluorescence staining: Localization of NFL and NFT immunofluorescence double staining in different groups of hippocampal regions, photographed at 400× mirror, scale length 50 μm, NFT (green light), NFL (red light).

Fig. 4. Effects of long-term atorvastatin intervention on NFL modification levels and associated proteins.

A The presence of ubiquitination modifications at the K272 locus of NFL was identified by database PhosphoSitePlus analysis. B Long-term atorvastatin intervention was found to up-regulate the level of ubiquitination modification of NFL by Co-IP. C Long-term atorvastatin intervention was found to down-regulate the level of crotonylation modification of NFL by Co-IP. D Long-term atorvastatin intervention increases the enzymatic activity of SIRT2. E Long-term atorvastatin intervention was found to improve the interactions between SIRT2 and NFL by Co-IP. F Long-term atorvastatin intervention did not affect HUWE1-NFL interactions found by Co-IP approach. *P < 0.05, **P < 0.01.

Fig. 5. Effects of SIRT2 inhibition on NFL ubiquitination and crotonylation in hippocampal neurons.

A Neuronal purity was determined by immunofluorescence detection of the expression of the neuron-specific marker MAP2 in hippocampal neurons cultured for 9 days. B Western blot detected a significant decrease in SIRT2 expression level and a significant increase in NFL expression after AGK2 intervention in hippocampal neuronal cells. C Decreased ubiquitination level of NFL was detected by Co-IP in AGK2-treated rat hippocampal neuronal cells. D Increased level of crotonylation of NFL was detected by Co-IP in AGK2-treated rat hippocampal neuronal cells. E Real-time PCR to detect the mRNA expression level of SIRT2 in cells; Western blot detection of protein concentration levels of SIRT2 in cells. F Real-time PCR to detect the mRNA expression level of NFL in cells; Western blot detection of protein concentration levels of NFL in cells. G The level of crotonylated modification of NFL in each group of cells was detected by Co-IP. H The level of ubiquitination modification of NFL in each group of cells was detected by Co-IP.

Fig. 6. Atorvastatin affects the level of crotonylation and ubiquitination in NFL via SIRT2.

A The CB-DOCK2 molecular docking results revealed that atorvastatin interacts with 31 amino acid residues of SIRT2. B WB and graph analysis of cellular thermal shift assay. C The level of crotonylated modification of NFL in each group of cells was detected by Co-IP. D The level of ubiquitination modification of NFL in each group of cells was detected by Co-IP.

Fig. 7. Probing changes in crotonylation levels and ubiquitination levels in the NFL after mutation at the NFL K272 locus.

A–C Real-time PCR and Western blot were used to verify that successful constructs have been constructed to construct mutant lentivirus 1 (NFL K272Q), mutant lentivirus 2 (NFL K272R), Lv-shNFL, and Lv-shNC. D The level of crotonylated modification of NFL was detected by CO-IP in each group of cells. E SIRT2 regulates through the NFL K272 locus, the NFL’s Ubiquitination level: the level of ubiquitination modification of NFL in each group of cells was detected by Co-IP. ****P < 0.0001. NFL K272Q: mutant lentivirus 1; NFL K272R: mutant lentivirus 2; NFL WT: wild-type NFL group; Lv-shNC: NFL control lentivirus; Lv-shNFL: NFL-infected lentivirus; HA-SIRT2: rat Sirt2 overexpressing lentivirus.

Fig. 8

Diagram of the scientific mechanism hypothesis.