Protective effect of C2 ceramide on dopaminergic neurons in a mouse model of Parkinson’s disease

doi:10.12307/2024.229

Abstract

Abstract: BACKGROUND: C2 ceramide reduces the formation of Alpha-Synuclein (α-Syn) oligomers as the protein phosphatase 2A agonist, which has an important regulatory effect on cell aging in the central nervous system.
OBJECTIVE: To investigate the protective mechanism of C2 ceramide on dopaminergic neurons.
METHODS: Twenty-five C57BL/6 mice were randomly divided into control group, model group, C2 ceramide low-, medium- and high-dose groups (n=5 per group). Except for the control group, a mouse model of Parkinson’s disease was established by injecting mutant A53T α-Syn oligomers into the left striatum in the other groups. On the 30th day after the striatal injection, three C2 ceramide groups were intragastrically administered with C2 ceramide (1, 5, 10 μg/g) dissolved in saline at one time, while the control and model groups were administered with the same amount of saline within 30-90 days after modeling, for a total of 60 days. Behavioral changes in each group of mice were observed during this period. On the 90th day after striatal injection, mouse brain tissue was extracted by perfusion under anesthesia, and the changes of dopaminergic neurons in the midbrain substantia nigra were analyzed by immunohistochemical staining. The levels of α-Syn oligomerization and phosphorylation in the midbrain of mice were detected by ELISA, and the changes of enzyme activities related to α-Syn phosphorylation were analyzed.
RESULTS AND CONCLUSION: C2 ceramide had an ameliorating effect on Parkinson’s disease-like dyskinesia in mice caused by the striatal injection of mutant A53T α-Syn oligomers. High-dose C2 ceramide showed better effects on dyskinesia in mice with Parkinson’s disease (P < 0.01). The mutant A53T α-Syn oligomers significantly reduced the number of dopaminergic neurons in the substantia nigra of mice (P < 0.01), while the number of dopaminergic neurons in the substantia nigra increased significantly in the C2 ceramide high-dose group (P < 0.01). The levels of α-Syn oligomers and phosphorylated α-Syn in the brain were significantly reduced in the C2 ceramide high-dose group compared with the model group (P < 0.01), while the level of ceramide was increased (P < 0.05) and the activity of protein phosphatase 2A was significantly upregulated (P < 0.01). To conclude, C2 ceramide can attenuate the neurotoxic effects induced by oligomerized α-Syn by the phosphorylation modification environment of α-Syn in mouse midbrain tissue and protect against the reduction in the number of nigrostriatal dopaminergic neurons in mice, thereby reducing the degree of dyskinesia in Parkinson’s disease.

Key words: C2 ceramide, dopaminergic neuron, Parkinson’s disease, alpha-synuclein, protein phosphatase 2A, oligomer, phosphorylation, protective effect

CLC Number:

Li Jiahui, Qi Xue, Zhu Yuanfeng, Yu Lu, Liu Lifeng, Wang Peng. Protective effect of C2 ceramide on dopaminergic neurons in a mouse model of Parkinson’s disease[J]. Chinese Journal of Tissue Engineering Research, 2024, 28(11): 1653-1659.

Figures/Tables 4

2.1 实验动物数量分析实验中的25只C57BL/6 系小鼠在实验过程中无脱失，最终25只小鼠全部纳入结果分析。 2.2 小鼠一般情况左侧纹状体注射造模后第30天，模型组小鼠开始出现肢体轻微震颤、行动迟缓，个别小鼠表现为竖毛、后肢张开、背部上弓以及步态不稳等帕金森病样行为学改变。而在造模后第90天，C2神经酰胺高剂量组小鼠较模型组相比，震颤明显减轻，且未见竖毛及后肢张开等异常表现。 2.3 行为学评估在纹状体注射后的第30，60，90天，各实验组小鼠的爬杆实验结果均与对照组无显著性差异(P > 0.05)，见图2A。悬挂实验和滚轴实验结果显示，注射后的第60天，模型组小鼠较对照组相比，出现运动能力的下降(P < 0.05)，随着造模时间的增加，第90天时，模型组小鼠运动能力较对照组相比明显降低(P < 0.01)。悬挂实验以检测小鼠上、下肢肌力为主要评分标准，滚轴实验则主要考察小鼠的反应能力及综合运动能力。结果表明，C2神经酰胺低剂量组(1 μg/g) 小鼠的运动能力与模型组相比无显著性差异，而中剂量组 (5 μg/g)小鼠在第90天时表现为较模型组运动能力的提升 (P < 0.05)。高剂量组小鼠(10 μg/g)则在第90 天时表现出较模型组相比更为明显的运动能力的恢复(P < 0.01)，其运动能力与对照组相比无显著性差异(P > 0.05)，见图2B，C。平衡木实验的结果表明：注射后30 d，模型组小鼠较对照组相比出现平衡能力的下降(P < 0.05)，且在第60，90天，模型组小鼠平衡能力降低更为明显(P < 0.01)。而在C2神经酰胺各剂量组中，中剂量组小鼠在第90天时出现了较模型组小鼠能力提升(P < 0.05)，但仍表现为较对照组小鼠平衡能力的下降(P < 0.01)；高剂量组小鼠在造模第30天时，并未表现出与对照组小鼠相比运动能力的显著性差异(P > 0.05)，而在第60，90天时，均表现出与模型组小鼠相比更为明显的运动能力的恢复(P < 0.01)，见图2D。"

References

[1] MONTEMURRO N, ALIAGA N, GRAFF P, et al. New targets and new technologies in the treatment of parkinson’s disease: a narrative review. Int J Environ Res Public Health. 2022;19(14):8799.
[2] COSTA HN, ESTEVES AR, EMPADINHAS N, et al. Parkinson’s disease: a multisystem disorder. Neurosci Bull. 2023;39(1):113-124.
[3] MYERS PS, O’DONNELL JL, JACKSON JJ, et al. Proteinopathy and longitudinal cognitive decline in Parkinson disease. Neurology. 2022;99(1):e66-e76.
[4] SIMON C, SOGA T, OKANO HJ, et al. α-Synuclein-mediated neurodegeneration in dementia with Lewy bodies: the pathobiology of a paradox. Cell Biosci. 2021;11(1):196.
[5] BARBA L, PAOLINI PAOLETTI F, BELLOMO G, et al. Alpha and beta synucleins: from pathophysiology to clinical application as biomarkers. Mov Disord. 2022;37(4):669-683.
[6] BELL R, THRUSH RJ, CASTELLANA-CRUZ M, et al. N-terminal acetylation of α-synuclein slows down its aggregation process and alters the morphology of the resulting aggregates. Biochemistry. 2022;61(17):1743-1756.
[7] SALAMON A, ZADORI D, SZPISJAK L, et al. The genetic background of Parkinson’s disease and novel therapeutic targets. Expert Opin Ther Targets. 2022;26(10):827-836.
[8] GUHATHAKURTA S, SONG MK, BASU S, et al. Regulation of αlpha-synuclein gene (SNCA) by epigenetic modifier TET1 in Parkinson disease. Int Neurourol J. 2022;26(Suppl 2):S85-S93.
[9] 王鹏,历春,王海鹏,等.Alpha-突触核蛋白寡聚体致帕金森病小鼠模型的行为学变化[J].中国老年学杂志,2016,36(21):5227-5230.
[10] GHANEM SS, MAJBOUR NK, VAIKATH NN, et al. α-Synuclein phosphorylation at serine 129 occurs after initial protein deposition and inhibits seeded fibril formation and toxicity. Proc Natl Acad Sci U S A. 2022;119(15):e2109617119.
[11] WANG R, WANG Y, QU L, et al. Iron-induced oxidative stress contributes to α-synuclein phosphorylation and up-regulation via polo-like kinase 2 and casein kinase 2. Neurochem Int. 2019;125:127-135.
[12] SHIN WH, CHUNG KC. Death-associated protein kinase 1 phosphorylates α-synuclein at Ser129 and exacerbates rotenone-induced toxic aggregation of α-synuclein in dopaminergic SH-SY5Y cells. Exp Neurobiol. 2020;29(3): 207-218.
[13] LASHUEL HA, MAHUL-MELLIER AL, NOVELLO S, et al. Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity. NPJ Parkinsons Dis. 2022; 8(1):136.
[14] DENT SE, KING DP, OSTERBERG VR, et al. Phosphorylation of the aggregate-forming protein alpha-synuclein on serine-129 inhibits its DNA-bending properties. J Biol Chem. 2022;298(2):101552.
[15] KAWAHATA I, FINKELSTEIN DI, FUKUNAGA K. Pathogenic impact of α-synuclein phosphorylation and its kinases in α-synucleinopathies. Int J Mol Sci. 2022;23(11):6216.
[16] UEDA S, NISHIHARA M, HIOKA Y, et al. Polo-like kinase 2 plays an essential role in cytoprotection against MG132-induced proteasome inhibition via phosphorylation of serine 19 in HSPB5. Int J Mol Sci. 2022;23(19):11257.
[17] ZHANG C, NI C, LU H. Polo-like kinase 2: from principle to practice. Front Oncol. 2022;12(8):956225.
[18] TIAN H, LU Y, LIU J, et al. Leucine carboxyl methyltransferase downregulation and protein phosphatase methylesterase upregulation contribute toward the inhibition of protein phosphatase 2A by α-synuclein. Front Aging Neurosci. 2018;10:173.
[19] DEHGHAN A, PINTO RC, KARAMAN I, et al. Metabolome-wide association study on ABCA7 indicates a role of ceramide metabolism in Alzheimer’s disease. Proc Natl Acad Sci U S A. 2022;119(43):e2206083119.
[20] ZHUO C, ZHAO F, TIAN H, et al. Acid sphingomyelinase/ceramide system in schizophrenia: implications for therapeutic intervention as a potential novel target. Transl Psychiatry. 2022;12(1):260.
[21] ZHANG Y, JI S, ZHANG X, et al. Human CPTP promotes growth and metastasis via sphingolipid metabolite ceramide and PI4KA/AKT signaling in pancreatic cancer cells. Int J Biol Sci. 2022;18(13):4963-4983.
[22] PLOTEGHER N, BUBACCO L, GREGGIO E, et al. Ceramides in Parkinson’s disease: from recent evidence to new hypotheses. Front Neurosci. 2019; 13:330.
[23] CLARK AR, OHLMEYER M. Protein phosphatase 2A as a therapeutic target in inflammation and neurodegeneration. Pharmacol Ther. 2019;201(9):181-201.
[24] CUSTODIA A, ARAMBURU-NÚÑEZ M, CORREA-PAZ C, et al. Ceramide metabolism and Parkinson’s disease-therapeutic targets. Biomolecules. 2021;11(7):945.
[25] 韩燕银.C2-神经酰胺抑制α-synuclein聚集的研究[D].桂林:桂林医学院,2019.
[26] 张天华,李永春,李宪伟,等.C2-神经酰胺对间皮瘤细胞裸鼠移植瘤生长的抑制作用[J].现代肿瘤医学,2014,22(9):2038-2043.
[27] CHOONG CJ, MOCHIZUKI H. Neuropathology of α-synuclein in Parkinson’s disease. Neuropathology. 2022;42(2):93-103.
[28] PAN L, LI C, MENG L, et al. Tau accelerates α-synuclein aggregation and spreading in Parkinson’s disease. Brain. 2022;145(10):3454-3471.
[29] TIJMS BM, GOBOM J, REUS L, et al. Pathophysiological subtypes of Alzheimer’s disease based on cerebrospinal fluid proteomics. Brain. 2020; 143(12):3776-3792.
[30] SEPPI K, RAY CHAUDHURI K, COELHO M, et al. Update on treatments for nonmotor symptoms of Parkinson’s disease-an evidence-based medicine review. Mov Disord. 2019;34(2):180-198.
[31] ELLIS TD, COLÓN-SEMENZA C, DEANGELIS TR, et al. Evidence for early and regular physical therapy and exercise in Parkinson’s disease. Semin Neurol. 2021;41(2):189-205.
[32] YOO H, LEE J, KIM B, et al. Role of post-translational modifications on the alpha-synuclein aggregation-related pathogenesis of Parkinson’s disease. BMB Rep. 2022;55(7):323-335.
[33] RAMALINGAM N, JIN SX, MOORS TE, et al. Dynamic physiological α-synuclein S129 phosphorylation is driven by neuronal activity. NPJ Parkinsons Dis. 2023;9(1):4.
[34] PENG WANG, XIN LI, XURAN LI, et al. Blood plasma of patients with Parkinson’s disease increases alpha-synuclein aggregation and neurotoxicity. Parkinsons Dis. 2016;2016:7596482.
[35] LUNGHI G, CASNNA EV, LOBERTO N, et al. β-glucocerebrosidase deficiency activates an aberrant lysosome-plasma membrane axis responsible for the onset of neurodegeneration. Cells. 2022;11(15):2343.
[36] SMITH L, SCHAPIRA AHV. GBA variants and Parkinson disease: mechanisms and treatments. Cells. 2022;11(8):1261.
[37] SMITH JK, MELLICK GD, SYKES AM. The role of the endolysosomal pathway in α-synuclein pathogenesis in Parkinson’s disease. Front Cell Neurosci. 2023;16:1081426.
[38] 王鹏,李昕,陈予东,等.α-突触核蛋白寡聚体抑制大鼠原代培养神经元突起早期生长[J].首都医科大学学报,2014,35(5):587-591.
[39] TRINGALI C, GIUSSANI P. Ceramide and sphingosine-1-phosphate in neurodegenerative disorders and their potential involvement in therapy. Int J Mol Sci. 2022;23(14):7806.
[40] DEN HOEDT S, CRIVELLI SM, LEIJTEN FPJ, et al. Effects of sex, age, and apolipoprotein e genotype on brain ceramides and sphingosine-1-phosphate in alzheimer’s disease and control mice. Front Aging Neurosci. 2021;13:765252.
[41] AYUB M, JIN HK, BAE JS. Novelty of sphingolipids in the central nervous system physiology and disease: focusing on the sphingolipid hypothesis of neuroinflammation and neurodegeneration. Int J Mol Sci. 2021;22(14):7353.
[42] MINGIONE A, PIVARI F, PLOTEGHER N, et al. Inhibition of ceramide synthesis reduces α-synuclein proteinopathy in a cellular model of Parkinson’s disease. Int J Mol Sci. 2021;22(12):6469.
[43] BHUMKAR A, MAGNAN C, LAU D, et al. Single-molecule counting coupled to rapid amplification enables detection of α-synuclein aggregates in cerebrospinal fluid of Parkinson’s disease patients. Angew Chem Int Ed Engl. 2021;60(21):11874-11883.
[44] POGGIOLINI I, GUPTA V, LAWTON M, et al. Diagnostic value of cerebrospinal fluid alpha-synuclein seed quantification in synucleinopathies. Brain. 2022; 145(2):584-595.
[45] ZHAO X, HE H, XIONG X, et al. Lewy body-associated proteins a-synuclein (a-syn) as a plasma-based biomarker for Parkinson’s disease. Front Aging Neurosci. 2022;14:869797.