Impaired cholesterol metabolism affecting subventricular zone neurogenesis in ob/ob mice

LI Rui-Ting; LEI Ling-Feng; YANG Na; XIANG Zhi-Yu; LI Ze-Kai; LU Li

doi:10.16098/j.issn.0529-1356.2023.02.006

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Acta Anatomica Sinica ›› 2023, Vol. 54 ›› Issue (2) : 165-174. DOI: 10.16098/j.issn.0529-1356.2023.02.006

Neurobiology

Impaired cholesterol metabolism affecting subventricular zone neurogenesis in ob/ob mice

LI Rui-ting¹ LEI Ling-feng¹ YANG Na¹ XIANG Zhi-yu² LI Ze-kai³ LU Li^1*

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Abstract

Objective To investigate the effect of cholesterol on the proliferation and differentiation of neural stem cells (NSCs) in ob/ob obese mice, and to explore the possible mechanism of central nervous systym dysfunction caused by obesity. Methods Selected 6 4-month-old ob/ob and wild type (WT) mice, and cell proliferation antigen (Ki67) and doublecortin (DCX) immunofluorescenct staining were used to detect ob/ob mice lateral ventricle subventricular zone (SVZ) neurogenesis level. Cultured SVZ NSCs isolated from 18 4-month-old ob/ob and WT mice, and BrdU incorporation experiment and β-Ⅲ-tubulin (Tuj1) immunofluorescent staining were employed to detect the self-renewal and differentiation ability of NSCs. Matrix-assisted laser desorption/ionization time of flight mass spectrometry（MALDI- MS）was used to detect the lipid distribution in 4-month-old ob/ob and WT mice brain tissues, and measure the changes of cholesterol(ST) content and the expression genes related to cholesterol synthesis. Cultured 15 WT postnatal day 0(P0) mouse SVZ NSCs in vitro and electrotransfected with the small interfering RNA(siRNA) sequence of cholesterol synthesis rate-limiting enzyme 3-hydroxy-3-methyl-glutaryl coenzyme A reductase（Hmgcr）verified the knockdown efficiency, to detecte the effect of Hmgcr gene knockdown on NSCs by BrdU incorporation experiment and Tuj1 immunofluorescent staining. Results Compared with the WT mice, the number of Ki67+ and DCX+ cells in the SVZ tissue of ob/ob mice decreased significantly (P<0.05); The result of in vitro experiments showed that the BrdU positive rate (P<0.05) and Tuj1 positive rate (P<0.05) of ob/ob mice NSCs were also significantly lower than that of WT mice NSCs. MALDI-MS test result showed that the lipid distribution of ST (26∶0) and ST (27∶3) reduced significantly on SVZ of ob/ob mice. Meanwhile, Kit assay result showed that the cholesterol content of ob/ob mice SVZ tissue and NSCs both reduced significantly (P<0.05). In addition, the Real-time PCR result showed that the mRNA expression levels of cholesterol synthesis-related genes Hmgcr, Lanosterol 14-alpha demethylase（Cyp51）, 3-beta-hydroxysteroid-Δ8, Δ7-isomerase（Ebp）and squalene synthase（Fdft1）in the SVZ tissue and NSCs of ob/ob mice were both significantly lower than those in WT mice (P<0.05), suggesting that ob/ob mice SVZ cholesterol synthesis disordered. Real-time PCR result showed that the interference efficiency of siRNA was 55.27%±8.768% (P<0.05), and the result of Western blotting showed that the interference efficiency of siRNA was 32.69%±8.056%(P<0.05). The result of BrdU incorporation experiment indicated that the positive rate of BrdU in the Hmgcr-siRNA group was significantly lower than that in the control group (P<0.05). After differentiation induced with 1% FBS, the positive rate of Tuj1 in the Hmgcr-siRNA group also decreased significantly compared with the control group (P<0.05), suggesting that impaired cholesterol synthesis inhibited the self-renewal and differentiation ability of neural stem cells. Conclusion Decreased cholesterol levels in the ob/ob mice brain can affect the proliferation and differentiation of SVZ NSCs.

Key words

Cholesterol / Subventricular zone / Neurogenesis / Neural stem cell / Immunofluorescence / Ob/ob mouse

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LI Rui-ting LEI Ling-feng YANG Na XIANG Zhi-yu LI Ze-kai LU Li. Impaired cholesterol metabolism affecting subventricular zone neurogenesis in ob/ob mice[J]. Acta Anatomica Sinica. 2023, 54(2): 165-174 https://doi.org/10.16098/j.issn.0529-1356.2023.02.006

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