U18666A Treatment Results in Cholesterol Accumulation, Reduced Na<sup>+</sup>, K<sup>+</sup>‐ATPase Activity, and Increased Oxidative Stress in Rat Cortical Astrocytes

Copetti-Santos, Daniela; Moraes, Vitória da Costa; Weiler, Dácio Franco; Mello, Alexandre Silva de; Machado, Fernanda de Souza; Marinho, Jéssica Pereira; Siebert, Cassiana; Kolling, Janaína; Funchal, Cláudia; Wyse, Ângela T. S.; Coelho, Janice Carneiro

doi:10.1007/s11745-015-4062-4

Cited by 5 publications

(4 citation statements)

References 60 publications

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“…The treatment with U18666A resulted in an accumulation of cholesterol as expected, but moreover we observed gliosis in the control cells, demonstrated by a significantly increased number of GFAP + /vimentin + cells, accompanied by a reduced amount of phosphorylated GFAP and vimentin. Recent studies described U1866A induced cholesterol accumulations in rat astrocytes influencing the metabolic pathway of these cells [ 56 , 57 ], but effects in regards of gliosis were not topic of these studies.…”

Section: Discussionmentioning

confidence: 99%

Activation of PKC triggers rescue of NPC1 patient specific iPSC derived glial cells from gliosis

Peter

Rost

Rolfs

et al. 2017

Orphanet J Rare Dis

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BackgroundNiemann-Pick disease Type C1 (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. The pathological mechanisms, underlying NPC1 are not yet completely understood. Especially the contribution of glial cells and gliosis to the progression of NPC1, are controversially discussed. As an analysis of affected cells is unfeasible in NPC1-patients, we recently developed an in vitro model system, based on cells derived from NPC1-patient specific iPSCs. Here, we asked if this model system recapitulates gliosis, observed in non-human model systems and NPC1 patient post mortem biopsies. We determined the amount of reactive astrocytes and the regulation of the intermediate filaments GFAP and vimentin, all indicating gliosis. Furthermore, we were interested in the assembly and phosphorylation of these intermediate filaments and finally the impact of the activation of protein kinase C (PKC), which is described to ameliorate the pathogenic phenotype of NPC1-deficient fibroblasts, including hypo-phosphorylation of vimentin and cholesterol accumulation.MethodsWe analysed glial cells derived from NPC1 patient specific induced pluripotent stem cells, carrying different NPC1 mutations. The amount of reactive astrocytes was determined by means of immuncytochemical stainings and FACS-analysis. Semi-quantitative western blot was used to determine the amount of phosphorylated GFAP and vimentin. Cholesterol accumulation was analysed by Filipin staining and quantified by Amplex Red Assay. U18666A was used to induce NPC1 phenotype in unaffected cells of the control cell line. Phorbol 12-myristate 13-acetate (PMA) was used to activate PKC.ResultsImmunocytochemical detection of GFAP, vimentin and Ki67 revealed that NPC1 mutant glial cells undergo gliosis. We found hypo-phosphorylation of the intermediate filaments GFAP and vimentin and alterations in the assembly of these intermediate filaments in NPC1 mutant cells. The application of U18666A induced not only NPC1 phenotypical accumulation of cholesterol, but characteristics of gliosis in glial cells derived from unaffected control cells. The application of phorbol 12-myristate 13-acetate, an activator of protein kinase C resulted in a significantly reduced number of reactive astrocytes and further characteristics of gliosis in NPC1-deficient cells. Furthermore, it triggered a restoration of cholesterol amounts to level of control cells.ConclusionOur data demonstrate that glial cells derived from NPC1-patient specific iPSCs undergo gliosis. The application of U18666A induced comparable characteristics in un-affected control cells, suggesting that gliosis is triggered by hampered function of NPC1 protein. The activation of protein kinase C induced an amelioration of gliosis, as well as a reduction of cholesterol amount. These results provide further support for the line of evidence that gliosis might not be only a secondary reaction to the loss of neurons, but might be a direct consequence of a reduced PKC activity due to the phenotypical choles...

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Section: Discussionmentioning

confidence: 99%

Activation of PKC triggers rescue of NPC1 patient specific iPSC derived glial cells from gliosis

Peter

Rost

Rolfs

et al. 2017

Orphanet J Rare Dis

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“…Since the function of the NPC2 protein is to control intracellular free cholesterol homeostasis [ 19 , 20 ], we next determined whether direct free cholesterol accumulation in HSCs would cause the same response toward PDGF-BB treatment like the NPC2 knockdown HSCs. U18666A, an inhibitor of free cholesterol transport, is a positive control to induce free cholesterol accumulation [ 26 , 27 , 28 , 29 ]. As shown in Figure 1 B,C, on the right, similar results were observed where U18666A-treated HSCs had a significant increase in cell proliferation when compared with the control, while PDGF-BB treatment did not influence the free cholesterol levels in HSCs ( Figure 1 D, left).…”

Section: Resultsmentioning

confidence: 99%

“…Besides, from our previous research, we found that knockdown NPC2 in HSC-T6 cells would increase 20% free cholesterol accumulation [ 24 ]. Since U18666A is a specific drug that induces free cholesterol sequestration in late endosomes/lysosomes and mimics an NPC phenotype [ 27 , 28 , 29 ], the efficiency of U18666A showed a more than 50% increase in cholesterol accumulation when compared to the control [ 26 , 58 ]. Together, these results suggest that increased TLR4 expression in free cholesterol accumulated HSCs may be a key factor in responding to LPS stimulation.…”

Section: Discussionmentioning

confidence: 99%

Niemann-Pick Type C2 Protein Regulates Free Cholesterol Accumulation and Influences Hepatic Stellate Cell Proliferation and Mitochondrial Respiration Function

Wang

Twu²,

Wang

et al. 2018

IJMS

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Liver fibrosis is the first step toward the progression to cirrhosis, portal hypertension, and hepatocellular carcinoma. A high-cholesterol diet is associated with liver fibrosis via the accumulation of free cholesterol in hepatic stellate cells (HSCs). Niemann-Pick type C2 (NPC2) plays an important role in the regulation of intracellular free cholesterol homeostasis via direct binding with free cholesterol. Previously, we reported that NPC2 was downregulated in liver cirrhosis tissues. Loss of NPC2 enhanced the accumulation of free cholesterol in HSCs and made them more susceptible to transforming growth factor (TGF)-β1. In this study, we showed that knockdown of NPC2 resulted in marked increases in platelet-derived growth factor BB (PDGF-BB)-induced HSC proliferation through enhanced extracellular signal-regulated kinases (ERK), p38, c-Jun N-terminal kinases (JNK), and protein kinase B (AKT) phosphorylation. In contrast, NPC2 overexpression decreased PDGF-BB-induced cell proliferation by inhibiting p38, JNK, and AKT phosphorylation. Although NPC2 expression did not affect caspase-related apoptosis, the autophagy marker light chain 3β (LC3B) was decreased in NPC2 knockdown, and free cholesterol accumulated in the HSCs. The mitochondrial respiration functions (such as oxygen consumption rate, ATP production, and maximal respiratory capacity) were decreased in NPC2 knockdown, and free cholesterol accumulated in the HSCs, while NPC2-overexpressed cells remained normal. In addition, NPC2 expression did not affect the susceptibility of HSCs to lipopolysaccharides (LPS), and U18666A treatment induced free cholesterol accumulation, which enhanced LPS-induced Toll-like receptor 4 (TLR4), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 phosphorylation, interleukin (IL)-1 and IL-6 expression. Our study demonstrated that NPC2-mediated free cholesterol homeostasis controls HSC proliferation and mitochondrial function.

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“…Previous study has implied that inhibition of LSS causes secondary apoptosis in neurons due to increase in reactive oxygen species (ROS) [ 30 ]. It also observed a decreased catalase (CAT) and superoxide dismutase (SOD) activity in astrocytes after inhibiting LSS activity, which causes the dysfunction of lipid metabolism in the cerebral cortex [ 31 , 32 ]. As a result, we speculate that LSS resists oxidation and after inhibiting LSS activity, oxidative stress increases and leads to dysfunctional lipid metabolism.…”

Section: Discussionmentioning

confidence: 99%

Lanosterol Synthase Pathway Alleviates Lens Opacity in Age-Related Cortical Cataract

Shen

Zhu

Kang

et al. 2018

Journal of Ophthalmology

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Purpose Lanosterol synthase (LSS) abnormity contributes to lens opacity in rats, mice, dogs, and human congenital cataract development. This study examined whether LSS pathway has a role in different subtypes of age-related cataract (ARC). Methods A total of 390 patients with ARC and 88 age-matched non-ARC patients were enrolled in this study. LSS expression was analyzed by western blot and enzyme-linked immunosorbent assay (ELISA). To further examine the function of LSS, we used U18666A, an LSS inhibitor in rat lens culture system. Results In lens epithelial cells (LECs), LSS expression in LECs increased with opaque degree C II, while it decreased with opaque degree C IV and C V. While in the cortex of age-related cortical cataract (ARCC), LSS expression was negatively related to opaque degree, while lanosterol level was positively correlated to opaque degree. No obvious change in both LSS and lanosterol level was found in either LECs or the cortex of age-related nuclear cataract (ARNC) and age-related posterior subcapsular cataract (ARPSC). In vitro, inhibiting LSS activity induced rat lens opacity and lanosterol effectively delayed the occurrence of lens opacity. Conclusions This study indicated that LSS and lanosterol were localized in the lens of human ARC, including ARCC, ARNC, and ARPSC. LSS and lanosterol level are only correlated with opaque degree of ARCC. Furthermore, activated LSS pathway in lens is protective for lens transparency in cortical cataract.

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U18666A Treatment Results in Cholesterol Accumulation, Reduced Na⁺, K⁺‐ATPase Activity, and Increased Oxidative Stress in Rat Cortical Astrocytes

Cited by 5 publications

References 60 publications

Activation of PKC triggers rescue of NPC1 patient specific iPSC derived glial cells from gliosis

Activation of PKC triggers rescue of NPC1 patient specific iPSC derived glial cells from gliosis

Niemann-Pick Type C2 Protein Regulates Free Cholesterol Accumulation and Influences Hepatic Stellate Cell Proliferation and Mitochondrial Respiration Function

Lanosterol Synthase Pathway Alleviates Lens Opacity in Age-Related Cortical Cataract

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U18666A Treatment Results in Cholesterol Accumulation, Reduced Na+, K+‐ATPase Activity, and Increased Oxidative Stress in Rat Cortical Astrocytes

Cited by 5 publications

References 60 publications

Activation of PKC triggers rescue of NPC1 patient specific iPSC derived glial cells from gliosis

Activation of PKC triggers rescue of NPC1 patient specific iPSC derived glial cells from gliosis

Niemann-Pick Type C2 Protein Regulates Free Cholesterol Accumulation and Influences Hepatic Stellate Cell Proliferation and Mitochondrial Respiration Function

Lanosterol Synthase Pathway Alleviates Lens Opacity in Age-Related Cortical Cataract

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U18666A Treatment Results in Cholesterol Accumulation, Reduced Na⁺, K⁺‐ATPase Activity, and Increased Oxidative Stress in Rat Cortical Astrocytes