Delineating pathological pathways in a chemically induced mouse model of Gaucher disease

Ayelet Vardi, Hila Zigdon, Anna Meshcheriakova, Andrés D Klein, Chen Yaacobi, Raya Eilam, Brandon M Kenwood, Ahad A Rahim, Giulia Massaro, Alfred H Merrill, Einat B Vitner, Anthony H Futerman
<span title="2016-07-05">2016</span> <i title="Wiley"> <a target="_blank" rel="noopener" href="" style="color: black;">Journal of Pathology</a> </i> &nbsp;
Great interest has been shown in understanding the pathology of Gaucher disease (GD), due to the recently discovered genetic relationship with Parkinson's disease. For such studies, suitable animal models of GD are required. Chemical induction of GD by inhibition of acid β-glucosidase (GCase) using the irreversible inhibitor, conduritol B-epoxide (CBE), is particularly attractive, although few systematic studies examining the effect of CBE on development of symptoms associated with neurological
more &raquo; ... forms of GD have been performed. We now demonstrate a correlation between the amount of CBE injected into mice and levels of accumulation of the GD substrates, glucosylceramide and glucosylsphingosine, and show that disease pathology, indicated by altered levels of pathological markers, depends on both levels of accumulated lipids and the time at which their accumulation begins. Gene array analysis shows a remarkable similarity in the gene expression profiles of CBE-treated mice and a genetic GD mouse model, the Gba flox/flox ;nestin-Cre mouse, with 120 of the 144 genes up-regulated in CBE-treated mice also up-regulated in Gba flox/flox ;nestin-Cre mice. We also demonstrate that various aspects of neuropathology and some behavioral abnormalities can be arrested upon cessation of CBE treatment during a specific time window. Together, our data demonstrate that injection of mice with CBE provides a rapid and relatively easy way to induce symptoms typical of neuronal forms of GD. This is particularly useful when examining the role of specific biochemical pathways in GD pathology, since CBE can be injected into mice defective in components of putative pathological pathways, alleviating the need for time-consuming crossing of mice. Accepted Article different ages, and demonstrate that pathology is very similar to the two genetic models discussed above. Moreover, characterization of mice injected with CBE provides novel mechanistic insight into disease pathology. We suggest that the CBE model, even though it has inherent limitations, as do all mouse models, is nevertheless a useful and rapid means to induce nGD. In addition, we demonstrate that cessation of CBE treatment can arrest some but not all symptoms of nGD, and discuss how this experimental approach could be used to test the efficacy of potential therapies. Materials and methods Mice From postnatal days 8 or 15, C57BL/6 mice (Harlan Laboratories, Israel) were injected intra-peritoneally (IP) daily with 25, 37.5, 50 or 100 mg CBE (Calbiochem Millipore, Darmstadt, Germany) per kg body weight, or with PBS. Gba flox/flox mice were crossed with Gba flox/WT ;nestin-Cre mice to generate Gba flox/flox ;nestin-Cre mice and Gba flox/WT ;nestin-Cre mice, which served as healthy controls. Genotyping was performed by polymerase chain reaction. Mice with a mixed genetic background (C57BL6/J and CBA with further backcrossings with C57BL6/J; Jackson Laboratories, USA) were also injected with CBE; this mouse expresses Thy1-YFP-H in some cortical neurons (18). Mice were maintained in the experimental animal center of the Weizmann Institute of Science. All animal experiments were approved by the Weizmann Institute Institutional Animal Care and Use Committee. The use of K14-lnl/lnl mice is documented in the Supporting Information. GCase activity assays and sphingolipid analysis GCase activity assay was performed as described, as was sphingolipid analysis by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) (8); for more details, see the Supporting Information. This article is protected by copyright. All rights reserved. Accepted Article RNA extraction and quantitative PCR RNA extraction and quantitative PCR were performed as described (9). A detailed description, along with the primers used for PCR, is given in the Supporting Information. Immunohistochemistry MAC2 staining was performed as described (7) and a detailed description, along with the methods used for GFAP and fluoro-Jade C staining, is given in the Supporting Information. Microarray analysis Starting from postnatal day 8, C57BL/6 mice were injected IP daily for 10 days with 25 mg CBE per kg body weight or with PBS. Microarray analysis was performed as documented in the Supporting Information. Behavioral experiments Behavioral tests were performed (23, 24) as documented in the Supporting Information. Results The effect of different concentrations of CBE on nGD pathology and comparison to Gba flox/flox ;nestin-Cre mice While previous studies have used a wide variety of CBE concentrations, ranging from 7.5 to 300 mg/kg body weight (Table 1) , little effort has been made to systematically compare the effects of CBE on development of nGD symptoms, and to determine how this correlates with levels of GlcCer and GlcSph accumulation. We injected C57BL/6 mice every day, starting on day 8, with different concentrations of CBE and measured the body weights of the mice and their life-spans. As might be predicted, the highest CBE concentration (100 mg/kg body weight) had the most dramatic effect, with C57BL/6 mice beginning to lose weight by This article is protected by copyright. All rights reserved.
<span class="external-identifiers"> <a target="_blank" rel="external noopener noreferrer" href="">doi:10.1002/path.4751</a> <a target="_blank" rel="external noopener" href="">pmid:27234572</a> <a target="_blank" rel="external noopener" href="">fatcat:lklqbjuos5etrp6qxf3hixv5wa</a> </span>
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