Glucosylceramide and Glucosylsphingosine Quantitation by Liquid Chromatography-Tandem Mass Spectrometry to Enable In Vivo Preclinical Studies of Neuronopathic Gaucher Disease

Hamler, Rick; Brignol, Nastry; Clark, Spencer K.; Morrison, Sean J.; Dungan, Leo B.; Chang, Hui Hua; Khanna, Richie; Frascella, Michelle; Valenzano, Kenneth J.; Elias, Benjamin; Boyd, Robert E.

doi:10.1021/acs.analchem.7b01442

Cited by 27 publications

(25 citation statements)

References 39 publications

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“…Quantification of GlcCer and GlcSph in the cerebellar brain extracts from 4L/PS-NA mice revealed dramatic and age-dependent increases both in the GlcCer and in GlcSph levels. Thus, GlcCer concentrations increased about 18 times in the cerebellum of 18 week old 4L/PS-NA mice as compared to controls at the same age, which is comparable to the reported 23 times increase in 20 week old 4L/PS-NA mice [30]. Even a higher, 32.6-fold increase in the levels of GlcCer d18:1/18:0 in the cerebellum of 18 week old 4L/PS-NA mice was reported [31].…”

Section: Neuronal Alterations In 4l/ps-na Micesupporting

confidence: 84%

“…Concentrations of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph) in the extracts from the mouse cerebellum were measured by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) following a slightly modified protocol as described elsewhere (Hamler et al, 2017). Glucosyl(β) ceramide (d18:1/18:0), galactosyl (β) ceramide (d18:1/18:0), glucosyl(β) ceramide-d5, glucosyl(β) sphingosine (d18:1), galactosyl (β) sphingosine (d18:1), glucosyl(β) sphingosine-d5, and galactosyl(β) sphingosine-d5 were purchased from Avanti Polar Lipids (Sigma-Aldrich, St. Louis, MO, USA).…”

Section: Glucosylceramide and Glucosylsphingosine Measurementsmentioning

confidence: 99%

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Characterization of the visceral and neuronal phenotype of 4L/PS-NA mice modeling Gaucher disease

et al. 2020

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Gaucher disease is caused by a deficiency in glucocerebrosidase that can result in non-neuronal as well as neuronal symptoms. Common visceral symptoms are an increased organ size, specifically of the spleen, and glucosylceramide as well as glucosylsphingosine substrate accumulations as a direct result of the glucocerebrosidase deficiency. Neuronal symptoms include motor deficits and strong alterations in the cerebellum. To evaluate the effect of new compounds for the treatment of this devastating disease, animal models are needed that closely mimic the human phenotype. The 4L/PS-NA mouse as model of Gaucher disease is shown to present reduced glucocerebrosidase activity similar to human cases but an in-depth characterization of the model was still not performed. We therefore analyzed 4L/PS-NA mice for visceral alterations, motor deficits and also neuronal changes like glucocerebrosidase activity, substrate levels and neuroinflammation. A special focus was set at pathological changes of the cerebellum. Our results show that 4L/PS-NA mice have strongly enlarged visceral organs that are infiltrated by enlarged leukocytes and macrophages. Furthermore, animals present strong motor deficits that are accompanied by increased glucosylceramide and glucosylsphingosine levels in the brain, astrocytosis and activated microglia in the cortex and hippocampus as well as reduced calbindin levels in the cerebellum. The latter was directly related to a strong Purkinje cell loss. Our results thus provide a detailed characterization of the 4L/PS-NA mouse model over age showing the translational value of the model and validating its usefulness for preclinical efficiency studies to evaluate new compounds against Gaucher disease.

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Section: Neuronal Alterations In 4l/ps-na Micesupporting

confidence: 84%

Section: Glucosylceramide and Glucosylsphingosine Measurementsmentioning

confidence: 99%

Characterization of the visceral and neuronal phenotype of 4L/PS-NA mice modeling Gaucher disease

et al. 2020

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“…3) association between NPC1 and components of the vATPase [76,78]. 4) the potential involvement of GlcSph, although this is less likely here as GBA2 inhibition does not correct GlcSph elevation [41,79,80]. 5) the possibility that NPC1 exports sphingosine [5,81,82], reported as an endogenous inhibitor of GBA2 [83]; recent molecular modelling work from our laboratory supports this [84].…”

Section: Accepted Manuscriptmentioning

confidence: 95%

Cytosolic glucosylceramide regulates endolysosomal function in Niemann-Pick type C disease

Wheeler

Haberkant

Bhardwaj

et al. 2019

Neurobiology of Disease

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Niemann-Pick type C disease (NPCD) is a neurodegenerative disease associated with increases in cellular cholesterol and glycolipids and most commonly caused by defective NPC1, a late endosomal protein. Using ratiometric probes we find that NPCD cells show increased endolysosomal pH. In addition U18666A, an inhibitor of NPC1, was found to increase endolysosomal pH, and the number, size and heterogeneity of endolysosomal vesicles. NPCD fibroblasts and cells treated with U18666A also show disrupted targeting of fluorescent lipid BODIPY-LacCer to high pH vesicles. Inhibiting non-lysosomal glucocerebrosidase (GBA2) reversed increases in endolysosomal pH and restored disrupted BODIPY-LacCer trafficking in NPCD fibroblasts. GBA2 KO cells also show decreased endolysosomal pH. NPCD fibroblasts also show increased expression of a key subunit of the lysosomal proton pump vATPase on GBA2 inhibition. The results are consistent with a model where both endolysosomal pH and Golgi targeting of BODIPY-LacCer are dependent on adequate levels of cytosolic-facing GlcCer, which are reduced in NPC disease. Niemann-Pick type C disease (NPCD) is a devastating neurodegenerative condition most commonly due to mutations in NPC1 [1,2] a protein of late endosomes and lysosomes [3]. (Due to difficulties in precisely distinguishing these two sets of organelles the term endolysosome will be used here to include both.) Mutations in NPC1 are associated with impaired endocytic transport via decreased endolysosomal calcium release [4,5]. In turn, endocytosis and luminal calcium are dependent on correct endolysosomal acidification [4] and have been found to be controlled by glycolipids in neurons [6], melanocytes [7], plant vacuoles [8] and C. elegans [9]. It is increasingly apparent that aberrant lysosomal GlcCer in Gaucher disease is associated with elevated endolysosomal pH [10-13]. Glycolipids, vital for mammals [14], are also implicated in membrane trafficking [10,15]. Similarly in yeast the NPC1 homologue ncr1 regulates both vacuolar pH [16] and glycolipid transport [17]. An overview of sphingolipid metabolism highlighting the connection with endocytosis is offered in Fig. S1. Does NPC1 affect endolysosomal pH? Conflicting evidence has been found both in disease fibroblasts [5,16,18-20] and in cells treated with U18666A, a putative inhibitor of NPC1 [5,21-23]. If endocytic traffic is delayed then probes which permeate all acidic organelles will show increased pH values even though fully mature lysosomes still acidify correctly. We used such a general probe to

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“…The analytical method used does not differentiate between GlcCer and GalCer. However, the peak was assigned as GlcCer, as using a Hydrophilic Interaction Liquid Chromatography (HILIC) method (54), fibroblasts were found to contain ∼20-fold more GlcCer than GalCer.…”

Section: Methodsmentioning

confidence: 99%

Glucosylceramide synthase inhibition with lucerastat lowers globotriaosylceramide and lysosome staining in cultured fibroblasts from Fabry patients with different mutation types

Welford

Mühlemann

Garzotti

et al. 2018

Human Molecular Genetics

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Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the GLA gene coding for α-galactosidase A (α-GalA). The deleterious mutations lead to accumulation of α-GalA substrates, including globotriaosylceramide (Gb3) and globotriaosylsphingosine. Progressive glycolipid storage results in cellular dysfunction, leading to organ damage and clinical disease, i.e. neuropathic pain, impaired renal function and cardiomyopathy. Many Fabry patients are treated by bi-weekly intravenous infusions of replacement enzyme. While the only available oral therapy is an α-GalA chaperone, which is indicated for a limited number of patients with specific ‘amenable’ mutations. Lucerastat is an orally bioavailable inhibitor of glucosylceramide synthase (GCS) that is in late stage clinical development for Fabry disease. Here we investigated the ability of lucerastat to lower Gb3, globotriaosylsphingosine and lysosomal staining in cultured fibroblasts from 15 different Fabry patients. Patients’ cells included 13 different pathogenic variants, with 13 cell lines harboring GLA mutations associated with the classic disease phenotype. Lucerastat dose dependently reduced Gb3 in all cell lines. For 13 cell lines the Gb3 data could be fit to an IC50 curve, giving a median IC50 [interquartile range (IQR)] = 11 μM (8.2–18); the median percent reduction (IQR) in Gb3 was 77% (70–83). Lucerastat treatment also dose dependently reduced LysoTracker Red staining of acidic compartments. Lucerastat’s effects in the cell lines were compared to those with current treatments—agalsidase alfa and migalastat. Consequently, the GCS inhibitor lucerastat provides a viable mechanism to reduce Gb3 accumulation and lysosome volume, suitable for all Fabry patients regardless of genotype.

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Glucosylceramide and Glucosylsphingosine Quantitation by Liquid Chromatography-Tandem Mass Spectrometry to Enable In Vivo Preclinical Studies of Neuronopathic Gaucher Disease

Cited by 27 publications

References 39 publications

Characterization of the visceral and neuronal phenotype of 4L/PS-NA mice modeling Gaucher disease

Characterization of the visceral and neuronal phenotype of 4L/PS-NA mice modeling Gaucher disease

Cytosolic glucosylceramide regulates endolysosomal function in Niemann-Pick type C disease

Glucosylceramide synthase inhibition with lucerastat lowers globotriaosylceramide and lysosome staining in cultured fibroblasts from Fabry patients with different mutation types

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