14 research outputs found

    Data_Sheet_2_A multifaceted evaluation of microgliosis and differential cellular dysregulation of mammalian target of rapamycin signaling in neuronopathic Gaucher disease.pdf

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    Neuronopathic Gaucher disease (nGD) is an inherited neurodegenerative disease caused by mutations in GBA1 gene and is associated with premature death. Neuroinflammation plays a critical role in disease pathogenesis which is characterized by microgliosis, reactive astrocytosis, and neuron loss, although molecular mechanisms leading to neuroinflammation are not well-understood. In this report, we developed a convenient tool to quantify microglia proliferation and activation independently and uncovered abnormal proliferation of microglia (∼2-fold) in an adult genetic nGD model. The nGD-associated pattern of inflammatory mediators pertinent to microglia phenotypes was determined, showing a unique signature favoring pro-inflammatory chemokines and cytokines. Moreover, highly polarized (up or down) dysregulations of mTORC1 signaling with varying lysosome dysfunctions (numbers and volume) were observed among three major cell types of nGD brain. Specifically, hyperactive mTORC1 signaling was detected in all disease-associated microglia (Iba1high) with concurrent increase in lysosome function. Conversely, the reduction of neurons presenting high mTORC1 activity was implicated (including Purkinje-like cells) which was accompanied by inconsistent changes of lysosome function in nGD mice. Undetectable levels of mTORC1 activity and low Lamp1 puncta were noticed in astrocytes of both diseased and normal mice, suggesting a minor involvement of mTORC1 pathway and lysosome function in disease-associated astrocytes. These findings highlight the differences and complexity of molecular mechanisms that are involved within various cell types of the brain. The quantifiable parameters established and nGD-associated pattern of neuroinflammatory mediators identified would facilitate the efficacy evaluation on microgliosis and further discovery of novel therapeutic target(s) in treating neuronopathic Gaucher disease.</p

    Data_Sheet_1_A multifaceted evaluation of microgliosis and differential cellular dysregulation of mammalian target of rapamycin signaling in neuronopathic Gaucher disease.pdf

    No full text
    Neuronopathic Gaucher disease (nGD) is an inherited neurodegenerative disease caused by mutations in GBA1 gene and is associated with premature death. Neuroinflammation plays a critical role in disease pathogenesis which is characterized by microgliosis, reactive astrocytosis, and neuron loss, although molecular mechanisms leading to neuroinflammation are not well-understood. In this report, we developed a convenient tool to quantify microglia proliferation and activation independently and uncovered abnormal proliferation of microglia (∼2-fold) in an adult genetic nGD model. The nGD-associated pattern of inflammatory mediators pertinent to microglia phenotypes was determined, showing a unique signature favoring pro-inflammatory chemokines and cytokines. Moreover, highly polarized (up or down) dysregulations of mTORC1 signaling with varying lysosome dysfunctions (numbers and volume) were observed among three major cell types of nGD brain. Specifically, hyperactive mTORC1 signaling was detected in all disease-associated microglia (Iba1high) with concurrent increase in lysosome function. Conversely, the reduction of neurons presenting high mTORC1 activity was implicated (including Purkinje-like cells) which was accompanied by inconsistent changes of lysosome function in nGD mice. Undetectable levels of mTORC1 activity and low Lamp1 puncta were noticed in astrocytes of both diseased and normal mice, suggesting a minor involvement of mTORC1 pathway and lysosome function in disease-associated astrocytes. These findings highlight the differences and complexity of molecular mechanisms that are involved within various cell types of the brain. The quantifiable parameters established and nGD-associated pattern of neuroinflammatory mediators identified would facilitate the efficacy evaluation on microgliosis and further discovery of novel therapeutic target(s) in treating neuronopathic Gaucher disease.</p

    Comprehensive evaluation of blood-brain barrier-forming micro-vasculatures: Reference and marker genes with cellular composition

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    <div><p>Primary brain microvessels (BrMV) maintain the cellular characters and molecular signatures as displayed in vivo, and serve as a vital tool for biomedical research of the blood-brain barrier (BBB) and the development/optimization of brain drug delivery. The variations of relative purities or cellular composition among different BrMV samples may have significant consequences in data interpretation and research outcome, especially for experiments with high-throughput genomics and proteomics technologies. In this study, we aimed to identify suitable reference gene (RG) for accurate normalization of real-time RT-qPCR analysis, and determine the proper marker genes (MG) for relative purity assessment in BrMV samples. Out of five housekeeping genes, β-actin was selected as the most suitable RG that was validated by quantifying mRNA levels of alpha-L-iduronidase in BrMV isolated from mice with one or two expressing alleles. Four marker genes highly/selectively expressed in BBB-forming capillary endothelial cells were evaluated by RT-qPCR for purity assessment, resulting in <i>Cldn</i>5 and <i>Pecam1</i> as most suitable MGs that were further confirmed by immunofluorescent analysis of cellular components. <i>Plvap</i> proved to be an indicator gene for the presence of fenestrated vessels in BrMV samples. This study may contribute to the building blocks toward overarching research needs on the blood-brain barrier.</p></div

    Verification of <i>Actb</i> as the best reference gene by <i>Idua</i> expression in BrMV of WT mice and Het mice.

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    <p><b>(A)</b> Standard curve for absolute quantification of <i>Idua</i> mRNA. A plasmid containing <i>Idua</i> cDNA was used for generating standard curve with copy numbers by qPCR. Data was derived from 2 sets of standard samples, each amplified three times in duplicate. Error bars, standard deviation. (B, C) <i>Idua</i> expression in BrMV isolated from either wild-type C57/Bl6 mice (WT) or littermates of heterozygous for <i>Idua</i> knock-out (Het) with normalization by RG candidates. Total RNA from 4 WT and 4 Het samples were examined by RT-qPCR and calculated either by absolute <i>Idua</i> standard curve for copy numbers per ng RNA (B), or by ΔΔCt method for relative <i>Idua</i> fold changes (C).</p

    Correlation of purities measured by both qPCR and immunofluorescent microscopy.

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    <p>The relative purities of BrMV samples were determined by real-time RT-qPCR with relative comparison of mRNA abundances using standard curves of different marker genes as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197379#pone.0197379.g004" target="_blank">Fig 4</a>, as well as by immunofluorescence analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197379#pone.0197379.g005" target="_blank">Fig 5</a>. Each symbol represents relative purity of individual BrMV sample derived from 3 RT-qPCR experiments in duplicate reactions for qPCR, as well as from evaluation of >200 DAPI<sup>+</sup> nuclei from cytospin slides by immunostaining analysis.</p

    Cellular components of BrMV isolates determined by immunostaining.

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    <p>(A) Representative pictures of immunofluorescence analysis for BrMV isolates. The brain BrMV isolates were stained with fluorescein-labeled (green) lectin for BBB-forming endothelial cells, and Alexa 568-conjugated (red) anti-mouse CD68 for brain macrophages, anti-mouse GFAP for astrocytes, anti-mouse NeuN for neurons, or anti-mouse PDGFR-β for pericytes, and followed by counter-staining with DAPI (blue) for nucleic. Scale bar represents 50 μm for all views. (B) Semi-quantifications of cellular composition for nine BrMV samples. Each sample was evaluated with a total of >500 nuclei. Pearson correlation coefficient between Lectin<sup>+</sup> and other cell types are -0.862 for CD68<sup>+</sup> cells, -0.837 for GFAP<sup>+</sup> cells, -0.776 for NeuN<sup>+</sup> and -0.190 for PDGFR+ cells. (C) Semi-quantification of main cell types in BrMV isolates. Data are derived from 4 BrMV samples with similar purities (ranging of 64–69%) as determined by immunostaining; error bars represent SD. CV, coefficient of variation.</p

    The expression stability of RG candidates among endothelial cell lines and brain samples.

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    <p>Total RNA was extracted and converted into cDNA by reverse-transcription at 25 ng/ul, and followed by real-time qPCR with 25 ng/reaction. (A) Distribution of cycle threshold (Ct) values for five RG candidates by quantitative RT-PCR in BMEC and bEnd3 cell lines. The experiments were repeated 3 times in duplicate reactions. Boxes showed the range of Ct values for each candidate gene. The central line indicated the median Ct; the extended upper and lower indicate 75 and 25 percentiles. (B) The average expression stability (M value) of RG candidates in two endothelial cell lines analyzed by geNorm. RG candidates were ranked from the least stable to the most stable (left to right). (C) Ct values for four RG candidates in BrMV and CDB. Data were derived from 10 BrMV isolation experiments with 4 from WT mice, 2 from Het and 4 from <i>Idua</i> knock-out mice. Each sample was tested 3 times in duplicate. **, p<0.01, and ***, p<0.001. (D) The expression stability of four reference genes among all BrMV and CDB samples analyzed by geNorm. (E) RG candidates were ranked in the order of their expression stability evaluated by Bestkeeper based on coefficient of variation (CV%) and SD.</p

    Quantification of RNA input using RG standard curves derived from 3T3 or primary CDB samples.

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    <p>Total RNA samples isolated from 3T3 cell line or CDB samples of C57/Bl6 mice were serially diluted, applied for reverse transcription, and followed by qPCR of 4 reference genes. (A) Standard curves of RNA amounts generated by qPCR of 4 RGs. Data were derived from 3 dilution sets with each amplified in triplicates. E, amplification efficiency for a combination of RT and qPCR steps calculated from the slope of each standard curve; R<sup>2</sup> range from 0.986 to 0.998. (B) Quantification of total RNA inputs of BrMV and CDB isolates from 10 isolation experiments using different standard curves. Reverse transcription was conducted at 25 ng/ul (by NanoDrop) for all RNA samples, and real-time qPCR was performed using 25 ng/reaction (and indicated as dashed line). Each symbol represents mean of calculated RNA amount derived from Ct value of triplicate qPCR reactions of one sample. Short lines represent mean ± SD of RNA amounts calculated using different standard curves from each of RGs.</p

    Progression of Behavioral and CNS Deficits in a Viable Murine Model of Chronic Neuronopathic Gaucher Disease

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    <div><p>To study the neuronal deficits in neuronopathic Gaucher Disease (nGD), the chronological behavioral profiles and the age of onset of brain abnormalities were characterized in a chronic nGD mouse model (9V/null). Progressive accumulation of glucosylceramide (GC) and glucosylsphingosine (GS) in the brain of 9V/null mice were observed at as early as 6 and 3 months of age for GC and GS, respectively. Abnormal accumulation of α-synuclein was present in the 9V/null brain as detected by immunofluorescence and Western blot analysis. In a repeated open-field test, the 9V/null mice (9 months and older) displayed significantly less environmental habituation and spent more time exploring the open-field than age-matched WT group, indicating the onset of short-term spatial memory deficits. In the marble burying test, the 9V/null group had a shorter latency to initiate burying activity at 3 months of age, whereas the latency increased significantly at ≥12 months of age; 9V/null females buried significantly more marbles to completion than the WT group, suggesting an abnormal response to the instinctive behavior and an abnormal activity in non-associative anxiety-like behavior. In the conditional fear test, only the 9V/null males exhibited a significant decrease in response to contextual fear, but both genders showed less response to auditory-cued fear compared to age- and gender-matched WT at 12 months of age. These results indicate hippocampus-related emotional memory defects. Abnormal gait emerged in 9V/null mice with wider front-paw and hind-paw widths, as well as longer stride in a gender-dependent manner with different ages of onset. Significantly higher liver- and spleen-to-body weight ratios were detected in 9V/null mice with different ages of onsets. These data provide temporal evaluation of neurobehavioral dysfunctions and brain pathology in 9V/null mice that can be used for experimental designs to evaluate novel therapies for nGD.</p></div

    Gait analysis in mice at different ages.

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    <p>Mice painted with different colors on front and hind paws were allowed walking through a clear tunnel at ages indicated. Footprints were measured for front-paw base width (A), hind-paw base width (B), stride length (C), and overlap between the left front and hind paws (D). *, p<0.05; **, p<0.01 (n = 7–25 mice per group) by Student’s t-test.</p
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