Human and mouse neuroinflammation markers in Niemann‐Pick disease, type C1

Niemann‐Pick disease, type C1 (NPC1) is an autosomal recessive lipid storage disorder in which a pathological cascade, including neuroinflammation occurs. While data demonstrating neuroinflammation is prevalent in mouse models, data from NPC1 patients is lacking. The current study focuses on identifying potential markers of neuroinflammation in NPC1 from both the Npc1 mouse model and NPC1 patients. We identified in the mouse model significant changes in expression of genes associated with inflammation and compared these results to the pattern of expression in human cortex and cerebellar tissue. From gene expression array analysis, complement 3 (C3) was increased in mouse and human post‐mortem NPC1 brain tissues. We also characterized protein levels of inflammatory markers in cerebrospinal fluid (CSF) from NPC1 patients and controls. We found increased levels of interleukin 3, chemokine (C‐X‐C motif) ligand 5, interleukin 16 and chemokine ligand 3 (CCL3), and decreased levels of interleukin 4, 10, 13 and 12p40 in CSF from NPC1 patients. CSF markers were evaluated with respect to phenotypic severity. Miglustat treatment in NPC1 patients slightly decreased IL‐3, IL‐10 and IL‐13 CSF levels; however, further studies are needed to establish a strong effect of miglustat on inflammation markers. The identification of inflammatory markers with altered levels in the cerebrospinal fluid of NPC1 patients may provide a means to follow secondary events in NPC1 disease during therapeutic trials.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147148/1/jimd0083.pd

Identification of Novel Pathways Associated with Patterned Cerebellar Purkinje Neuron Degeneration in Niemann-Pick Disease, Type C1

Niemann-Pick disease, type C1 (NPC1) is a lysosomal disease characterized by progressive cerebellar ataxia. In NPC1, a defect in cholesterol transport leads to endolysosomal storage of cholesterol and decreased cholesterol bioavailability. Purkinje neurons are sensitive to the loss of NPC1 function. However, degeneration of Purkinje neurons is not uniform. They are typically lost in an anterior-to-posterior gradient with neurons in lobule X being resistant to neurodegeneration. To gain mechanistic insight into factors that protect or potentiate Purkinje neuron loss, we compared RNA expression in cerebellar lobules III, VI, and X from control and mutant mice. An unexpected finding was that the gene expression differences between lobules III/VI and X were more pronounced than those observed between mutant and control mice. Functional analysis of genes with anterior to posterior gene expression differences revealed an enrichment of genes related to neuronal cell survival within the posterior cerebellum. This finding is consistent with the observation, in multiple diseases, that posterior Purkinje neurons are, in general, resistant to neurodegeneration. To our knowledge, this is the first study to evaluate anterior to posterior transcriptome-wide changes in gene expression in the cerebellum. Our data can be used to not only explore potential pathological mechanisms in NPC1, but also to further understand cerebellar biology

Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds

Niemann-Pick disease type C1 (NPC1) is a rare autosomal recessive lysosomal storage disease primarily caused by mutations in NPC1. NPC1 is characterized by abnormal accumulation of unesterified cholesterol and glycolipids in late endosomes and lysosomes. Common signs include neonatal jaundice, hepatosplenomegaly, cerebellar ataxia, seizures and cognitive decline. Both mouse and feline models of NPC1 mimic the disease progression in humans and have been used in preclinical studies of 2-hydroxypropyl-β-cyclodextrin (2HPβCD; VTS-270), a drug that appeared to slow neurological progression in a Phase 1/2 clinical trial. However, there remains a need to identify additional therapeutic agents. High-throughput drug screens have been useful in identifying potential therapeutic compounds; however, current preclinical testing is time and labor intensive. Thus, development of a high-capacity in vivo platform suitable for screening candidate drugs/compounds would be valuable for compound optimization and prioritizing subsequent in vivo testing. Here, we generated and characterize two zebrafish npc1-null mutants using CRISPR/Cas9-mediated gene targeting. The npc1 mutants model both the early liver and later neurological disease phenotypes of NPC1. LysoTracker staining of npc1 mutant larvae was notable for intense staining of lateral line neuromasts, thus providing a robust in vivo screen for lysosomal storage. As a proof of principle, we were able to show that treatment of the npc1 mutant larvae with 2HPβCD significantly reduced neuromast LysoTracker staining. These data demonstrate the potential value of using this zebrafish NPC1 model for efficient and rapid in vivo optimization and screening of potential therapeutic compounds. This article has an associated First Person interview with the first author of the paper

Quantitative proteomic analysis of Niemann-Pick disease, type C1 cerebellum identifies protein biomarkers and provides pathological insight.

Niemann-Pick disease, type C1 (NPC1) is a fatal, neurodegenerative disorder for which there is no definitive therapy. In NPC1, a pathological cascade including neuroinflammation, oxidative stress and neuronal apoptosis likely contribute to the clinical phenotype. While the genetic cause of NPC1 is known, we sought to gain a further understanding into the pathophysiology by identifying differentially expressed proteins in Npc1 mutant mouse cerebella. Using two-dimensional gel electrophoresis and mass spectrometry, 77 differentially expressed proteins were identified in Npc1 mutant mice cerebella compared to controls. These include proteins involved in glucose metabolism, detoxification/oxidative stress and Alzheimer disease-related proteins. Furthermore, members of the fatty acid binding protein family, including FABP3, FABP5 and FABP7, were found to have altered expression in the Npc1 mutant cerebellum relative to control. Translating our findings from the murine model to patients, we confirm altered expression of glutathione s-transferase α, superoxide dismutase, and FABP3 in cerebrospinal fluid of NPC1 patients relative to pediatric controls. A subset of NPC1 patients on miglustat, a glycosphingolipid synthesis inhibitor, showed significantly decreased levels of FABP3 compared to patients not on miglustat therapy. This study provides an initial report of dysregulated proteins in NPC1 which will assist with further investigation of NPC1 pathology and facilitate implementation of therapeutic trials

Summary of 2D-GE Differential Protein Spots.

<p>Venn diagram depicting gel spots that were identified at each time point. Numbers noted in parenthesis are the total number spots with differential intensity identified at that specific time point. Within each time point the breakdown of spots is provided in which the gel intensity suggested either increased (up arrow) or decreased (down arrow) expression relative to the control. A total of 109 spots were differentially expressed. In week one, 22 were unique to this time point whereas 8 were unique to week three and 37 were unique to week five. From the MS analysis, a total of 77 unique proteins were identified in both the mutant and control (paired) gel spots. The protein identifications included 49 increased and 22 decreased.</p

Protein Characterization.

<p>Distribution of differentially expressed proteins by cellular location (A), and biological process (B).</p

CSF-FABP3 concentration in NPC1 patients and controls.

<p>(A) Comparison of FABP3 concentration in untreated (n = 27) and miglustat treated NPC1 patients (n = 18) (p<0.01, t-test, unpaired, Welch's correction). (B) Serial change in FABP3 levels pre- and post-miglustat treatment. Lines connect measurements from the same patient before and after miglustat initiation where one patient was followed serially before miglustat treatment. (C) Percent change in CSF-FABP3 concentration pre- and post-miglustat treatment. (D) Percent change of FABP3 concentration over time in the untreated, treated and pre- post-groups. A one-way ANOVA was used to determined significance (p<0.0001) of the FABP3 concentration change following miglustat initiation.</p

2D-GE Image of CHM2A Protein Spot.

<p>Representative 2D-GE images of CHM2A expression over the time course study. Arrows indicate the protein spot of interest. Spots for each genotype and at each time point represent triplicate analysis of a protein pool comprised of n = 4 to 6 cerebella lysates.</p

KEGG Pathway Analysis of Differentially Expressed Proteins.

<p>KEGG pathway analysis of the top ten significant pathways altered in the <i>Npc1^−/−</i> mouse cerebella. The pathway along with the number of proteins associated with the pathway and the p-value are provided.</p

Expression of fatty acid binding protein 3 (FABP3).

<p>(A) Representative 2D-GE images of FABP3 showing the over-expression in the mutant mouse cerebellum compared to the control tissue at the week five time point (top) and graphically displayed (bottom). Arrows note the protein spot of interest. (B) FABP3 levels measured in CSF from control (n = 30) and NPC1 patients (n = 42). Data is represented as average ± standard error of the mean. Significance was determined using an unpaired t-test with Welch's correction, p<0.0001.</p