Proteomic analysis of mucopolysaccharidosis I mouse brain with two-dimensional polyacrylamide gel electrophoresis

doi:10.1016/j.ymgme.2016.10.001

Molecular Genetics and Metabolism

Volume 120, Issues 1–2, January–February 2017, Pages 101-110

https://doi.org/10.1016/j.ymgme.2016.10.001 Get rights and content

Highlights

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We conducted 2D PAGE analysis of MPS I mice brain.
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The proteins identified in this study would provide potential biomarkers for diagnostic and therapeutic studies of MPS I.
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Metabolism pathways, intracellular ionic homeostasis and the cytoskeleton are implicated in the neuropathology of MPS I disease.

Abstract

Mucopolysaccharidosis type I (MPS I) is due to deficiency of α-l-iduronidase (IDUA) and subsequent storage of undegraded glycosaminoglycans (GAG). The severe form of the disease, known as Hurler syndrome, is characterized by mental retardation and neurodegeneration of unknown etiology. To identify potential biomarkers and unveil the neuropathology mechanism of MPS I disease, two-dimensional polyacrylamide gel electrophoresis (PAGE) and nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) were applied to compare proteome profiling of brains from MPS I and control mice (5-month old). A total of 2055 spots were compared, and 25 spots (corresponding to 50 different proteins) with a fold change ≥ 3.5 and a p value < 0.05 between MPS I and control mice were further analyzed by nanoLC-MS/MS. These altered proteins could be divided into three major groups based on Gene Ontology (GO) terms: proteins involved in metabolism, neurotransmission and cytoskeleton. Cytoskeletal proteins including ACTA1, ACTN4, TUBB4B and DNM1 were significantly downregulated. STXBP1, a regulator of synaptic vesicle fusion and docking was also downregulated, indicating impaired synaptic transmission. Additionally, proteins regulating Ca^2 + and H⁺ homeostasis including ATP6V1B2 and RYR3 were downregulated, which may be related to disrupted autophagic and endocytotic pathways. Notably, there is no altered expression in proteins associated with cell death, ubiquitin or inflammation. These results for the first time highlight the important role of alterations in metabolism pathways, intracellular ionic homeostasis and the cytoskeleton in the neuropathology of MPS I disease. The proteins identified in this study would provide potential biomarkers for diagnostic and therapeutic studies of MPS I.

Introduction

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disease that results from deficiency of α-l-iduronidase (IDUA, E.C.3.2.1.76), which degrades the glycosaminoglycans (GAG) heparan sulfate and dermatan sulfate. The widespread accumulation of GAG leads to progressive cellular damage and organ dysfunction, with the central nervous system (CNS) being one of the primary sites of pathology. The CNS pathology in MPS I patients is manifested by learning delays, dementia, hydrocephalus and mental retardation. The etiology of neurological dysfunction in MPS I is unclear. It has been reported that neurons and glial cells accumulate GAG [1] and gangliosides [2]. Activation of glial cells [3] and alterations in oxidative status [4] in the cortex and cerebellum has been reported. It has been previously shown that MPS I mice had difficulty to habituate in the repeated open field test [5] and impaired long-term memory for aversive training [6]. Interestingly, although one study [7] reported abnormal performance of MPS I mice in Morris water maze tests, another study found inconclusive results [8]. In more recent studies, MPS I mice showed impaired learning behaviors in water T maze test [9] and spatial memory skills in the Barnes maze test [10]. These findings describe abnormal cognitive and neuropathology in MPS I, but the mechanisms are likely to be complex and remain to be elucidated.

Development of proteomics technology includes two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), which has provided a powerful tool to study the complicated biological processes in cells and rapidly profiling the global protein expression alterations. 2D-PAGE is powerful in identifying proteins and protein isoforms that may be neglected by other methods such as in-solution digestion and nanoLC-MS/MS or 1D-PAGE and nanoLC-MS/MS. As a mass spectrometric screening method, 2D-PAGE offers many advantages: high throughput, broad dynamic range, good reproducibility and adequate sensitivity. Although combing 2D-PAGE with mass spectrometry is a common proteomic approach for high throughput screening of putative biomarkers in several disorders [11], [12], it has not been used in lysosomal diseases.

In this study, a comparative analysis of the proteome of MPS I and wildtype mouse brains using two dimensional gel electrophoresis (2D-GE) and nanoLC-ESI-MS/MS were performed. We identified 50 proteins that were differentially expressed in brains of MPS I versus wildtype mice. Bioinformatics analyses using Database for Annotation, Visualization, and Integrated Discovery (DAVID) [13], Protein ANalysis THrough Evolutionary Relationships (PANTHER) [14] and Search Tool for the Retrieval of Interacting genes (STRING) [15] databases allowed for functional classification of the detected proteins and highlighted MPS I-relevant biological pathways. This approach of screening identified potential biomarkers of MPS I that may reveal specific protein signatures indicating MPS I risk. These might also prove useful to assess prognosis, and provide outcome measures for assessing response to therapies.

Section snippets

Animals and sample collection

MPS I knockout mice (idua-/-), a kind gift from Dr. Elizabeth Neufeld, UCLA, has been generated by insertion of neomycin resistance gene into exon 6 of the 14-exon IDUA gene on the C57BL/6 background. MPS I mice (idua-/-) and wildtype were genotyped by PCR. All mouse care and handling procedures were in compliance with the rules of the Institutional Animal Care and Use Committee (IACUC) of the University of Minnesota. The whole mouse brains were collected from one MPS I and one wildtype mouse

2D-PAGE of brain samples from MPS I and wildtype mice

Brain samples from MPS I and wildtype mice (5-month old) were collected for identifying putative differentially expressed proteins using 2D-PAGE combined with nanoLC-LC-MS/MS. In all, 2 coomassie and 4 silver-stained (Fig. S1) gels of 2D-PAGE of the brain samples (250 μg protein in each gel) were run for reasons of reproducibility. A total of 2055 spots were compared (Fig. 1) and only those spots with a fold change ≥ 3.5 and a p value < 0.05 between MPS I and wildtype mice were picked for in-gel

Functional classification of dysregulated proteins

In this study, 50 proteins were revealed as differentially expressed between MPS I and wildtype mouse brains. These proteins function in diverse biological processes, and some functional groups are formed through cluster and pathway analysis. We discuss below the different pathways and their interdependency, as well as some interesting proteins.

Acknowledgements

The authors thank Kendrick Labs (Madison, WI, http://www.kendricklabs.com/) for 2D-PAGE and quantitative analyses. All authors declare that they have no conflicts of interest related to this work. This work is supported by NIH grant P01HD032652. Dr. Li Ou is a fellow of the Lysosomal Disease Network (U54NS065768). The Lysosomal Disease Network is a part of the Rare Diseases Clinical Research Network (RDCRN), an initiative of the Office of Rare Diseases Research (ORDR), and NxCATS. This

References (37)

G. Baldo et al.
Evidence of a progressive motor dysfunction in Mucopolysaccharidosis type I mice
Behav. Brain Res.
(Jul 15, 2012)
S.U. Walkley
Secondary accumulation of gangliosides in lysosomal storage disorders
Semin. Cell Dev. Biol.
(Aug, 2004)
S.D. Hartung et al.
Correction of metabolic, craniofacial, and neurologic abnormalities in MPS I mice treated at birth with adeno-associated virus vector transducing the human alpha-l-iduronidase gene
Mol. Ther.
(Jun, 2004)
G.K. Reolon et al.
Long-term memory for aversive training is impaired in Idua(-/-) mice, a genetic model of mucopolysaccharidosis type I
Brain Res.
(Mar 3, 2006)
D.A. Wolf et al.
Direct gene transfer to the CNS prevents emergence of neurologic disease in a murine model of mucopolysaccharidosis type I
Neurobiol. Dis.
(Jul, 2011)
D. Pan et al.
Progression of multiple behavioral deficits with various ages of onset in a murine model of Hurler syndrome
Brain Res.
(Jan 10, 2008)
L. Ou et al.
High-dose enzyme replacement therapy in murine Hurler syndrome
Mol. Genet. Metab.
(Feb, 2014)
L. Ou et al.
ZFN-mediated liver-targeting gene therapy corrects systemic and neurological disease of mucopolysaccharidosis type I
Mol. Ther.
(May, 2016)
P.K. Smith et al.
Measurement of protein using bicinchoninic acid
Anal. Biochem.
(Oct, 1985)
P.H. O'Farrell
High resolution two-dimensional electrophoresis of proteins
J. Biol. Chem.
(May 25, 1975)

B.R. Oakley et al.

A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels

Anal. Biochem.

(Jul 1, 1980)

D.S. Spellman et al.

Stable isotopic labeling by amino acids in cultured primary neurons: application to brain-derived neurotrophic factor-dependent phosphotyrosine-associated signaling

Mol. Cell. Proteomics

(Jun, 2008)

A. Kamal et al.

Connecting vesicle transport to the cytoskeleton

Curr. Opin. Cell Biol.

(Aug, 2000)

M.A. Samie et al.

Lysosomal exocytosis and lipid storage disorders

J. Lipid Res.

(Jun, 2014)

K. Bhaskar et al.

Co-purification and localization of Munc18-1 (p67) and Cdk5 with neuronal cytoskeletal proteins

Neurochem. Int.

(Jan, 2004)

E.F. Spence et al.

Actin out: regulation of the synaptic cytoskeleton

J. Biol. Chem.

(Nov 27, 2015)

M.A. Philips et al.

Myg1-deficient mice display alterations in stress-induced responses and reduction of sex-dependent behavioural differences

Behav. Brain Res.

(Feb 11, 2010)

D.L. Medina et al.

Transcriptional activation of lysosomal exocytosis promotes cellular clearance

Dev. Cell

(Sep 13, 2011)

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Proteomic analysis of mucopolysaccharidosis I mouse brain with two-dimensional polyacrylamide gel electrophoresis

Highlights

Abstract

Introduction

Section snippets

Animals and sample collection

2D-PAGE of brain samples from MPS I and wildtype mice

Functional classification of dysregulated proteins

Acknowledgements

Behav. Brain Res.

Semin. Cell Dev. Biol.

Mol. Ther.

Brain Res.

Neurobiol. Dis.

Brain Res.

Mol. Genet. Metab.

Mol. Ther.

Anal. Biochem.

J. Biol. Chem.

Anal. Biochem.

Mol. Cell. Proteomics

Curr. Opin. Cell Biol.

J. Lipid Res.

Neurochem. Int.

J. Biol. Chem.

Behav. Brain Res.

Dev. Cell