Proteomic profiling in MPTP monkey model for early Parkinson disease biomarker discovery

https://doi.org/10.1016/j.bbapap.2015.01.007Get rights and content

Highlights

  • Proteomic profiling in a chronic MPTP-induced non-human primate model of parkinsonism

  • Complementary quantitative iTRAQ-based, glyco- and phosphoproteomics approaches

  • Monkey states were verified by neuroimaging and postmortem immunohistological measurements.

  • > 300 differentially expressed proteins IDed in asymptomatic and symptomatic monkeys

  • Several identified proteins were translated for their usefulness in human PD diagnosis.

Abstract

Identification of reliable and robust biomarkers is crucial to enable early diagnosis of Parkinson disease (PD) and monitoring disease progression. While imperfect, the slow, chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced non-human primate animal model system of parkinsonism is an abundant source of pre-motor or early stage PD biomarker discovery. Here, we present a study of a MPTP rhesus monkey model of PD that utilizes complementary quantitative iTRAQ-based proteomic, glycoproteomics and phosphoproteomics approaches. We compared the glycoprotein, non-glycoprotein, and phosphoprotein profiles in the putamen of asymptomatic and symptomatic MPTP-treated monkeys as well as saline injected controls. We identified 86 glycoproteins, 163 non-glycoproteins, and 71 phosphoproteins differentially expressed in the MPTP-treated groups. Functional analysis of the data sets inferred the biological processes and pathways that link to neurodegeneration in PD and related disorders. Several potential biomarkers identified in this study have already been translated for their usefulness in PD diagnosis in human subjects and further validation investigations are currently under way. In addition to providing potential early PD biomarkers, this comprehensive quantitative proteomic study may also shed insights regarding the mechanisms underlying early PD development. This article is part of a Special Issue entitled: Neuroproteomics: Applications in neuroscience and neurology.

Introduction

Parkinson disease (PD) is a chronic, progressively debilitating neurodegenerative disorder affecting 1–2% of persons over the age of 65 years worldwide [1], [2]. It is currently incurable, and treatment attempts are complicated by the advanced loss of neurons in the substantia nigra pars compacta (SNpc) present even at the earliest clinical manifestation of motor dysfunction [1], [2], [3], [4]. Although PD has a prolonged prodromal phase during which non-motor clinical features as well as physiological abnormalities may be present [5], current PD diagnosis still largely relies on the more apparent classical clinical motor symptoms, which occur at later stages of the disease. At present, few diagnostic tools for unequivocal identification of PD patients at early stages are available [2], [6], [7]. This prevents early treatment that would potentially improve prognosis and impedes the progress of research toward treatments aimed at the preclinical population. Furthermore, there is currently no effective biomarker to predict or monitor PD progression.

Clinical premotor features, including olfactory disturbance, excessive daytime sleepiness, rapid eye movement behavior disorder, constipation, and depression, have been strongly linked to PD [5]. However, none of these signs alone are specific and sensitive enough for identifying premotor PD, thus limiting their clinical utilities to identifying high-risk individuals. The most sensitive tests developed to date as early PD biomarkers are based on imaging modalities, which can detect functional and structural abnormalities before the onset of motor dysfunction [6], [7]. However, the usefulness of neuroimaging techniques is limited by high cost, limited accessibility, and is subject to confounding factors such as medication and compensatory responses. Thus, a current major focus of early PD biomarker research is to identify biochemical marker candidates in the brain or body fluids, which might reflect the state of the disease. We have already investigated several potential cerebrospinal fluid (CSF) markers (known to be important in sporadic PD) in a cohort of symptomatic and asymptomatic subjects carrying one of the strongest risk factors for PD — the leucine rich repeat kinase 2 (LRRK2) gene mutations, and identified that some CSF protein levels correlated with the progressive loss of striatal dopaminergic functions as determined by positron emission tomography (PET) [8], [9]. The emergence of several -omics techniques, including transcriptomics, proteomics and metabolomics, has also greatly enhanced our ability to identify novel pathways and potential biomarkers for PD [7], [10], [11].

In the current study, using a non-human primate animal model of parkinsonism [12], [13], [14] and advanced unbiased proteomic profiling technologies, we intended to further identify key brain region proteins that are altered during the early disease stages and may be used as early PD biomarker candidates. In contrast to previous proteomic profiling studies in rodent models [15], [16], [17], [18], [19], [20], [21], we used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administered to monkeys via a slow intoxication protocol to produce a gradual development of nigral and striatal lesions mimicking the typical, chronic evolution of PD in humans [12], [13]. Additionally, we selectively enriched the glycoproteins and phosphoproteins and compared their proteomic profiles in the putamen samples collected at different states that mimic early stage parkinsonism, where no motor symptoms are apparent (asymptomatic) and later stage parkinsonism, where the cardinal motor symptoms are apparent (symptomatic). Differentially expressed proteins were identified between these states that were verified by behavioral, neuroimaging, and postmortem immunohistological measurements.

Section snippets

Animals and experimental design

Fifteen adult female rhesus monkeys (Macaca mulatta, 4.5–8.5 kg) from the Yerkes National Primate Research Center colony were used in this study, in accordance with the guidelines from the National Institutes of Health. All procedures were approved by the Animal Care and Use Committee at Emory University (IACUC#: 256-2007Y, date: 08/2007). The animals were housed in a temperature-controlled room and exposed to a 12-h light/dark cycle. They were fed twice daily with monkey chow supplemented with

MPTP treatment effects

Consistent with our previous findings [12], [13], [22], all MPTP-treated monkeys in the symptomatic (SYM) group displayed moderate to severe parkinsonian symptoms after 20–22 weeks of MPTP treatment, while monkeys in the saline control (CON) and asymptomatic (ASYM) groups did not show significant changes in their parkinsonism rating scores at the termination of the experiment. The different behavioral responses were correlated with the 18F-FECNT uptake in all striatal regions of interest (see

Discussion

To our knowledge, our study represents the first large-scale proteomic analysis of striatum in the MPTP monkey model of PD. In the investigation we have identified quantitatively altered proteins in this model of parkinsonism with potential utility as early disease diagnostic biomarkers and biomarker candidates for the subtle changes that mark the progression from early to advanced stages. It should be noted that although animal models provide valuable contributions to biomarker discovery and

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Acknowledgements

This study was supported by generous grants from the National Institutes of Health (NIH) (R01 NS057567, U01 NS082137, P50 NS062684-6221, P30 ES007033-6364, R01 AG033398, R01 ES016873, and R01 ES019277 to JZ). It was also supported in part by the University of Washington's Proteomics Resource (UWPR95794), a NIH infrastructure grant to the Yerkes National Primate Research Center (P51 OD011132), the Emory UDALL Center of Excellence for Parkinson's Disease (P50 NS071669), and the National Natural

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    This article is part of a Special Issue entitled: Neuroproteomics: Applications in neuroscience and neurology.

    1

    These authors contributed equally to this work.

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