MSACL 2016 EU Abstract

Changes of Phospholipids in Mouse Model of Parkinson’s Disease by Using MALDI Imaging and UPLC-MS

Geul Bang (Presenter)
Korea Basic Science Institute

Authorship: Geul Bang(1), Semin Park(1), Sujung Kim(2), Junyoung Heo(2), Young Hwan Kim(1*)
(1) Biomedical Omics Group, Korea Basic Science Institute, Cheongju, Korea, (2) Department of Biochemistry, School of Medicine, Chungnam National University, Daejeon, Korea

Short Abstract Parkinson’s disease (PD) is a common neurodegenerative disease whose pathologic substrate is nigrostriatal dopaminergic degeneration due to the neuronal loss in the pars compacta of the substantia nigra (SN). Thus, it is of great importance to determine early neuronal changes that may contribute to disease progression. In this study, we utilized MALDI Imaging and UPLC-ESI-MS/MS to identify phospholipids related with PD within SN of mouse brain tissue. The SN regions of tissues were collected using laser capture microdissection (LCM). The two phospholipid species (PC 34:1 and PC 32:0) were significantly down-regulated in SN region of PD mouse model.

Long Abstract

Introduction

Parkinson’s disease (PD) is a common neurodegenerative disease whose pathologic substrate is nigrostriatal dopaminergic degeneration due to the neuronal loss in the pars compacta of the substantia nigra (SNc). Thus, it is of great importance to determine early neuronal changes that may contribute to disease progression. The behavioral symptoms appear after 50–60% of neuronal loss, cell death and degeneration of the substantia nigra (SN). The mechanisms responsible for dopaminergic cell loss are still incomprehensible. Recent interest has focused on lipids and their role in both cell-signaling pathways that govern survival during neurodegeneration disease. Also, lipid is an important mediator in various structural and signaling roles, having previously been implicated in many processes, such as cellular growth and survival. However, in many cases it is still unclear as to whether the effects are due to direct signaling by the lipids themselves as second messengers, or due to hyper- or hypoactivity of their associated enzymes. And, information regarding the lipid alterations in animal models of PD is lacking.

Molecular profiling of phospholipids can be performed directly on or near the surface of brain tissue slices with high specificity and sensitivity by utilizing matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). MALDI-IMS is a unique tool that integrates molecular and histological information together with information attained using traditional optical microscopy. This technology allows us to directly determine the localization, structure, and relative quantity of molecules in a single experimental tissue. IMS has proven to be a powerful tool for analyzing and visualizing different classes of biological molecules, including metabolites, peptides, and proteins, in tissue sections.

In this study, because it is possible to use LCM to capture substantia nigra region of rats subjected to our mouse model of PD with high purity, the SN regions of tissues were collected using laser capture microdissection (LCM). We analyzed the lipid profiles evident in the substantia nigra region.

Methods

The experiment was performed with control (B6 mouse) group (3 mice) and a saline group (3 mice) and a MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) group (3 mice) and Parkinson's disease transgenic group (3 mice). All brain samples (n = 3 in each group) were dissected from mice, immediately transferred to liquid nitrogen, and stored at -80°C. Serial sections (10 ìm thick) were sectioned from each samples and freeze–thaw mounted onto an indium titanium oxide (ITO)-coated glass slide (Bruker) with a cryostat (Leica Microsystems) using an optimum cutting temperature. The matrix application step has an important impact on spectral quality and requires optimization of multiple parameters depending on the kind of tissue as well as the analyte of interest. 2,5-DHB (2,5-Dihydroxybenzoic acid) was chosen to detect in positive-ion mode and 9-AA (9-Aminoacridine) in negative-ion mode, since it is most commonly used for MALDI-MS and MALDI-IMS with high vacuum chamber.. The brain samples were also subjected to matrix application by sublimation deposition method, with 600 mg of DHB sublimated at 180 °C and 9-AA sublimated at 220 °C for the deposition to reach 1.0 ìm thickness, using iMLayer (Shimadzu, Japan). Both profiling and IMS experiments on tissue sections were performed using rapifleX (Bruker) and iMSope (Shimadzu) MALDI imaging instruments. MALDI imaging data measurements were performed with spatial resolution of 50 ìm and 10 ìm. The substantia nigra (SN) was isolated from the extracted brain by laser capture microdissection (LCM). For UPLC-ESI-MS/MS analysis, total lipids in mouse SN region were extracted by modified Bligh and Dyer method with MTBE and analyzed using UPLC/Q-TOF MS and data-dependent acquisition MS/MS coupled on CSH C18 AQUITY column.

Results

In the present study, we examined the phospholipids profiles directly on brain tissue sections obtained from a mouse model of PD utilizing mass spectrometry. Profiling MS was used to measure the spatial arrangement and quantification of phospholipids in the biological samples. To determine the overall changes in phospholipid composition, we took tissue sections from the substantia nigra (SN) regions of brains from MPTP and Homo mice age-matched controls at 10 weeks of age. The MALDI imaging molecular ion images showed differential distribution in all sample groups. Especially, PC 34:1, [M+K]+ at m/z 798, PC 34:1, [M+Na]+ at m/z 782 and PC 32:0, [M+K]+ at m/z 772 among the phospholipids identified in positive-ion mode showed unique distributions in the brain tissues of the groups. In the negative-ion mode, there was a reduction of phosphatidic acid (PA), phosphatidylinositol (PI) and sulfatde (ST) species in brain tissues of both MPTP and Homo PD mouse models. According to UPLC-ESI-MS/MS results, the two phospholipid species (PC 34:1 and PC 32:0) were also significantly down-regulated in PD mice models.

Conclusions

The two PC species are significantly decreasesd within the SN region of Parkinson's disease mouse models. These lipid changes may contribute to structural reconfiguration, immune system signaling, and pro- and anti-apoptotic pathways. The decrease of PC species in the PD brains observed in positive-ion MS analysis directly resulted from the activation of PLA2, which converts PC into LPC and is hyperactivated in various disease conditions such as including Parkinson’s disease, ischemia, spinal cord trauma, and head injury. It was also reported that increased neuronal activity of PLA2 stimulates glial cells and subsequently induces neuroinflammation. This broad lipidomic profiling may be used to further investigate the role of specific lipid species in the etiology and progression of PD, as well as providing potential biomarkers for PD progression. Our results suggest that this method is a powerful tool for analyzing the distribution and quantification of various phospholipids and providing novel insight into the forecast of disease.

References & Acknowledgements:

This work is supported by a grant of Ministry of Trade, Industry and Energy.

Financial Disclosure

IP Royalty: no

Planning to mention or discuss specific products or technology of the company(ies) listed above:

no