Western blot detection of brain phosphoproteins after performing Laser Microdissection and Pressure Catapulting (LMPC)

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Abstract

The Central Nervous System (CNS) is constituted of complex and specific anatomical regions that cluster together and interact with each other with the ultimate objective of receiving and delivering information. This information is characterized by selective biochemical changes that happen within specific brain sub-regions. Most of these changes involve a dynamic balance between kinase and phosphatase activities. The fine-tuning of this kinase/phosphatase balance is thus critical for neuronal adaptation, transition to long-term responses and higher brain functions including specific behaviors. Data emerging from several biological systems may suggest that disruption of this dynamic cell signaling balance within specific brain sub-regions leads to behavioral impairments. Therefore, accurate and powerful techniques are required to study global changes in protein expression levels and protein activities in specific groups of cells. Laser-based systems for tissue microdissection represent a method of choice enabling more accurate proteomic profiling. The goal of this study was to develop a methodological approach using Laser Microdissection and Pressure Catapulting (LMPC) technology combined with an immunoblotting technique in order to specifically detect the expression of phosphoproteins in particular small brain areas.

Highlights

ā–ŗ The anatomical complexity of the CNS allows highly specific brain-cell responses. ā–ŗ Specific brain proteomic responses occur within functional brain sub-regions. ā–ŗ The fine-tuning of kinase activities is critical for brain neural functions. ā–ŗ The characterization of kinase-mediated neural changes in brain areas is complex. ā–ŗ LMPC/Western blot is a method of choice to obtain accurate CNS kinase profiles.

Introduction

The anatomical complexity of the Central Nervous System (CNS) allows highly specific and selective cellular responses of the brain. However, since these changes in cellular activities are located in small brain areas, their characterization is extremely difficult. Dissecting these sub-regions of interest thus bypassing the problem of the functional heterogeneity of the cerebral tissue is therefore a challenge. Indeed, cerebral structures are often composed of sub-regions with specific functional properties. For example, the hippocampal formation divided into distinct subfields referred to as CA1, CA2, CA3 and DG (Dentate Gyrus) plays a prominent role in some of the hippocampus-mediated behaviors (Abrous et al., 2005, Martin and Clark, 2007). Another example is the nucleus accumbens (NAc) which can be divided into two structures referred to as the Core and the Shell. These structures also have different morphologies and functions (Marinelli and Piazza, 2002, Di Chiara, 2002, Ikemoto, 2007).

Consequently, molecular mechanisms of protein-mediated physiologies/physiopathologies can only be understood once the specific cellular targets of these proteins have been identified. In this context, laser-assisted tissue microdissection is a method of choice which is much more precise than conventional manual dissection (Emmert-Buck et al., 1996). Two main laser-based microscope-aided systems of tissue microdissection have been developed namely laser capture microdissection and laser cutting microdissection. They are now marketed respectively by Life Technologies/Applied Biosystems, initially Arcturus a company formerly based in the USA (website: http://www.appliedbiosystems.com) and European-based companies such as Zeiss (website: http://www.zeiss.de/microdissection), Leica (website: http://www.leica-microsystems.com) or Molecular Machines & Industries (website: http://www.molecular-machines.com/products/lasermicrodissection.html), for review see Murray (2007) and Burgemeister (2005).

Thus, laser-based methods of microdissection coupled with real time quantitative PCR (qPCR) analysis has already been successfully applied to study messenger RNA (mRNA) expression from different tissue (Fend et al., 1999, Vincent et al., 2002, Jacquet et al., 2005, Bernard et al., 2009, Ou et al., 2010).

However, changes in the molecular activities of a cell or group of cells are not limited to changes in the quantities of mRNA but also involve changes in protein levels. Finally, a third level of regulation involves posttranslational modifications of proteins allowing the functional diversification of the proteome (i.e., phosphorylation, glycosylation, methylation, acetylation, etc.) (Han and Martinage, 1992, Walsh et al., 2005). Protein phosphorylation is mediated by the largest class of posttranslational modifying enzymes named protein kinases. This superfamily of proteins, also known as the kinome, is involved in one of the most popular cell signaling transduction mechanisms controlling complex processes both in prokaryotic and eukaryotic cells. Indeed, numerous data from the literature show that neuronal activity involves the modulation of kinase activities and that the disruption of the dynamic cell signaling balance between kinase and phosphatase activities leads to behavioral impairments (Greengard et al., 1993, Jovanovic et al., 1996, Atkins et al., 1998, Revest et al., 2005, Revest et al., 2010a, Revest et al., 2010b, Barik et al., 2010). Consequently, accurate and powerful techniques such as laser-based systems of tissue microdissection are required to specifically isolate the cellular targets where these molecular events take place.

In this study, we used the well-adapted Western blot technique to study the phosphorylation of proteins. Detection of protein phosphorylation first requires dissecting sufficient quantities of material for further proteomic analysis and then developing an experimental protocol that preserves the functional integrity of the activated cells. Here, we describe a sensitive Western blot protocol coupled with the LMPC process guided by histological staining of the tissue sections that both preserves phosphorylation and allows the detection of phosphorylated proteins within small areas of the brain.

Section snippets

Cell culture

The PC12 cell line (ATCC CRL-1721) derived from a transplantable rat pheochromocytoma was used. The PC12 cells were cultured in fresh, antibiotic-free medium (10% Foetal Bovine Serum) then trypsinized and counted up to the appropriate concentration (50Ā Ć—Ā 103, 10Ā Ć—Ā 103, 5Ā Ć—Ā 103, 1Ā Ć—Ā 103 cells/lane of gels) using a Malassez chamber.

Brain tissue samples

For all the experiments, 5ā€“6 week-old Spragueā€“Dawley male rats (Charles River, Arbresle, France) were given an overdose of pentobarbital (0.1Ā ml/kg, Bayer, Germany). Brains

Results

We first studied protein expression by comparing their patterns of expression from an in vitro versus an in vivo situation (Fig. 1). We compared protein expression from PC12 cells and brain hypothalamic structure dissected by LMPC. PC12 cells were chosen as an in vitro model since they are widely used to detect proteins that are also expressed within the brain (Das et al., 2004, Carpentier et al., 2008, Revest et al., 2010b). In order to optimize protein electrophoresis and then Western blot

LMPC and proteomic-combined approach

Laser-assisted microdissection has been used extensively in combination with analysis at the DNA/RNA levels (Fend et al., 1999, Vincent et al., 2002, Jacquet et al., 2005, Bernard et al., 2009, Ou et al., 2010). Data from the literature are now starting to show that laser-assisted microdissection is also an appropriate technology for performing proteomic analysis, by eliminating the general impression that laser treatment or histochemical staining might damage protein samples (Craven et al.,

Acknowledgments

We are grateful to F. Levet for his bioinformatic support using ImageJ software at the Bordeaux Imaging Center (BIC) at the Neurosciences Institute of the University of Bordeaux 2. The authors thank Dr. DN. Abrous for her anatomical expertise on brain tissue. We are grateful to A. Le Roux for technical help. This work was supported by the INSERM, the Agence Nationale pour la Recherche (ANR; contract HICOMET (2008), NeuroRelaps (2010), TIMMS (2010)), MILDT/INCa/Inserm (2008), Bordeaux Institute

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