Isolation of the Brain Secretome from Ex Vivo Brain Slice Cultures
Summary
The brain secretome plays a pivotal role in the development and normal functioning of the central nervous system. In this article, we provide a detailed protocol for isolation of the brain secretome from ex vivo brain slice cultures.
Abstract
The brain secretome consists of proteins either actively secreted or shed from the cell surface by proteolytic cleavage in the extracellular matrix of the nervous system. These proteins include growth factor receptors and transmembrane proteins, among others, covering a broad spectrum of roles in the development and normal functioning of the central nervous system. The current procedure to extract the secretome from cerebrospinal fluid is complicated and time-consuming, and it is difficult to isolate these proteins from experimental animal brains. In this study, we present a novel protocol for isolating the brain secretome from mouse brain slice cultures. First, the brains were isolated, sliced, and cultured ex vivo. The culture medium was then filtered and concentrated for isolating proteins by centrifugation after a few days. Finally, the isolated proteins were resolved using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently probed for purity characterization by western blot. This isolation procedure of the brain secretome from ex vivo brain slice cultures can be used to investigate the effects of the secretome on a variety of neurodevelopmental diseases, such as autism spectrum disorders.
Introduction
The extracellular matrix of the nervous system consists of proteins that are either actively secreted or shed from the cell surface, called the "secretome". The brain secretome contains proteins such as growth factor receptors and transmembrane proteins1, which encompass a broad spectrum of roles in the development and normal functioning of the central nervous system. A large number of proteins secreted by the glial cells, including microglia and astrocytes in particular, reflect a wide variety of glial conditions, including neuroinflammation in the central nervous system2. Protein secretome has been demonstrated to have both neuroprotective and neurotoxic effects. For example, genes encoding neuroligin, a transmembrane protein present at post-synapses that regulates the structure and function of synaptic junctions3, have been found to be a risk factor for autism spectrum disorders. Neuroligin undergoes a process called ectodomain shedding, in which the ectodomains of transmembrane proteins are released from the cell surface by cleavage from the membrane in the form of secretome4,5.
Many proteins in the secretome within the brain can be detected in cerebrospinal fluid; thus, proteomic analysis of this fluid can help identify novel physiological and pathological mechanisms and biomarkers of neurological diseases6,7. Many of the physiological functions of brain secretome proteins remain unknown and require further exploration. The establishment of an effective procedure to extract the brain secretome is critical for these studies. However, the current procedure to extract the secretome from cerebrospinal fluid is complicated and time-consuming, and it is difficult to isolate these proteins from experimental animal brains8. In this article, we present a new protocol for isolating the secretome from mouse brain slices.
Brain slices cultured in vitro contain the same cell types and three-dimensional cell structure as brain tissue, preserving normal and intact synaptic circuits, receptor distribution, transmitter transmission, and other physiological functions. The model mimics the growth and functioning of nerve cells in mice when brain slices are placed in an appropriate culture medium to ensure sustained cell growth and survival. In this study, the mouse brain slices continued to secrete neurosecretory proteins9, and the isolated brain was dissected and placed on a filter insert under sterile conditions. Filter inserts were placed in 6-well plates containing the culture medium. Given that proteins secreted by neurons and glial cells can penetrate the membrane of the insert but not the cells, the brain secretome can thus be collected from the conditional culture medium.
This article describes the basic procedures for (i) the isolation of brain slices, (ii) the collection of brain slices, (iii) the collection of brain slice cultures from conditional medium, (iv) western blot analyses, and (v) proteomic analysis (Figure 1).
Protocol
Representative Results
Discussion
The brain secretome refers to the collection of signaling molecules, known as neurosecretory or neuropeptide products, that are released by neurons or glial cells into the extracellular environment. The brain secretome holds critical functions in many biological and physiological processes in the nervous system17,18. Understanding the brain secretome and its functions is important for gaining insight into the mechanisms underlying brain function and disorders. Mo…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This research was supported by Shenzhen Clinical Research Center for Mental Disorders (20210617155253001), Shenzhen Fund for Guangdong Provincial High-level Clinical Key Specialties (SZGSPO13), Shenzhen Science and Technology Innovation Committee (JCYJ20200109150700942), Key-Area Research and Development Program of Guang Dong Province (2019B030335001), and Shenzhen Key Medical Discipline Construction Fund (SZXK042) We are grateful for the assistance of Guangdong Provincial Key Laboratory of Advanced Biomaterials.
Materials
| 0.22 μm Non-Sterile Millex Syringe Filters with PES Membrane | Millipore | SLGPR33RB | |
| 1,4-Dithio-DL-threitol (DTT) | Merck (Sigma Aldrich) | DTT-RO | |
| 6-well plates | CORNING | 3516 | |
| Actin antibody | Cell Signaling Technology | #3700 | |
| APP Antibody | Cell Signaling Technology | 2452 | |
| BioRad Mini-Protean TGX precast gels | BioRad | #1610185 | |
| Bovine serum albumin (BSA) | Merck (Sigma Aldrich) | 10735094001 | |
| Bromophenol Blue | Merck (Sigma Aldrich) | B0126 | |
| Brush pen | Deli | 1.00008E+11 | |
| CaCl2 | Merck (Sigma Aldrich) | C1016 | |
| CF3COOH | Merck (Sigma Aldrich) | 302031 | |
| CH3CN | Merck (Sigma Aldrich) | 34851 | |
| Clusterin antibody | Cell Signaling Technology | #42143 | |
| D-Glucose | Merck (Sigma Aldrich) | G8270 | |
| Easy-nLC1200 | Thermo Fisher Scientific | Easy-nLC1200 | |
| Filter paper | ROHU | ROHU-DLLZ00105 | |
| GAPDH antibody | Cell Signaling Technology | # 5174S | |
| Glycerol | Merck (Sigma Aldrich) | G5516-100ML | |
| Hanks' Balanced Salt Solution (HBSS) | Invitrogen (Gibco) | 14175095 | |
| Human/Rat NLGN2 Antibody | R&D Systems | AF5645 | |
| ICH2CONH2 | Merck (Sigma Aldrich) | I6125 | |
| Immun-Blot PVDF membranes | BIO-RAD | #1620177 | |
| KCl | Merck (Sigma Aldrich) | P3911 | |
| L-Ascorbic Acid | Merck (Sigma Aldrich) | A92902 | |
| Map2 antibody | Cell Signaling Technology | # 4542S | |
| MgSO4 | Merck (Sigma Aldrich) | M7506 | |
| m-IgGκ BP-HRP | SANTA CRUZ BIOTECHNOLOGY | sc-516102 | |
| Millicell-CM 0.4 μm | Millipore | PIHP03050 | |
| Millipore AmiconUltra-0.5ML | Millipore | UFC5010 | |
| mouse anti-rabbit IgG-HRP | SANTA CRUZ BIOTECHNOLOGY | sc-2357 | |
| NaH2PO4 | Merck (Sigma Aldrich) | S0751 | |
| NaHCO3 | Merck (Sigma Aldrich) | S5761 | |
| NH4HCO3 | Merck (Sigma Aldrich) | A6141 | |
| Parenzyme | Thermo Fisher Scientific | R001100 | |
| Penicillin-Streptomycin Solution(P/S) | Invitrogen (Gibco) | 15070063 | |
| Pierce BCA Protein Assay Kit | Thermo Fisher Scientific | 23225 | |
| QEactive | Thermo Fisher Scientific | IQLAAEGAAPFALGMAZR | |
| Rabbit Anti-Goat IgG (H&L)-HRP Conjugated | Easybio | BE0103-100 | |
| Razor blade | BFYING EAGLE | HX-L146-1H | |
| Sodium chloride NaCl | Merck (Sigma Aldrich) | S6150 | |
| Sodium dodecylsulfate (SDS) | Merck (Sigma Aldrich) | V900859 | |
| Sodium Pyruvate | Merck (Sigma Aldrich) | P2256 | |
| SpeedVac SRF110 Refrigerated Vacuum Concentrator (German) | Thermo Fisher Scientific | SRF110P2-115 | |
| Sucrose | Merck (Sigma Aldrich) | G8270 | |
| Tissue grinder | SCIENTZ | SCIENTZ-48 | |
| Tris (Hydroxymethyl) | Merck (Sigma Aldrich) | TRIS-RO | |
| TritonX-100 | Merck (Sigma Aldrich) | T8787 | |
| Tween-20 | Merck (Sigma Aldrich) | P1379 | |
| Vibratory slicer | Leica | Leica VT 1000S |
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