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 secretome^4,5.

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 diseases^6,7. 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

This protocol was approved and follows the animal care guidelines set by the Southern University of Science and Technology Animal Care and Use Committee (SUSTech-JY202112006). Adult male and female C57BL/6J mice (6-8 weeks old, 22-30 g) were used in this study. Mice were housed at 22-25 °C on a circadian cycle of 12 h of light and 12 h of darkness, with ad libitum access to food and water. The steps of the protocol are listed as follows.

1. Isolation of brain slices

1. Prepare the surgical tools: ophthalmic bending tweezers, beakers (250 mL, 500 mL), filter paper, scissors, razor blade, single-sided blade, and brush pen. Place these tools in a foam box filled with crushed ice to cool.
2. Open the valve for the mixed gas containing 95% O₂ and 5% CO₂. Clean the gas pipe head by placing it into a beaker of ultra-pure water.
3. Prepare the ice-cold dissection buffer using the following components: 225 mM sucrose, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, 11 mM D-glucose, 5 mM L-ascorbic acid, 3 mM sodium pyruvate, 7 mM MgSO4, and 0.5 mM CaCl₂. Adjust the pH to 7.3-7.4.
4. Prepare the culture solution with the following components: 122 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, 11 mM D-glucose, 7 mM MgSO4, and 0.5 mM CaCl₂. Adjust the pH to 7.3-7.4, then filter and add 10% penicillin-streptomycin solution (P/S) into the culture solution.
5. Oxygenate the culture solution and dissection solution for 30 min before sacrificing the mice. Break the razor blade to take half of the slice, and then mount it on the slicer. Install the base and tool holder of the slicer.
6. Sacrifice the mice by cervical dislocation, or by using 2%-3% isofluorane to anesthetize.
   1. Use scissors to cut off the skull at the neck. Cut the skin in the middle of the skull to expose the whole skull.
   2. Use ophthalmic scissors to cut the skull along the median line and use curved eye forceps to cut the skull apart.
   3. Finally, use curved forceps to reach the base of the skull and cut off the base nerve; the entire brain can now be removed.
7. Rapidly place the brain into an ice-cold dissection buffer. Use a sharp, sterilized, single-sided blade to cut away excess parts of the brain, including the bottom part.
8. Stick the brain tissue into the tank using appropriate glue. Pour the slicing solution into the tank, ensuring that the brain tissue is covered, and continue to oxygenate.
9. Adjust the parameters of the vibratome to slice the tissue into 300 µm thick sections. The speed and frequency of the slicer used in this study were 0.3 mm/s and 70 Hz, respectively.
10. Use a brush to pick out the brain slices and place them into a dish with the culture solution. Then, transfer two to four slices onto each 30 mm, 0.4 µm pore size tissue culture insert in 6-well plates containing 1.5 mL of the culture solution, at a distance that does not allow the slices to grow into each other.
11. Incubate the tissues at 37°C, 5% CO₂, and 95% humidity for 14 h.

2. Brain slice collection

1. Aspirate the culture medium from the coverslip and gently transfer the brain slice into the 1.5 mL tube with a brush.
2. Add 200-400 µL of Hanks' balanced salt solution (HBSS) containing 1 mmol/L phenylmethylsulfonyl fluoride (PMSF) to the tube. Rigorously oscillate and mix to form a homogeneous solution using a tissue grinder. The frequency and time used in this study were 800 Hz and 60 s, respectively.
3. Centrifuge the suspension at 700 x g for 10 min at 4 °C. Then, collect the supernatant into a new 1.5 mL centrifuge tube.
4. Add 20-40 µL of 20% Triton X-100 to the supernatant and rotate the sample with a rocking shaker at 4°C for 1 h.
5. Centrifuge the suspension at 13,000 x g for 20 min at 4 °C and collect the supernatant. Normalize all the brain slice samples to a protein concentration of 2 µg/µL using a bicinchoninic acid (BCA) protein assay kit.
6. Mix the supernatant in 5x sodium dodecyl sulfate (SDS) loading buffer (10% SDS, 10 mM dithiothreitol [DTT], 250 mM Tris-HCl [pH 6.8], 30% [v/v] glycerol, and 0.05% [w/v] bromophenol blue dye) and boil the sample for 5 min at 95 °C.
7. Freeze the prepared samples at -20 °C. They will subsequently be subjected to detection by electrophoresis and western blotting methods.

3. Collection of brain slice culture conditioned medium (CM)

1. Aspirate the culture medium after 14 h and filter the CM using a 0.22 µm filter.
2. Concentrate the conditioned medium up to 100 µL with a 10 kDa ultrafiltration centrifuge tube at 9,000 x g for 50 min at 4 °C. Normalize all medium samples to a protein concentration of 0.8 µg/µL using the BCA protein assay kit.
3. Elute the conditioned medium in 5x SDS loading buffer and boil for 5 min at 95 °C.
4. Freeze the prepared samples at -20 °C. They will subsequently be subjected to detection by electrophoresis and western blotting methods.

4. Western blot analyses

1. Prepare sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE )gel as previously described¹⁰. Load the brain slice and medium sample onto precast SDS-PAGE gels. Separate the samples at a constant voltage of 80 V for 30 min initially, and then at a constant voltage of 120 V for 70 min.
2. Transfer the proteins to polyvinylidene difluoride (PVDF) membranes and block with 5% bovine serum albumin (BSA) in tris-buffered saline-Tween 20 (TBST; 150 mM NaCl, 20 mM Tris, 0.1% Tween-20, pH 7.4) for 1 h.
3. Add primary antibodies in the TBST as follows: mouse anti-sAPP-α (antibody dilution ratio of 1:5,000), rabbit anti-clusterin (antibody dilution ratio of 1:5,000), mouse anti-MAP2 (antibody dilution ratio of 1:5,000), mouse anti-GAPDH (antibody dilution ratio of 1:5,000), and mouse anti-actin (antibody dilution ratio of 1:5,000), and incubate overnight at 4 °C on a rocker.
4. Wash the membrane three times with TBST for 15 min each.
5. Add an appropriate secondary antibody in TBST buffer as follows: m-IgGκ BP-HRP (1:5,000 dilution), mouse anti-rabbit IgG-HRP (secondary antibody dilution ratio of 1:5,000). Incubate for 1-2 h.
6. Wash the membrane three times using TBST for 15 min each.
7. Scan and image the membranes using an appropriate imaging system.

5. Proteomic analysis

1. In this study, collect the culture medium (CM) in accordance with step 3.1 and subsequently concentrate it to a final volume of 80 µL by using a 3 kDa concentration tube at 9,000 x g for 40 min at 4 °C. Store the CM sample on ice, awaiting mass spectrometry analysis.
2. Conduct the proteomic detection and analysis methods for samples following the protocols as described in previous papers^11,12,13.

Results

To quantify the secretion of extracellular proteins in brain slices, we examined protein concentrations of the brain slice samples and conditioned medium samples through BCA assay experiments. Brain slice samples and conditioned medium samples all had high protein concentrations (Table 1 and Figure 2). We found that a large number of extracellular proteins were secreted into the medium, and the average protein concentrations in the conditioned medium were ca...

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 system^17,18. Understanding the brain secretome and its functions is important for gaining insight into the mechanisms underlying brain function and disorders. Mo...

Materials

Name	Company	Catalog Number	Comments
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