Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Summary

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

Abstract

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Introduction

Synaptic transmission relies on the physical integrity of the synapse, a concept that was envisaged as early as 1897 by Foster and Sherrington¹. Thus, understanding the distribution of key neurotransmission components (e.g., ion channels, receptors, etc.) is essential to elucidate synaptic function, both in normal and pathological conditions. Electron microscopy (EM) has contributed enormously to the current ultrastructural notion of prototypical central nervous system (CNS) synapses. In such a way, EM has finely established the differences between pre- and postsynaptic densities, which are separated by a cleft of a rather uniform distance (~25 nm)². Interestingly, the postsynaptic apparatus exhibits a relatively continuous, electron-dense thickening below its plasma membrane, the so-called postsynaptic density, or PSD². Conversely, at the presynaptic apparatus, a noticeable discontinuous cytomatrix network is arranged just beneath the plasma membrane, which is essential to the alignment and docking of synaptic vesicles to the plasma membrane active zone³. Hence, EM constitutes the golden experimental approach to survey the distribution of proteins within structurally preserved CNS synapses. However, the information provided by electron micrographs is static. Indeed, accumulating evidences show that in vivo synapses are extremely dynamic, thus experiencing dramatic structural changes upon sustained synaptic transmission. In addition, the morphology and composition of synapses can change throughout different CNS regions and upon development, maturation, aging, and the development of neuropathological conditions. Overall, a protocol focused on isolating proteins belonging to different synaptic compartments in physiological conditions represents a valuable tool for a more comprehensive study of synaptic functioning.

Here, we describe this kind of complementary experimental approach, which allows for the preparative biochemical enrichment of the different synaptic membrane compartments-namely, extra-, pre- and postsynaptic membrane domains. This membrane fractionation method, first described by Philips et al. (2001)⁴, is based on a pH shift that weakens the adhesive interactions occurring within the pre- and postsynaptic apparatus. First, by using mild detergents at pH 6.0, it is possible to discern the adherens junction that holds the pre- and postsynaptic apparatus and that is maintained from the extrasynaptic membrane domain, which is solubilized and thus can be extracted from the synaptic contacts. Subsequently, raising the pH from 6.0 to 8.0 in the presence of mild detergents weakens the strength of the adherens junction that keeps the presynaptic active zone tightly bound to the postsynaptic density. Hence, the presynaptic compartment is solubilized and can be separated from the postsynaptic density, which is mostly preserved because the concentration of detergent used does not promote its solubilization⁴. Interestingly, the fractionation efficiency, eventually higher than 90%, can be confirmed by different subsynaptic markers: i) synaptosomal-associated protein 25 (SNAP-25), from the presynaptic active zone; ii) synaptophysin, from the extrasynaptic fraction (i.e., outside the active zone and including microsomes); and iii) postsynaptic density protein 95 (PSD-95), from the postsynaptic density. Notably, this brain membrane fractionation method has been used successfully. Accordingly, it has been possible to precisely determine the subsynaptic localization of different receptors, such as alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors⁵, adenosine A₁ receptor (A₁R)⁶, adenosine A_2A receptor (A_2AR)⁷, adenosine triphosphate (ATP) P2 receptors⁸, nicotinic acetylcholine receptor subunits⁹, and Parkinson's disease-associated receptor GPR37¹⁰. However, a number of limitations may impede the proper assessment of the synaptic distribution of a particular neuronal protein. Thus, in this procedure, we not only fully describe the entire protocol, but we also highlight some critical points to be considered, such as the rather large amount of tissue needed, the low protein yield, and the mandatory requirement to validate the efficiency of each separation before performing the definite experiment.

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Protocol

All animal experimental procedures were approved by the University of Barcelona Committee on Animal Use and Care (CEEA), in compliance with the guidelines described in the Guide for the Care and Use of Laboratory Animals¹¹ and following the European Community, law 86/609/CCE, FELASA, and ARRIVE guidelines. Thus, mice are housed in standard cages, with ad libitum access to food and water, and are maintained under controlled standard conditions (12 h dark/light cycle starting at 7:30 AM, 22 °C temperature, and 66% humidity).

1. Obtaining Mouse Brain Synaptosomes Using a Discontinuous Sucrose Gradient

Note: This method was reported previously¹⁰.

1. Prepare fresh isolation buffer (IB), pH 7.4; 2 M sucrose; 1 M sucrose/0.1 mM CaCl₂; and 0.1 mM CaCl₂ (see Table 1). Chill the solutions on ice.
2. Sacrifice five mice by cervical dislocation. Decapitate and rapidly remove the brain of each animal. Dissect the brain region of interest (i.e., the hippocampus, 0.25-0.35 g of fresh tissue) and place it in a 5 mL Potter-Elvehjem glass tube on ice with 1 mL of IB.
3. Homogenize the tissue using a homogenizing stirrer (10 strokes at 700-900 rotations per min), preferentially in an ice bath to preclude sample warming.
4. Place the homogenized tissue (1 mL) into a 15 mL tube containing 6 mL of 2 M sucrose and 2.5 mL of 0.1 mM CaCl₂ at 4 °C. Mix slowly by inverting the tube.
   Note: The addition of calcium is essential to maintaining the adhesive interactions between organelles and thus to preserving the structure of the different "densities."
5. Place the solution in a 12 mL centrifuge tube and slowly add 2.5 mL of 1 M sucrose/0.1 mM CaCl₂ on top of each tube to form the sucrose gradient.
   Note: Label the position of the gradient interface on the centrifuge tube using a permanent marker.
6. Weigh and equilibrate the centrifuge tubes with IB solution within their corresponding steel supports and with their closing lids.
7. Centrifuge the tubes for 3 h at 100,000 x g and 4 °C using a swinging bucket rotor centrifuge. Completely fill the rotor with all the steel supports (even empty, if required).
8. Discard the top layer containing myelin. With a Pasteur pipette, collect the white ring between the 1.25-M and 1-M sucrose interphase corresponding to the synaptosome fraction.
9. Dilute the synaptosomes with 9X their volume using IB in a centrifuge tube.
10. Weigh and equilibrate the centrifuge tubes with IB solution within their corresponding steel supports and with their closing lids.
11. Centrifuge the tubes for 30 min at 15,000 x g and 4 °C using a swinging bucket rotor centrifuge.
12. Discard the supernatant and resuspend the pellet with 1.1 mL of IB solution. Collect 100 µL of this synaptosomal solution and centrifuge for 5 min at 11,000 x g and 4 ºC.
13. Resuspend the synaptosomal pellet in 5% sodium dodecyl sulfate (SDS) and store this sample at -20 ºC.
    Note: This sample will correspond to the total synaptosome fraction for further Western-blot analysis. The remaining synaptosomal fraction (~1 mL) can either be frozen down at -20 ºC until use or processed for subsequent subsynaptic fractionation. The synaptosomes or purified synapses represent between 1 and 2% of the total hippocampal volume¹².

2. Pre-, Post- and Extrasynaptic-isolation

1. Prepare 2x fresh solubilization buffer, pH 6.0; 1x solubilization buffer, pH 6.0; solubilization buffer, pH 8.0; and 0.1 mM CaCl₂ (see Table 1). Chill the solutions on ice.
   Note: The pH of these solutions should be accurately adjusted to achieve a good subsynaptic fractionation.
2. Slowly dilute the 1-mL resuspended synaptosomal fraction (see step 1.12) with 5 mL of 0.1 mM CaCl₂.
3. Add the same volume (5 mL) of ice-cold 2x solubilization buffer, pH 6.0, and incubate for 50 min on ice under high agitation in a beaker on ice.
   Note: While 1% of Triton X-100 at pH 6.0 solubilizes all plasma membrane proteins, it preserves proteins within the synaptic contacts or junctions (i.e., pre- and postsynaptic structures)⁴.
4. Place the solution in a centrifuge tube. Weigh and equilibrate the tubes with equivalent volumes of 0.1 mM CaCl₂ and 2x solubilization buffer solution, pH 6.0, within their corresponding steel supports and with their closing lids.
5. Centrifuge the tubes for 30 min at 40,000 x g and 4 °C using a swinging bucket rotor centrifuge. The resulting supernatant represents the extrasynaptic fraction, and the pellet corresponds to the synaptic junctions (i.e., pre- and postsynaptic fractions).
6. Concentrate the supernatant containing the extrasynaptic fraction to a final 200 µL volume using a 15 mL 10K filter tube centrifuged at 4,000 x g and 4 ºC.
7. Precipitate the resulting concentrated extrasynaptic fraction (200 µL) with 5 volumes (1 mL) of pre-chilled (-20 °C) acetone overnight at -20 °C.
8. The next day, centrifuge the extrasynaptic fraction for 30 min at 18,000 x g and -15 °C. After centrifugation, discard the acetone supernatant and dry the pellet to remove any trace of acetone.
9. Finally, resuspend the pellet containing the extrasynaptic proteins with 200 µL of 5% SDS. Sonicate the pellet, if required.
10. Without disrupting it, carefully wash the pellet containing the pre- and postsynaptic fractions with 2 mL of 1x solubilization buffer, pH 6.0. Discard the buffer.
11. Resuspend the pellet with 10 mL of 1x solubilization buffer, pH 8.0, using a glass Pasteur pipette.
    Note: While 1% of Triton X-100 at pH 8.0 solubilizes the presynaptic specialization, it preserves the insoluble postsynaptic density⁴.
12. Incubate the suspension for 50 min on ice under high agitation in a beaker on ice.
13. Place the solution in a centrifuge tube. Weigh and equilibrate the tubes with 1x solubilization buffer solution, pH 8.0, within their corresponding steel supports and with their closing lids.
14. Centrifuge the tubes for 30 min at 40,000 x g and 4 °C using a swinging bucket rotor centrifuge; the resulting supernatant corresponds to the presynaptic fraction and the pellet to the postsynaptic fraction.
15. Process the supernatant containing the presynaptic fraction, as described in steps 2.6-2.9.
16. Resuspend the pellet containing postsynaptic fraction with 200 µL of 5% SDS. Sonicate the pellet, if required.

3. Analyze the samples by immunoblot to validate the membrane fractionation

1. Determine the amount of protein in each fraction (i.e., the total, extra-, pre- and post-synaptic fractions) using the bicinchoninic acid protein assay.
2. Take 20 µg of protein from each fraction and dilute it to a final volume of 50 µL in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. Boil for 5 min at 100 ºC.
3. Separate the proteins by SDS-PAGE electrophoresis 10%, with a 4% concentrating gel under reducing conditions. Electrophorese at constant voltage of 80 V until the dye enters the lower gel and then increase the voltage to 120 V.
4. Transfer the proteins to a PVDF membrane and block with IB blocking solution for 45 min at room temperature under continuous shaking.
5. Incubate the membrane overnight at 4 ºC with the indicated primary antibody (i.e., anti-SNAP-25, anti-PSD-95, anti-synaptophysin, or anti-GPR37) diluted in IB blocking solution under continuous shaking.
6. Wash the membrane three times (10 min each) with IB washing solution to eliminate unbound primary antibody.
7. Incubate with the indicated secondary antibody for 90 min in dark conditions at room temperature under continuous shaking.
8. Wash the membrane three times (10 min each) with IB washing solution to eliminate unbound secondary antibody.
9. Incubate the membrane with chemiluminescent substrate (prepare the mix under dark conditions and following the 1:1 proportion of solution A and B provided by the manufacturer).
10. Analyze the membrane in a chemiluminescent detection device.

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Results

The described methodology has been largely used for the subsynaptic analysis of neuronal proteins in general and for the isolation and biochemical characterization of synaptic receptors^5,6,7,8,9 in particular. Interestingly, the representative result displayed here show the usefulness of this experimental procedure for the anal...

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Discussion

The protocol presented here constitutes a powerful biochemical tool for the study of the subsynaptic distribution of specific proteins within any brain region. However, there are some drawbacks inherent to the technique that deserve to be highlighted here. For instance, one of the main limitations is the relatively large amount of tissue needed to purify a reasonable amount of protein in order to perform the immunoblot analysis of all subsynaptic fractions. This issue might be related to the fact that synapses (i.e.,...

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Materials

Name	Company	Catalog Number	Comments
Sucrose	Pancreac Química SL, Barcelona, Spain	1,316,211,211
CaCl2	Pancreac Química SL, Barcelona, Spain	2,112,211,210
MgCl2·6H2O	Pancreac Química SL, Barcelona, Spain	1,313,961,210
Protease inhibitor cocktail Set III	Millipore, Darmstadt, Germany	535140
Trizma Base	Sigma, St. Louis, MO, USA	T1503
Tris-HCl	Pancreac Química SL, Barcelona, Spain	1,236,541,209
Triton X-100	Sigma, St. Louis, MO, USA	X100
SDS	Sigma, St. Louis, MO, USA	L3771
Glycerol	Sigma, St. Louis, MO, USA	G5516
Bromophenol Blue	Pancreac Química SL, Barcelona, Spain	1,311,651,604
Dithiothreitol	Sigma, St. Louis, MO, USA	D0632
Tween 20	Sigma, St. Louis, MO, USA	P2287
Non fat dry milk
NaCl	Pancreac Química SL, Barcelona, Spain	1,216,591,211
KCl	Pancreac Química SL, Barcelona, Spain	1,314,941,210
KH2PO4	Merck	4873
Na2HPO4	Pancreac Química SL, Barcelona, Spain	1,316,781,211
Basic 20 pH	Crison, Alella, Spain
Polytron VDI 12 Adaptable Homogenizer	VWR, Radnor, PA, USA.
Ultra-Clear Tubes (14x89mm)	Beckman Coulter, Hospitalet de Llobregat, Barcelona	344059	Tubes should be filled almost completely when used to prevent collapsing due to ultracentrifugation.
Amicon Ultra-15 Centrifugal filters Ultracel -10K	Merck Millipore, Darmstadt, Germany	UFC901008
Centrifuge 5430R	Eppendorf, Hamburgo, Germany
Optima L-90K Ultracentrifuge	Beckman Coulter, Hospitalet de Llobregat, Barcelona
Sonifier 250	Branson, Danbury, Connecticut
Amersham Imager 600	GE Healthcare Europe GmbH, Barcelona, Spain
Disposable Glass Pasteur Pippetes 230 mm	VWR, Radnor, PA, USA	612-1702
Compact Balance EK-610	A&D, Tokyo, Japan
Pierce™ BCA Protein Assay Kit	Pierce Biotechnology, Rockford, IL, USA
SuperSignal west pico chemiluminescent substrate	Thermo Fisher Scientific Inc., Waltham, MA, USA
GR 200 Precision Balance	A&D, Tokyo, Japan
Anti-GPR37	Homemade antibody anti-GPR37 produced and validated in Francisco Ciruela Laboratory.		Primary antibodies used at a final concentration of 0.250 μg/mL
Anti-SNAP-25, anti-PSD-95, anti-synaptophysin	Abcam, Cambridge, United Kingdom		Primary antibodies diluted 1:10,000
HRP-conjugated goat anti-mouse IgG	Thermo Fisher Scientific, Waltham, MA, USA.		Secondary antibody diluted 1:10,000
HRP-conjugated goat anti-rabbit IgG	Thermo Fisher Scientific, Waltham, MA, USA.		Secondary antibody diluted 1:30,000