Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Summary

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

Abstract

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960’s, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

Introduction

Neurons communicate through synapses, and the quality of this communication is regulated to a large extent by alterations in the composition of proteins at the synapse. In particular, the proteins located in the post-synaptic density participate in neuronal communication by intimately scaffolding neurotransmitter receptors with their signal transduction systems¹. Furthermore, lasting changes in the strength of synaptic efficacy are controlled by the addition or removal of receptors at the post-synaptic density^1-6. Therefore, the isolation and quantification of synaptic proteins is a necessary and useful technique to gain insight into the ways that neurons respond to stimuli and alter synaptic efficacy⁷. This article describes a common technique to isolate synaptic proteins from rodent brain tissue by ultracentrifugation on discontinuous sucrose gradients. The synaptic plasma membrane fraction can be enriched and isolated based on its density in sucrose, which has been empirically determined to be similar to 1.2 M sucrose.

Depending on the biological question, subcellular fractions can be separated by continuous or discontinuous gradients of either sucrose or Percoll. Continuous gradients allow for the separation of proteins into multiple fractions; this can be particularly useful to demonstrate the co-localization of proteins within a given fraction⁸. However, the preparation of continuous gradients is more laborious and is unnecessary for many applications. Discontinuous gradients are comparatively easier to prepare and can be used to separate proteins into a few, generally-defined fractions. Discontinuous gradients that are composed of three sucrose layers of increasing molarity have been widely used to isolate proteins associated with the synaptic plasma membrane (SPM). This synaptic plasma membrane fraction can be further processed to the post-synaptic density fraction (PSD) by detergent treatment and isolation of the detergent-insoluble fraction.

When this process was first described in the 1960’s^9,10, electron microscopy was used to demonstrate the organelles and membranes that roughly define the synaptic plasma membrane and post-synaptic density fractions^9-14. These studies demonstrated the inclusion of pre- and post-synaptic membranes and synaptic vesicles in the SPM fraction; after detergent treatment primarily the electron-dense, post-synaptic densities were visible. In the procedure, a hypotonic shock is used to pinch off the synaptic processes from the cell body¹⁰. This step takes advantage of the fact that mitochondria are more resistant to osmotic shock and remain intact, and so they sediment at the bottom of the sucrose gradient (Figure 1).

Using this same enrichment technique, the SPM and PSD fractions were first biochemically defined by polyacrylamide gel electrophoresis and sequencing of the major protein components^15-17. Subsequently western blot analysis has been used to detect and quantify the levels of synaptic proteins and further define these fractions (Figure 2). We have used this technique in our laboratories to quantify changes in the synaptic levels of the dopamine transporter that occur when the Slc6a3 locus is duplicated in mice¹⁸. We have also used this technique in NMDA receptor deficient mice to uncover synapse-specific reductions in proteins that are part of the DISC1 interactome¹⁹.

It is evident from western blot analysis that SPM fractions contain synaptic vesicle membrane proteins, endosome markers, mitochondrial proteins, membrane associated synthetic enzymes and signal transduction molecules, as well as integral components of the post-synaptic density and synaptic plasma membranes^20-23. Even PSD fractions can have contamination with abundant mitochondrial proteins and it may be necessary to perform a second gradient sedimentation or additional purification steps to remove them¹³. Recently, quantitative mass spectrometry has provided a list of over 100 proteins in the post-synaptic density alone, as well as an indication of the relative abundance of these components^24,25.

Protocol

The following protocol conforms to the guidelines of the Canadian Council of Animal Care and has been approved by the Faculty of Medicine and Pharmacy Animal Care Committee at the University of Toronto.

1. Prepare Required Reagents as Described in Table 1

1. Add protease and phosphatase (if necessary) inhibitors to all sucrose solutions, buffers and ddH₂O at concentrations outlined in Table 1. IMPORTANT: Perform all of the steps at 4 °C and pre-cool all of the reagents and equipment prior to the start of the experiment. Label all tubes, including ultracentrifuge tubes, with permanent marker.

2. Dissection of the Appropriate Brain Region

1. Sacrifice the animal by cervical dislocation or decapitation. House and euthanize animals in accordance with institutional and governmental policies on animal care.
2. Rapidly remove the brain from the skull and place on an chilled dissection block.
3. Dissect the appropriate brain region from fresh tissue and proceed to Step 3.

3. Tissue Homogenization with Motor Driven Glass-Teflon Homogenizer

1. Place 50-100 mg (for SPM), or 100-200 mg (for PSD) of the dissected tissue in a labeled 13 ml polypropylene tube containing 4 ml of 0.32 M HEPES-buffered sucrose solution. Transfer the sample to a 15 ml tapered glass-Teflon tissue grinder/homogenizer, set the motor drive to 900 rpm (setting 7) and homogenize the sample with 12 strokes over a 30 sec period. Use a different glass-Teflon homogenizer between samples, or rinse the homogenizer with cold distilled water between samples and wipe dry with a Kimwipe. NOTE: Up to 4 g of tissue can be homogenized in 4 ml of 0.32 M HEPES-buffered sucrose solution, however it is advisable to use less tissue to ensure proper fractionation.
2. Transfer the homogenized sample back to the same 13 ml polypropylene tube. Reserve 100 μl of homogenate and store at -80 °C for subsequent protein quantification and western blot analysis of the total protein fraction.

4. Low Speed Centrifugation to Remove the Nuclear Fraction (Yields Supernatant S1)

1. Centrifuge the homogenate in a fixed angle rotor at 900 x g for 10 min at 4 °C. Transfer the supernatant (S1) to a new, labeled 13 ml tube and resuspend the nuclear fraction pellet (P1) in 500 μl of 0.32 M HEPES-buffered sucrose. NOTE: The (P1) fraction can be stored at -80 °C and used for western blot analysis of the nuclear fraction.

5. Enrichment of the Crude Synaptosomal Fraction (Yields Pellet P2)

1. Centrifuge the supernatant (S1) at 10,000 x g for 15 min at 4 °C. Remove the supernatant (S2) and reserve 500 μl in a microcentrifuge tube to store at -80 °C for subsequent protein quantification and western blot analysis of the cytosolic/light membrane fraction. Resuspend the remaining crude synaptosomal fraction pellet (P2) in 1 ml of 0.32 M HEPES-buffered sucrose solution and then add another 3 ml of 0.32 M HEPES-buffered sucrose solution.
2. Centrifuge at 10,000 x g for 15 min at 4 °C. Remove the supernatant (S2’) and reserve 500 μl in a microcentrifuge tube to store at -80 °C for subsequent protein quantification and western blot analysis of the cytosolic/light membrane fraction. Save the washed crude synaptosomal pellet (P2’) in the polypropylene tube.

6. Lysing (Hypoosmotic Shock) of the Crude Synaptosomal Fraction

1. Lyse the crude synaptosomal pellet (P2’) in the polypropylene tube by resuspending it in 1 ml of ddH₂O. Add another 3 ml of ddH₂O, transfer to a glass-Teflon tissue homogenizer, and homogenize by hand with 3 strokes. NOTE: It is important to perform this step as rapidly as possible and quickly proceed to step 6.2.
2. Transfer samples from the homogenizer back into the same 13 ml polypropylene tube and rapidly adjust the sample back to 4 mM HEPES with 16 μl of 1 M HEPES solution, invert to mix.
3. Rotate samples at 4 °C for 30 min to ensure complete lysing.

7. Enrichment of the Synaptosomal Membrane Fraction (P3)

1. Centrifuge at 25,000 x g for 20 min at 4 °C. Remove the crude vesicular fraction supernatant (S3) and reserve it in a 5 ml tube to store at -80 °C for subsequent protein quantification and western blot analysis. Resuspend the synaptosomal membrane fraction (P3) in 1 ml of 0.32 M HEPES-buffered sucrose solution.

8. Preparation of the Discontinuous Sucrose Gradient

1. Pipet exactly 3.5 ml of 1.2 M HEPES-buffered sucrose solution and exactly 3.0 ml each of 1.0 M and 0.8 M HEPES-buffered sucrose solution into separate 6 ml snap cap tubes. NOTE: This is done to pre-measure the volumes of the buffers that are used to make the sucrose gradient. Exact measurement of the volumes is important to ensure that the resulting gradients will be balanced for ultracentrifugation.
2. Using a glass Pasteur pipet and bulb, transfer 1.2 M HEPES-buffered sucrose solution to the 12 ml polyallomer ultracentrifuge tube. Carefully layer the 1.0 M HEPES-buffered sucrose solution on top of the 1.2 M HEPES-buffered sucrose solution. Finally, layer the 0.8 M HEPES-buffered sucrose solution on top of the 1.0 M HEPES-buffered sucrose solution. Use a fresh Pasteur pipet for each sucrose solution and take care not to disturb the sucrose gradient. Vibrations from a bench vortex or microcentrifuge will disturb the integrity of the gradient.
3. Using a Pasteur pipet, layer the resuspended synaptosomal membrane fraction (P3) on top of the prepared discontinuous sucrose gradient. Ensure that the gradients are balanced by weighing them inside the bucket of the swinging bucket rotor. If the buckets are not balanced, use a P200 pipet to carefully add 0.32 M HEPES-buffered sucrose to the top of the gradient and balance the rotor buckets.

9. Fractionation of the Synaptic Plasma Membrane (SPM)

1. Ultracentrifuge in a swinging bucket rotor at 150,000 x g for 2 hr at 4 °C. Carefully remove the sucrose gradients from the ultracentrifuge buckets. Using an 18 G needle and a 1 ml syringe, puncture the tube at the bottom of the 1.0 M / 1.2 M HEPES-buffered sucrose solution interphase and withdraw the synaptic plasma membrane layer (SPM).
2. Make note of the volume that each SPM sample occupies in the 1 ml syringe. Place the collected SPM layer in 3.5 ml thick-wall ultracentrifuge tubes, add exactly 2.5 volumes of 4 mM HEPES to adjust the sucrose concentration from 1.2 M to 0.32 M. Once each sample has been adjusted to 0.32 M sucrose, balance the tubes with 0.32 M HEPES-buffered sucrose solution.
3. Ultracentrifuge in a fixed angle rotor at 200,000 x g for 30 min at 4 °C. Remove and discard the supernatant. Resuspend the synaptic plasma membrane pellet (SPM) in 300 μl of 50 mM HEPES / 2 mM EDTA solution. Store samples at -80 °C until used for western blotting.

10. Preparation of the Postsynaptic Density Fraction (PSD)

1. Thaw SPM samples on ice.
2. Make a solution of 0.54% Triton X-100 in 50 mM HEPES / 2 mM EDTA solution.
3. In a 5 ml polystyrene tube, combine 2.7 ml of Triton X-100/HEPES/EDTA solution and 300 μl SPM fraction and rotate the sample for 15 min at 4 °C
4. Transfer samples to 3.5 ml thick-wall ultracentrifuge tubes and centrifuge samples at 32,000 x g for 20 min at 4 °C.
5. Reserve the supernatant (Triton X-100 soluble fraction [TS]) and store at -80 °C. NOTE: At this stage, the pellet represents the “PSD-1T” fraction. A second treatment with detergent will produce a “PSD-2T” fraction. If a PSD-1T fraction is desired, proceed to step 10.6.
   1. To produce a PSD-2T fraction, resuspend the pellet in 3 ml of 0.5% Triton X-100 in 50 mM HEPES / 2 mM EDTA solution. Rotate samples for 15 min at 4 °C. Centrifuge samples at 32,000 x g for 20 min at 4 °C and proceed to step 10.6.
6. Discard the supernatant and resuspend the postsynaptic (PSD) pellet in 50 mM HEPES / 2 mM EDTA solution. NOTE: The resuspension volume may depend on the size of the pellet; a volume of 50-75 μl is recommended. Because the PSD fraction is a detergent-insoluble fraction, it is often necessary to add 1-2 μl of 0.5% SDS to enable complete resuspension of protein. Store samples at -80 °C until used for western blotting.

Results

The preparation of the sucrose density gradient should result in a clear separation of the three molar solutions of sucrose (0.8, 1.0, and 1.2 M sucrose). See Figure 3A for an example of the gradient before the protein sample is added. If the gradient is prepared too much in advance, or if it is prepared on a bench surface with vibration from other equipment, the gradient will be compromised and proper separation will not be achieved. If a clear separation of the three solutions is not visible, it is adv...

Discussion

There are several steps in the procedure that are critical for a successful outcome. In step 3, it is important that a consistent degree of homogenization is achieved for each sample. When homogenizing tissue with the motor driven homogenizer, a constant speed is used not only for the rotation of the pestle, but also with the number of strokes. The incubation time during the osmotic shock should be precise, since extended homogenization or incubation in the hypotonic solution will lyse mitochondria and contaminate the SP...

Materials

Name	Company	Catalog Number	Comments
1M HEPES, pH 7.4	BioShop	HEP003.100
HEPES	BioShop	HEP001.500
Sucrose	BioShop	SUC507.1
EDTA	BioBasic	EB0185
PMSF	BioShop	PMS123.5
Aprotinin	BioShop	APR600.1
Leupeptin	BioShop	LEU001.1
Pepstatin	BioShop	PEP605.5
Benzamidine	BioShop	BEN601.25
Sodium fluoride	BioShop	SFL001.100
Sodium pyrophosphate	BioShop	SPP310.100
Sodium orthovanadate	BioShop	SOV664.10
β-glycerophosphate	BioShop	GYP001.10
Triton X-100	BioShop	TRX506.500
18G x 1 ½” needle	BD	305196
1cc syringe	BD	309659
Name of Equipment	Company	Catalog number
Glass Teflon homogenizer Kontes Duall 23	VWR	KT885450-0023
IKA Model RW 16 Basic Stirrer	IKA Works	2572100
Sorvall SM-24 Fixed Angle Rotor	ThermoScientific	29017
Sorvall RC 6 Plus Centrifuge	ThermoScientific	46910
Thinwall Polyallomer Tubes (13.2mL)	Beckman Coulter	331372
SW 41 Ti Rotor Swinging Bucket	Beckman Coulter	331362
Beckman L-80 Floor Ultracentrifuge	Beckman Coulter
Thickwall Polycarbonate Tubes (3.5mL)	Beckman Coulter	349622
TLA-100.3 Fixed Angle Rotor	Beckman Coulter	349481
Beckman TL-100 Tabletop Ultracentrifuge	Beckman Coulter