Protein Methods Articles:
Thermo Fisher Scientific, Rockford, IL
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Method to isolate functional synaptosomes
Enrich neuronal synaptic proteins while maintaining phosphoprotein integrity.
Hai-Yan Wu , Ph.D.;
Kay Opperman, Ph.D.;
Barbara Kaboord, Ph.D.;
In the mammalian brain, synaptic function is governed by complexes held together by protein-protein, protein-lipid and lipid-lipid interactions. The interplay of synaptic proteins controls functions such as learning, memory, sensory integration, motor coordination, and emotional responses. There is evidence that the functional loss or dysregulation of various synaptic proteins is associated with neurodegenerative diseases.1 The ability to isolate and observe molecular changes in protein composition and function at synapses is important in understanding these disease mechanisms.
An enriched fraction of synaptic proteins can be obtained from isolated nerve terminals (i.e., synaptosomes) created during nerve tissue homogenization (Figure 1). Synaptosomes contain the complete presynaptic terminal, including mitochondria and synaptic vesicles, with the postsynaptic membrane and the postsynaptic density. Synaptosomes are commonly used to study synaptic function because they contain functional ion channels, receptors, enzymes and proteins, as well as the intact molecular machinery for the release, uptake and storage of neurotransmitters. Because signal transmission is highly regulated by transient phosphorylation of neuronal proteins at the synapse,2 preservation of this protein modification during synaptosome preparation is essential.
Figure 1. Schematic of a synaptosome formed from the detached nerve terminal and part of the postsynaptic membrane during mechanical homogenization.
In this study, we show that the Thermo Scientific Syn-PER Synaptic Protein Isolation Reagent is effective for isolating functional synaptosomes containing active synaptic proteins from neuronal tissue. Additionally, the Syn-PER Reagent facilitates the study of labile or transient neuronal protein phosphorylation events by stabilizing or preserving these modifications during tissue disruption.
RESULTS and DISCUSSION:
Synaptic protein extraction and enrichment
To determine the efficiency of the extraction process, we compared the total synaptic protein yields of samples prepared with Syn-PER Reagent or a standard homebrew buffer using a general Dounce extraction protocol for fresh mouse brain tissues (Figure 2). The synaptic protein yield in samples obtained using the Syn-PER Reagent was about three-fold higher compared to samples prepared with the homebrew reagent (Figure 3). The total protein recovered in the Syn-PER Reagent preparation was 9.7 ± 1.0µg/mg of brain tissue. Using the homebrew reagent, we obtained 3.4 ± 0.8µg/mg of brain tissue.
Figure 2. Protocol for isolating synaptosomes from mouse brain. The procedure requires approximately 1 hour from tissue homogenization to collection of the synaptosomal fraction.
Figure 3. Higher total protein yield is obtained using the Thermo Scientific Syn-PER Synaptic Protein Extraction Reagent than a homebrew reagent. The protein yield in synaptosome suspensions produced from fresh mouse brain (200mg) was quantified using the Thermo Scientific Pierce BCA Protein Assay (Part No. 23225). The total protein yield was three-fold greater using the Syn-PER Reagent compared to a representative homebrew reagent.
To determine specificity of the synaptosome extraction procedure, we performed Western blot analyses to detect individual synaptic proteins N-methyl-D-aspartate receptor 2B subunit (NMDAR2B), PSD95, GluR2/3/4 of α-amino-3hydroxy-5-methyl-4-ioxazolepropionic acid (AMPA) receptor and synaptophysin (Figure 4). In samples prepared with the Syn-PER Reagent, the signal is more intense for each synaptic protein in the synaptosome suspension than in the homogenate; however, in samples prepared with the homebrew reagent the signal in the synaptosome suspension is similar to that in the homogenate, indicating minimal to no enrichment. The purity of the synaptosome suspension was further confirmed by probing for the cytosolic proteins calcineurin and CDK5, and the nuclear marker protein HDAC2. Although calcineurin and CDK5 were detected in the synaptosome fraction, the signals were similar to the homogenate. In contrast, the bands for these proteins were stronger in the cytosolic fraction. As expected, HDAC2 was not detected in the synaptosome sample because nuclei were effectively excluded from this fraction.
Figure 4. Greater enrichment of synaptic proteins is achieved in samples prepared using the Thermo Scientific Syn-PER Reagent than a homebrew reagent. Total protein (10µg) from mouse brain tissue homogenates (H), cytosol (C) fraction, and synaptosome suspension (Syn) were analyzed by Western blot. The pre- and post-synaptic protein markers evaluated include synaptophysin, post-synaptic density protein 95 (PSD95), NMDA receptor 2B subunit, and AMPA receptors (GluR2/3/4). Calcineurin, Cdk5, and HDAC2 were purity controls and β-actin served as a loading control.
Functional synaptosome isolation
Synaptic transmission relies on the stimulation of neurotransmitter release via exocytosis of synaptic vesicles and subsequent vesicle retrieval by endocytosis. To determine if synaptosomes prepared with the Syn-PER Reagent are functional, synaptic vesicle endocytosis and exocytosis was measured by monitoring the uptake and release of FM* 2-10 Dye, a lipophilic styryl fluorescent dye.3 The FM 2-10 Dye is nearly nonfluorescent in solution but becomes fluorescent when internalized in synaptosomes (Ex506/Em620nm). When FM 2-10 Dye was incubated with the synaptosome suspension prepared with the Syn-PER Reagent, FM 2-10 Dye was internalized by endocytotic vesicles resulting in the synaptosomes becoming fluorescent. In the presence of calcium, KCl stimulation induced the release of accumulated FM 2-10 Dye into solution, resulting in a slow decay of FM 2-10 Dye fluorescence for 18 minutes (Figure 5). This result indicates that the Syn-PER Reagent-prepared synaptosome suspension is capable of endocytotic uptake and exocytotic release of the FM 2-10 Dye. Because endocytosis and exocytosis are highly controlled biological processes regulated by a variety of synaptic proteins, the results of this assay demonstrated that proteins in the synaptosome suspension were functional.
Figure 5. Functional synaptosomes show release of a fluorescent dye upon stimulation. Synaptosome suspensions prepared using the Syn-PER Reagent were incubated with FM 2-10 Dye, a styryl fluorescent dye. In the presence of 1.2mM calcium, fluorescence (Ex506/Em620nm) slowly decreased for 18 minutes after adding 30mM KCl. Each point is the mean ± SD of two samples.
Because synaptic transmission is highly regulated by protein phosphorylation, preserving the population of synaptic phosphoproteins is critical to understanding the functional dynamics of the synapse. We compared protein phosphorylation levels of samples prepared with the Syn-PER Reagent to other commercial lysis buffers. Western blot analyses using antibodies specifically recognizing phospho-ERK (Thr202/Tyr204), phospho-GluR2 (Try869/Tyr873/Try876) of AMPA receptor and phospho-PSD95 (Tyr236/Tyr240) resulted in markedly higher signals for each phosphorylated protein in the brain homogenates and synaptosome suspensions (Figure 6) prepared with the Syn-PER Reagent than in those prepared with the other commercial lysis buffer. The total ERK signals were constant, indicating that the ERK protein was present in equivalent amounts in both preparations. These data revealed that protein phosphorylation was better preserved when samples were prepared using the Syn-PER Reagent compared to the other buffer.
Figure 6. The Thermo Scientific Syn-PER Reagent preserves phosphorylation better than other commercial lysis buffers. Western blot analysis of the phosphoproteins p-PSD95, p-GluR2 of AMPA receptor, and p-ERK1/2 was performed on mouse brain homogenates (H), cytosolic fractions (C) and synaptosome suspensions (Syn).
The Syn-PER Reagent can be used to prepare functional synaptosomes from fresh neuronal tissue. The Syn-PER Reagent prevents phosphoprotein degradation during the homogenization and isolation process, thereby facilitating more accurate assessments of labile or transient neuronal protein phosphorylation events.
Synaptosome preparation: Whole brain or one hemisphere, excluding the cerebellum, (~200-400mg) was homogenized in 10 volumes of the Syn-PER Reagent or a homebrew reagent4 (protease inhibitors included; Part No. 87785) using a 7mL Dounce tissue grinder with 10 up-and-down even strokes. The homogenate was centrifuged at 1200 × g for 10 minutes to remove cell debris, and the supernatant was centrifuged at 15,000 × g for 20 minutes. The pellets, containing synaptosomes, were gently resuspended in the respective reagent.
Western blots: The protein concentration of each sample was determined using the Pierce BCA Protein Assay (Part No. 23225). Equal amounts of total protein (10-20μg/lane) were resolved on denaturing 2-10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 3% bovine serum albumin and incubated with primary antibody overnight at 4°C. Blots were incubated with goat anti-rabbit or goat anti-mouse horseradish peroxidase-conjugated secondary antibody (Part No. 31430)(Part No. 31460) for 1 hour at room temperature and then washed. Bands were visualized using the Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Part No. 34080) and exposed to film.
FM 2-10 Dye uptake and release assay: Freshly prepared synaptosome suspensions (600µg in 500mL calcium-containing HBSS) were incubated with 100µM of FM 2-10 Dye (Sigma-Aldrich Co.) at room temperature. To stimulate the uptake of the dye, the synaptosomes were incubated with 30mM KCl. After 10-15 minutes to allow for dye internalization, synaptosomes were pelleted by centrifuging for 5 minutes at 15,000 × g and washed twice with HBSS to remove excess dye. Synaptosomes were resuspended in HBSS either with or without 1.2mM CaCl2. The release of the dye was induced with 30mM KCl and monitored at Ex506/Em620nm.
Animal care: C57BL/J6 mice (8-12 weeks old, mixed gender), from Charles River and housed in the University of Illinois College of Medicine at Rockford animal facility, were used to obtain brain tissues. Experiments were performed exactly as approved by the Animal Care and Use Committee in the University of Illinois College Of Medicine at Rockford, and conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
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