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Mar 2017

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Preparation, Stimulation and Other Uses of Adult Rat Brain Synaptosomes
成年大鼠脑突触体的制备、刺激及其他用途   

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Abstract

In this paper, our protocol for preparation of brain synaptosomes is described. Synaptosomes are a valuable model system for analysis of structural components of the synapse as well as for investigation of synaptic function. Synaptosomal preparations are necessary for understanding molecular changes at synapses where critical post-translational modifications of synaptic proteins may occur. Not only are synaptosomes rich in synaptic proteins, but they can be used for analyzing uptake of neurotransmitters into synaptic vesicles and for analysis of the involvement of neurotransmitter synthesis and release. Synaptosomes can be stimulated with increased calcium influx to release neurotransmitters. Synaptosomal preparations have been used in characterizing calcium dependent phosphorylation and activation of the GABA synthesizing enzyme GAD65 (L-glutamic acid decarboxylase with molecular weight of 65 kDa). By examining protein complexes on the membrane of synaptic vesicles obtained from synaptosomal preparations, it was possible to characterize the role of GAD65 in the coupled synthesis and vesicular uptake of GABA (γ-aminobutyric acid) culminating in GABA vesicular release, which contributes in an important way to fine-tuning of GABAergic neurotransmission.

Keywords: GAD65 (GAD65), Synaptic Vesicle (SV) (突触囊泡(SV)), GABA (GABA), Synaptosomes (突触体)

Background

Synaptosomal preparation methods were established in neuroscience research laboratories over 40 years ago and have been of great value in scientific innovations relating to neurotransmitter release with respect to elevated extracellular potassium as well as responses to increased intracellular calcium. In addition to elucidating the process of neurotransmitter release, synaptosomal preparations have been a valuable source of synaptic vesicles. Studies on synaptic vesicles have for example been used in the characterization of protein components that participate in coupled neurotransmitter synthesis and vesicular release. Synaptosomal preparations have also been very valuable as an intermediate in isolation of synaptic vesicles which are then analyzed in terms of protein complexes located on the vesicular membrane that include neurotransmitter synthesizing enzymes. Of key significance in this regard is the vesicular membrane GAD65 complex which includes CSP (Cystine-String Protein), VGAT (Vesicular GABA transporters) and CaMKII (calcium/calmodulin-dependent protein kinase II). Regulation of GAD65 activity has been further characterized in terms of GAD65 cleavage by calpain and activation of membrane bound GAD through phosphorylation as well as accelerated anchoring of GAD65 to SV by palmitoylation.

The basic methodology for vesicular preparation presented in this paper does not significantly differ from the originally established technology from the 1970s but the applications have been greatly extended over this time and therefore expanded to include analytical investigations on neurotransmitter synthesizing enzymes, on signaling complexes associated with these enzymes, on mechanisms of regulation of these enzymes and on functional outcomes of enzyme activation including vesicular neurotransmitter release in response to high extracellular potassium levels or to increased calcium influx.

Materials and Reagents

  1. Pipette tips (VWR, catalog number: 82028-570 )
  2. Centrifuge tubes (round bottom–50 ml) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3118-0050 )
  3. 15 ml Falcon tubes (VWR, catalog number: 10026-076 )
  4. Glass tube (DWK Life Sciences, Kimble, catalog number: 885451-0022 )
  5. Scintillation vial (Fisher Scientific, catalog number: 03-337-20 )
  6. Experiments involving animals:
    The use of animals and animal tissues in this protocol was approved by and in accordance with the requirements of Institutional Animal Care and Use Committee, Florida Atlantic University. The Public Health Service animal welfare assurance number is A3883-01. For all experiments, Adult 250-300 g Sprague-Dawley male rats (Harlan, IL, USA) were used. Two animals per cage were housed and maintained at 22 °C with an alternating 12-h light/dark cycle. The animals were allowed a minimum stabilization period of at least 3 days after which they were utilized for experimentation.
  7. Scintillation cocktail (RPI, catalog number: 111175 )
  8. Sucrose (Fisher Scientific, catalog number: BP220-212 )
  9. HEPES-NaOH (RPI, catalog number: H2393 )
  10. Tris-HCl (Fisher Scientific, catalog number: BP152-1 )
  11. Calcium chloride dihydrate (CaCl2·2H2O) (Fisher Scientific, catalog number: C79-500 )
  12. Disodium phosphate (Na2HPO4) (Sigma-Aldrich, catalog number: 255793-50G )
  13. Monopotassium phosphate (KH2PO4) (Fisher Scientific, catalog number: P286-1 )
  14. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761 )
  15. Sodium chloride (NaCl) (VWR, catalog number: BDH0286 )
  16. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: EDS-100G )
  17. Triton X-100 (Sigma-Aldrich, catalog number: X100-100ML )
  18. Mammalian Protease Inhibitor (MPI) (Sigma-Aldrich, catalog number: P8340 )
  19. Phosphatase inhibitor (PhI) (HaltTM) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 78426 )
  20. 2-Aminoethylisothio-uronium bromide (AET) (Sigma-Aldrich, catalog number: A5879 )
  21. Pyridoxal 5’ Phosphate (PLP) (Sigma-Aldrich, catalog number: P9255-25G )
  22. Dipotassium phosphate (K2HPO4) (Sigma-Aldrich, catalog number: P8281 )
  23. Pyruvate kinase (Sigma-Aldrich, catalog number: P9136-25KU )
  24. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P3911-500G )
  25. Phospho(enol) pyruvate (Sigma-Aldrich, catalog number: P7127-250MG )
  26. Adenosine 5’-triphosphate (ATP) (Sigma-Aldrich, catalog number: A26209-10G )
  27. Magnesium sulfate (MgSO4) (Fisher Scientific, catalog number: M65-500 )
  28. γ-Amino-n-Butyric acid (GABA) (Sigma-Aldrich, catalog number: A5835 )
  29. Glutamate (Glu) (Sigma-Aldrich, catalog number: G5889-100G )
  30. [3H] Glutamate (Amersham Biosciences, catalog number: TRK445 )
  31. [14C] GABA (PerkinElmer, catalog number: NEC290E250UC )
  32. Oxirane acrylic beads (Sigma-Aldrich, catalog number: O9754 )
  33. HEPES buffered sucrose solution (see Recipes)
  34. Krebs ringer phosphate (KRP) buffer (see Recipes)
  35. Brain lysis buffer (see Recipes)
  36. Standard GAD buffer (see Recipes)
  37. GPBS buffer (see Recipes)
  38. ATP mixture (see Recipes)

Equipment

  1. Pipettes (VWR, catalog number: 89079-976 )
  2. Guillotine (Stoelting, catalog number: 51330 )
  3. Con-torque homogenizer (Eberbach, model: E2355 , catalog number: 7265)
  4. Avanti J-25 centrifuge (Beckman Coulter, model: Avanti J-25 , catalog number: 363102)
  5. Optima L-90 K centrifuge (Beckman Coulter, model: Optima L-90K , catalog number: 365672)
  6. Tri-Carb 2900TR Liquid Scintillation Analyzer (PerkinElmer, model: Tri-Carb 2900TR )

Software

  1. ImageJ software (ImageJ)
  2. Prism software (GraphPad Software)
  3. SPSS software

Procedure

  1. Isolation of synaptosomes (Figure 1) (Wu et al., 1973; Wu, 1982)


    Figure 1. Schematic diagram for protocol

    1. Add HEPES buffered sucrose solution (4 ml/g, see Recipe 1; use freshly prepared sucrose solution, and it should be cooled on ice to 4 °C before use) to freshly collected adult rat brain. Discard the cerebellum and meninges. A rat brain is approximately 4 g.
      Note: All steps are performed on ice (4 °C) unless otherwise specified.
    2. Homogenize thoroughly: Using con-torque homogenizer and glass tubes the mixture is subjected to vigorous manual homogenization (15 strokes) (Figure 1).
    3. Centrifuge at 1,000-1,500 x g for 10 min to remove the nuclear debris (use Avanti J-25 centrifuge and centrifuge tubes [round bottom]).
    4. The resulting supernatant (S1) is transferred to a new tube while the pellet (P1) is further homogenized with an equal volume of sucrose buffer (Recipe 1) and re-centrifuged under the same conditioned to yield supernatant (S1’) and pellet (P1’).
    5. The supernatants S1 and S1’ are combined and centrifuged at 20,000 x g for 30 min (Use Avanti J-25 centrifuge and centrifuge tubes [round bottom]).
    6. The resulting pellet (P2) is crude synaptosome fraction (Figure 1).
    7. Discard S2 and note the discarded volume. The pellet (P2) is washing by gently swirling it in fresh sucrose buffer whose volume is equivalent to the discarded supernatant (S2).
      Note: Don’t pipette up and down, but only gently swirl the tube to resuspend P2.
    8. The solution is then centrifuged again at 20,000 x g for 30 min to yield the wash crude synaptosomes (P2’). From one rat brain, it is about 1 ml. Use Avanti J-25 centrifuge and centrifuge tubes (round bottom).
      Note: Store the P2’ at -80 °C if you don’t use it immediately; always keep it on ice if you carry on for further steps.

Uses of isolated synaptosomes
Synaptosomal preparations can be used for investigation of neurotransmitter release (Figure 1) (neuronal depolarization) upon stimulation with high potassium as well for analysis of mechanisms of neurotransmitter release from a presynaptic neuron which can be calcium dependent or calcium independent (Wu et al., 1973; Wu, 1982; Bao et al., 1995). Furthermore, synaptic vesicles may be prepared from synaptosomes for analysis of GABA uptake into vesicles as well as analysis of the GAD complex interacting with the vesicular membrane (Jin et al., 2003; Wu et al., 2007; Buddhala et al., 2012). Modification of GAD function can be investigated in terms of regulation of GAD cleavage and regulation of GAD interaction with synaptic vesicles (Sha et al., 2005; Buddhala et al., 2012). Different uses of synaptosomes are outlined below.

  1. Synaptosomal stimulation with high K+    
    1. Freshly prepared, washed crude synaptosomes(P2’) are re-suspended in 15 ml Falcon tubes in an equal volume of 2x stimulation buffer (freshly prepared cold Kreb’s ringer phosphate (KRP) buffer, see Recipe 2).
    2. Divide the synaptosomes into 5 equal fractions–Unstimulated, stimulated (Stim) 5’, Stim 10’, Stim 30’ and Stim 60’ (use 15 ml Falcone tubes).
    3. Aliquots of the synaptosomes in KRP buffer are stimulated by the addition of KCl at either 10, 50 or 100 millimolar and incubated for 37 °C at 45 min.
    4. A non-stimulated sample serves as the control (maintained at 4 °C or on ice).
    5. Further study of synaptosomal lysate for immuno-blotting or radioactive GAD activity assays, the synaptosomes in each aliquot are lysed in 1 ml of brain lysis buffer (see Recipe 3).
    6. The lysates are then rocked at 4 °C for 30 min and are centrifuged at 25,000 x g for 30 min (using Avanti J-25 centrifuge). After this step, the supernatant is collected and stored at -80 °C or else, for immediate preparation of synaptic vesicles, the supernatant is kept on ice or at 4 °C. The supernatant is collected and the protein concentrations are normalized to serve as the samples for subsequent steps.

  2. Release of neurotransmitters is calcium dependent (Wu et al., 1973; Fon and Edwards, 2001)
    Neurotransmitter discharged from a presynaptic neuron in response to increased neural activity diffuses across the synaptic cleft and transduces the physiological signal by attaching to postsynaptic receptors.
    The receptors therefore define the nature of the physiological signal. Classical neurotransmitters such as γ-aminobutyric acid (GABA), glutamate, and acetylcholine (ACh) activate ion channels and thus facilitate quick synaptic transmission. In comparison, neuromodulators, such as monoamines and peptides (as well as GABA, glutamate, and ACh), activate G-protein–coupled receptors, which stimulate second messengers and act on an extended time scale. The neurotransmitter cycle comprises transmitter biosynthesis, storage, reuptake, and degradation. The synaptic vesicle cycle includes targeting vesicles to the nerve terminal where docking, fusion, endocytosis, and recycling take place. High rates of exocytosis involve the well-organized packaging of neurotransmitter into SVs (vesicular neurotransmitter transporters) as well as fast recycling of SVs at the nerve terminal, either straight from the plasma membrane or through an endosomal intermediate. The local recycling comprises sorting of SV proteins from plasma membrane proteins, clathrin-mediated endocytosis, on top of docking of SVs at the plasma membrane. Regulated fusion follows in response to locally elevated Ca2+ arriving at the nerve terminal through voltage-gated Ca2+ channels, which cluster near the site of vesicle fusion.

  3. Preparation of synaptic vesicles (Figure 1) (Jin et al., 2003)
    1. Synaptosomes (P2’) are rapidly osmolysed in 10 volumes of ice cold Nano pure water containing a 1:100 dilution of mammalian protease inhibitor (MPI) cocktail and phosphatase inhibitors (PhI) (use centrifuge tubes with round bottom).
    2. The diluted synaptosomal lysate is homogenized as described above and incubated on ice for 45 min.
    3. Centrifuge at 47,000 x g for 15 min (use Optima L-90 K centrifuge and centrifuge tubes (round bottom)) to remove large membrane fractions and mitochondria.
    4. Collect the supernatants (S3) without disturbing the pellet and centrifuge at 200,000 x g for 2 h (use Optima L-90 K centrifuge and centrifuge tubes (round bottom)).
    5. The pellet obtained in this way is crude synaptic vesicles (P4).
    6. Add 500 μl of standard GAD buffer (see Recipe 4) to each fraction to remove any contaminating diluting cytosolic fractions (S3) and tight pellet (P4) are washed three times. The pellets are to be used for the steps which are listed below in the details of GABA uptake assay.

  4. GABA uptake method (Jin et al., 2003)
    1. Crude SVs are purified as described above and mixed with anti-GAD65 IgG-coupled oxirane acrylic beads (Sigma O9754) for purification of GABAergic specific SVs. AntiGAD65 antibody -SV beads (2 mg of protein per ml) are incubated in 100 microliters total volume with 4 mg/ml of SV (final concentration: 2 mg/ml), 120 µg/ml pyruvate kinase (final concentration: 60 µg/ml in GPBS buffer (see Recipe 5)].
    2. Incubate SV mixture at 32 °C for 2 min.
    3. Add the equal volume of ATP mixture (the concentration of SV mixture with ATP mixture at 1:1 of ratio) (2 mg/ml; see Recipe 6) and pipette to combine.
    4. The combination is further incubated at 32 °C, and an aliquot of 30 µl of the reaction mixture is removed and vacuum-filtered through nitrocellulose membrane at 1-, 5-, 10-, 15-, and 20-min intervals.
    5. The membranes are washed two times with 5 ml of ice-cold GPBS buffer.
    6. After the membrane is air dried, put the membrane in scintillation vial, add scintillation cocktail, vortex the vial and leave it overnight.
    7. The radioactivity persisting on the membrane is calculated based on scintillation counter measurements (dpm). The count in Disintegrations Per Minute (dpm) is measured using equipment Tri-Carb 2900TR Liquid Scintillation Analyzer (PerkinElmer, MA, USA)
    8. For experiments involving the GAD inhibitor, hydrazine, or aminooxyacetate, the inhibitor is first preincubated with SV mixture for 10 min before the addition of ATP mixture. For uptake assays involving both [3H] Glu and [14C] GABA, the conditions are the same as explained above (Steps E1 to E4) except that both [3H] Glu and [14C] GABA are included in the reaction mixture.

  5. Complex with GAD on the vesicular membrane (Jin et al., 2003; Buddhala et al., 2012; Modi et al., 2015)
    GAD65 is attached to SVs by creating a protein complex (first with Heat Shock Cognate: HSC70) that was discovered through the attachment to proteins on SVs, e.g., CSP, VGAT, and (anti-calmodulin-dependent kinase II) CaMKII. This protein complex functions as a machine to verify that GABA biosynthesis and packaging into the SV is exactly coupled. This illustrates a functional and structural coupling of GABA synthesis, regulation, and packaging into SVs.
    The physiological events initiated by neuronal stimulation through activation of SV-associated GAD and the subsequent packaging of GABA into the SV can be explained as below.
    GABA is distributed by exocytosis after the onset of an action potential. The SV is recycled by a process involving clathrin-coated pits. The clathrin coat is then separated from the vesicles through interaction with HSC70. Vesicles are then returned to the resting state of SVs, where the proton gradient is returned by V-ATPase. GAD65 is stimulated through protein phosphorylation by a proton gradient dependent protein kinase. One of the candidates for the protein kinase is CaMKII. GABA that has been newly synthesized by GAD65 is then carried into SVs by VGAT. These refilled GABA-containing SVs are prepared to be released upon onset of a new action potential. SGAD is activated by calcineurin-mediated dephosphorylation and inhibited by protein kinase A-mediated protein phosphorylation. When GABA neurons are excited, the influx of Ca2+ into the terminal results in dephosphorylation and activation of SGAD/GAD67 (L-glutamic acid decarboxylase with molecular weight of 67 kDa). GABA synthesized by SGAD/GAD67 in the cytosol may also be transferred into SVs, although it represents a minor pathway. Cytosolic GABA may also be metabolized to create ATP through the GABA shunt pathway, which may be utilized to preserve the electrochemical proton gradient for GABA transport. GAD65 is mainly responsible for the synthesis of GABA to be utilized as a neurotransmitter, whereas GAD67 is employed for GABA to be used for other functions such as serving as a signaling molecule in growth, a source of increased cell viability, and a source of GABA released via nonvesicular mechanism.

  6. Regulation of GABA synthesis through control of GAD activity (Bao et al., 1995; Sha et al., 2005; Wei and Wu, 2005; Sha et al., 2008; Buddhala et al., 2012; Modi et al., 2014; Chou et al., 2017)
    The stimulation of the glutamic acid decarboxylase (GAD) enzymes GAD65 and GAD67 switches GABA neurotransmission at the pre-synaptic site. Transcription or mRNA splicing controls concentrations of GAD65 and GAD67. Post-translational modifications include proteolytic cleavage, phosphorylation and palmitoylation, that regulate the activities of these key enzymes (Wei et al., 2004). A truncated form of GAD65 (tGAD65) is more dynamic than full-length GAD65 (fGAD65) although, by comparison, truncated GAD67 (tGAD67) is less active than full-length GAD67 (fGAD67). The protein mu-calpain is responsible for cleaving of fGAD65 and fGAD67.
    GABA neurotransmission is dependent on whether GAD is associated with synaptic vesicles (SV) and calpain carries out a critical function by producing the highly active tGAD65 causing in increased GABA synthesis and packaging uptake into SV.
    It is known that reversible protein phosphorylation is a key mechanism responsible for the regulation of protein functions. Neurotransmitter systems regulated by phosphorylation of their synthesizing enzymes has also been demonstrated. Protein phosphorylation and dephosphorylation play a vital role in the regulation of GAD activity in the brain. Soluble GAD (sGAD) is inactivated upon phosphorylation, most likely by protein kinase A (PKA), and stimulated upon dephosphorylation by protein phosphatase 2B (PP2B, calcineurin). By the contrast, membrane-bound GAD (mGAD) is activated by phosphorylation and disabled by dephosphorylation. The kinase responsible for the phosphorylation of mGAD is not yet known. Although it is commonly assumed that GAD65 is in mGAD preparations and that GAD67 is principally present in sGAD preparation, GAD65 and GAD67 have been shown to exist in both soluble and membrane fractions of the brain preparations.
    PKCε regulates GAD65 phosphorylation while GAD67 is inhibited through phosphorylation by PKA. Cysteine residues 455 and 446 in GAD67 and GAD65 alone are vital for full-length GAD regulation.
    T91 was shown to be a key phosphorylation site for GAD67 and T95 in hGAD65 was shown to be phosphorylated by kinase C ε (PKCε) by analysis using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Cooperation with the cofactor PLP at these respective targets controls the switch between PLP-bound active holoGAD and an unbound active apo GAD form. Temporary switching to the PLP bound active holoGAD is vital to GABA neurotransmission. Specific to GAD65 but not GAD67 is palmitoylation by (Huntingtin-interacting protein) HIP14 which accelerates GAD65 anchoring to SV and increases the input of vesicular GABA to neurotransmission. 

Data analysis

Data for all experiments are analyzed with Prism or SPSS software. The statistical significance of the data is determined by t-tests or one-way analysis of variance (ANOVA). P values of less than 0.05 are considered significant. The data appears to be normally distributed with similar standard deviations and standard errors observed between and within experimental groups.

Recipes

Note: All the following solutions (Recipes 1-6) can be stored at 4 °C for up to 7 days.

  1. HEPES buffered sucrose solution
    0.32 M sucrose
    4 mM HEPES-NaOH
    Adjust pH to 7.4 (10 ml/g of brain w/v)
    Store at 4 °C
  2. Kreb’s ringer phosphate (KRP) buffer
    10 mM Tris-HCl
    2.2 mM CaCl2
    0.5 mM Na2HPO4
    0.4 mM KH2PO4
    4 mM NaHCO3
    80 mM NaCl
    Adjust pH to 7.5
    Store at 4 °C
  3. Brain lysis buffer
    50 mM Tris-HCl
    150 mM NaCl
    2 mM EDTA
    Adjust pH to 8
    1% Triton X-100
    1% MPI & PhI
    Store at 4 °C
  4. Standard GAD buffer
    50 mM potassium phosphate
    1 mM 2 amino ethylisothiouronium bromide (AET)
    0.2 mM pyridoxal 5’ phosphate
    Adjust pH to 7.2
    Store at 4 °C
  5. GPBS buffer
    9.5 mM KH2PO4
    40.5 mM K2HPO4
    8 mM KCl
    86.6 mM potassium gluconate
    Adjust pH to 7.4
    Store at 4 °C
  6. ATP mixture
    2 mM ATP
    4.4 mM MgSO4
    12 mM phospho(enol) pyruvate
    50 µM GABA
    2 mM Glu
    110.1 µCi/µl [3H] Glu
    Store at 4 °C

Acknowledgments

This work was funded by Grant 6JK08 from the Florida Department of Health and by a grant from the AEURA Trust. There are no conflicts of interest or competing interests.

References

  1. Bao, J., Cheung, W. Y. and Wu, J. Y. (1995). Brain L-glutamate decarboxylase. Inhibition by phosphorylation and activation by dephosphorylation. J Biol Chem 270(12): 6464-6467.
  2. Buddhala, C., Suarez, M., Modi, J., Prentice, H., Ma, Z., Tao, R. and Wu, J. Y. (2012). Calpain cleavage of brain glutamic acid decarboxylase 65 is pathological and impairs GABA neurotransmission. PLoS One 7(3): e33002.
  3. Chou, C. C., Modi, J. P., Wang, C. Y., Hsu, P. C., Lee, Y. H., Huang, K. F., Wang, A. H., Nan, C., Huang, X., Prentice, H., Wei, J. and Wu, J. Y. (2017). Activation of brain L-glutamate decarboxylase 65 isoform (GAD65) by phosphorylation at threonine 95 (T95). Mol Neurobiol 54(2): 866-873.
  4. Fon, E. A. and Edwards, R. H. (2001). Molecular mechanisms of neurotransmitter release. Muscle Nerve 24(5): 581-601.
  5. Jin, H., Wu, H., Osterhaus, G., Wei, J., Davis, K., Sha, D., Floor, E., Hsu, C. C., Kopke, R. D. and Wu, J. Y. (2003). Demonstration of functional coupling between γ-aminobutyric acid (GABA) synthesis and vesicular GABA transport into synaptic vesicles. Proc Natl Acad Sci U S A 100(7): 4293-4298.
  6. Modi, J. P., Prentice, H. and Wu, J. Y. (2014). Regulation of GABA Synthesis and Transport. Nova Science Publishers ISBN: 978-1-63321-838-3.
  7. Modi, J. P., Prentice, H. and Wu, J. Y. (2015). Regulation of GABA Neurotransmission by glutamic acid decarboxylase (GAD). Curr Pharm Des 21(34): 4939-4942.
  8. Sha, D., Jin, Y., Wu, H., Wei, J., Lin, C. H., Lee, Y. H., Buddhala, C., Kuchay, S., Chishti, A. H. and Wu, J. Y. (2008). Role of μ-calpain in proteolytic cleavage of brain L-glutamic acid decarboxylase. Brain Res 1207: 9-18.
  9. Sha, D., Wei, J., Wu, H., Jin, Y. and Wu, J. Y. (2005). Molecular cloning, expression, purification, and characterization of shorter forms of human glutamic decarboxylase 67 in an E. coli expression system. Brain Res Mol Brain Res 136(1-2): 255-261.
  10. Wei, J., Davis, K. M., Wu, H. and Wu, J. Y. (2004). Protein phosphorylation of human brain glutamic acid decarboxylase (GAD)65 and GAD67 and its physiological implications. Biochemistry 43(20): 6182-6189.
  11. Wei, J. and Wu, J. Y. (2005). Structural and functional analysis of cysteine residues in human glutamate decarboxylase 65 (GAD65) and GAD67. J Neurochem 93(3): 624-633.
  12. Wu, H., Jin, Y., Buddhala, C., Osterhaus, G., Cohen, E., Jin, H., Wei, J., Davis, K., Obata, K. and Wu, J. Y. (2007). Role of glutamate decarboxylase (GAD) isoform, GAD65, in GABA synthesis and transport into synaptic vesicles-Evidence from GAD65-knockout mice studies. Brain Res 1154: 80-83.
  13. Wu, J. Y. (1982). Purification and characterization of cysteic acid and cysteine sulfinic acid decarboxylase and L-glutamate decarboxylase from bovine brain. Proc Natl Acad Sci U S A 79(14): 4270-4274.
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简介

在这篇论文中,我们描述了制备脑突触体的方案。突触体是用于分析突触的结构组分以及用于调查突触功能的有价值的模型系统。突触体制备对于理解可能发生突触蛋白的关键翻译后修饰的突触处的分子变化是必需的。突触小体不仅含有丰富的突触蛋白,还可用于分析神经递质向突触小泡的摄取和神经递质合成和释放的参与分析。可以用增加的钙内流刺激突触体释放神经递质。突触体制剂已被用于表征钙依赖性磷酸化和GABA合成酶GAD65(分子量为65kDa的L-谷氨酸脱羧酶)的活化。通过检查从突触体制剂获得的突触小泡膜上的蛋白质复合物,可以表征GAD65在GABA囊泡释放的偶联合成和囊泡摄取中的作用,这最终导致GABA囊泡释放GABA能神经传递的微调方法。

【背景】突触体制备方法在40多年前在神经科学研究实验室中建立,并且在涉及神经递质释放相关的细胞外钾升高以及对细胞内钙增加的应答方面具有极大的价值。除了阐明神经递质释放的过程之外,突触体制剂已经成为突触囊泡的有价值的来源。关于突触小泡的研究已经被用于表征参与偶联的神经递质合成和囊泡释放的蛋白质组分。突触体制剂作为突触囊泡分离的中间体也是非常有价值的,然后根据位于包含神经递质合成酶的囊泡膜上的蛋白质复合物进行分析。在这方面关键的意义在于包括CSP(胱氨酸 - 丝氨酸蛋白),VGAT(囊泡GABA转运蛋白)和CaMKII(钙/钙调蛋白依赖性蛋白激酶II)的囊泡膜GAD65复合物。通过钙蛋白酶对GAD65进行切割和通过磷酸化激活膜结合的GAD以及通过棕榈酰化将GAD65加速锚定到SV,进一步表征了GAD65活性的调节。

本文中介绍的囊泡制备的基本方法与20世纪70年代最初建立的技术没有显着差异,但是在这个时候应用已经大大扩展,因此扩展到包括对神经递质合成酶的分析研究,酶,调节这些酶的机制以及酶激活的功能性结果,包括响应于高的胞外钾水平或增加的钙流入而引起的囊泡神经递质释放。


关键字:GAD65, 突触囊泡(SV), GABA, 突触体

材料和试剂

  1. 移液器吸头(VWR,目录号:82028-570)
  2. 离心管(圆底50ml)(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:3118-0050)
  3. 15毫升Falcon管(VWR,目录号:10026-076)
  4. 玻璃管(DWK Life Sciences,Kimble,目录号:885451-0022)
  5. 闪烁小瓶(Fisher Scientific,目录号:03-337-20)
  6. 涉及动物的实验:
    本议定书中动物和动物组织的使用得到了佛罗里达大西洋大学机构动物护理和使用委员会的批准。公共卫生服务动物福利保证号码是A3883-01。对于所有实验,使用成年人250-300g Sprague-Dawley雄性大鼠(Harlan,IL,USA)。每笼2只动物饲养并保持在22℃,交替12小时光照/黑暗循环。允许动物至少3天的最小稳定期,之后将它们用于实验。
  7. 闪烁鸡尾酒(RPI,目录号:111175)
  8. 蔗糖(Fisher Scientific,目录号:BP220-212)
  9. HEPES-NaOH(RPI,目录号:H2393)
  10. Tris-HCl(Fisher Scientific,目录号:BP152-1)
  11. 氯化钙二水合物(CaCl 2•2H 2 O)(Fisher Scientific,目录号:C79-500)
  12. 磷酸二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:255793-50G)
  13. 磷酸二氢钾(KH2PO4)(Fisher Scientific,目录号:P286-1)
  14. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S5761)
  15. 氯化钠(NaCl)(VWR,目录号:BDH0286)
  16. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:EDS-100G)
  17. Triton X-100(Sigma-Aldrich,目录号:X100-100ML)
  18. 哺乳动物蛋白酶抑制剂(MPI)(Sigma-Aldrich,目录号:P8340)
  19. 磷酸酶抑制剂(PhI)(Halt TM)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:78426)
  20. 2-氨基乙基异硫脲 - 脲(AET)(Sigma-Aldrich,目录号:A5879)
  21. 5'磷酸吡哆醛(PLP)(Sigma-Aldrich,目录号:P9255-25G)
  22. 磷酸二钾(KH 2 HPO 4)(Sigma-Aldrich,目录号:P8281)
  23. 丙酮酸激酶(Sigma-Aldrich,目录号:P9136-25KU)
  24. 氯化钾(KCl)(Sigma-Aldrich,目录号:P3911-500G)
  25. 磷酸(烯醇)丙酮酸(Sigma-Aldrich,目录号:P7127-250MG)
  26. 腺苷5'-三磷酸(ATP)(Sigma-Aldrich,目录号:A26209-10G)
  27. 硫酸镁(MgSO 4)(Fisher Scientific,目录号:M65-500)
  28. γ-氨基 - 正丁酸(GABA)(Sigma-Aldrich,目录号:A5835)
  29. 谷氨酸(Glu)(Sigma-Aldrich,目录号:G5889-100G)
  30. [3 H]谷氨酸(Amersham Biosciences,目录号:TRK445)
  31. [14C] GABA(PerkinElmer,目录号:NEC290E250UC)
  32. 环氧丙烷珠(Sigma-Aldrich,目录号:O9754)
  33. HEPES缓冲蔗糖溶液(见食谱)
  34. 克雷布斯磷酸盐(KRP)缓冲液(见食谱)
  35. 脑裂解缓冲液(见食谱)
  36. 标准的GAD缓冲区(见食谱)
  37. GPBS缓冲区(见食谱)
  38. ATP混合物(见食谱)

设备

  1. 移液器(VWR,目录号:89079-976)
  2. 断头台(Stoelting,目录号:51330)
  3. 扭矩均化器(Eberbach,型号:E2355,目录号:7265)
  4. Avanti J-25离心机(Beckman Coulter,型号:Avanti J-25,目录号:363102)
  5. Optima L-90K离心机(Beckman Coulter,型号:Optima L-90K,目录号:365672)
  6. Tri-Carb 2900TR液体闪烁分析仪(PerkinElmer,型号:Tri-Carb 2900TR)

软件

  1. ImageJ软件(ImageJ)
  2. 棱镜软件(GraphPad软件)
  3. SPSS软件

程序

  1. 突触体的分离(图1)(Wu et al。,1973; Wu,1982)


    图1.协议示意图

    1. 添加HEPES缓冲的蔗糖溶液(4ml / g,参见配方1;使用新鲜制备的蔗糖溶液,并且在使用之前应该在冰上冷却到4℃)添加到新收集的成年大鼠脑中。丢弃小脑和脑膜。大鼠脑约4克。
      注意:除非另有说明,否则所有步骤均在冰上(4°C)进行。
    2. 均匀匀化:使用力矩均化器和玻璃管,将混合物剧烈地手动均化(15次)(图1)。
    3. 1000-1,500×g离心10分钟以除去核碎片(使用Avanti J-25离心机和离心管(圆底))。
    4. 将得到的上清液(S1)转移到新管中,同时用等体积的蔗糖缓冲液(配方1)使团块(P1)进一步均化,并在相同的条件下重新离心以产生上清液(S1')和沉淀P1')。
    5. 将上清液S1和S1'合并,并以20000×g离心30分钟(使用Avanti J-25离心机和离心管[圆底])。
    6. 得到的小球(P2)是粗糙的突触体部分(图1)。
    7. 丢弃S2并注意丢弃的体积。通过在体积等于丢弃的上清液(S2)的新鲜蔗糖缓冲液中轻轻旋转来洗涤沉淀(P2)。
      注意:不要上下移动,只能轻轻旋动管道以重新悬浮P2。
    8. 然后将溶液再次以20000×g离心30分钟以产生清洗粗突触体(P2')。从一只大鼠脑中,大约有1毫升。使用Avanti J-25离心机和离心管(圆底)。
      如果您不立即使用P2',请将P2'存放在-80°C;如果您继续进行下一步操作,请始终将其置于冰面上。

孤立突触体的用途
突触体制剂可用于研究高钾刺激后的神经递质释放(图1)(神经元去极化),以及分析从可以是钙依赖性或钙依赖性的突触前神经元释放神经递质的机制(Wu等人1973; Wu,1982; Bao等人,1995)。此外,突触小泡可由突触体制备,用于分析GABA摄入囊泡以及分析与囊泡膜相互作用的GAD复合物(Jin等人,2003; Wu等人。,2007; Buddhala et al。。,2012)。 GAD功能的修饰可以通过调节GAD切割和调节GAD与突触小泡的相互作用来研究(Sha等人,2005; Buddhala等人,2012 )。下面概述了突触体的不同用途。

  1. 高K +突触体的刺激    
    1. 将新鲜制备的洗过的粗突触体(P2')重新悬浮在等体积的2x刺激缓冲液(新鲜制备的冷Kreb氏磷酸盐缓冲液(KRP)缓冲液,见方案2)的15ml Falcon管中。
    2. 将突触体分成5个相同的部分 - 未刺激,刺激(Stim)5',刺激10',刺激30'和刺激60'(使用15ml Falcone管)。
    3. KRP缓冲液中的突触体的等分试样通过加入10,50或100毫摩尔的KCl来刺激,并在45分钟时孵育37℃。
    4. 一个未受刺激的样品作为对照(保持在4℃或在冰上)。
    5. 进一步研究突触体裂解物进行免疫印迹或放射性GAD活性测定,将每个等分试样的突触体溶解在1ml脑裂解缓冲液中(见方案3)。
      6.然后将裂解物在4℃摇动30分钟,并在25,000×g下离心30分钟(使用Avanti J-25离心机)。此步骤后,收集上清液并于-80℃保存,或者为了立即制备突触囊泡,将上清液保存在冰上或4℃下。收集上清液,将蛋白质浓度标准化,作为后续步骤的样品。

  2. 神经递质的释放是依赖于钙的(Wu等,1973; Fon and Edwards,2001)
    响应于增加的神经活性而从突触前神经元释放的神经递质扩散穿过突触间隙并通过附着至突触后受体来转导生理信号。
    受体因此定义生理信号的性质。经典的神经递质如γ-氨基丁酸(GABA),谷氨酸盐和乙酰胆碱(ACh)激活离子通道,从而促进快速的突触传递。相比之下,神经调节剂(如单胺和多肽(以及GABA,谷氨酸和ACh))激活G蛋白偶联受体,刺激第二信使并在延长的时间范围内起作用。神经递质循环包括递质生物合成,储存,再摄取和降解。突触小泡循环包括将囊泡靶向到发生对接,融合,内吞和再循环的神经末梢。胞吐作用的高速率涉及将神经递质组装良好的SVs(囊泡神经递质转运蛋白)以及在神经末端SVs的快速再循环,无论是直接从质膜还是通过内体中间体。局部再循环包括在血浆膜上的SV对接之上从血浆膜蛋白,网格蛋白介导的内吞作用中分选SV蛋白质。通过在囊泡融合部位附近簇集的电压门控Ca 2 + +通道到达神经末端的局部升高的Ca 2+响应受调节的融合。 />
  3. 突触小泡的制备(图1)(Jin et al。,2003)
    1. 突触体(P2')在含有哺乳动物蛋白酶抑制剂(MPI)混合物和磷酸酶抑制剂(PhI)的1:100稀释度(使用圆底离心管)的10体积冰冷的纳米纯水中快速渗透。
    2. 如上所述匀浆稀释的突触体裂解物并在冰上孵育45分钟。
    3. 离心47000×g 15分钟(使用Optima L-90 K离心机和离心管(圆底))去除大的膜组分和线粒体。
    4. 收集上清液(S3)而不干扰沉淀,并在200,000×gg下离心2小时(使用Optima L-90K离心机和离心管(圆底))。
    5. 用这种方法获得的颗粒是粗糙的突触小泡(P4)。
    6. 加入500μl标准GAD缓冲液(参见配方4)至每个级分以除去任何污染的稀释胞质级分(S3),并且将紧密小球(P4)洗涤三次。该颗粒将被用于以下在GABA摄取测定的细节中列出的步骤。

  4. GABA吸收方法(Jin等,2003)
    1. 如上所述纯化粗SV,并与抗GAD65IgG偶联的环氧乙烷丙烯酸珠(Sigma O9754)混合以纯化GABA能特异性SV。用4mg / ml SV(终浓度:2mg / ml),120μg/ ml丙酮酸激酶(最终浓度:60μg/ ml)在100微升总体积中孵育抗GAD65抗体-SV珠(2mg蛋白/ ml) ml的GPBS缓冲液(见方法5)]。

    2. 在32°C孵育SV混合物2分钟。
    3. 加入等体积的ATP混合物(SV混合物与ATP混合物的比例为1:1的比例)(2 mg / ml;见6)和移液管混合。
    4. 将组合物在32℃下进一步温育,取出30μl反应混合物的等分试样,以1,5,10,15和20分钟的间隔通过硝酸纤维素膜进行真空过滤。

    5. 用5ml冰冷的GPBS缓冲液洗涤膜两次
    6. 膜空气干燥后,将膜置于闪烁小瓶中,加入闪烁鸡尾酒,涡旋小瓶并放置过夜。
    7. 基于闪烁计数器测量值(dpm)计算持续在膜上的放射性。使用设备Tri-Carb 2900TR液体闪烁分析仪(PerkinElmer,MA,USA)测量每分钟崩解次数(dpm)
    8. 对于涉及GAD抑制剂,肼或氨氧基乙酸盐的实验,首先将抑制剂与SV混合物预温育10分钟,然后加入ATP混合物。对于涉及[3 H] Glu和[14 C] GABA两者的摄取测定,条件与上述(步骤E1至E4)解释的相同,除了[在反应混合物中包括GABA和[14 H] Glu和[14 C] GABA。

  5. 与囊泡膜上的GAD复合(Jin等人,2003; Buddhala等人,2012; Modi等人,2015)
    通过与SVs上的蛋白质,例如CSP,VGAT和(抗钙调蛋白依赖性蛋白酶)结合而发现的蛋白质复合物(首先是热休克同源物:HSC70)依赖性激酶II)CaMKII。这种蛋白质复合体起到一个机器的作用,可以证实GABA的生物合成和包装到SV中是完全耦合的。这说明了GABA合成,调节和包装到SV中的功能和结构耦合。
    通过激活SV相关的GAD和随后将GABA包装到SV中的神经元刺激引发的生理事件可以解释如下。
    GABA在动作电位发生后通过胞吐作用分配。 SV通过涉及网格蛋白涂层的工艺回收。然后通过与HSC70的相互作用将网格蛋白外壳与囊泡分开。然后将囊泡返回到SV的静息状态,其中质子梯度由V-ATP酶返回。通过质子梯度依赖性蛋白激酶通过蛋白质磷酸化刺激GAD65。蛋白激酶的候选者之一是CaMKII。然后通过GAD65新合成的GABA被VGAT载入SV。这些重新填充的含GABA的SV准备在新的动作电位发生时释放。 SGAD被钙调磷酸酶介导的去磷酸化所激活,并被蛋白激酶A介导的蛋白质磷酸化所抑制。当GABA神经元兴奋时,Ca2 +进入末端导致SGAD / GAD67(分子量为67kDa的L-谷氨酸脱羧酶)的去磷酸化和活化。在细胞溶胶中由SGAD / GAD67合成的GABA也可以转移到SV中,尽管它代表了次要途径。胞质GABA也可以被代谢以通过GABA分路途径产生ATP,其可以用于保持GABA转运的电化学质子梯度。 GAD65主要负责合成用作神经递质的GABA,而GAD67用于GABA用于其他功能,例如用作生长中的信号分子,细胞活力增加的来源和GABA来源

  6. 通过控制GAD活性来调节GABA合成(Bao等人,1995; Sha等人,2005; Wei和Wu,2005; Sha等人, 2008; Buddha等人,2012; Modi等人,2014; Chou等人,2017)
    谷氨酸脱羧酶(GAD)酶GAD65和GAD67的刺激在突触前位点转换GABA神经传递。转录或mRNA剪接控制GAD65和GAD67的浓度。翻译后修饰包括蛋白水解切割,磷酸化和棕榈酰化,这些调节这些关键酶的活性(Wei等人,2004)。 GAD65(tGAD65)的截短形式比全长GAD65(fGAD65)更具动态性,但相比之下,截短的GAD67(tGAD67)比全长GAD67(fGAD67)活性低。蛋白质mu-calpain负责切割fGAD65和fGAD67。
    GABA神经传递依赖于GAD是否与突触囊泡(SV)相关联,并且钙蛋白酶通过产生高活性的tGAD65来实现关键功能,所述高活性的tGAD65引起增加的GABA合成和包装摄取到SV中。
    已知可逆蛋白磷酸化是负责调节蛋白功能的关键机制。神经递质系统受其合成酶的磷酸化调控也已被证实。蛋白质磷酸化和去磷酸化在调节大脑中的GAD活性中起关键作用。可溶性GAD(sGAD)在磷酸化后最有可能被蛋白激酶A(PKA)灭活,并被蛋白磷酸酶2B(PP2B,钙调磷酸酶)去磷酸化刺激。相比之下,膜结合GAD(mGAD)被磷酸化激活并被去磷酸化抑制。负责mGAD磷酸化的激酶还不知道。尽管通常认为GAD65在mGAD制剂中,并且GAD67主要存在于sGAD制剂中,但GAD65和GAD67已经显示存在于大脑制剂的可溶性和膜级分中。
    PKCε调节GAD65磷酸化,而GAD67通过PKA磷酸化被抑制。 GAD67和GAD65单独的半胱氨酸残基455和446对于全长GAD调节是至关重要的。
    显示T91是GAD67的关键磷酸化位点,并且hGAD65中的T95通过使用基质辅助激光解吸/电离飞行时间(MALDI-TOF)质谱的分析显示被激酶Cε(PKCε)磷酸化。在这些目标上与辅助因子PLP的合作控制了在PLP绑定的主动holoGAD和未绑定的主动apo GAD之间的切换。暂时切换到PLP绑定的主动holoGAD对GABA神经传递至关重要。特异于GAD65而不是GAD67的是棕榈酰化(Huntingtin相互作用蛋白)HIP14,其加速GAD65锚定到SV并增加囊泡GABA向神经传递的输入。

数据分析

用Prism或SPSS软件分析所有实验的数据。数据的统计学显着性由测试或单因素方差分析(ANOVA)确定。小于0.05的P 值被认为是显着的。数据似乎是正态分布的,在实验组之间和实验组之间观察到类似的标准偏差和标准误差。

食谱

注意:以下所有解决方案(配方1-6)都可以在4°C下保存7天。

  1. HEPES缓冲蔗糖溶液
    0.32 M蔗糖
    4mM HEPES-NaOH
    调节pH至7.4(10毫升/克的脑W / V)
    在4°C储存
  2. 克雷布的磷酸氢钠(KRP)缓冲液
    10mM Tris-HCl
    2.2 mM CaCl 2 2/2 0.5mM Na 2 HPO 4 4 0.4mM KH 2 PO 4 4 4mM NaHCO 3•/ 2 80 mM NaCl
    调整pH值到7.5
    在4°C储存
  3. 脑裂解缓冲液
    50mM Tris-HCl
    150 mM NaCl
    2 mM EDTA
    调整pH值到8
    1%Triton X-100
    1%MPI& PhI
    在4°C储存
  4. 标准的GAD缓冲区
    50 mM磷酸钾
    1 mM 2氨基乙基异硫脲溴化物(AET)
    0.2mM吡哆醛5'磷酸盐
    调整pH值到7.2
    在4°C储存
  5. GPBS缓冲区
    9.5 mM KH 2 PO 4 4/2 40.5mM K 2 HPO 4 4 8 mM KCl
    86.6毫米葡萄糖酸钾
    调整pH值到7.4
    在4°C储存
  6. ATP混合物
    2 mM ATP
    4.4mM MgSO 4
    12 mM磷酸(烯醇)丙酮酸
    50μMGABA
    2 mM Glu
    110.1μCi/μl[3 H] Glu
    在4°C储存

致谢

这项工作由佛罗里达州卫生部的Grant 6JK08和AEURA信托基金资助。
没有利益冲突或利益冲突。

参考

  1. Bao,J.,Cheung,Y.和Wu,J.Y。(1995)。 脑L-谷氨酸脱羧酶。通过去磷酸化作用抑制磷酸化和激活。“生物化学杂志”270(12):6464-6467。
  2. Buddhala,C.,Suarez,M.,Modi,J.,Prentice,H.,Ma,Z.,Tao,R.和Wu,J.Y。(2012)。 钙蛋白酶切割脑谷氨酸脱羧酶65是病理性的并且损害GABA的神经传递。 > PLoS One 7(3):e33002。
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引用:Modi, J., Prentice, H. and Wu, J. (2017). Preparation, Stimulation and Other Uses of Adult Rat Brain Synaptosomes. Bio-protocol 7(24): e2664. DOI: 10.21769/BioProtoc.2664.
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