The authors appreciate efforts by prior laboratory members Kendra Mackett, Sandra Slusher, and Jie Chen who worked to optimize the method. The authors also thank the National Institutes of Health, Michael J. Fox Foundation, Texas Tech University Health Sciences Center El Paso Scholarly Activity Research Project (SARP), Lizanell and Colbert Coldwell Foundation, Multiple System Atrophy Coalition, Hoy Family Research, and Anna Mae Doyle Gift Funds for financial support of this research.

1. Preparation of Buffers and Reagents
<ol>
	<li>Preparation of p-nitrophenyl phosphate (pNPP) buffer
	<ol>
		<li>Prepare 50 mL of pNPP buffer as follows.</li>
		<li>Weigh out 0.30285 g Tris base (see the Table of Materials) and dissolve it in 25 mL of ultrapure deionized water.
		<ol>
			<li>Add 41.6 &#181;L of 120 mM CaCl2. Adjust to pH 7.0 with 1 M HCl and bring the final solution volume to 50 mL with ultrapure deionized water.</li>
			<li>Ensure that the final concentration is 50 mM Tris-HCl, 100 mM CaCl2, pH 7.0.</li>
			<li>Store pNPP buffer at 4 oC.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>For malachite green solutions, prepare the following.
	<ol>
		<li>Prepare solution A by adding 32.8 mL of concentrated HCl to 67.2 mL of ultrapure deionized water to create a 4 M HCl solution. 
		NOTE: Malachite solution is prepared in a fume hood using disposable gloves, face-mask, and safety goggles.</li>
		<li>Prepare solution B by weighing out 4.2 g of ammonium molybdate and adding it to 95.8 mL of solution A.</li>
		<li>Prepare solution C by weighing out 0.045 g malachite green oxalate salt and adding ultrapure deionized water to bring the final total volume to 100 mL.</li>
		<li>Prepare solution D by mixing solutions B and C at 1:3 (v/v) ratio, stir for 1 h at room temperature .</li>
		<li>Filter Solution D through a 0.22 &#181;m pore size nitrocellulose membrane filter unit using a 50 mL syringe.</li>
		<li>Store prepared malachite solution at 4 oC protected from light.</li>
	</ol>
	</li>
	<li>Preparation of activator
	<ol>
		<li>To prepare a 1% tween 20 solution, pipette 10 &#181;L of tween 20 into 990 &#181;L of ultrapure deionized water (v/v) then vortex until the tween 20 dissolves completely.</li>
	</ol>
	</li>
	<li>Preparation of malachite green Tween 20 (MGT) working solution.
	<ol>
		<li>Mix the activator (step 1.3) with the malachite green solution (solution D, step 1.2.5) in a ratio of 1:100 (e.g. 1 &#181;L activator to 99 &#181;L malachite green solution).</li>
	</ol>
	</li>
	<li>Preparation of threonine phosphopeptide (KRpTIRR) (pT) substrate.
	<ol>
		<li>To prepare a 2 mM stock, place a 1 mg phosphopeptide vial on ice, add 550 &#181;L of ultrapure deionized water, and vortex gently to dissolve.
		<ol>
			<li>Make ~10 aliquots and store pT at -20oC.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Synuclein Preparation
	<ol>
		<li>Prepare synucleins in a fume hood using disposable gloves, face-mask, and safety goggles.</li>
		<li>Resuspend 1 mg of recombinant synuclein in 1 mL ultrapure deionized water for a final concentration of 1 mg/mL.</li>
		<li>Vortex gently to dissolve and place on ice.</li>
		<li>Prepare 50 &#181;L aliquots and store at -80 oC.</li>
	</ol>
	</li>
	<li>Preparation of phosphate standards
	<ol>
		<li>Prepare the 0.1 mM phosphate stock solution as follows.
		<ol>
			<li>Weigh out 0.1361 g KH2PO4 and place in a beaker with 80 mL of ultrapure deionized water. Add a stir bar and mix to dissolve. Transfer all of the solution to a graduated cylinder and bring the final total volume to 100 mL with ultrapure deionized water.</li>
			<li>Filter the entire solution through a 0.22 &#181;m pore size nitrocellulose membrane filter unit using a 50 mL syringe; the final concentration is 10 mM.</li>
			<li>Dilute the solution (10 mM) 1:100; the final concentration will be 0.1 mM phosphate (PO4) to be used for assay standards.</li>
			<li>Store the phosphate stock solution at 4 oC.</li>
		</ol>
		</li>
		<li>See Table 1 for limiting dilutions used to make PO4 standards with a final total volume of 1250 &#181;L each. Add the appropriate amount of phosphate and ultrapure deionized water to 1.5 mL tubes as per Table 1.
		<ol>
			<li>Store prepared PO4 standards at -20 oC.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="7">Phosphate Standards&#160;</td>
		</tr>
		<tr>
			<td>0.1 mM PO4 solution (mL)</td>
			<td>0</td>
			<td>75</td>
			<td>150</td>
			<td>300</td>
			<td>600</td>
			<td>1,200</td>
		</tr>
		<tr>
			<td>Ultrapure deionized water (mL)</td>
			<td>1,250</td>
			<td>1,175</td>
			<td>1,100</td>
			<td>950</td>
			<td>650</td>
			<td>50</td>
		</tr>
		<tr>
			<td>Final concentration of PO4 [pmol]</td>
			<td>0</td>
			<td>150</td>
			<td>300</td>
			<td>600</td>
			<td>1,200</td>
			<td>2,400</td>
		</tr>
	</tbody>
</table>
Table 1: Phosphate Standards.
2. PP2A Assay in Response to Recombinant Synucleins
NOTE: The total final volume for each reaction will be 60 &#181;L, so the experimenter must adjust the initial volume of pNPP buffer appropriately to accommodate the volume of synucleins used for each sample. In most cases, the volume of pNPP buffer will be between 40 and 44 &#181;L.
<ol>
	<li>Prepare each PP2Ac reaction as follows.
	<ol>
		<li>In a 1.5 mL tube, add 39 &#181;L of pNPP buffer and place on ice.</li>
		<li>Add 1 &#181;L (105 ng/&#181;L or 0.0007 U/&#181;L) of human recombinant PP2Ac (rPP2Ac).</li>
		<li>Add 4 &#181;L of 0, 1.5, 7.5, 15 or 30 &#181;M synucleins to rPP2Ac in pNPP buffer for final concentrations of 0, 0.1, 0.5, 1.0 and 2.0 &#181;M and incubate at 4 &#176;C for 30 min on a rotating unit; the final volume will be 60 &#181;L.</li>
		<li>After 30 min, move samples to ice and add 16 &#181;L of 2 mM pT substrate.</li>
		<li>Incubate each PP2A assay mixture for 10 min at 30 &#176;C in a water bath with intermittent shaking.</li>
		<li>To a flat bottom 96-well plate, add 25 &#181;L of each PO4 standard (0, 150, 300, 600, 1,200, and 2,400 pmol) in duplicate wells from low to high.</li>
		<li>Next add 25 &#181;L of each experimental sample (PP2A assay &#177; synuclein), prepared in steps 2.1.1 to 2.1.6, to duplicate wells on the same 96-well plate.</li>
		<li>Next, add 75 &#181;L of MGT working solution to each well containing standards and samples.</li>
		<li>Leave plate at room temperature for 10 min.</li>
		<li>Observe a color change in samples having measurable differences in phosphate levels.</li>
	</ol>
	</li>
</ol>
3. Photometric Analysis
<ol>
	<li>Analyze the 96-well plate containing standards and samples using a spectrophotometric plate reader with the wavelength set to 630 nm29.</li>
	<li>Plot the absorbance curve produced (samples read at 630 nm) and note that it remains linear up to 2,400 pmol concentration of PO4.</li>
</ol>

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Dr. Perez was a member of the faculty in the Department of Neurology at the University of Pittsburgh School of Medicine from 1999 &#8211; 2011.&#160;

This malachite green assay with pT was used to measure PP2Ac activity because it is easier to perform than the classical method that uses 32P-labelled substrates. Another advantage of this assay over radioactive assays is that radioisotopes pose some risk and can be costly, especially 32P-labelled molecules that have short half-lives, which limits the number of assays one can perform before reagents expire. Using malachite green rather than radioactivity also eliminates the need to dispose of 32P waste. Malachite green assays are also quite sensitive when measuring the activity of serine/threonine phosphatases when compared to another colorimetric assay that uses the substrate p-nitrophenylphosphate (pNPP), which is non-selective as it can be dephosphorylated by a broad number of enzymes32.
Commercial malachite green kits to measure PP2A activity have been available for some time, and were previously used in the laboratory. However, when doing many malachite green assays, such kits became prohibitively expensive. In addition, commercial kits for PP2A activity are stable only for one year, while having reagents available in powdered form and with proper storage, provide readily available assay components that are structurally stable for long periods of time.
Before testing bSyn and gSyn, aSyn was used as a positive control to stimulate PP2Ac activity and to determine the amount of PP2Ac required for the assays, and also to confirm that resulting data remain on the scale with the standard curve (150 - 2400 pmol of PO4). It is noteworthy that if aSyn stimulates PP2Ac too strongly, though the reaction is typically linear, data will go off scale and chemicals in the MGT solution precipitate, making it impossible to measure accurate amounts of released free-PO4 with a plate reader.
Given that aSyn contributes to PP2Ac activity in vivo and in vitro12,19,21,23,25, and that all synucleins have considerable homology, it had been hypothesized that all synuclein family members may be able to stimulate PP2Ac in cell free assays. Using the colorimetric assay described here, it was found that indeed, all three recombinant synucleins (aSyn, bSyn, gSyn) stimulated PP2Ac activity, raising the possibility that all three synucleins may perform similar functions in the nervous system. However, brain from Parkinson&#39;s or Dementia with Lewy Body cases have less PP2Ac activity in tissues containing highly aggregated aSyn23. Mouse tissues harboring Lewy body-like pathology also exhibit reduced PP2Ac activity25. This, and new data in Figure 3 as well as prior data from aSyn knockout mice12, strongly suggest that neither bSyn nor gSyn can typically compensate for the loss of soluble aSyn in brain, or alternatively that those synuclein homologs may not associate with PP2Ac as strongly as aSyn as shown (Figure 3). The Allen Brain Atlas shows colocalization of all three synuclein mRNAs in many brain areas, and though triple synuclein knockout mice have been generated9,33, PP2Ac activity has not been assessed in that model. Interestingly, aSyn that is phosphorylated on Ser129 by Polo-like kinase 2, is less able to activate PP2Ac12, but evidence shown here for bSyn suggest that bSyn phosphorylation is unlikely to impact PP2Ac activity, as very little bSyn came down with PP2Ac in mouse brain (Figure 3). Taken together, the data suggest that endogenous PP2Ac activity modulators, like aSyn, likely contribute to wellness and disease.
The protocol outlined here is a simple yet powerful technique to determine the capacity of PP2Ac to dephosphorylate a phosphopeptide substrate in response to various treatments. For instance, this same methodology can be used to test other PP2Ac activators, such as ceramides as well as potential novel therapeutics34, or to assess the impact of PP2Ac inhibitors in vitro. It is also important to point out that similar assays can measure the activity of PP2Ac that has been isolated by 1D6 immunoprecipitation from cell extracts or body tissues, as previously described by the authors12,19,25. Future applications for the malachite green assay exist beyond measuring PP2Ac activity, as ATP activity can also be determined using such an assay.
Note, a standard curve must be generated each time for every independent PP2Ac assay. It is mandatory to ensure that all reagents used are phosphate free, as free phosphates artificially increase background signal and produce erroneous results. On the day of the assay it is essential to prepare enough fresh MGT solution for all samples to be assayed. Also, MGT is only good the day that it is made and tends to precipitate after a few hours. PP2Ac purchased from a vendor must be aliquotted and stored frozen soon after it is received to prevent freeze thaw cycles that reduce its activity. As other phosphatases can dephosphorylate the pT substrate (e.g. PP1 and PP535), when analyzing cell or tissue extracts in which PP2Ac has not been isolated by immunoprecipitation, samples must be passed over columns to remove free phosphates and specific inhibitors of phosphatases must be used to confirm phosphatase specificity, as previously described18.

An erratum was issued for: <a href="https://www.jove.com/video/55361/recombinant-synucleins-stimulate-protein-phosphatase-2a-catalytic">Recombinant &#945;- &#946;- and &#947;-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays</a>
One of the authors&#39; names was corrected from:
Brian Marcus
to:
Brian S. Marcus

An erratum was issued for: <a href="https://www.jove.com/video/55361/recombinant-synucleins-stimulate-protein-phosphatase-2a-catalytic">Recombinant &#945;- &#946;- and &#947;-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays</a>

Erratum: Recombinant &#945;- &#946;- and &#947;-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays

Studies defining the distribution of synucleins in brain and spinal cord show that all three synucleins are enriched in neural tissues, although varying patterns of localization have been reported1. For instance, aSyn is most abundant at presynaptic sites in cerebral cortex and basal ganglia2,3, bSyn has a more uniform distribution in brain and spinal cord4,5, and gSyn is more abundant in spinal cord and peripheral ganglia6. Although some embryonic expression of all synucleins may occur, the three homologs become more abundant during the neonatal period4,5,6,7,8. Remarkably, data from synuclein triple knockout mice, indicate that loss of all three synucleins causes age onset neuronal dysfunction9, supporting important synuclein functions in the central nervous system (CNS)10,11. It is not yet known if synuclein triple knockout mice show changes in PP2Ac distribution or activity. Data from single synuclein knockout mice reveal that loss of aSyn alone is able to significantly reduce PP2Ac activity in brain12. In addition to the CNS, all synucleins localize to other tissues throughout the body13,14,15.
Because of its well-established genetic links to neurodegeneration16,17, aSyn is among the best studied of the three synucleins. The Perez laboratory has long worked to define normal functional activities of aSyn, especially in the CNS11,12,18,19,20,21,22,23 and more recently in peripheral tissues24,25. Among these, was the discovery that aSyn plays a key regulatory role in stimulating PP2Ac activity19. PP2A activity contributes widely to normal brain function, including for the regulation of dopamine production26. The PP2A holoenzyme consists of three independent subunits, the catalytic C subunit, PP2Ac, a scaffolding A subunit, and one of several different regulatory/targeting B subunits that help localize PP2A to specific subcellular microdomains, thus providing PP2A functional diversity27. Evidence shows that aSyn interactions with PP2Ac contribute significantly to its phosphatase activity in vitro and in vivo12,19,21,23,25. When aSyn accumulates in protein aggregates, as it does when Lewy bodies form inside neurons of Parkinson&#39;s disease and Dementia with Lewy bodies (DLB), tissues with aggregated aSyn have diminished PP2Ac activity23,25, when levels of soluble aSyn diminish. Given that all three synucleins have significant homology28 (aSyn shares 66% identity with bSyn and 56% with gSyn) and that aSyn contributes to the activation of PP2Ac, it was hypothesized that all members of the synuclein protein family may be able to increase PP2Ac activity, at least in vitro. To assess this, PP2Ac activity was measured in response to recombinant aSyn, bSyn, and gSyn using a simple colorimetric assay, modeled after the method of Fathi et al.29. This method and the novel findings are detailed herein.

<table><tbody><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1 M Trizma Base</td><td>Sigma-Aldrich</td><td>T15003</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2 &lt;/sub&gt;(calcium chloride)</td><td>Fisher Scientific</td><td>C70-500</td><td></td></tr><tr><td>Concentrated HCl&amp;nbsp;&amp;nbsp;</td><td>JT Baker</td><td>9535-33</td><td></td></tr><tr><td>Ammonium molybdate</td><td>Sigma-Aldrich</td><td>A1343</td><td></td></tr><tr><td>Malachite green oxalate salt</td><td>Sigma-Aldrich</td><td>M6880</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P9416</td><td></td></tr><tr><td>Threonine phosphopeptide (KRpTIRR)</td><td>New England peptide LLC</td><td>BP12-011</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (potassium phosphate)</td><td>Sigma-Aldrich</td><td>795488</td><td></td></tr><tr><td>Recombinant human PP2A C subunit</td><td>Cayman Chemical&amp;nbsp;</td><td>10011237</td><td></td></tr><tr><td>Recombinant human &amp;alpha;-synuclein</td><td>rPeptide</td><td>S-1001-2</td><td></td></tr><tr><td>Recombinant human &amp;beta;-synuclein</td><td>rPeptide</td><td>S-1003-2</td><td></td></tr><tr><td>Recombinant human &amp;gamma;-synuclein</td><td>rPeptide</td><td>S-1007-2</td><td></td></tr><tr><td>PP2Ac 1D6 antibody</td><td>Millipore</td><td>05-421</td><td></td></tr><tr><td>PP2Ac antibody</td><td>Novus Biologicals</td><td>NB100-92217</td><td></td></tr><tr><td>aSyn antibody C-20</td><td>Santa Cruz Biotechnology</td><td>sc-7011-R</td><td></td></tr><tr><td>bSyn antibody EP1537Y</td><td>Novus Biologicals</td><td>NB100-79903</td><td></td></tr><tr><td>gSyn antibody E-20</td><td>Santa Cruz Biotechnology</td><td>sc-10698</td><td></td></tr><tr><td>Zeba Spin Desalting Columns, 7K MWCO, 0.5 mL</td><td>Thermo-Fisher Scientific</td><td>89882</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment/Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Multiskan Spectrum Plate Reader</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>51118650</td><td></td></tr><tr><td>pH Meter&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>13-636-AB15P</td><td></td></tr></tbody></table>

recombinant &#945;- &#946;- and &#947;-synucleins stimulate protein phosphatase 2a catalytic subunit activity in cell free assays

&#945;-Synuclein (aSyn), &#946;-Synuclein (bSyn), and &#947;-Synuclein (gSyn) are members of a conserved family of chaperone-like proteins that are highly expressed in vertebrate neuronal tissues. Of the three synucleins, only aSyn has been strongly implicated in neurodegenerative disorders such as Parkinson&#39;s disease, Dementia with Lewy Bodies, and Multiple System Atrophy. In studying normal aSyn function, data indicate that aSyn stimulates the activity of the catalytic subunit of an abundantly expressed dephosphorylating enzyme, PP2Ac in vitro and in vivo. Prior data show that aSyn aggregation in human brain reduces PP2Ac activity in regions with Lewy body pathology, where soluble aSyn has become insoluble. However, because all three synucleins have considerable homology in the amino acid sequences, experiments were designed to test if all can modulate PP2Ac activity. Using recombinant synucleins and recombinant PP2Ac protein, activity was assessed by malachite green colorimetric assay. Data revealed that all three recombinant synucleins stimulated PP2Ac activity in cell-free assays, raising the possibility that the conserved homology between synucleins may endow all three homologs with the ability to bind to and activate the PP2Ac. Co-immunoprecipitation data, however, suggest that PP2Ac modulation likely occurs through endogenous interactions between aSyn and PP2Ac in vivo.

Protein function and activity can be altered by post translational modifications, such as phosphorylation. Many proteins can be phosphorylated by a kinase, or dephosphorylated by a phosphatase. In the case of protein phosphatase 2A (PP2A), this multisubunit phosphatase facilitates the dephosphorylation of serine and threonine residues on a broad number of phosphoproteins. Abnormal PP2A activity can lead to dysregulation of protein function which occurs in many diseases, including Parkinson&#39;s disease where aSyn can become hyperphosphorylated, and in Alzheimer&#39;s disease where tau protein can become hyperphosphorylated. This provided justification for optimizing an in-house procedure to rapidly evaluate PP2Ac activity and determine if abnormal PP2Ac activity may lead to cellular dysfunction.
Using this cell-free colorimetric assay, PP2Ac activity was measured by quantifying the amount of free PO4 cleaved from the pT substrate (Figure 1A) and compared to known picomolar amounts of PO4 in the standard curve (Figure 1B). PP2Ac activity was also measured in response to 0.0, 0.1, 0.5, 1.0, and 2.0 &#181;M synucleins (Figure 1A), as compared to baseline PP2Ac activity in the absence of added synuclein (at the large arrow in Figure 1A).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55361/55361fig1.jpg" /> 
Figure 1: PP2A activation by recombinant synucleins, is measured relative to known levels of phosphate in the standards. In (A) aSyn, bSyn, and gSyn were evaluated in a range from 0 - 2 &#181;M protein concentrations, to compare to 0 - 2400 pmol PO4 standards. (B) A standard curve for the malachite green PP2Ac assay was generated to provide known amounts of phosphate against which values obtained in experimental conditions can be calculated. In all cases, when PP2Ac activity increases, the amount of free PO4 cleaved from the pT substrate increases and produces a darker green color. <a href="https://cloudflare.jove.com/files/ftp_upload/55361/55361fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The results show that addition of synucleins to PP2Ac in the initial incubation step could increase the amount of free PO4 measured, thereby demonstrating increased PP2Ac activity. The statistical analysis after many repetitions show that all three recombinant synucleins (aSyn, bSyn, gSyn) significantly increase PP2Ac activity (Figure 2). These results suggest that changes in the amount of soluble cellular synucleins can alter PP2Ac activity; and thus may impact cellular homeostasis. It is not known, however, if all three synucleins contribute to the activation of PP2Ac in vivo, though data in Figure 3 suggest that aSyn likely contributes to PP2Ac activity in vivo.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55361/55361fig2.jpg" /> 
Figure 2: PP2Ac activity is stimulated by all three synucleins. Graphical representation of PP2Ac activation in response to varying concentrations of recombinant aSyn, bSyn or gSyn is shown. All three recombinant synucleins stimulated PP2Ac activity. aSyn in most experiments appeared to be slightly more potent, though ANOVAs performed using statistical software were remarkably similar overall. Data represent the mean &#177; SEM of 4-9 independent experiments. ns, not significant; * p &#60;0.05; ** p &#60;0.01; **** p &#60;0.0001. <a href="https://cloudflare.jove.com/files/ftp_upload/55361/55361fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To assess which synuclein(s) can interact with PP2Ac in brain, co-immunoprecipitation (Co-IP) assays were performed using wild type mouse brain and the PP2Ac-specific-antibody, 1D6. Proteins were then separated by SDS-PAGE and blots were probed for PP2Ac, aSyn, bSyn, and gSyn using established methods19. Recombinant synuclein controls demonstrate aSyn, bSyn and gSyn antibody specificity (Figure 3A). Co-IP data reveal that the major synuclein associated with PP2Ac in brain was aSyn, as much less bSyn came down in the co-IP (Figure 3B), and no gSyn came down in the Co-IP (data not shown). This suggests a distinctive role for endogenous aSyn in the regulation of PP2Ac in brain. It also raises the possibility that PP2Ac modulation with bSyn-based-therapies could have potential to restore PP2A activity in those with synucleinopathy, as bSyn is known to be nonamyloidogenic yet as shown here can stimulate PP2Ac30,31.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55361/55361fig3v2.jpg" /> 
Figure 3: Interaction of PP2Ac with endogenous synucleins in mouse brain. (A) Recombinant aSyn, bSyn and gSyn were probed with synuclein specific antibodies as described below. (B) Using mouse brain homogenate Co-IP was performed with antibody 1D6, then samples were analyzed on immunoblots. Data are shown for PP2Ac, aSyn, and bSyn, but not for gSyn which did not Co-IP with PP2Ac. Start samples show relative levels of PP2Ac (lane 1), aSyn (lane 4), and bSyn (lane 7) in the initial homogenates. End samples show the remaining levels of PP2Ac (lane 2), aSyn (lane 5), and bSyn (lane 8) in homogenates after the 1D6 PP2Ac Co-IP. Significant levels of PP2Ac were immunoprecipitated using 1D6 antibody (lane 3), which also brought down ample amounts of aSyn (lane 6, at arrow) but very little bSyn (lane 9, strongly overexposed to show faint bSyn signal, at arrow). *Asterisks and brackets mark the&#160;non-specific signals on blots. See the Table of Materials for details on antibodies used. <a href="https://cloudflare.jove.com/files/ftp_upload/55361/55361fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Recombinant Î±- Î²- and Î³-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays at JoVE.com

Recombinant &#945;- &#946;- and &#947;-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays

This publication presents a protocol showing how to measure the activity of recombinant protein phosphatase 2A catalytic subunit (PP2Ac) in response to recombinant &#945;-synuclein, &#946;-synuclein, or &#947;-synuclein proteins using a simple colorimetric assay. We show findings regarding &#945;-synuclein specificity toward PP2Ac in mouse brain.

&#945;-Synuclein (aSyn), &#946;-Synuclein (bSyn), and &#947;-Synuclein (gSyn) are members of a conserved family of chaperone-like proteins that are highly ...

recombinant-synucleins-stimulate-protein-phosphatase-2a-catalytic

Research

JoVE Journal

Neuroscience

6.7K Views.  Texas Tech University Health Sciences Center El Paso. Deze publicatie presenteert een protocol waarin wordt beschreven hoe voor het meten van de activiteit van recombinante eiwitten fosfatase 2A katalytische subeenheid (PP2Ac) in reactie op recombinante α-synuclein, β-synuclein, of γ-synuclein eiwitten met behulp van een eenvoudige colorimetrische bepaling. Bevindingen met betrekking tot de specificiteit van de α-synuclein naar PP2Ac laten we zien in de hersenen van de muis.

Video: Recombinant α- β- and γ-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Biochemistry

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T &#945;S and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of &#945;SD (disease-associated &#945;S) in these mice, an ELISA was designed to specifically quantify &#945;SD in sick mice. Analysis of the central nervous system in this mouse model showed the presence of &#945;SD mainly in the caudal brain regions and the spinal cord. There were no differences in &#945;SD distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of &#945;SD immunoreactivity using an antibody against Ser129 phosphorylated &#945;S by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of &#945;S. The propagation of &#945;SD, suggesting the involvement of a &#8220;prion-like&#8221; mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

Medicine

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

Quantitative Measurement of &#947;-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms

We have developed a pair of cell-based reporter gene assays to quantitatively measure &#947;-secretase cleavage of distinct substrates. This manuscript describes procedures that may be used to monitor &#947;-secretase-mediated cleavage of either APP-C99 or Notch, using a Gal4 promoter-driven firefly luciferase reporter system. These assays were established by stably co-transfecting HEK293 cells with the Gal4-driven luciferase reporter gene and either the Gal4/VP16-tagged C-terminal fragment of APP (APP-C99; CG cells), or the Gal4/VP16-tagged Notch-&#916;E (N&#916;E; NG cells). Using these reporter assays in parallel, we have demonstrated that an ErbB2 inhibitor, CL-387,785, can preferentially suppress &#947;-secretase cleavage of APP-C99 in CG cells, but not N&#916;E in NG cells. The differential responses exhibited by the CG and NG cells, when treated with CL-387,785, represent a preferred characteristic for &#947;-secretase modulators, and these responses are in stark contrast to the pan-inhibition of &#947;-secretase induced by DAPT. Our studies provide direct evidence that &#947;-secretase activities toward different substrates can be differentiated in a cellular context. These new assays may therefore be useful tools in drug discovery for improved AD therapies.

We have successfully generated two substrate-specific &#947;-secretase assays. Both cell-based assays presented here are designed to quantify &#947;-secretase enzymatic activities via the output of firefly luciferase reporters.

We have developed a pair of cell-based reporter gene assays to quantitatively measure &#947;-secretase cleavage of distinct substrates. This manuscript ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Biology

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson&#39;s disease synucleinopathy and nigrostriatal degeneration. The standardization of &#945;-syn PFF generation and in vivo application is critical in order to ensure consistent, robust &#945;-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric &#945;-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of &#945;-syn PFFs into rats or mice. Starting with monomeric &#945;-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed &#945;-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated &#945;-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.&#160;

The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

Stereotaxic Intracranial Delivery of Chemicals, Proteins or Viral Vectors to Study Parkinson's Disease

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in the substantia nigra. Affected brain areas display intraneuronal fibrillar inclusions consisting mainly of alpha-synuclein (asyn) proteins. No animal model thus far has recapitulated all characteristics of this disease. Here, we describe the use of stereotaxic injection to deliver chemicals, proteins, or viral vectors intracranially in order to mimic various aspects of PD. These methods are well-established and widely used throughout the PD field. Stereotaxic injections are incredibly flexible; they can be adjusted in concentration, age of animal used for injection, brain area targeted and in animal species used. Combinations of substances allow for rapid variations to assess treatments or alter severity of the pathology or behavioral deficits. By injecting toxins into the brain, we can mimic inflammation and/or a severe loss of dopaminergic neurons resulting in substantial motor phenotypes. Viral vectors can be used to transduce cells to mimic genetic or mechanistic aspects. Preformed fibrillar asyn injections best recapitulate the progressive phenotype over an extended period of time. Once these methods are established, it can be economical to generate a new model compared to creating a new transgenic line. However, this method is labor intensive as it requires 30 minutes to four hours per animal depending on the model used. Each animal will have a slightly different targeting and therefore create a diverse cohort which on one hand can be challenging to interpret results from; on the other hand, help mimic a more realistic diversity found in patients. Mistargeted animals can be identified using behavioral or imaging readouts, or only after being sacrificed leading to smallercohort size after the study has already been concluded. Overall, this method is a rudimentary but effective way to assess a diverse set of PD aspects.

We describe how to successfully inject solutions into specific brain areas of rodents using a stereotaxic frame. This survival surgery is a well-established method used to mimic various aspects of Parkinson's disease.

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in ...

Streamlined Protocol for Leishmania Translational Extract (LTE) Production

Production and Optimization of LTE, a Leishmania tarentolae Derived Cell-Free Protein Expression System for Recombinant Protein Production

This protocol outlines the production and optimization of a eukaryotic Cell-Free Protein Expression System (CFPS) derived from the unicellular flagellate Leishmania tarentolae, referred to as Leishmania Translational Extract or LTE. Although this organism originally evolved as a parasite of geckos, it can be cultivated easily and inexpensively in flasks or bioreactors. Unlike Leishmania major, it is non-pathogenic to humans and does not require special laboratory precautions. Another advantage of using Leishmania for CFPS is that the addition of a single antisense oligonucleotide to the CFPS, targeting a conserved splice leader sequence on the 5'-end of all protein-coding RNAs, can suppress endogenous protein expression. We provide procedures for cell disruption and lysate processing, which have been simplified and improved compared to previous versions. These procedures start with simple flask cultures. Additionally, we explain how to introduce genetic information using vectors containing species-independent translation initiation sites (SITS) and how to perform straightforward batch optimization and quality control to ensure consistent protein expression quality.

Leishmania Translational Extract (LTE) is a eukaryotic cell-free protein expression system derived from the single-celled parasite, Leishmania tarentolae. This optimized protocol makes LTE simple and cost-effective to manufacture. It is suitable for various applications focused on the multiparallel expression and study of complex eukaryotic proteins and their interactions.

This protocol outlines the production and optimization of a eukaryotic Cell-Free Protein Expression System (CFPS) derived from the unicellular flagellate ...

Recombinant α- β- and γ-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays

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