I thank Dr Adamantios Mamais for extracting &#945;-synuclein from human tissue and running of blots. This work was supported in part by a MRC Grant (MR/L010933/1) and in part by the Wellcome Trust/MRC Joint Call in Neurodegeneration award (WT089698) to the UK Parkinson's Disease Consortium (UKPDC) whose members are from the UCL Institute of Neurology, the University of Sheffield and the MRC Protein Phosphorylation Unit at the University of Dundee. RB received funding from MJFox foundation for this work and is also funded by the Reta Lila Weston Trust.

Post-mortem brain tissue was donated to Queen Square Brain Bank for neurological disorders, University College London, Institute of Neurology using ethically approved protocols and stored for research under a license issued by the human tissue authority (HTA) UK (no. 12198). See list of cases used for this protocol (Table 1).
1. Preparation of Buffers
<ol>
	<li>Prepare 1X TBS (Tris-buffered saline) buffer:
	<ol>
		<li>Prepare 10X TBS as stock solution with 500 mM Tris-HCl pH 7.4 and 1,500 mM NaCl.
		<ol>
			<li>Weigh out 60.5 g Tris-Cl pH 7.6 and 87.6 g of NaCl in 800 ml of high purity water. Adjust pH with 1 M HCl and make the final stock volume to 1 L.</li>
		</ol>
		</li>
		<li>Prepare the final concentration of 1X TBS by diluting the stock 10 times in distilled water.</li>
		<li>Add protease inhibitors (PI), (1 tablet per 50 ml buffer) and Phos-stop phosphatase inhibitor tablets (1 tablet per 10 ml of buffer) to abrogate the effects of non-specific enzymatic actions of proteases and phosphatases. 
		Note: Here we have used the enzyme inhibitor tablets from Roche but equally other commercially available inhibitors can be used according to the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>Prepare TBS-SDS buffer by adding 5% w/v Sodium dodecyl sulphate (SDS) to 1X TBS (pH 7.4) buffer. Heat the solution to 60 &#176;C with constant stirring using a magnetic stirrer to solubilize SDS.</li>
	<li>Prepare TBS-SDS-Urea buffer by adding urea to a final concentration of 8 M w/v to 1X TBS-SDS buffer (pH 7.4). Heat the solution to 60 &#176;C with constant stirring on a magnetic stirrer to dissolve urea.</li>
</ol>
2. Sample Homogenization and Differential Ultracentrifugation
Note: The following part of the work involves handling of human brain tissue according to the rules of HTA UK. Local standard operating procedures are followed at all times. Brain samples are dissected from frozen brain blocks and tissue material homogenized in a microcabinet maintained at negative pressure. Disposable gowns gloves, face-masks, over-shoe protectors and safety goggles are worn throughout the procedure. Human tissue waste is discarded after autoclaving at 121 &#176;C for 20 min for safe disposal. Sharps (scalpel blades) are disposed in an appropriate sharps container for containment and safe disposal.
<ol>
	<li>Take a chunk of frozen human tissue (approximately 0.5 g in weight from the basal ganglia region) and rapidly mince into small fragments (approximately 1 mm2) on ice using a small petri-dish and a sterile scalpel blade. Use a separate petri-dish and scalpel blade for each sample. Collect the minced tissue in a 15 ml tube and add 10 volumes of ice-cold 1XTBS buffer to the tube.</li>
	<li>Homogenize the samples using a mechanical homogenizer at 20,000 rpm for 10 sec. Cool on ice for 2 min. Repeat this step three times. 
	Note: This is done so that the samples are not heated up while homogenizing.</li>
	<li>Clarify by centrifugation at 1,000 x g for 5 min at 4 &#176;C. Discard pellet of non-homogenized material.</li>
	<li>Take supernatant (crude homogenate), add to polycarbonate centrifuge tubes (size of tube depends on rotor size) and balance carefully. Centrifuge at 100,000 x g for 1 hr at 4 &#176;C.</li>
	<li>Retain supernatant as this is the TBS-soluble fraction.</li>
	<li>Wash pellet twice in five volumes of 1X TBS buffer and centrifuge each time at 100,000 x g for 15 min at 4 &#176;C. Discard the supernatants.
	<ol>
		<li>Resuspend the final pellet by sonicating the pellet for 10 sec at 20 KHz in approximately 5 volumes of 1X TBS-SDS at RT. 
		Note: Before addition of the SDS containing buffer samples should be brought at 10 &#176;C to avoid SDS precipitation.</li>
		<li>Ultracentrifuge at 100,000 x g for 30 min at 25 &#176;C.</li>
	</ol>
	</li>
	<li>Retain supernatant as this is the SDS soluble fraction.
	<ol>
		<li>Wash pellet twice in 5 volumes of 1X TBS-SDS buffer at RT. Centrifuge each time at 100,000 x g for 15 min at 25 &#176;C.</li>
		<li>Solubilize the final pellet in 50 &#956;l of 1X TBS-SDS-UREA buffer using a sonicator set at 20 KHz for 10 sec&#160;to attain complete solubilization; this is termed the urea soluble fraction.</li>
	</ol>
	</li>
	<li>Assay each fraction for total protein content using a commercial protein assay kit according to manufacturer&#39;s protocol. Dilute TBS-SDS-Urea samples 1:1 with 1X TBS buffer to allow compatibility with protein assay reagents. Freeze samples in small aliquots (20 &#956;l) at -80 &#176;C to reduce freeze-thaw cycles. 
	Note: Adding 10% glycerol to samples is recommended for long-term storage at -80 &#176;C.</li>
</ol>
3. Immunoblotting of Samples and Analyzing Results
<ol>
	<li>Run samples from TBS, TBS-SDS and TBS-SDS-Urea extracts on 10-well 4-12% Bis-Tris polyacrylamide gel with MOPS as running buffer using standard techniques. See Gallagher and Chakravarti (2008)29 for a detailed western blot protocol.</li>
	<li>Load 10 &#181;g of protein from each sample onto each lane along with a molecular weight marker for TBS and SDS and urea fractions.</li>
	<li>Run gel at 200 V for 1 hr or until the blue dye from loading buffer has reached the bottom of the gel.</li>
	<li>Transfer proteins from gels electrophoretically onto nylon membranes28, pre-wetted first in 100% methanol and then into transfer buffer containing 20% methanol. Provide an identification mark on the blot to determine the protein side and the orientation of the Molecular-Weight size markers.
	<ol>
		<li>Sandwich the gel with the membrane alongside wet filter paper. Correct placement of membrane is crucial with the membrane between the gel and the anode. Perform the transfer for 2 hr at 40 V.</li>
		<li>Make up 1X PBS-tween (1X PBS-T) buffer: Dissolve 1 tablet of PBS in 200 ml water to yield 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4.</li>
		<li>Block the membrane in 5% BSA solution in 1X PBS-T for 30 min with shaking at 70 rpm. This prevents non-specific binding of primary antibody to the membrane.</li>
	</ol>
	</li>
	<li>Probe the protein blot with 6 ml of &#945;-syn primary antibody Syn-1 (BD Biosciences; mouse monoclonal antibody) at 1:750 dilution (O/N at 4 &#176;C) followed by a series of washes with 1X PBS-T (3 x 5 min each time)28.</li>
	<li>Incubate the blot with the appropriate HRP conjugated secondary antibody at 1:2,000 dilution for 30 min. Perform a series of washes (3 x 5 min each time) with 1X PBS-T.
	<ol>
		<li>Immerse blots in enhanced chemiluminescence solution for 15 sec&#160;in a dark room. Cover blots with a cling film and place autoradiography films against the blot with protein side up in a light-proof cassette to capture the appropriate signals (appropriate size bands).</li>
		<li>Develop autoradiography films in an automated developer. 
		Note: Blots can also be developed manually using developer and fixer from a commercial source in case an automated developer machine is not available.</li>
	</ol>
	</li>
	<li>Incubate the western blot with 6 ml of western blot stripping buffer for 10 min with constant shaking to strip the blot of &#945;-syn antibody signal. Follow steps 3.43 through to 3.6.2 using beta-actin primary antibody (Mouse monoclonal) at 1: 8,000 dilution.</li>
	<li>Scan and convert the final image into a Tiff image and measure densities using NIH ImageJ for quantitation purposes (a free downloadable software). 
	Note: The software allows reading of densities of relative area of interest (the appropriate size bands) and the data can be expressed either as a ratio of &#945;-syn density / &#946;-actin (a house-keeping gene) as arbitrary units or on its own when a house keeping gene is not valid (as in the case of urea-soluble samples).</li>
</ol>

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</ol>

The author has nothing to disclose.

This article describes a biochemical protocol for extraction and examination of the differential solubility properties of monomeric, oligomeric and aggregated &#945;-syn from diseased brains using denaturing gel running conditions. This is a well-established technique to study aggregated &#945;-syn17, 18, 20, 21 a protein that is normally soluble in its native form but gain amyloidogenic or increased aggregation properties with disease progression or with genetic mutations. Nonetheless, some groups have used slight variations in SDS concentrations in their buffers (8% or 10% instead of 5%)21,22, 30. The SDS is an anionic detergent and solubilises &#945;-syn oligomers that can include membrane bound forms of &#945;-syn, whereas urea, a chaotropic reagent denatures the insoluble aggregated and fibrillar or amyloidogenic forms of &#945;-syn20. In this regard a systematic study by Paleologou et al31 examined the stability of &#945;-syn oligomers and fibrils with varying concentrations of urea (6.5 - 8 M) or SDS (0.25-2%) and reported that oligomers stable in SDS concentrations but not with high concentrations of urea. Their FILA-1 antibody, specific for &#945;-syn oligomers and fibrils detected higher concentrations of fibrils at 6.5 M urea concentrations under non-denaturing conditions. The buffer concentrations used in this protocol closely mirrors what has been described in Culvenor et al. (1999)32.
The anatomical brain regions for biochemical extraction of insoluble &#945;-syn should be carefully selected and this should reflect &#945;-syn LB and LN load depending on disease progression33,34. The cases used here are basal ganglia samples from the neocortical and limbic subtypes of PD reflecting late and intermediate stages of PD progression according to McKeith ctriteria34. At the Queen Square Brain Bank, one half of the brain is routinely fixed in formalin for histochemical analysis and the other half is carefully dissected into different anatomical regions and flash frozen and stored at -80 oC freezers for further biochemical and DNA/RNA analysis studies. Following formalin fixation of the brains and dissection by neuropathologists, immunohistochemistry is performed using &#945;-syn antibody and results archived. Also as a routine procedure, the pH of the brain tissue is measured upon arrival as a measure of agonal state of the brain tissue. This forms a firm basis for the quality of tissue preservation for biochemical analysis.
Our data here shows that there are more SDS soluble &#945;-syn monomer in PD cases compared to controls; in particular the neocortical PD cases has higher &#945;-syn load compared to the limbic PD variety. The neocortical cases represent greater progression of PD &#945;-syn pathology in the neocortical regions such as the frontal and parietal cortices33,34. The limbic PD cases have higher LB scores in the basal forebrain/limbic regions areas such as the amygdala, transentorhinal and the cingulate regions. For meaningful data and statistical analysis, one must run at least 4 cases from each cohort. Likewise we have not attempted statistical analysis of the blot data presented here.
It must also be noted that different antibodies may recognize different forms/composition of higher order &#945;-syn oligomers and each may have its own relative sensitivity and preference. This is evident from the results of Tong et al.29 where they show that 4 different &#945;-syn antibodies (Syn-1, SS, Onco and LB509) give changeable results at least for &#945;-syn oligomers/aggregates. The &#945;-syn monomers were recognized to similar extents by all 4 antibodies although these can have variation depending on whether the &#945;-syn antibody epitope is located either in the N-terminal or C-terminal29. Similarly, specific antibodies for phospho-alpha synuclein determine distinct high-molecular weight &#945;-synuclein species20,21. In the protocol described here, the highly characterized antibody Syn-1 has been used19-21.
However, it is important to consider validating any new antibody on western blots through antibody preabsorption experiments and also overexpression and knockdown of the protein in cells.
It is important to consider other variations that have been applied by researchers to determine smaller fragments of &#945;-syn and or unstable tetrameric &#945;-syn forms. This could include mild-fixation of membranes with 0.4% PFA in PBS to retain truncated forms of &#945;-syn35 or treatment of sample homogenates with cross linkers to stabilize tetramers36.
Accurate biochemical evaluation of proteins using post-mortem tissue requires minimization of enzymatic protein degradation and modification. It is therefore advisable to use tissue with the shortest post-mortem delays and /or select matched samples within the case cohorts. Immunohistochemistry using standard antibodies for &#945;-syn immunohistochemistry should be performed prior to starting the isolation protocol. The extent of &#945;-syn pathology is highly variable and can vary according to regions selected. It is advisable to select regions with abundant pathology as a positive control for optimal yield with every run. The use of protease inhibitors and if necessary phosphatase inhibitors to prevent undesired enzymatic degradation after release of intracellular proteases or phosphatases occurring during cell lysis and maintaining the samples at 4 &#176;C is crucial.
It is important that all of the samples within the batch of experiments are handled evenly and consistently to avoid intra-user variations; multiple freeze-thaw cycles should be avoided as this may potentially retract post-translational modifications such as phosphorylation. A critical point to bear in mind when examining a large number of samples is that the data from immunoblots should be normalized to one sample, which should run in parallel to all other samples on the gels, and all data referenced to that sample. This is done to ensure normalization of immunoblot data between several blots. This is the method adopted in our recent study22.
It should be noted that in order to keep the background ECL low, the blots should not be allowed to dry out at any time during the western blot protocol. In addition, very long exposures (&#62;10 min) on autoradiography films should be avoided as this will lead to saturation of ECL signal and will lead to inaccurate quantitation.
This above protocol is a relatively straightforward technique using common laboratory reagents and instruments and can be a valuable tool for investigating the pathophysiology of &#945;-syn protein in PD research. This methodology can not only be extended with appropriate modifications to the study of &#945;-syn biology in &#945;-syn transgenic animals but also to other neurodegenerative diseases featuring abnormal deposits of aggregated proteins such as AD, MSA, DLB, HD and frontotemporaldementias. However the western blot data described here could also be validated using enzyme-linked immunosorbent assays using antibodies for specific forms of &#945;-syn31.

One of the key pathological features common amongst several neurodegenerative diseases is the formation of abnormal protein aggregates that occurs in a protein/disease specific manner1. Thus, there are the characteristic extraneuronal amyloid-beta plaques and aggregated hyperphosphorylated tau deposits within neurons in Alzheimer&#39;s disease (AD); the aggregated &#945;-syn deposits in Lewy Bodies (LBs) which are intraneuronal inclusions, and also within dystrophic neuronal processes called Lewy neurites (LNs) in PD, PD dementia and Dementia with Lewy bodies (DLBs)2-4. Abnormal &#945;-syn deposits are also seen in hallmark lesions of glial cytoplasmic inclusions in MSA5. In Huntington&#39;s disease, there are the characteristic Poly-Q deposits, and abnormal Tar DNA binding protein-43 and fused in sarcoma proteins (FUS) are deposited in frontotemporal dementias. Importantly for PD, &#945;-syn gene mutations have been found to cause autosomal dominant PD6-11. Additionally, &#945;-syn gene triplication12 and duplication13 also causes familial PD thus linking increased &#945;-syn expression to the pathomechanisms of PD. Notably, both sporadic and familial PD cases harbour abnormal deposits of &#945;-syn pathology14. Current available treatments for PD are only palliative and have no effect on halting or delaying the inexorable disease progression.
&#945;-syn is abundantly expressed in the human brain and makes up about 1% of the total brain protein. It is present more specifically within the neurons but can exist albeit in lower amounts within glial cells. A contentious point is the native structure of &#945;-syn which can exist as a random monomer15 or as a folded tetramer16. During disease, &#945;-syn changes its conformation that allows it to get misfolded and this process is thought to be the cause of disease pathogenesis. Despite several years of investigations into the pathophysiological aspects of &#945;-syn and disease, the exact causes of disease mechanisms have remained elusive14.
Studies using post-mortem analysis of Parkinsonian brains have thus far provided important clues regarding normal and abnormal biology of &#945;-syn both in sporadic PD and also PD associated with mutations in the LRRK2 gene17-22. Here, a biochemical extraction protocol (see scheme presented in Figure 1) for increasingly insoluble &#945;-syn deposits from human post-mortem PD brain tissue that harbour &#945;-syn aggregates to varying degrees has been described. This technique can also be adapted with modifications in buffer compositions to study aggregate proteins from other neurodegenerative diseases23,24 and also from transgenic animal brain tissue25-27. The main consideration for the adaptations are the differences in solubility and the total amounts of the respective proteins some of which are mainly nuclear and may need high-salt buffers for optimal extraction as shown for FUS28.

<table><tbody><tr><td>Tris-HCL</td><td>Sigma</td><td>T5912</td><td></td></tr><tr><td>sodium chloride</td><td>VWR</td><td>27800.291</td><td></td></tr><tr><td>SDS</td><td>Fluka</td><td>71727</td><td></td></tr><tr><td>Urea</td><td>Sigma</td><td>U5378</td><td></td></tr><tr><td>Protease inhibitor tablets&amp;nbsp;</td><td>Roche</td><td>04 693 116001</td><td></td></tr><tr><td>Phosphatase inhibitor tablets&amp;nbsp;</td><td>Roche&amp;nbsp;</td><td>04 906 837001</td><td></td></tr><tr><td>Phosphate buffered saline tablets&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>P4417</td><td></td></tr><tr><td>Tween-20&amp;nbsp;</td><td>Sigma</td><td>P9416</td><td></td></tr><tr><td>NuPAGE MOPS SDS running buffer</td><td>Invitrogen</td><td>NP0001</td><td></td></tr><tr><td>NuPAGE transfer buffer</td><td>Invitrogen</td><td>NP0006-1</td><td></td></tr><tr><td>Hybond-P membrane&amp;nbsp;</td><td>GE Healthcare</td><td>RPN303F</td><td></td></tr><tr><td>Whatman 3MM filter paper</td><td>Sigma</td><td>WHA30306185</td><td></td></tr><tr><td>Bis-tris gels 4-12%&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td>NP0322BOX</td><td></td></tr><tr><td>Supersignal West Pico Chemiluminescent substrate</td><td>Pierce</td><td>34080</td><td></td></tr><tr><td>Bio-RAD DC protein assay kit&amp;nbsp;</td><td>Bio-Rad</td><td>500-0112</td><td></td></tr><tr><td>Thickwall Beckman Polycarbonate tubes</td><td>Beckman</td><td>355630</td><td></td></tr><tr><td>Western blot stripping buffer</td><td>Thermo scientific</td><td>46430</td><td></td></tr><tr><td>Syn-1 primary antibody (mouse monoclonal)</td><td>BD Biosciences</td><td>610786</td><td></td></tr><tr><td>beta-actin primary antibody (mouse monoclonal&amp;nbsp;</td><td>Sigma</td><td>A5441</td><td></td></tr><tr><td>HRP cojugated goat anti-mouse secondary antibody</td><td>Santa Cruz</td><td>sc-2031</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A7030</td><td></td></tr><tr><td>Instrument</td><td>Company</td><td>Rotor/ model no&amp;nbsp;</td><td></td></tr><tr><td>Benchtop Ultracentrifuge Optima Max</td><td>Beckman</td><td>MLA-80</td><td></td></tr><tr><td>Sonicator</td><td>Heat Systems</td><td>XL20-20</td><td></td></tr><tr><td>Homogeniser</td><td>Jenke and Kunkel</td><td>TP18/10</td><td></td></tr></tbody></table>

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.

TBS, SDS and Urea soluble proteins extracted from basal ganglia following the protocol shown in Figure 1 were run on gels and immunoblotted with the Syn-1 mouse monoclonal &#945;-syn primary antibody. The TBS soluble fractions displayed the presence of monomeric &#945;-syn (~14 KDa species) in the PD and control cases examined (Figure 2A). The SDS soluble fractions also showed abundant monomeric &#945;-syn in all three subtypes of cases studied as displayed in the low exposure blot (Figure 2C). The PD cases also showed higher molecular weight (MW) &#945;-syn species as seen on the high exposure blot, which are likely to be oligomers (Figure 2C). The urea soluble fractions from PD cases showed elevated amounts of &#945;-syn monomers, oligomers and aggregated species compared to control cases. The neocortical idiopathic PD cases demonstrate higher amounts of aggregated &#945;-syn species, whilst the limbic cases show intermediate levels of insoluble &#945;-syn. Both the SDS and urea fractions from idiopathic PD cases demonstrate variable levels of truncated &#945;-syn products at 12 kDa and 6 kDa as described 20. In contrast, the control cases did not show any insoluble or aggregated &#945;-syn (Figure 2E). Semi-quantitative density measures reflect the quantity of LB load as categoriesd using &#945;-syn immunohistochemistry demonstrating the insoluble nature of aggregated &#945;-syn (Figure 2B, 2D, 2F) in neocortical and limbic PD cases.
<img alt="Figure 1" src="/files/ftp_upload/53415/53415fig1.jpg" /> 
Figure 1. Schematic diagram of insoluble &#945;-syn extraction procedure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53415/53415fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53415/53415fig2.jpg" /> 
Figure 2. Representative immunoblots of &#945;-syn levels from PD and control human tissue. TBS, SDS and urea soluble fractions from basal ganglia (a region that harbors &#945;-syn pathology in PD cases) of idiopathic PD cases and neurologically normal (control) brains were analysed for the presence of &#945;-syn using western immunoblot. 10 &#956;g of protein was loaded onto each lane. In TBS fractions (A), mainly &#945;-syn monomers are present in all cases. In SDS fractions (C), some oligomers of &#945;-syn are seen along with monomers mainly in the PD tissue. In urea fractions (C), monomers, oligomers and aggregated &#945;-syn are variably expressed in the PD cases reflecting their respective LB load. The neurologically normal control samples show the presence of only small amounts of monomeric &#945;-syn. Please note possible degradation products of &#945;-syn are seen in some of the cases with high LB load. The solid arrow head represents the monomeric form of &#945;-syn. The * possibly represents a C-terminally truncated form of &#945;-syn as previously described20,26. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53415/53415fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Case&#160;</td>
			<td>Sex</td>
			<td>age at death yrs</td>
			<td>post-mortem delay (hrs)</td>
			<td>pH of tissue&#160;</td>
		</tr>
		<tr>
			<td>Neocortical 1</td>
			<td>M</td>
			<td>82</td>
			<td>40</td>
			<td>6.3</td>
		</tr>
		<tr>
			<td>Neocortical 2</td>
			<td>F</td>
			<td>75</td>
			<td>35.3</td>
			<td>6.4</td>
		</tr>
		<tr>
			<td>Limbic 1</td>
			<td>M</td>
			<td>81</td>
			<td>28.5</td>
			<td>6.2</td>
		</tr>
		<tr>
			<td>Limbic 2</td>
			<td>F</td>
			<td>79</td>
			<td>36.5</td>
			<td>6.2</td>
		</tr>
		<tr>
			<td>Control 1</td>
			<td>M</td>
			<td>80</td>
			<td>38</td>
			<td>6.1</td>
		</tr>
		<tr>
			<td>Control 2</td>
			<td>F</td>
			<td>83</td>
			<td>32.5</td>
			<td>6.3</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Limited demographics of the cases used&#160;

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

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, ...

sequential-extraction-soluble-insoluble-alpha-synuclein-from

Nanyang Technological University

Research

JoVE Journal

Neuroscience

17.2K Views.  UCL Institute of Neurology. Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (α-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 α-syn aggregation in diseased and non-diseased tissue can be examined.

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

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain. The ionic detergent N-lauryl-sarcosine (sarkosyl) effectively solubilizes natively folded proteins in brain tissue allowing the enrichment of detergent-insoluble protein aggregates from a wide range of neurodegenerative proteinopathies, such as Alzheimer&#39;s disease (AD), Parkinson&#39;s disease and amyotrophic lateral sclerosis, and prion diseases. Human control and AD postmortem brain tissues were homogenized and sedimented by ultracentrifugation in the presence of sarkosyl to enrich detergent-insoluble protein aggregates including pathologic phosphorylated tau, the core component of neurofibrillary tangles in AD. Western blotting demonstrated the differential solubility of aggregated phosphorylated-tau and the detergent-soluble protein, Early Endosome Antigen 1 (EEA1) in control and AD brain. Proteomic analysis also revealed enrichment of &#946;-amyloid (A&#946;), tau, snRNP70 (U1-70K), and apolipoprotein E (APOE) in the sarkosyl-insoluble fractions of AD brain compared to those of control, consistent with previous tissue fractionation strategies. Thus, this simple enrichment protocol is ideal for a wide range of experimental applications ranging from Western blotting and functional protein co-aggregation assays to mass spectrometry-based proteomics.

An abbreviated fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain is described.

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

Biochemistry

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson&#8217;s disease and beta-amyloid and tau in Alzheimer&#8217;s disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.

Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in ...

Bioengineering

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

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 ...

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (&#945;Syn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of &#945;Syn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting &#945;Syn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark &#945;Syn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...

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 ...

Analyzing the Parkinson's Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &#945;-Synuclein

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, consequently, the progressive loss of control of voluntary movements. This neurodegenerative process is triggered by the deposition of protein aggregates in the brain, which are mainly constituted of &#945;-synuclein. Several studies have indicated that neuroinflammation is required to develop the neurodegeneration associated with Parkinson&#39;s disease. Notably, the neuroinflammatory process involves microglial activation as well as the infiltration of peripheral T cells into the substantia nigra (SN). This work analyzes a mouse model of Parkinson&#39;s disease that recapitulates microglial activation, T-cell infiltration into the SN, the neurodegeneration of nigral dopaminergic neurons, and motor impairment. This mouse model of Parkinson&#39;s disease is induced by the stereotaxic delivery of adeno-associated viral vectors encoding the human wild-type &#945;-synuclein (AAV-h&#945;Syn) into the SN. The correct delivery of viral vectors into the SN was confirmed using control vectors encoding green fluorescent protein (GFP). Afterward, how the dose of AAV-h&#945;Syn administered in the SN affected the extent of h&#945;Syn expression, the loss of nigral dopaminergic neurons, and motor impairment were evaluated. Moreover, the dynamics of h&#945;Syn expression, microglial activation, and T-cell infiltration were determined throughout the time course of disease development. Thus, this study provides critical time points that may be useful for targeting synuclein pathology and neuroinflammation in this preclinical model of Parkinson&#39;s disease.

This work analyzes the vector dose and exposure time required to induce neuroinflammation, neurodegeneration, and motor impairment in this preclinical model of Parkinson&#39;s disease. These vectors encoding the human &#945;-synuclein are delivered into the substantia nigra&#160;to recapitulate the synuclein pathology associated with Parkinson&#39;s disease.

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, ...

&#945;-Synuclein Propagation: Intranasal Administration in Parkinson&#39;s Disease

Technique for Intranasal Administration of &#945;-Synuclein Aggregates

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the presence of Lewy bodies, which are aggregates of &#945;-synuclein (&#945;-Syn). Recently, the disease was proposed to develop and progress through the prion-like propagation of &#945;-Syn aggregates from the olfactory bulb (OB) or dorsal nucleus of the vagus nerve. Although the origin of &#945;-Syn aggregates in the OB remains unclear, their propagation from the olfactory mucosa has been recently suggested. We previously showed that intranasal administration of &#945;-Syn aggregates in a mouse model induced &#945;-Syn pathology in the OB of mice. In this study, we present a method of intranasal administration of &#945;-Syn aggregates that induced &#945;-Syn pathology in the OB of mice. Intranasal administration of &#945;-Syn aggregates is a very simple and straightforward method, and we believe it will be a useful tool in the research for elucidating the origin of &#945;-Syn pathology in the OB and the pathway of &#945;-Syn propagation through the olfactory system.

The present protocol describes a method for intranasal administration of &#945;-synuclein aggregates. This method provides insights into &#945;-synuclein propagation from the olfactory mucosa to the olfactory bulb in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the presence of Lewy bodies, which are aggregates of &#945;-synuclein ...

An Immunofluorescence Assay to Detect Alpha-Synuclein Aggregates in a Skin Biopsy Section

Source: Vacchi, E., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59558">Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay.</a> J. Vis. Exp. (2019).
This video demonstrates an immunofluorescence assay to detect neurodegenerative disease-specific alpha-synuclein aggregates on skin biopsy sections. Free-floating immunostaining is employed to fluorescently label the carboxyl-terminal hydrolase and the alpha-synuclein aggregates in the dermal neurons. Under a confocal microscope, cells with fluorescence from both antibodies indicate the presence of alpha-synuclein aggregates in the dermal neurons.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines,
1. Skin Biopsy Collection

	Let a ...

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

Samenvatting

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