Name: targeting alpha synuclein aggregates in cutaneous peripheral nerve fibers by free-floating immunofluorescence assay
Uploaded: 2019-06-25T13:30:00.000+00:00
Duration: 8 min 33 s
Description: Watch this Scientific Journal Video about Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay at JoVE.com

We thank Parkinson Schweiz and ABREOC (the Scientific Research Advisory Board of the Ente Ospedaliero Cantonale) for their financial support of this study.

The protocol has been approved by the Cantonal Ethics Committee and all enrolled subjects gave written informed consent to the study.
1. Skin Biopsy Collection
<ol>
	<li>Let a qualified physician perform the skin biopsy in an appropriate clinical setting.</li>
	<li>Choose the area to perform the skin biopsy and clean it with an alcohol swab.</li>
	<li>Prepare the anesthetic solution with 1 cc of lidocaine 2%.</li>
	<li>With the needle parallel to the area, inject local anesthesia subcutaneously.</li>
	<li>After checking the effect of anesthesia, take the 3 mm disposable punch and apply rotational and delicate downward pressure to rotate it down through the epidermis and dermis, until the subcutaneous fat is reached.</li>
	<li>Withdraw the punch.</li>
	<li>Use disposable forceps to gently pull the skin plug up.</li>
	<li>Use scissors to cut out the base of the specimen at the level of the fatty tissue.</li>
	<li>Place the specimen in a tube containing 10 mL of periodate-lysine-paraformaldehyde (PLP) fixative solution.</li>
	<li>Disinfect the area and cover it with an adhesive bandage.</li>
</ol>
2. Tissue Fixation and Storage
<ol>
	<li>Make fresh PLP fixative solution (see Table 1).</li>
	<li>Immediately after the collection of the skin biopsy, submerge it in a tube containing 10 mL of PLP fixative solution and incubate it overnight (O/N) at 4 &#176;C.</li>
	<li>The day after, under the fume hood, remove the PLP fixative gently and in the same tube, wash the biopsy 3 times for 5 min with 5 mL of 0.1 M Sorensen&#8217;s solution (Table 1).</li>
	<li>Discard the Sorensen&#8217;s solution and incubate the biopsy with 5 mL of cryo-protectant solution O/N at 4 &#176;C.</li>
	<li>Store the biopsy at 4 &#176;C if the cut with a cryotome is performed within 1 week; store the biopsy at -20 &#176;C if the cut is performed within 3 months; embed the biopsy in a cryomold (steps 3.2-3.4) and store it at -80 &#176;C to conserve it for a longer period of time.</li>
</ol>
3. Tissue Cut with a Cryotome
<ol>
	<li>Set the cryotome at -20 &#176;C.</li>
	<li>Take a cryomold and fill it up with the cryo-embedding medium. Avoid creating bubbles.</li>
	<li>Using tweezers immerse the biopsy into the cryo-embedding medium with the longitudinal axis (epidermis - dermis) parallel to the bottom of the cryomold.</li>
	<li>Snap freeze the sample with liquid nitrogen to obtain a solid cube of cryo-embedding medium containing the biopsy in the right orientation.</li>
	<li>Put the sample in the cryostat and wait for 30 min to allow the biopsy to acclimatize.</li>
	<li>Fix the sample on the cryostat and cut 50 &#181;m sections.</li>
	<li>With the help of a little brush, transfer the cryo-sections in a 96 well plate containing 200 &#181;L of antifreeze solution in each well. One section per well.</li>
	<li>Store at -20 &#176;C. 
	NOTE: If the analyze the entire biopsy is not analyzed, cut it only partially. In this case, the sections must be stored at -20 &#176;C and the remaining part of the biopsy at -80 &#176;C to conserve it for a longer period of time.</li>
</ol>
4. Immunofluorescence Staining
<ol>
	<li>Fill a 96 well plate, with 100 &#181;L of washing solution.</li>
	<li>Transfer the sections to be analyzed from the storage plate to the new one containing the washing solution. 1 section per each well (4 sections per anatomical site, per patient: usually a total of 12 sections per patient).</li>
	<li>Leave the section in the washing solution for 10 min at room temperature (RT). Transfer the section into another well containing the same solution and repeat the wash.</li>
	<li>Move the sections into new wells containing 100 &#181;L of Blocking solution and incubate for at least 90 min and for a maximum of 4 h at RT.</li>
	<li>Dilute the primary antibodies anti-PGP9.5 (Rabbit polyclonal, 1:1000) and anti-5G4 (Mouse monoclonal, 1:400) in the working solution.</li>
	<li>Transfer the sections into new wells containing 100 &#181;L of the working solution of the primary antibodies and incubate them O/N at RT.</li>
	<li>As step 4.3, wash the sections 2 times for at least 10 min at RT, with 100 &#181;L of washing solution.</li>
	<li>Dilute the secondary antibodies (conjugate with different fluorophores) Goat anti-Rabbit to detect PGP9.5 (1:700) and a Goat anti-Mouse to detect 5G4 (1:700) in the working solution.</li>
	<li>Transfer the sections into new wells containing 100 &#181;L of the working solution of the secondary antibodies for 90 min at RT. From this point, cover the 96 well plate with aluminum foil to avoid the bleaching of fluorophore conjugated with secondary antibody/ies.</li>
	<li>As step 4.3, wash the sections 2 times for at least 10 min, with 100 &#181;L of the washing solution.</li>
	<li>Transfer the sections into new wells containing 100 &#181;L of DAPI (diluted 1:5,000 in PBS 1x) for 5 min at RT.</li>
	<li>As step 4.3, wash the sections 2 times for at least 10 min, with 100 &#181;L of the washing solution.</li>
	<li>Mount the sections on a slide in the correct position avoiding misfolding.</li>
	<li>Add a few drops of the mounting medium on the slide and cover with a coverslip.</li>
	<li>Let slides dry O/N before using the confocal/fluorescence microscope.</li>
	<li>Store the slides in an appropriate box at 4 &#176;C avoiding the light exposure. If accurately stored, the signal will be visible for about 6 months. 
	NOTE: The transfer of a section from the 96 well plate containing the newly cut slices to the 96 well plate containing the washing solution (step 4.1) is performed using a small brush that helps to pick up the biopsy. After the transfer of the section into the first well, every time the slice needs to be incubated with a different solution, move it from well to well with the help of the brush.</li>
</ol>
5. Immunofluorescence Imaging
<ol>
	<li>View sections under an inverted fluorescence microscope or a confocal microscope (20X, 40x or higher magnification, use successive frames of 2 &#181;m increments on a Z-stack plan for best results).</li>
	<li>Acquire images by a microscope connected camera</li>
	<li>Use an adequate imaging software (i.e. ImageJ) to analyze positive signals in sections in terms of spatial distribution and intensity of the signal. To do this perform the steps mentioned below.
	<ol>
		<li>Open the file with ImageJ software.</li>
		<li>Click on Image &#62; color &#62; split channels in order to analyze each color channel.</li>
		<li>Click Image &#62; stack &#62; Z project to obtain a merge of multiple section acquisitions.</li>
		<li>Click Image &#62; color &#62; merge channels to obtain a merge of different color channels of the same acquisition.</li>
	</ol>
	</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Antifreeze solution 
			(store at 4 &#176;C for up to 6 months)</td>
			<td>30% Glycerol 
			30% Ethylene glycol 
			30% dH2O 
			10% 2x Phosphate buffer</td>
		</tr>
		<tr>
			<td>Blocking solution 
			(prepare at the moment)</td>
			<td>4% Normal Goat Serum 
			1% Triton X-100 
			in Washing solution</td>
		</tr>
		<tr>
			<td>Cryo-protectant 
			(store at 4 &#176;C for up to 6 months)</td>
			<td>20% Glycerol 
			80% Sorensen&#39;s solution</td>
		</tr>
		<tr>
			<td>Disodium hydrogen phosphate solution 
			(store at RT up to 6 months)</td>
			<td>0.45M Disodium hydrogen phosphate (Na2HPO4) in dH2O 
			Filter in a sterile bottle</td>
		</tr>
		<tr>
			<td>Lysine solution 
			(store at 4 &#176;C for up to 3 weeks)</td>
			<td>50% of 0.3M L-Lysine monohydrochloride solution 
			50% of 0.1M Sorensen&#39;s solution pH7.6 
			pH 7.4, filter in a sterile bottle</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA) 8% 
			(prepare under fume hood and store at 4 &#176;C for up to 1 month)</td>
			<td>2.6M PFA in dH2O (to 55&#176;C_do not exceed 60 &#176;C<nr> to avoid formic acid formation) 
			filter in a sterile bottle</nr></td>
		</tr>
		<tr>
			<td>Phosphate buffer 2x 
			(store at 4 &#176;C for up to 6 months)</td>
			<td>6% Monosodium phosphate (NaH2PO4) solution 
			45% Disodium phosphate (Na2HPO4) solution in dH2O</td>
		</tr>
		<tr>
			<td>PLP fixative solution 
			(prepare at the moment, under fume hood )</td>
			<td>25% Paraformaldehyde 8% 
			0.01M Sodium (meta)periodate 
			75% Lysine solution&#60;</td>
		</tr>
		<tr>
			<td>Sodium Dihydrogen Phosphate Monohydrate 
			(store at RT up to 6 months)</td>
			<td>0.52M Sodium Dihydrogen Phosphate Monohydrate (NaH2PO4*H2O) in dH2O 
			Filter in a sterile bottle.</td>
		</tr>
		<tr>
			<td>Sorensen&#39;s solution 
			(store at 4 &#176;C for up to 1 month)</td>
			<td>2.5% Monosodium phosphate solution 
			18.7% Disodium phosphate solution 
			pH7.6, in dH2O</td>
		</tr>
		<tr>
			<td>Washing solution 
			(prepare at the moment)</td>
			<td>0.25M Trizma base 
			0.26M NaCl 
			pH7.6, in dH2O</td>
		</tr>
		<tr>
			<td>Working solution 
			(prepare at the moment)</td>
			<td>50% Blocking solution 
			50% Washing solution</td>
		</tr>
	</tbody>
</table>
Table 1: Required solutions. List of required solutions for the protocol and brief description of how to prepare it.

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The authors have nothing to disclose.

We describe a free-floating immunofluorescence assay for skin biopsies for the diagnosis of PD: it exploits double immunostaining with anti-PGP9.5 antibody, a panaxonal marker, and anti-5G4, a conformation specific antibody that recognizes the aggregated form of &#945;Syn.
The great advantages of skin biopsy for diagnostic purpose in PD and possibly in other protein conformational disorders are: 1) the direct access to nervous tissue prone to disease by a mildly invasive technique and thus wit...

Skin biopsy has acquired a great importance as the diagnostic and research tool in the field of neurological disorders1. Indeed, epidermis and dermis contain abundant somatic sensory nerve fibers (myelinated and unmyelinated), nociceptive free nerve endings, sensory receptors and autonomic innervation of sweat glands, vessels, sebaceous glands and muscle arrector pilorum2.
In the mid-20th century, the setup for immunohistochemistry of PGP9.5 antibody allowed the evidence of an extensive innervation of human epidermis mammalian skin3. PGP9.5 is a carboxyl-terminal hydrolase equally distributed along axons of both the central and peripheral nervous system (PNS). The availability of this antibody allowed not only to clarify the morphology and anatomy of PNS in the skin but also implemented the study of diseases associated with it3,4. Skin biopsy contributed to defining a new clinical entity: the small fiber neuropathy. Several international groups demonstrated the association between the loss of intraepidermal nerve fibers and symptoms/signs of small fiber neuropathy5 by skin biopsy analysis and provided standardized protocols for nerve morphometry as well as normative reference values to be used in the clinical practice6,7,8.
Recently a large amount of evidence has shown that neurodegenerative diseases, characterized by misfolded protein accumulations in the central nervous system, are multi-system pathologies9. Indeed, PD is characterized by &#945;Syn accumulation in the dopaminergic neuron of substantia nigra, but it has been demonstrated that &#945;Syn and its pathological form, phosphorylated &#945;Syn (P-&#945;Syn), could be detected also in the peripheral tissues. Gastro-intestinal mucosa10, salivary glands11, skin autonomic fibers surrounding sweat glands and pilomotor muscles12,13,14, show immunoreactivity to pathogenic forms of &#945;Syn, in accordance with Braak hypothesis that intriguingly postulate that &#945;Syn pathology may begin in PNS well in advance, before its accumulation in the brain15. Further, the presence of p-&#945;Syn has been demonstrated in skin nerves of patients with REM Behavior Disorders that are considered prodromal PD16,17 thus skin pathologic &#945;Syn can be considered a promising early peripheral histopathological marker of synucleinopathy.
The association of small fiber neuropathy in PD has been demonstrated previously and it has been found that intraepidermal nerve fibers density reflects disease progression18,19. Hence, the skin biopsy is a useful tool for studying neurodegeneration in PD and for establishing a pre-mortem histopathological diagnosis of the disease. Indeed, skin biopsy has a great advantage of being an easily accessible and minimally invasive procedure, allowing the analysis of nervous tissue prone to the pathology. Finally, the possibility of repeating the skin biopsy in the course of follow-up of the same patients allows studying the longitudinal correlation with disease progression.
In our laboratory, exploiting a double immuno-staining with PGP9.5 and the conformation-specific monoclonal 5G4 antibody, that recognizes disease specific forms of &#945;Syn including small aggregates20,21, we were able to show the presence of &#945;Syn aggregates in skin nerves with a promising high diagnostic efficiency19. Immunofluorescence analysis of the skin biopsy in conformational diseases stands out as a promising source of biomarkers by combining both the detection of protein aggregates and the measure of the neurodegeneration in vivo. Hereafter, we illustrate an easy and versatile protocol on handling the skin biopsy and performing the free-floating immunofluorescence staining for detecting &#945;Syn aggregates. Moreover, this protocol can be adapted for targeting any other protein of interest expressed in skin PNS.
The following study protocol has been used to evaluate the diagnostic utility of aggregated &#945;Syn analysis in the PNS of PD by skin biopsy19. Inclusion criteria for PD were: a definite clinical diagnosis according to the UK Brain Bank diagnostic criteria, disease duration at least 3 years, no family history, and no major cognitive impairment or major dysautonomic symptoms in the history. Exclusion criteria were known causes of neuropathy (glycated hemoglobin, creatinine, vitamin B12, TSH, serum immunofixation, HIV, HCV, syphilis, and borreliosis). Each subject underwent to 3 mm-diameter skin biopsies at three anatomical sites (neck at C8 dermatomal level, thigh 10 cm above the knee, leg 10 cm above lateral malleolus) on the side, which was clinically more affected. In general, the following protocol is about handling the skin biopsy and performing the free-floating immunofluorescence staining and analysis. Hence it can be adapted and used for the detection of other proteins of interest in skin tissue.

<table><tbody><tr><td>5G4 (anti human &amp;alpha;Syneclein 5G4)</td><td>Analytik Jena Roboscreen</td><td>847-0102004001</td><td>Mouse monoclonal&amp;nbsp;</td></tr><tr><td>AlexaFluor 488 Goat anti Rabbit IgG&amp;nbsp;</td><td>Invitrogen</td><td>1971418</td><td>2 mg/mL</td></tr><tr><td>AlexaFluor 594 Goat anti Mouse IgG&amp;nbsp;</td><td>Invitrogen</td><td>1922849</td><td>2 mg/mL</td></tr><tr><td>Disodium hydrogen phosphate solution</td><td>Merk Millipore</td><td>106586</td><td></td></tr><tr><td>Ethylene Glycol</td><td>Sigma-Aldrich</td><td>324558</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G7757</td><td></td></tr><tr><td>L-Lysine monohydrochloride</td><td>Sigma-Aldrich</td><td>L5626</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Aldrich Chemistry</td><td>441244</td><td></td></tr><tr><td>PGP9.5</td><td>Abcam</td><td>ab15503</td><td>Rabbit polyclonal</td></tr><tr><td>Sodium Chloride</td><td>Sigma&amp;nbsp;</td><td>S3014</td><td></td></tr><tr><td>Sodium Dihydrogen Phosphate Monohydrate</td><td>Merck Millipore</td><td>106346</td><td></td></tr><tr><td>Sodium (meta)periodate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S1878</td><td></td></tr><tr><td>Trizma Base</td><td>Sigma&amp;nbsp;</td><td>T1503</td><td></td></tr><tr><td>Tryton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Vectashield&amp;nbsp;</td><td>Vector Laboratories</td><td>H-1000</td><td>Mounting medium</td></tr></tbody></table>

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.

Following the described procedure (Figure 1), we detected &#945;Syn aggregates, labeled with 5G4 antibody, in dermal nerve fascicles innervating autonomic structures of PD patients. Morphology of alpha-synuclein deposits appears as a dotted signal along the axons of dermal nerves (Figure 2). Indeed, exploiting this protocol in 19 PD patients and 17 controls in skin biopsies at three anatomical site (cervical, thigh and distal leg) we found that 5G4 had 81% of se...

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

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 ...

targeting-alpha-synuclein-aggregates-cutaneous-peripheral-nerve

Research

JoVE Journal

Neuroscience

8.2K Views.  Neurocenter of Southern Switzerland. De diagnose van de ziekte van Parkinson is nog steeds afhankelijk van klinische tekenen van motorische betrokkenheid die later verschijnen in de loop van de ziekte, wanneer de meeste neuronen van de substantia nigra al verloren zijn gegaan. Dit protocol maakt het mogelijk de huidzenuwen analyse door huidbiopsie, dat een potentiële nieuwe biomarker van de ziekte vertegenwoordigt, te worden gebruikt in klinische proeven. Huidbiopsie is een gemakkelijk te beoordelen, niet-invasieve procedure di...

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

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, the olfactory bulb (OB), the dorsal motor nucleus of the vagus (DMV)3, the intermediolateral nucleus of the spinal cord 4 and the substantia nigra, providing the basis for the neuropathological staging of the disease4,5. The ENS and the OB are the most exposed nervous structures and the first ones to be affected. Interestingly, PD has been related to pesticide exposure6-8. Here we show in detail two methods used in our previous study 9. In order to analyze the effects of rotenone acting locally on the ENS, we administered rotenone using a gavage to one-year old C57/BL6 mice. Rotenone is a widely used pesticide that strongly inhibits mitochondrial Complex I 10. It is highly lipophylic and poorly absorbed in the gastrointestinal tract 11. Our results showed that the administration of 5 mg/kg of rotenone did not inhibit mitochondrial Complex I activity in the muscle or the brain. Thus, suggesting that using our administration method rotenone did not cross the hepatoportal system and was acting solely on the ENS. Here we show a method to administer pesticides using a gavage and the image analysis protocol used to analyze the effects of the pesticide in alpha-synuclein accumulation in the ENS. The first part shows a method that allows intragastric administration of pesticides (rotenone) at a desired precise concentration. The second method shows a semi-automatic image analysis protocol to analyze alpha-synuclein accumulation in the ENS using an image analysis software.

Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

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, ...

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83+/-:Gfap-luc+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83+/-:Gfap-luc+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83+/-:Gfap-luc+/-) mice with a median incubation time of 229 &#177;17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83+/-:Gfap-luc+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

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 ...

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 ...

Immunostaining of Neurons Treated with Alpha-Synuclein Aggregates

Source: Panattoni, G., et al. <a href="https://app.jove.com/v/57884">Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation.</a> J. Vis. Exp. (2018)
This video demonstrates the immunostaining of neurons treated with alpha-synuclein (&#945;S) aggregates. The neurons are first treated with &#945;S aggregates. They are then fixed, permeabilized, and treated with a blocking solution. Primary antibodies are added, followed by a fluorescently labeled secondary antibodies and a nuclear stain. The sample is then mounted and observed under a microscope.

1. Neurons Treatment
Note: The treatment has been performed at Division (DIV) 7. All the steps are carried on under a cell culture hood in sterile ...

JoVE Encyclopedia of Experiments

Neuropathology

Neurodegenerative Diseases

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

Source: Vacchi, E., et al. <a href="https://app.jove.com/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 ...

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

Studying Alpha-Synuclein Accumulation in Primary Embryonic Mouse Dopamine Neurons

Source: Er, S. et al., <a href="https://app.jove.com/v/61118">Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons.</a>&#160;J. Vis. Exp. (2020).
This video demonstrates the process of quantifying alpha-synuclein accumulation in embryonic mouse dopamine neurons pre-treated with alpha-synuclein pre-formed fibrils. It outlines the steps for fixing, staining, and quantifying the dopamine neurons containing alpha-synuclein aggregates using immunofluorescence.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1.&#160;Preparation

	Prepare ...

Detection of Phosphorylated Alpha-Synuclein Using Western Blotting

Begin with CSF proteins, including phosphorylated alpha-synuclein, a biomarker of neurodegenerative diseases.
Add a loading buffer containing a detergent, a reducing agent, and a tracking dye.
Heat to denature the proteins, then load them into an SDS-PAGE gel.
Perform electrophoresis to separate the proteins by molecular weight into distinct bands.
Incubate the gel in a blotting buffer with agitation.
Then, electro-transfer the protein bands from the gel onto the membrane.
Treat the membrane with a fixative to retain the protein on the membrane, then wash.
Incubate with a blocking solution containing primary antibodies and phosphatase inhibitors to maintain alpha-synuclein&#39;s phosphorylation.
The antibodies exclusively interact with phosphorylated alpha-synuclein, and the blocking reagents block nonspecific interaction sites, then wash.
Introduce enzyme-conjugated secondary antibodies that interact with primary antibodies, then wash.
Add a chemiluminescent substrate, interacting with enzymes and generating a chemiluminescence.
A chemiluminescent band confirms the presence of phosphorylated alpha-synuclein.

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

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