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

The diagnosis of Parkinson's disease still relies on clinical signs of motor involvement that appear later on in the course of the disease, when most of the neurons of the substantia nigra are already lost. This protocol allows the dermal nerves analysis by skin biopsy, that represents a potential new biomarker of the disease, to be used in clinical trials. Skin biopsy is an easily assessable, not-invasive procedure that allows the analysis of peripheral nervous tissue that is prone to the pathology.

Alpha synuclein aggregates detection by skin biopsy can be used for diagnostic purposes, possibly at early phases, when potential cure is most effective. Follow-up skin biopsies in the same patient allow a time and special course analysis of alpha synuclein aggregate spreading in human cutaneous nervous system. These can shed light on the pathogenesis of disease.

To begin this procedure, prepare a fresh PLP fixative solution as outlined in Table One of the text protocol. Immediately after collecting skin biopsy, submerge it in a tube containing 10 milliliters of the fixative solution. Incubate overnight at four degrees Celsius.

The next day, in a fume hood, remove the PLP fixative gently. In the same tube, wash the biopsy three times for five minutes each, using five milliliters of 0.1 molar Sorensen's solution for each wash. Discard the Sorensen's solution, and add five milliliters of cryoprotectant solution.

Incubate overnight at four degrees Celsius. The next day, store the biopsy at four degrees Celsius if the cut with a cryotome is to be performed within one week. First, set the cryotome to minus 20 degrees Celsius.

Take a cryomold and fill it up with cryo-embedding medium. Using tweezers, immerse the biopsy into the cryo-embedding medium, with the longitudinal axis parallel to the bottom of the cryomold. Snap freeze the sample with liquid nitrogen, to obtain a solid cube of cryo-embedding medium containing the biopsy in the right orientation.

Put the sample in the cryostat, and wait for 30 minutes to allow the biopsy to acclimatize. Then, fix the sample on the cryostat and cut into 50-micrometer sections. Using a small brush, transfer the cryo-sections to a 96-well plate containing 200 microliters of antifreeze solution in each well.

After this, store the plate at minus 20 degrees Celsius. To begin, fill some of the wells of a fresh 96-well plate with 100 microliters of washing solution. Transfer the sections to be analyzed from the storage plate to the one containing the washing solution.

Leave the sections in the washing solution for 10 minutes at room temperature. Then, transfer each section into an empty well containing the same washing solution, and repeat the wash. Next, add washing solution to the sections, and transfer them into new wells containing 100 microliters of blocking solution.

Incubate at room temperature for at least 90 minutes, and a maximum of four hours. Dilute the primary antibodies, anti-PGP9.5 and anti-5G4, in the working solution. Transfer the sections into new wells containing 100 microliters of the working solution of the primary antibodies, and incubate overnight at room temperature.

The next day, wash the sections in wells containing washing solution as previously described. Dilute the secondary antibodies, Goat anti-Rabbit to detect PGP9.5, and Goat anti-Mouse to detect 5G4, in the working solution. Transfer the washed sections into new wells containing 100 microliters of the working solution of the secondary antibodies.

Cover the plate with aluminum foil to avoid the bleaching of fluorophore conjugated with secondary antibodies, and incubate at room temperature for 90 minutes. After this, wash the sections two times in washing solution as previously described. Transfer the washed sections into new wells containing 100 microliters of DAPI for five minutes at room temperature.

Wash the sections two times in washing solution as previously described. Then, mount the sections on a slide in the correct position, avoiding misfolding. Add a few drops of mounting medium to the slide and cover the section with a cover slip.

Let the slides dry overnight. The next day, store the slides in an appropriate box at four degrees Celsius, making sure to avoid light exposure. Using an inverted fluorescence microscope or a confocal microscope, view the sections.

Acquire the images by a camera connected to the microscope. Then, use a fluorescence microscope or a confocal microscope to analyze positive signals in sections in terms of spatial distribution and intensity of the signal as outlined in the text protocol. Following the described method, alpha synuclein aggregates labeled with 5G4 antibody are detected in dermal nerve fascicles innervating autonomic structures of PD patients.

The morphology of alpha synuclein deposits appears as a dotted signal along the axons of dermal nerves. Representative use of this protocol in 19 PD patients and 17 controls in skin biopsies at three anatomical sites shows that 5G4 has a sensitivity of 81%and a specificity of 86%when compared to healthy controls. Phosphorylated alpha synuclein has a sensitivity of 56%and a specificity of 100%5G4 and phosphorylated alpha synuclein-positive deposits are found mainly around sweat glands, but are also found in muscle arrector pili, small vessels, and subepidermal and dermal plexus, but never in intraepidermal nerve fibers.

Generally, within the sweat glands lumen, it is possible to observe a nonspecific signal that could be misinterpreted as 5G4 or phosphorylated alpha synuclein positivity. This type of signal is dotted, spherical, and it is due most probably to intraluminal autofluorescent material as demonstrated in technical controls without primary and secondary antibodies. Colocalization with PGP9.5 that marks the nerve fibers, which morphologically are filamentous and elongated, helps to identify the correct signal.

Therefore, the specificity of 5G4 signal is highly increased by a double immunostaining with an axonal marker. An accurate fixation of the biopsy is necessary for producing a good-quality immunofluorescent staining and for the reliable interpretation of the fluorescent signals. If the fixative is not correctly prepared, or if an over-fixation has occurred, the result will be a high autofluorescence that will mask the signal of nerve fibers crossing the dermal-epidermal junction, or innervate the main dermal structures.

The most critical step of this procedure is tissue fixation. Pay attention to the pH of PLP fixative solution, and timing of incubation, to avoid autofluorescence and a specific signal.