Skin sympathetic fiber α-synuclein deposits

Abstract

Our study demonstrated that a search for neuritic inclusions of phosphorylated α-synuclein in the skin sympathetic nerve fibers could provide a sensitive in vivo biomarker for degenerative peripheral autonomic neuropathy and may shed more light on the pathogenesis of PAF.

Skin nerve α-synuclein deposits

“…31 This deposition pattern differed from that found in pure autonomic failure where abnormal deposits were selectively found in autonomic fibers. 18 Unlike pure autonomic failure, current evidence also suggests a potential phosphorylated a-synuclein spread from spinal ganglia along the peripheral nervous system in IPD. This conclusion is made because, first, phosphorylated a-synuclein deposits showed a proximaldistal gradient with the highest rate of positivity in the cervical site and lowest rate in the leg.…”

“…Twelve free-floating sections were incubated overnight with a panel of primary antibodies, including the mouse or rabbit panneuronal marker protein gene product (PGP) 9.5 (1:800; Biogenesis, Poole, UK), mouse collagen IV (1:800; Chemicon, Temecula, CA), and autonomic markers such as rabbit dopamine-b-hydroxylase (DbH) (1:150; Chemicon) to identify the noradrenergic fibers 17 and rabbit vasoactive intestinal peptide (VIP) (1:1,000; Incstar, Stillwater, MN), colocalized in the sudomotor cholinergic fibers. 16 Sections were then washed and secondary antibodies, labeled with cyanine dye fluorophores 2 and 3.18 (1:400; Jackson ImmunoResearch, West Grove, PA), were added for an overnight incubation (see Donadio et al 18 for further details). Sections were initially viewed under a Zeiss fluorescent microscope (model Axioskop 40; Zeiss, Jena, Germany).…”

“…Additional 10-mm sections from the same skin sample were obtained during the freezing sliding microtome session to evaluate a-synuclein deposits. 18 Usually 4 thin sections 50 mm apart were analyzed for each skin sample. They were double-immunostained overnight with a panel of primary antibodies including mouse phosphorylated a-synuclein at Ser 129 (p-syn, 1:1,000; Wako Chemicals, Richmond, VA), rabbit DbH, VIP, or PGP (see above).…”

“…Sections were then washed, and secondary antibodies were added for an incubation of 1 hour. As secondary antibodies, an anti-rabbit Alexa Fluor 488 (1:200; Jackson ImmunoResearch, West Grove, PA) and antimouse Jackson cyanine dye fluorophores 3.18 (1:200) were used (see Donadio et al 18 for further details). Sections were initially viewed and analyzed under a Zeiss fluorescent microscope.…”

“…The extreme variability of the area or degree of innervation of skin structures showing a synuclein staining in the majority of patients (i.e., nerve bundles or arterioles) hampered the use of a relative automated quantification of phosphorylated a-synuclein staining with a possible standardization among different patients as previously made. 18 For a 3-dimensional study of the innervation pattern (50-mm sections) and a colocalization analysis (10-mm sections doublestained with a-synuclein and DbH or VIP), digital images were also acquired using a laser-scanning confocal microscope (Leica DMIRE 2, TCS SL; Leica Microsystems, Heidelberg, Germany). Each image was collected in successive frames of 1-to 2-mm increments on a Z-stack plan at the appropriate wavelengths for secondary antibodies with a 320 or 340 plan apochromat objective and subsequently projected to obtain a double-stained, 3-dimensional digital image by a computerized system (LCS Lite; Leica Microsystems).…”