Synuclein Analysis in Adult Xenopus laevis

Abstract

The α-, β- and γ-synucleins are small soluble proteins expressed in the nervous system of mammals and evolutionary conserved in vertebrates. After being discovered in the cartilaginous fish Torpedo californica, synucleins have been sequenced in all vertebrates, showing differences in the number of genes and splicing isoforms in different taxa. Although α-, β- and γ-synucleins share high homology in the N-terminal sequence, suggesting their evolution from a common ancestor, the three isoforms also differ in molecular characteristics, expression levels and tissue distribution. Moreover, their functions have yet to be fully understood. Great scientific interest on synucleins mainly derives from the involvement of α-synuclein in human neurodegenerative diseases, collectively named synucleinopathies, which involve the accumulation of amyloidogenic α-synuclein inclusions in neurons and glia cells. Studies on synucleinopathies can take advantage of the development of new vertebrate models other than mammals. Moreover, synuclein expression in non-mammalian vertebrates contribute to clarify the physiological role of these proteins in the evolutionary perspective. In this paper, gene expression levels of α-, β- and γ-synucleins have been analysed in the main organs of adult Xenopus laevis by qRT-PCR. Moreover, recombinant α-, β- and γ-synucleins were produced to test the specificity of commercial antibodies against α-synuclein used in Western blot and immunohistochemistry. Finally, the secondary structure of Xenopus synucleins was evaluated by circular dichroism analysis. Results indicate Xenopus as a good model for studying synucleinopathies, and provide a useful background for future studies on synuclein functions and their evolution in vertebrates.

Keywords: Western blot; Xenopus laevis; qRT-PCR; recombinant proteins; synuclein.

Figure 1

Alignment of syn amino acid sequences. Comparisons among human and Xenopus α- (a), β- (b) and γ- (c) syns, among Xenopus α- and β-syns (d), and among α- and γ-syns (e). (a) Conserved repeats of the apolipoprotein lipid-binding motif [EGS]-KT-K-[EQ]-[GQ]-V-XXXX are underlined. The non-amyloid-component (NAC) region of α-syn, the phosphorylatabletyrosines and serines are highlighted in grey, green and yellow, respectively. The methionines representing binding sites for Mn(II) and other metals are highlighted in red. Negative amino acids in the CT region are indicated in bold. The amino acids that in humans are involved in the pathological mutations linked to Parkinson’s disease are shown in red. The aa stretch GVTAVAQKTVE that is directly involved in the formation of human amyloid fibrils is double underlined. The sequences were aligned with Clustal Omega. Asterisks indicate identity of amino acids; double dots indicate amino acids with the same polarity or size; dots indicate semiconserved substitutions. The epitope recognized by the ab27766 antibody is dotted underlined.

Figure 2

Syn gene expression in the major organs of adult Xenopus. qRT-PCR analysis of α- (a), β- (b) and γ- (c) syn gene expression in the main organs of adult Xenopus. Expression levels were normalized against GAPDH and expressed as fold change relative to brain sample. Br: brain, SC: spinal cord, E: eye, Mu: muscle, He: heart, St: stomach, In: intestine, Li: liver, Sp: spleen Ki: kidney, Lu: lung, Sk: skin.

Figure 3

SDS-PAGE analysis of purified recombinant Xenopus α-syn (Xsynα). (Left panel), purified GST-Xsynα before and after treatment with thrombin. (Right panel), GSH-Sepharose chromatography fractions: GST-Xsynα treated with thrombin, flow-through (FT), Xsynα and GST recovered in the wash and GSH-eluted fractions, respectively; MW: molecular weight markers.

Figure 4

Western blot analysis of α-syn expression by ab27766 antibody. Validation of the antibody on Xenopus (Xα, Xβ, Xγ) and Cyprinus carpio (Cβ and Cγ) recombinant syns (a,b). α-syn expression in the main Xenopus organs (c,h). Red ponceau staining is shown in (a,c,e). α-syn immunolabelling in (b,d,f,g). Negative control (primary antibody omitted) in (h). Br: brain; Ey: eye; He: heart; In: intestine; Ki: kidney; Li: liver; Lu: lung; Mu: skeletal muscle; Ne: nerve; SC: spinal cord; Sk: skin; Sp: spleen; St: stomach; Xα, Xβ and Xγ: Xenopus recombinant α-, β- and γ-syn, respectively; Cβ and Cγ: carp recombinant β- and γ-syn, respectively.

Figure 5

Immunohistochemical analysis of the α-syn distribution. Xenopus brain coronal sections (a–d). Strong α-syn immunoreactivity was found in the interpeduncular nucleus (a,c) and in the visual projections, tractus opticus marginalis (d). Retina (e,f). The strongest α-syn immunoreactivity was found in the thick inner plexiform layer (white arrow) and in the outer plexiform layer (white arrowhead) (e). No immunoreactivity was found in control sections (b,f). The α-syn immunoreactivity was found in motor nerve endings within skeletal muscle (longitudinal (g), and transverse section (h), arrows) and heart muscle (i,j). α-syn immunolabelled nerve fibres were found also within all layers of the stomach wall (k,l). Some sections have been counterstained with Nuclear Fast Red Solution. IN: interpeduncular nucleus; optma: tractus opticus marginalis. Bar = 100 µm.

Figure 6

Fluorescence spectra of purified recombinant Xenopus syns. Protein concentration was 0.11 mg/mL for α- and β-syn, and 0.24 mg/mL for γ-syn.

Figure 7

CD spectra of purified recombinant Xenopus syns. The proteins were diluted in 10 mM potassium phosphate buffer pH 7, containing 50 mM Na₂SO₄ (a); SDS was added at 10 mM (b), while CuSO₄ was added at 100 µM final concentration (c). The spectra are normalized for protein concentration.

Figure 8

CD spectra of Xenopus α-syn. The protein (10 μM) in 10 mM potassium phosphate buffer pH 7, containing 50 mM Na₂SO₄, was incubated at 37 °C for the specified times.

Figure 9

Xenopus laevis: a potential model for the study of synucleins.