Co-Transmission of Alpha-Synuclein and TPPP/p25 Inhibits Their Proteolytic Degradation in Human Cell Models

Abstract

The pathological association of alpha-synuclein (SYN) and Tubulin Polymerization Promoting Protein (TPPP/p25) is a key factor in the etiology of synucleinopathies. In normal brains, the intrinsically disordered SYN and TPPP/p25 are not found together but exist separately in neurons and oligodendrocytes, respectively; in pathological states, however, they are found in both cell types due to their cell-to-cell transmission. The autophagy degradation of the accumulated/assembled SYN has been considered as a potential therapeutic target. We have shown that the hetero-association of SYN with TPPP/p25 after their uptake from the medium by human cells (which mimics cell-to-cell transmission) inhibits both their autophagy- and the ubiquitin-proteasome system-derived elimination. These results were obtained by ELISA, Western blot, FACS and immunofluorescence confocal microscopy using human recombinant proteins and living human cells; ANOVA statistical analysis confirmed that TPPP/p25 counteracts SYN degradation by hindering the autophagy maturation at the stage of LC3B-SQSTM1/p62-derived autophagosome formation and its fusion with lysosome. Recently, fragments of TPPP/p25 that bind to the interface between the two hallmark proteins have been shown to inhibit their pathological assembly. In this work, we show that the proteolytic degradation of SYN on its own is more effective than when it is complexed with TPPP/p25. The combined strategy of TPPP/p25 fragments and proteolysis may ensure prevention and/or elimination of pathological SYN assemblies.

Anti-Aggregative Effect of the Antioxidant DJ-1 on the TPPP/p25-Derived Pathological Associations of Alpha-Synuclein

“…TPPP/p25 and SYN are co-enriched and co-localized in the inclusion bodies of the post-mortem brain tissue of patients with parkinsonism due to their cell-to-cell transmission [ 11 , 38 ]. Recently, we have revealed that the excess SYN and TPPP/p25 can be degraded by autophagy and/or ubiquitin–proteasome system, and their eliminations are inhibited in the presence of specific inhibitors, such as chloroquine and MG132, respectively, indicating their intracellular degradation [ 34 ]. In addition, we have reported that the hetero-association of the two hallmark proteins counteract their degradation [ 34 ].…”

“…In this set of experiments, the effect of DJ-1 on the degradation of the excess SYN complexed with TPPP/p25 was detected and quantified. SYN and/or TPPP/p25 were taken up from the medium by human cells to mimic the pathological circumstances (cell-to-cell transmission) [ 34 ]. Now, both HeLa and SH-SY5Y cells were used as cell models to monitor the anti-aggregative effect of DJ-1.…”

“…For cellular experiments, 5 × 10 4 HeLa or SH-SY5Y cells (in 24-well plates) were transfected with DJ-1-expressing plasmid using the Turbofect (Thermo Fisher Scientific) transfection reagent according to the manufacture’s protocol. As the control, non-transfected cells were used as described previously [ 34 ]. The human recombinant SYN and/or TPPP/p25 were taken up from the medium, and then the cells were incubated for a further 4 h. The concentrations of TPPP/p25 and SYN were 80 nM and 800 nM, respectively.…”

“…The visualization of the hetero-associations of the proteins was archived by detecting the BiFC signal originated from transfections of coverslip-plated HeLa cells with the mVenus BiFC constructs of the proteins, as described previously [ 28 , 34 ]. Nuclei were counterstained with Hoescht 33342.…”

“…For the visualization and quantification of the protein bands, specific antibodies were used as follows: a rabbit polyclonal GFP antibody (for mVenus-DJ-1), a rabbit polyclonal SYN antibody, a rat polyclonal TPPP/p25 antibody and a mouse monoclonal beta-actin antibody, sequentially ( Table 1 ). Antibody binding was visualized by the appropriate IgG-peroxidase conjugate by a Bio-Rad ChemiDoc MP Imaging system (Bio-Rad, Hercules, CA, USA) using Immobilon Western substrate (Merck), as described previously [ 34 ]. For densitometric analysis, the intensity of the bands was analyzed by ImageJ 1.49.…”