A β-Wrapin Targeting the N-Terminus of α-Synuclein Monomers Reduces Fibril-Induced Aggregation in Neurons

Abstract

Reducing α-synuclein pathology constitutes a plausible strategy against Parkinson’s disease. As we recently demonstrated, the β-wrapin protein AS69 binds an N-terminal region in monomeric α-synuclein, interferes with fibril nucleation, and reduces α-synuclein aggregation in vitro and in a fruit fly model of α-synuclein toxicity. The aim of this study was to investigate whether AS69 also reduces α-synuclein pathology in mammalian neurons. To induce α-synuclein pathology, primary mouse neurons were exposed to pre-formed fibrils (PFF) of human α-synuclein. PFF were also injected into the striatum of A30P-α-synuclein transgenic mice. The extent of α-synuclein pathology was determined by phospho-α-synuclein staining and by Triton X-100 solubility. The degeneration of neuronal somata, dendrites, and axon terminals was determined by immunohistochemistry. AS69 and PFF were taken up by primary neurons. AS69 did not alter PFF uptake, but AS69 did reduce PFF-induced α-synuclein pathology. PFF injection into mouse striatum led to α-synuclein pathology and dystrophic neurites. Co-injection of AS69 abrogated PFF-induced pathology. AS69 also reduced the PFF-induced degeneration of dopaminergic axon terminals in the striatum and the degeneration of dopaminergic dendrites in the substantia nigra pars reticulata. AS69 reduced the activation of astroglia but not microglia in response to PFF injection. Collectively, AS69 reduced PFF-induced α-synuclein pathology and the associated neurodegeneration in primary neurons and in mouse brain. Our data therefore suggest that small proteins binding the N-terminus of α-synuclein monomers are promising strategies to modify disease progression in Parkinson’s disease.

Constitutively active STING causes neuroinflammation and degeneration of dopaminergic neurons in mice

“…To quantify the number of dopaminergic neurons in the substantia nigra (SN), every fifth section throughout the entire SN was stained for tyrosine hydroxylase (TH) as previously ( Szegő et al, 2021 ). In brief, after blocking (2% bovine serum albumin, 0,3% Triton X-100 in TBS; 1 hr RT), sections were incubated with the primary antibody in blocking solution (two overnights), followed by the fluorescently labelled secondary antibody (Alexa 488 conjugated donkey anti-sheep, 1:2000, overnight).…”

Constitutively active STING causes neuroinflammation and degeneration of dopaminergic neurons in mice

“…t-test, Mann-Whitney test or two-way ANOVA were performed using GraphPad Prism (Versions 5.01 and 9.0.0). Linear regression was performed using R. For generalized linear mixed-effects model (Szegő et al, 2021), animal, section and image were used as random effects nested within each other (R package: lme4). p values are indicated in the graphs by symbols with * representing p<0.05, ** representing p<0.01, *** representing p<0.001.…”

“…To quantify the number of dopaminergic neurons in the substantia nigra (SN), every fifth section throughout the entire SN was stained for tyrosine hydroxylase (TH) as previously (Szegő et al, 2021). In brief, after blocking (2 % bovine serum albumin, 0,3 % Triton X-100 in TBS; 1 h RT), sections were incubated with the primary antibody in blocking solution (two overnights), followed by the fluorescently labelled secondary antibody (Alexa 488 conjugated donkey anti-sheep, 1:2000, overnight).…”

“…ttest, Mann-Whitney test or two-way ANOVA were performed using GraphPad Prism (Versions 5.01 and 9.0.0). Linear regression was performed using R. For generalized linear mixed-effects model (Szegő et al, 2021)…”

Alpha-Synuclein Aggregation Pathway in Parkinson’s Disease: Current Status and Novel Therapeutic Approaches

“…To stabilize partially structured α-Syn monomers, engineered proteins such as β-wrapin AS69, which binds to the α-Syn N-terminus with high affinity, were employed [ 97 , 232 ]. β-Wrapins are proteins that can bind to α-Syn, generated by random mutagenesis in the gene encoding ZAβ3, a homodimer linked via the S–S bridge that can bind to the amyloid-β peptide.…”