Aug 2025· bioRxiv· Vol 17· 1 citation· 98 references
MedicineBiology
TL;DR
The findings indicate that RNA aptamers, such as 1R6, may represent promising therapeutic candidates for synucleinopathies, thus opening new avenues in the treatment of these diseases.
Abstract
The aggregation of α-synuclein (αSyn), a 140-mer protein, has been implicated in the pathogenesis of Parkinson’s disease, multiple system atrophy, and dementia with Lewy bodies. KTKEGV repeats (KR) of αSyn are key mediators of prion-like propagation and neurodegeneration. Despite the availability of symptomatic treatments, no current therapy effectively delays disease progression. Here we report a 77-nucleotide (nt) RNA aptamer (1R6) with potent affinity and selectivity for αSyn1-95 (KD = 18 nM) through in vitro selection. 1R6 significantly inhibited αSyn oligomerization and β-sheet-rich fibril assembly and promoted disaggregation of preformed fibrils. Additionally, 1R6 suppressed αSyn seeding, as determined by FRET-based cellular biosensor cell assay. Cellular studies revealed that 1R6 cotransfection completely prevented αSyn-induced cytotoxicity. To assess the protective effects of 1R6 in vivo, we used a Drosophila melanogaster model expressing human αSyn in neurons. Flies fed with 1R6 showed improved locomotor defects, reduced photoreceptor degeneration, and decreased αSyn levels in the head. Structural characterization through 1H-15N heteronuclear multiple quantum correlation nuclear magnetic resonance experiments demonstrated that 1R6 targets KR motifs, a finding further supported by in silico simulations. Our findings indicate that RNA aptamers, such as 1R6, may represent promising therapeutic candidates for synucleinopathies, thus opening new avenues in the treatment of these diseases.
Accumulation of misfolded α-synuclein protein in intracellular inclusion bodies of dopaminergic neurons underlies the pathogenesis of Synucleinopathies, which include Parkinson’s Disease (PD) and Dementia with Lewy Bodies (DLB). Therefore, preventing the accumulation of misfolded α-synuclein in dopaminergic neurons or their clearance could in principle offer intervention strategies against Synucleinopathies, which currently remain untreatable. In this study, we employ the Affinity-directed PROtein Missile (AdPROM) system consisting of the substrate receptor of the CUL2-E3 ligase complex VHL and a nanobody selectively recognising the human α-synuclein protein and demonstrate targeted degradation of endogenous α-synuclein from human cell lines with remarkable selectivity. We further demonstrate that targeted degradation of α-synuclein impedes the pre-formed fibril (PFF)-induced aggregation of α-synuclein in primary neurons derived from rats expressing human α-synuclein. This approach also represents the first demonstration of nanobody-guided proteasomal degradation of some reported clinically relevant α-synuclein variants.
Bill Carton, Géraldine Gelders, Gajanan Sathe et al.· npj Parkinson's Disease· 0 citations
α-Synucleinopathies are devastating neurodegenerative diseases characterized by pathological accumulation of a neuronal protein, α-synuclein (αSyn). Lowering soluble αSyn levels is a promising therapeutic strategy to limit aggregation and neurotoxicity, but directly targeting this protein is hindered by its intrinsically disordered structure and other factors, such as its conformational heterogeneity and intracellular drug delivery barriers. Consequently, increasing attention has been directed toward targeting the SNCA transcript, which encodes αSyn. Here, we developed phosphorodiamidate morpholino oligonucleotide (PMO)-based RNA-degrading chimeras (RDCs) that selectively bind the 5' untranslated region of SNCA messenger RNA (mRNA) and recruit RNase L for targeted RNA degradation. Through the systematic evaluation of nine RDCs, we identified and optimized 4-D1, which effectively reduced SNCA mRNA and αSyn protein expression in HEK293T cells in an RNase L-dependent manner. 4-D1 lowered SNCA transcript and αSyn protein levels in both primary cortical neurons from humanized SNCA mice and in human induced pluripotent stem cell-derived cortical neurons. This reduction prevented prion-like seeding induced by patient-derived αSyn fibrils and protected neurons from fibril-induced cytotoxicity. Finally, in vivo studies confirmed the efficacy of 4-D1 in reducing αSyn mRNA expression in humanized SNCA mice. These findings indicate that PMO-based RDCs may represent a promising therapeutic modality for α-synucleinopathies.
Ning Wang, Shalakha Hegde, Zhichao Tang et al.· Proceedings of the National...· 0 citations
Alpha-synuclein (αS) is a neuronal protein implicated in synaptic regulation, whose pathological aggregation is a hallmark of synucleinopathies, including Parkinson's disease. Despite its physiological importance, its intrinsically disordered nature and conformational plasticity complicate both mechanistic understanding and therapeutic targeting. Here, we investigate the use of protein-functionalized nanoparticles (NPs) as tools to modulate and probe αS assembly pathways, including aggregation and liquid-liquid phase separation. We designed three nanoconjugates by covalently attaching single-cysteine αS variants at positions 18, 76, and 140, spanning the protein's three functional regions, onto PEG-coated silica NPs. These conjugates preserved the disordered character of αS, exhibited low cytotoxicity, and were internalized into neuronal cells. Functional assays revealed site-dependent effects on aggregation: NP-αSA76C significantly delayed αS fibril formation and further inhibited aggregation of the amyloidogenic tau protein, suggesting interference with nucleation mechanisms, while NP-αSA18C modestly accelerated aggregation and showed strong affinity for mature fibrils. All nanoconjugates partitioned into αS condensates, though without marked site-specific differences, and interacted with model lipid membranes. Overall, αS-functionalized NPs influence protein aggregation and condensate interactions, with context-dependent site specificity. These findings highlight the importance of conjugation position in tuning NP-protein interactions and underscore the potential of these nanoconjugates for investigating and modulating pathological protein assembly in neurodegenerative diseases.
Marco Carrello, Filippo Busatta, Francisco Ramos-Martín et al.· International Journal of Bio...· 0 citations
The structure of α-synuclein is predominantly α-helical when bound to cellular membranes. However, under pathological or destabilizing conditions, this α-helical structure transitions into β-sheet-rich conformations, promoting protein aggregation and formation of Lewy bodies (LBs) in neurodegenerative disorders such as Parkinson's disease dementia (PDD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), and Alzheimer's disease (AD). DJ-1 is a multifunctional brain protein acting as a chaperone or protease under oxidative stress and associating with abnormal protein aggregates. Although previous studies suggested DJ-1 inhibits α-synuclein aggregation, the structural basis remained elusive. Here, we elucidate the direct interaction between α-synuclein and DJ-1 using size-exclusion chromatography (SEC), fluorescence spectroscopy, and multi-angle light scattering (MALS), and determined their crystal structure by X-ray diffraction. The interaction interface was mapped to residues Q24, E28, A29, and N65 of α-synuclein and K148, N173, and Q180 of DJ-1, with Q180 forming strong hydrogen bonds (2.60-2.97 Å) with α-synuclein. Transmission electron microscopy (TEM) demonstrated DJ-1 suppresses α-synuclein fibril formation. Based on the structure, we designed a DJ-1-derived peptide (173-180) that significantly inhibited α-synuclein aggregation in TEM and ELISA assays, suggesting its potential as a therapeutic candidate for α-synucleinopathies.
Hyeon Jin Kim, Da Hye Kim, C. Han et al.· International Journal of Bio...· 0 citations
Parkinson’s disease (PD) is a progressive neurodegenerative disorder that affects over 12 million people worldwide. A central pathological feature is the accumulation of aggregated alpha-synuclein (αS) in Lewy bodies and Lewy neurites. Engineered αS variants such as 3K (E35K+E46K+E61K) and KLK (KTKEGV→KLKEGV in 6 repeats) have been shown to enhance membrane binding and aggregation propensity, contributing to cellular toxicity. To further investigate the impact of 3K and KLK on αS biology, we developed a sensitive assay in a human neuroblastoma model to assess expression levels and cytotoxicity. Relative to wild-type αS, the 3K mutant exhibited reduced steady-state expression and increased toxicity, consistent with prior reports. In contrast, the KLK mutant showed no marked change in protein expression but induced significantly higher toxicity, more than the 3K variant. Furthermore, stearoyl-CoA desaturase (SCD) inhibition partially rescued the toxicity of 3K and KLK αS, with 3K showing greater restoration of cell growth than KLK, validating the sensitivity of our live-cell assay to detect pharmacological responses. These findings underscore the utility of our assay in dissecting disease-relevant mechanisms and highlight the potential of engineered αS variants to model pathogenic features of PD. This platform offers a versatile tool for evaluating therapeutic strategies targeting αS aggregation and toxicity in PD and related synucleinopathies.
Bao-Yi Li, A. K. Anderson, Heba Alnakhala et al.· Journal of Molecular Neurosc...· 0 citations
Synucleinopathies are a group of neurodegenerative disorders characterized by the accumulation of aggregated α-synuclein (α-syn), including Parkinson’s disease, Dementia with Lewy Bodies, and Multiple System Atrophy. These diseases are marked by locomotor and non-motor impairments, as well as mitochondrial dysfunction and the loss of dopaminergic (DA) neurons. We have developed several anti-α-syn single-domain antibodies (sdAbs) and demonstrated the diagnostic imaging potential of two of them and the acute therapeutic benefit of one in clearing α-syn in a mouse model. However, whether these sdAbs can suppress α-syn-mediated neuronal loss and locomotor impairment in vivo remains unclear. We evaluated the therapeutic potential of five anti-α-syn sdAbs to clear pathological α-syn in mouse neuronal culture and then demonstrated their in vivo efficacy in a Drosophila model of synucleinopathy. The sdAbs differed in their efficacy to lower levels of phospho-serine 129 α-syn, prevent loss of DA neurons, alleviate mitochondrial dysfunction, improve motor function, and prolong survival in synucleinopathy flies. The most effective sdAb, 2H1, has not been reported before. It binds strongly to the aggregation prone region of α-syn and robustly improves all these disease parameters. Additionally, that sdAb is associated with α-syn in the fly neurons, as shown through proximity dependent turboID biotinylation assays. The sdAb-turboID also biotinylated α-syn-associated proteins involved in synapse/vesicle trafficking pathways, pinpointing the location of their intracellular interaction. Our findings provide an insight into the therapeutic mechanism of action of these sdAbs and strongly support their clinical development.
Pragati, E. Congdon, Yixiang Jiang et al.· bioRxiv· 0 citations
