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ESCRT-mediated uptake and degradation of brain-targeted ?-synuclein single chain antibody attenuates neuronal degeneration in vivo.

ABSTRACT: Parkinson's disease and dementia with Lewy bodies are neurodegenerative disorders characterized by accumulation of ?-synuclein (?-syn). Recently, single-chain fragment variables (scFVs) have been developed against individual conformational species of ?-syn. Unlike more traditional monoclonal antibodies, these scFVs will not activate or be endocytosed by Fc receptors. For this study, we investigated an scFV directed against oligomeric ?-syn fused to the LDL receptor-binding domain from apolipoprotein B (apoB). The modified scFV showed enhanced brain penetration and was imported into neuronal cells through the endosomal sorting complex required for transport (ESCRT) pathway, leading to lysosomal degradation of ?-syn aggregates. Further analysis showed that the scFV was effective at ameliorating neurodegenerative pathology and behavioral deficits observed in the mouse model of dementia with Lewy bodies/Parkinson's disease. Thus, the apoB modification had the effect of both increasing accumulation of the scFV in the brain and directing scFV/?-syn complexes for degradation through the ESCRT pathway, leading to improved therapeutic potential of immunotherapy.

SUBMITTER: Spencer B

PROVIDER: S-EPMC4428402 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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ESCRT-mediated uptake and degradation of brain-targeted α-synuclein single chain antibody attenuates neuronal degeneration in vivo.

Spencer Brian B Emadi Sharareh S Desplats Paula P Eleuteri Simona S Michael Sarah S Kosberg Kori K Shen Jay J Rockenstein Edward E Patrick Christina C Adame Anthony A Gonzalez Tania T Sierks Michael M Masliah Eliezer E

Molecular therapy : the journal of the American Society of Gene Therapy 20140710 10

Parkinson's disease and dementia with Lewy bodies are neurodegenerative disorders characterized by accumulation of α-synuclein (α-syn). Recently, single-chain fragment variables (scFVs) have been developed against individual conformational species of α-syn. Unlike more traditional monoclonal antibodies, these scFVs will not activate or be endocytosed by Fc receptors. For this study, we investigated an scFV directed against oligomeric α-syn fused to the LDL receptor-binding domain from apolipopro ...[more]

PMID: 25008355

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Project description:BackgroundThe aggregation and spread of α-synuclein (α-Syn) protein and related neuronal toxicity are the key pathological features of Parkinson's disease (PD) and Lewy body dementia (LBD). Studies have shown that pathological species of α-Syn and tau can spread in a prion-like manner between neurons, although these two proteins have distinct pathological roles and contribute to different neurodegenerative diseases. It is reported that the low-density lipoprotein receptor-related protein 1 (LRP1) regulates the spread of tau proteins; however, the molecular regulatory mechanisms of α-Syn uptake and spread, and whether it is also regulated by LRP1, remain poorly understood.MethodsWe established LRP1 knockout (LRP1-KO) human induced pluripotent stem cells (iPSCs) isogenic lines using a CRISPR/Cas9 strategy and generated iPSC-derived neurons (iPSNs) to test the role of LRP1 in α-Syn uptake. We treated the iPSNs with fluorescently labeled α-Syn protein and measured the internalization of α-Syn using flow cytometry. Three forms of α-Syn species were tested: monomers, oligomers, and pre-formed fibrils (PFFs). To examine whether the lysine residues of α-Syn are involved in LRP1-mediated uptake, we capped the amines of lysines on α-Syn with sulfo-NHS acetate and then measured the internalization. We also tested whether the N-terminus of α-Syn is critical for LRP1-mediated internalization. Lastly, we investigated the role of Lrp1 in regulating α-Syn spread with a neuronal Lrp1 conditional knockout (Lrp1-nKO) mouse model. We generated adeno-associated viruses (AAVs) that allowed for distinguishing the α-Syn expression versus spread and injected them into the hippocampus of six-month-old Lrp1-nKO mice and the littermate wild type (WT) controls. The spread of α-Syn was evaluated three months after the injection.ResultsWe found that the uptake of both monomeric and oligomeric α-Syn was significantly reduced in iPSNs with LRP1-KO compared with the WT controls. The uptake of α-Syn PFFs was also inhibited in LRP1-KO iPSNs, albeit to a much lesser extent compared to α-Syn monomers and oligomers. The blocking of lysine residues on α-Syn effectively decreased the uptake of α-Syn in iPSNs and the N-terminus of α-Syn was critical for LRP1-mediated α-Syn uptake. Finally, in the Lrp1-nKO mice, the spread of α-Syn was significantly reduced compared with the WT littermates.ConclusionsWe identified LRP1 as a key regulator of α-Syn neuronal uptake, as well as an important mediator of α-Syn spread in the brain. This study provides new knowledge on the physiological and pathological role of LRP1 in α-Syn trafficking and pathology, offering insight for the treatment of synucleinopathies.

Project description:α-synucleinopathy is driven by an imbalance of synthesis and degradation of α-synuclein (αSyn), causing a build up of αSyn aggregates and post-translationally modified species, which not only interfere with normal cellular metabolism but also by their secretion propagates the disease. Therefore, a better understanding of αSyn degradation pathways is needed to address α-synucleinopathy. Here, we used the nerve growth factor-differentiated catecholaminergic PC12 neuronal cell line, which was conferred α-synucleinopathy by inducible expression of αSyn and tubulin polymerization-promoting protein p25α. p25α aggregates αSyn, and imposes a partial autophagosome-lysosome block to mimic aspects of lysosomal deficiency common in neurodegenerative disease. Under basal conditions, αSyn was degraded by multiple pathways but most prominently by macroautophagy and Nedd4/Ndfip1-mediated degradation. We found that expression of p25α induced strong p38MAPK activity. Remarkably, when opposed by inhibitor SB203580 or p38MAPK shRNA knockdown, endolysosomal localization and degradation of αSyn increased, and αSyn secretion and cytotoxicity decreased. This effect was specifically dependent on Hsc70 and the endosomal sorting complex required for transport machinery, but different from classical microautophagy, as the αSyn Hsc70 binding motif was unnecessary. Furthermore, in a primary neuronal (h)-αSyn seeding model, p38MAPK inhibition decreased pathological accumulation of phosphorylated serine-129-αSyn and cytotoxicity. In conclusion, p38MAPK inhibition shifts αSyn degradation from various forms of autophagy to an endosomal sorting complex required for transport-dependent uptake mechanism, resulting in increased αSyn turnover and cell viability in p25α-expressing cells. More generally, our results suggest that under conditions of autophagolysosomal malfunction, the uninterrupted endosomal pathway offers a possibility to achieve disease-associated protein degradation.

Project description:BackgroundThere are currently no disease-modifying therapeutics for Parkinson's disease (PD). Although extensive efforts were undertaken to develop therapeutic approaches to delay the symptoms of PD, untreated α-synuclein (α-syn) aggregates cause cellular toxicity and stimulate further disease progression. PROTAC (Proteolysis-Targeting Chimera) has drawn attention as a therapeutic modality to target α-syn. However, no PROTACs have yet shown to selectively degrade α-syn aggregates mainly owing to the limited capacity of the proteasome to degrade aggregates, necessitating the development of novel approaches to fundamentally eliminate α-syn aggregates.MethodsWe employed AUTOTAC (Autophagy-Targeting Chimera), a macroautophagy-based targeted protein degradation (TPD) platform developed in our earlier studies. A series of AUTOTAC chemicals was synthesized as chimeras that bind both α-syn aggregates and p62/SQSTM1/Sequestosome-1, an autophagic receptor. The efficacy of Autotacs was evaluated to target α-syn aggregates to phagophores and subsequently lysosomes for hydrolysis via p62-dependent macroautophagy. The target engagement was monitored by oligomerization and localization of p62 and autophagic markers. The therapeutic efficacy to rescue PD symptoms was characterized in cultured cells and mice. The PK/PD (pharmacokinetics/pharmacodynamics) profiles were investigated to develop an oral drug for PD.ResultsATC161 induced selective degradation of α-syn aggregates at DC50 of ~ 100 nM. No apparent degradation was observed with monomeric α-syn. ATC161 mediated the targeting of α-syn aggregates to p62 by binding the ZZ domain and accelerating p62 self-polymerization. These p62-cargo complexes were delivered to autophagic membranes for lysosomal degradation. In PD cellular models, ATC161 exhibited therapeutic efficacy to reduce cell-to-cell transmission of α-syn and to rescue cells from the damages in DNA and mitochondria. In PD mice established by injecting α-syn preformed fibrils (PFFs) into brain striata via stereotaxic surgery, oral administration of ATC161 at 10 mg/kg induced the degradation of α-syn aggregates and reduced their propagation. ATC161 also mitigated the associated glial inflammatory response and improved muscle strength and locomotive activity.ConclusionAUTOTAC provides a platform to develop drugs for PD. ATC161, an oral drug with excellent PK/PD profiles, induces selective degradation of α-syn aggregates in vitro and in vivo. We suggest that ATC161 is a disease-modifying drug that degrades the pathogenic cause of PD.

Dataset Information

ESCRT-mediated uptake and degradation of brain-targeted ?-synuclein single chain antibody attenuates neuronal degeneration in vivo.

Publications

ESCRT-mediated uptake and degradation of brain-targeted α-synuclein single chain antibody attenuates neuronal degeneration in vivo.

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