Chemoselective Semisynthesis of Covalent Nanobody-Guided Protein Degraders in Neurons

Chemoselective Semisynthesis of Covalent Nanobody-Guided Protein Degraders in Neurons | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Chemoselective Semisynthesis of Covalent Nanobody-Guided Protein Degraders in Neurons Nam Chu, Nhat Le, Niyi Adelakun, Ouada Nebie, Upendra Nayek, Ryejun Na, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8652640/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The selective removal of pathological proteins represents a promising strategy for treating neurodegenerative diseases driven by protein aggregation and synaptic dysfunction. Here, we present a chemoselective semisynthesis platform that uses expressed protein ligation (EPL) to generate nanobody-guided degraders (nanodegraders, NDs) incorporating SuFEx (sulfur–fluoride exchange) covalent modules for proximity-enabled crosslinking, azido-lysine handles for E3-ligand conjugation, and a chaperone-mediated autophagy (CMA1) motif for lysosomal targeting. Subsequent attachment of a cell-penetrating peptide (CPP) enables intracellular delivery, yielding α-synuclein (α-Syn) NDs that engage lysosomal, proteasomal, or dual-proteolytic degradation pathways. Dual-proteolytic NDs revealed route competition rather than synergy, defining design limits for multi-route degradation in neurons. The optimized covalent lysosome-targeting ND efficiently internalized into human iPSC-derived A53T α-Syn neurons, cleared aggregated α-Syn, restored calcium alterations and synaptotoxic effects, and remained stable and selective in vivo. This work establishes a modular chemical strategy for engineering nanobody-based degraders that dismantle neurotoxic proteins through multiple proteolytic systems. Health sciences/Diseases/Neurological disorders/Neurodegenerative diseases Biological sciences/Chemical biology/Chemical modification Biological sciences/Chemical biology/Protein folding/Protein aggregation Biological sciences/Chemical biology/Chemical tools Biological sciences/Chemical biology/Protein design Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The accumulation of misfolded and aggregation-prone proteins is a defining feature of many neurodegenerative disorders, including Parkinson’s, Alzheimer’s, Huntington’s and prion diseases. Aberrant protein aggregation disrupts proteostasis, impairs synaptic function, and drives neuronal death 1 . While pharmacological strategies suppress the expression or activity of individual proteins, these approaches rarely eliminate the existing pathological protein species that drive neurodegeneration 2,3 . Developing chemical biology strategies that enable the selective clearance of these toxic species therefore remains a major unmet challenge in neurodegenerative disease research 4,5 . Targeted protein degradation (TPD) has emerged as a powerful paradigm for removing specific proteins by co-opting the cellular degradation machinery. Proteolysis-targeting chimeras (PROTACs) 6 exemplify this concept by linking a ligand for the protein of interest to an E3 ligase recruiting moiety, promoting ubiquitination and proteasomal degradation. Related approaches, including molecular glues 7,8 , dTAG (degradation tag) 9 , or TRIM-Away 10 , similarly rely on proximity-inducing or adaptor-mediated mechanisms to recruit cytosolic E3 ligases and trigger proteasomal clearance of target proteins. More recently, TPD strategies have expanded beyond the proteasome to engage lysosomal degradation pathways. Lysosome-targeting chimeras (LYTACs) 11 and KineTACs 11 exploit receptor-mediated endocytosis to direct extracellular or membrane-associated proteins to lysosomes, while proteolysis-targeting antibodies such as AbTACs 12 and ProTabs 13 induce lysosomal elimination of cell-surface targets. Despite their success in oncology and cell signaling biology, current TPD modalities remain constrained by the availability of small-molecule binders or extracellular accessibility, and show limited applicability to undruggable proteins 14 , including the intrinsically disordered and aggregation-prone species central to neurodegeneration. Such proteins often lack stable tertiary structure or well-defined binding pockets, rendering them inaccessible to traditional small-molecule ligands and poorly recognized by proteasomal systems 15,16 . In addition, the tendency of misfolded proteins to form dense and compact aggregates can physically exclude traditional PROTACs that depend exclusively on proteasomal degradation 6 , underscoring the need for strategies that engage lysosomal pathways for aggregate clearance. Thus, there is an urgent need for advanced molecular platforms that can recognize, engage, and dismantle misfolded proteins within the complex intracellular environment of neurons. Recent progress in Nanobody® technology provides a promising foundation for this goal. Nanobodies are small, stable antibody fragments that retain full antigen-binding capability while highly soluble and structurally compact (~15 kDa, ~2.5nm x 4 nm). They exhibit exceptional specificity and can recognize conformational epitopes that are inaccessible to small molecules, making them powerful tools for targeting misfolded or aggregated proteins inside cells 17 . Chemical strategies to convert nanobodies into degraders have begun to emerge. Notably, the GlueTAC platform introduced proximity-enabled SuFEx chemistry to covalently lock nanobodies to membrane proteins, enhancing target retention during endocytosis and promoting lysosome-mediated degradation 18,19 . While these works established the feasibility of covalent nanobody chimeras, existing approaches rely on genetic encoding of reactive amino acids and are largely restricted to membrane-associated targets and single degradation pathways. An advanced strategy is needed to improve GlueTACs by enhancing cell penetration while combining lysosomal targeting with proteasome-based degradation for broader and more efficient protein clearance. To address these limitations, we developed a chemoselective protein semisynthesis platform for constructing nanobody-guided degraders (nanodegraders, NDs) that merge nanobody precision with chemically programmable degradation. Our strategy combines expressed protein ligation (EPL) 20 with SuFEx proximity-enabled crosslinking 21 to introduce covalent stabilizing modules and azido-lysine handles for click conjugation of diverse E3 ligase ligands, including thalidomide (CRBN) 22 and AHPC (Von Hippel-Lindau, VHL) 23 . In parallel, incorporation of a chaperone-mediated autophagy (CMA) targeting motif provides a sequence-encoded route for lysosomal engagement 24 . Subsequent cell-penetrating peptide (CPP) 25 attachment enables efficient intracellular delivery, generating nanodegraders capable of recruiting both proteasomal and lysosomal degradation pathways in a programmable manner. As a disease-relevant application, we applied this platform to target alpha-synuclein (α-Syn), a presynaptic protein whose pathological aggregation underlies Parkinson’s disease 26 . Mutations such as A53T promote α-Syn misfolding, aggregation, and neurotoxicity 27 . Using human iPSC-derived neurons carrying the A53T mutation, we show that NDs internalize, selectively covalently engage A53T α-Syn, and promote their degradation, thereby restoring synaptic integrity. Extending these findings in vivo , optimized NDs mediated α-Syn degradation up to 72 hours post-injection in mice, indicating favorable stability and translational potential. Together, these findings establish a generalizable chemical biology framework for reprogramming protein degradation pathways through modular nanobody semisynthesis, offering a new route to degrade pathogenic protein species in neurodegenerative disease. Nanobody® is a registered trademark of Sanofi or an affiliate. Results 1. Design of nanobody-guided protein degraders To establish a versatile and modular platform for constructing nanobody-guided degraders (nanodegraders, NDs), we employed a protein semisynthesis approach called expressed protein ligation (EPL) 20 . In this strategy, a recombinant protein fragment is fused to an intein domain and treated with a reactive thiol, such as MESNA, to generate a C-terminal thioester intermediate. This intermediate can then be chemoselectively ligated to a synthetic peptide bearing an N-terminal cysteine, forming a native amide bond through native chemical ligation. The N-terminal cysteine also supports Cys-Maleimide conjugation of the cR10 cell-penetrating peptide 25,28 , enabling efficient intracellular delivery. The EPL method provides precise control over nanobody modification and enables the incorporation of synthetic peptides with defined chemical functionalities. To test and optimize our strategy, we selected the ALFA tag nanobody 29 ( ALFA Nb) as a model scaffold, which binds the ALFA epitope with low-picomolar affinity and high selectivity. We generated various ALFA ND constructs (Fig. 1a) designed to degrade ALFA-tagged enhanced green fluorescent protein (ALFA-eGFP) inducibly expressed in HeLa, as well as in human iPSC-derived neurons. To direct ALFA Nb-bound eGFP to the lysosomal degradation pathway, a peptide motif that promotes chaperone-mediated autophagy 24,30 (CMA1) was C-terminally fused to the nanobody. The CMA1 motif was rationally designed based on conserved lysosome-targeting sequences identified in known CMA substrates, including RNase A, α-synuclein, and Tau, and corresponds to the amino acid sequence K FER Q VKKD QK DRV Q . This sequence contains the canonical K FER Q -like motif recognized by the Hsc70-LAMP2A machinery, enabling selective recruitment to the lysosomal compartment 24,30 . The resulting ALFA Nb-CMA1 fusion protein, which also contained an HA tag for detection, was recombinantly expressed and purified (Fig. 1b). For visualization and biochemical tracking, we synthesized a tetramethylrhodamine (TMR)-labeled N-ter Cysteine peptide and ligated it to the intein-mediated ALFA Nb–CMA1 thioester, yielding fluorescently labeled ALFA Nb through EPL (Fig. 1b). We next equipped the nanobody with a cell-penetrating peptide (CPP) to facilitate cellular uptake. We used a cyclized deca-arginine peptide (cR10) that contains an N-terminal maleimide group linked through a PEG2 spacer to the cyclic sequence (KrRrRrRrRrRE) 25 . The cR10 peptide was synthesized by solid-phase peptide synthesis with purity greater than 95% (Extended Data Fig. 10a). Covalent conjugation of cR10 to the cysteine residue of ALFA Nb-CMA1 via maleimide-thiol coupling generated the final construct, cR10- ALFA Nb-CMA1, hereafter referred to as the ALFA nanodegrader ( ALFA ND) (Fig. 1c-d). Importantly, the N-terminal maleimide did not interfere with nanobody structure and function (Extended Data Fig. 1a) 31 . We next examined whether ALFA ND could efficiently enter cells, bind ALFA-eGFP, and induce lysosomal degradation. Live-cell imaging revealed rapid uptake of ALFA ND, colocalize to ALFA-eGFP and then direct to lysosomes within 10 minutes after treatment (Fig. 1e). Over the following hours, HA-tag immunostaining confirmed cytoplasmic distribution of ALFA ND and colocalization with ALFA-eGFP in HeLa cells (Extended Data Fig. 1a) and human neurons (Fig. 1f-g). ALFA ND localized to lysosomal compartments together with ALFA-eGFP indicates successful targeting of the nanobody-bound substrate to the lysosomal degradation pathway. Notably, in human neurons, ALFA ND accumulated at dendritic spines (Fig. 1g), suggesting potential for clearing supersaturated or misfolded synaptic proteins. Following 16 hours of treatment, ALFA ND at 0.5 μM induced pronounced degradation of ALFA-eGFP, reducing fluorescence intensity by approximately 90% in HeLa cells (Extended Data Fig. 1a-b) and 68% in human neurons (Fig. 1g). Time-course analysis revealed that ALFA-eGFP degradation became evident between 4 and 16 hours after ALFA ND administration (Extended Data Fig. 1b-c). These findings confirm that the combination of lysosome-targeting CMA1 motif and cR10-mediated delivery enables efficient cellular internalization of the nanodegrader and robust lysosomal degradation of the target protein. Together, these results validate the EPL-based semisynthesis approach as a powerful and modular method to design nanobody-guided degraders. The ALFA model system establishes a proof of concept for constructing programmable nanodegraders capable of achieving selective, lysosome-dependent degradation of intracellular targets. 2. Modeling Parkinson’s disease-associated A53T α-Synuclein mutation using human iPSCs. To establish a disease-relevant neuronal system, we differentiated human induced pluripotent stem cells (hiPSCs) into cortical neurons using a rapid neurogenin-2 (NGN2)-induced protocol 32,33 . Stable transgenic hiPSC lines expressing doxycycline-inducible NGN2 were generated using Tet-on system, enabling efficient and scalable neuronal differentiation within 14 days (Extended Data Fig. 2a). The resulting neurons exhibited mature morphology, with dendrites and axons decorated by synaptic spines, and expressed neuronal and synaptic markers including MAP2, VGLUT1, and PSD95 (Extended Data Fig. 2a). We differentiated both wild-type (WT) and A53T α-synuclein (α-Syn) mutant hIPSC lines into cortical neurons. After 35 days of differentiation, A53T neurons displayed hallmark features of Parkinson’s disease, including elevated total and phosphorylated α-Syn (pSer129) 34 , accumulation of higher-molecular-weight species, and punctate cytoplasmic aggregates detected by immunostaining (Fig. 2a, b). Furthermore, consistent with previous models of α-Syn-induced toxicity, A53T neurons exhibited synaptic and structural impairments 35 . Synaptophysin levels were markedly reduced compared with WT neurons (Fig.2d, e). Phalloidin staining of F-actin, which is enriched in dendritic spine regions, was used to visualize spines and revealed a significant decrease in dendritic spine density (Fig. 2d, e). In addition, calcium imaging with Fluo-4 indicated dysregulated intracellular Ca²⁺ homeostasis in A53T neurons relative to WT controls (Extended Data Fig. 2b). Finally, we assessed endo-lysosomal function using a fluorescent endocytic reporter. A53T neurons showed abnormal lysosomal activity, characterized by reduced fluorescence in dendrites compared to WT neurons (Extended Data Fig. 2d), consistent with impaired cargo degradation and lysosomal trafficking (Extended Data Fig. 2c-d). Together, these data demonstrate that our human iPSC-derived cortical neurons recapitulate key molecular and cellular features of A53T α-Syn pathology, including aggregation, synaptic dysfunction, calcium imbalance, and dysregulated proteolysis activities, establishing a robust platform for testing nanodegrader-mediated clearance of pathogenic α-Syn. 3. Development of lysosome-directing α-Syn nanodegraders To demonstrate the versatility of our nanobody semisynthesis platform in a disease-relevant setting, we designed a nanodegrader targeting α-Syn) a key pathogenic driver of Parkinson’s disease 36 . We used α-Syn nanobody 2 (SynNb2) 37 as the targeting module and appended the previously described CMA1 lysosome-directing motif to its C terminus. To enable neuronal uptake, the construct was further equipped with the cell-penetrating peptide cR10 via cysteine-maleimide conjugation. The resulting conjugate, cR10-SynNb2-CMA1, referred to as nanodegrader 21 (ND21) (Extended Data Fig 3a), was purified by size-exclusion chromatography (Extended Data Fig 3b) and verified by MALDI-MS (Extended Data Fig 3c). ND21 represents the first lysosome-directing α-Syn nanodegrader generated using this modular chemoselective assembly pipeline. We next assessed ND21 uptake and activity in human iPSC-derived neurons carrying the A53T α-Syn mutation. Neurons were treated ND21 for 24 hours. Immunofluorescence staining for HA-taged ND21 and α-Syn showed internalization and clear colocalization of ND21 with α-Syn aggregates in both soma and dendritic regions (Fig. 3a). Quantitative imaging revealed degradation of α-Syn within the soma, with 41.5 % reduction at 100 nM ND21, while dendritic α-Syn levels remained largely unchanged compared to Mock treatment (Fig. 3a). This spatially restricted degradation pattern parallels the distribution of lysosomal activity, which is concentrated in the neuronal soma but markedly reduced in dendrites in A53T α-Syn neurons (Extended Data Fig. 2c-d). To confirm that efficient ND21-guided α-Syn degradation requires cell penetration, α-Syn recognition and lysosomal directing abilities of ND21, we evaluated a series of nonfunctional control constructs. The α-Syn Nb2-CMA1 construct, which lacks the cell-penetrating peptide cR10, failed to enter human neurons and showed no detectable α-Syn binding (Extended Data Fig. 4a). In contrast, the ALFA ND control, which contains the CMA1 motif but does not recognize α-Syn, readily internalized yet displayed diffuse cytoplasmic distribution without colocalization to α-Syn aggregates (Extended Data Fig. 4a). The third construct consisting of SynNb2 equipped with cR10 peptide but lacking the CMA1 motif, together with these two control constructs showed no efficient α-Syn degradation in A53T neurons (Extended Data Fig. 4b). Despite its partial spatial restriction, ND21 treatment led to robust recovering of spine loss in A53T neurons. Quantification of dendritic spines revealed ~97% recovery of spine density relative to WT α-Syn neuron controls (Fig. 3d-e and Table 1), suggesting that selective reduction of A53T α-Syn is sufficient to alleviate synaptic defects. Together, these results establish ND21 as a proof-of-concept lysosome-directing α-Syn nanodegrader that achieves selective degradation and functional rescue in human neurons and provide a framework for designing next-generation nanodegraders that harness lysosomal pathways to eliminate pathogenic α-Syn in this A53T neuronal model. 4. SuFEx-enabled covalent α-synuclein nanodegraders While ND21 efficiently engaged α-Syn and promoted lysosome-dependent degradation in the neuronal soma, its noncovalent interaction with α-Syn limited complex stability and degradation efficiency (~41.5%). To enhance the durability of ND-target association and enable covalent capture of α-Syn, we next incorporated sulfur fluoride exchange (SuFEx) proximity-enabled crosslinking chemistry 21 into the ND design. The SuFEx reaction provides a proximity-enabled irreversible crosslinking of interacting proteins through selective sulfur-fluoride exchange at nucleophilic residues (Tyr, His, Lys), thereby enhancing complex stability and residence time. This approach allows formation of stable covalent linkages between the ND and its substrate upon proximity-induced SuFEx reaction, thereby increasing complex lifetime and potentially improving degradation yield. We first validated proximity-enabled crosslinking ability of SuFEX-functionalized nanobodies and their intracellular targets in live cells using the ALFA nanobody. Intein-mediated ALFA Nb-thioester was ligated by EPL to an N-terminal cysteine peptide bearing unnatural SuFEx-reactive lysine (K-FSY, where FSY denotes the aryl fluorosulfate SuFEx handle), then conjugated to cR10 via cysteine-maleimide coupling to yield cR10- ALFA Nb-K-FSY (Extended Data Fig. 5a). Live cells expressing ALFA-tagged eGFP were incubated with cR10- ALFA Nb-K-FSY, followed by lysis and co-IP of ALFA-eGFP. Immunoblotting detected the nanobody in the precipitate and revealed a higher-molecular-weight band corresponding to a covalent cR10- ALFA Nb-K-FSY::ALFA-eGFP adduct (Extended Data Fig. 5b), demonstrating proximity-enabled intracellular crosslinking between SuFEX-functionalized nanobody, hereafter termed a covalent nanobody and its targeted protein. We next applied this semisynthetic strategy to α-synuclein nanobody 2 (SynNb2) fused to the CMA1 motif. Synthetic N-Cys peptides containing either K-FSY or K-PEG₂-FSY were ligated to SynNb2-CMA1 via EPL (Extended Data Fig. 5c), producing two covalent lysosome-directing nanodegraders, ND21F and ND21P2F, respectively. The PEG 2 spacer was introduced to improve solubility and provide linker flexibility of NDs. We then evaluated target engagement of covalent nanodegraders in human A53T α-Syn neurons. Immunofluorescence imaging showed that HA-tagged ND21F and ND21P2F co-localized extensively with α-Syn within soma and neurite locations of A53T neurons (Fig. 3c). The degree of co-localization was significantly higher than that observed with non-SuFEx NDs such as ND21 (Fig. 3c, d), indicating that FSY modification enhances nanobody-target engagement, likely by stabilizing the α-Syn-ND complex. Immunoprecipitation of α-Syn followed by western blot analysis further supported this observation: only the covalent-binding ND21P2F was co-immunoprecipitated with α-Syn (Extended Data Fig. 5d), confirming most durable interaction with the A53T α-Syn compared to other NDs. In addition, in vitro crosslinking assay between ND21F and purified A53T α-Syn demonstrates that covalent binding occurred after 1 hour (Extended Data Fig. 5e). Quantitative imaging revealed increase degradation of α-Syn, with 66 % and 69.5 % reduction at 100 nM ND21F and ND21P2F, respectively (Fig 3a, b and Table 1). 5. Proteasome-recruiting and dual-proteolytic nanodegraders targeting α-synuclein: Proteasome-recruiting α-Syn nanodegraders: To extend nanobody-guided degradation beyond lysosomal routing, we generated proteasome-recruiting α-Syn nanodegraders by introducing an E3-ligase ligand into SynNb2 using the EPL strategy. SynNb2 was ligated to a synthetic N-terminal cysteine peptide containing a PEG 12 linker for enhanced solubility and a Thalidomide moiety conjugated to a lysine side chain via NHS-ester amidation. The ligation proceeded efficiently (>90 %) and yielded a purified monomeric product after size-exclusion chromatography (Extended Data Fig.6a-b). The cR10 cell-penetrating peptide was subsequently coupled to generate the final construct ND2Tha, which was confirmed by MALDI-MS analysis (Extended Data Fig. 6c). We then treated ND2Tha with A53T neuron cultures, this proteasome-directing ND construct reduced α-Syn levels by ~38 % and partially restored dendritic spine density (~52 % of wild-type) at 100 nM (Fig. 4a, b and Table 1). These results demonstrate that EPL-based installation of an E3-ligase ligand, Thalidomide-CRBN 22 , via a flexible PEG 12 linker can redirect nanobody-guided degraders toward the ubiquitin-proteasome pathway in neurons, though with moderate efficacy relative to lysosomal designs. Dual-proteolytic α-Syn nanodegraders Dual engagement of lysosomal and proteasomal systems has been shown to improve clearance of misfolded or aggregation-prone proteins 38 . To test whether concurrent recruitment of lysosomal and proteasomal machinery could enhance α-Syn clearance, we next introduced the CMA1 lysosomal motif into the Thalidomide-bearing design, generating ND21Tha. This construct integrates lysosomal routing (CMA1), proteasome recruitment (Thalidomide-CRBN), and cR10-mediated neuronal delivery. ND21Tha reduced α-Syn levels by ~41 % and restored spine density to ~47 % of wild-type levels at 100 nM (Fig. 4a, b and Table 1). Comparing with single proteolytic pathway directing constructs, proteasome-directing ND2Tha and lysosome-directing ND21, ND21Tha shows no additive effect from dual-proteolytic pathway signaling. To harness both dual-proteolytic directing and SuFEx proximity-enabled chemistry within a single modular framework, we designed dual-proteolytic α-syn nanodegraders that integrate multiple degradation cues and orthogonal chemical handles. Building on our optimized expressed protein ligation (EPL) platform, we incorporated SuFEx chemical handle K-PEG₂-FSY together with a Lysine-azide residue in the synthetic peptide, providing orthogonal reactivity for the site-specific attachment of E3-ligase ligands. The recombinant fragment SynNb2-CMA1 was ligated to the synthetic N-terminal cysteine peptide containing both K-PEG 2 -FSY (SuFEx) and Lys(N₃) functionalities (Extended Data Fig. 7a). The resulting semisynthetic nanobody fragment was subsequently subjected to azide-alkyne cycloaddition with DBCO-functionalized thalidomide (for cereblon, CRBN) or AHPC (for von Hippel-Lindau, VHL) ligands, yielding ND21P2F-Tha and ND21P2F-VHL (Extended Data Fig. 7b), respectively. Each construct retained the CMA1 motif for lysosomal engagement, thereby creating nanodegraders capable of dual proteolytic targeting through both the lysosomal and ubiquitin-proteasome pathways. Although these dual-proteolytic, SuFEx-functionalized NDs were efficiently assembled and covalently stabilized with α-Syn, they reduced α-Syn by only ~52 % and ~47 %, with corresponding spine recovery of ~57 % and ~59 %, respectively (Fig. 4c, d and Table 1). The results indicate that degradation by these constructs reveals proteolytic pathway competition rather than additive synergy. Together, these findings suggest that incorporating multiple degradation signals within a single ND can introduce kinetic interference and pathway bias instead of enhanced turnover. The data define practical design constraints for multi-pathway degraders in neurons and emphasize that pathway-specific modularity is more effective than multi-route fusion for the selective elimination of pathogenic α-Syn. 6. In vitro efficacy and in vivo stability, selectivity, and distribution of ND21P2F We selected ND21P2F to evaluate the in vivo stability, distribution, and target selectivity of the ND strategy in the mouse brain. ND21P2F shows its most efficacy in both degradation of α-Syn, restore spine density and synaptic functions of A53T neuron culture in vitro (Fig. 3 and Table 1). Live-cell calcium imaging with Fluo-4 AM of WT neurons displayed strong, synchronous calcium influx upon depolarization, whereas A53T neurons showed blunted responses, indicating impaired calcium entry and reduced excitability (Extended Data Fig. 2b). ND21P2F treatment enhanced calcium transients, restoring fluorescence amplitudes to near WT levels (Fig. 5a-b and Extended Data Fig. 8a). Furthermore, the time course treatment confirmed the ability of ND21P2F to effectively degrade α-syn in A53T neurons. The treatment of young A53T neurons with ND21P2F significantly reduced α-synuclein levels in a time-dependent manner (Extended Data Fig. 8d-e) For in vivo experiments, we performed stereotaxic injections into the mouse striatum and analyzed tissues at 24 hours and 72 hours post-injection. Confocal imaging of coronal sections stained for the HA tag revealed clear ND21P2F localization at the injection site after 24 hours, with fluorescence extending into adjacent parenchyma (Fig. 5c). After 72 hours, HA signals persisted with comparable intensity, indicating high in-tissue stability and limited degradation of the injected ND over this period. Moderate diffusion into neighboring regions was also observed, suggesting controlled spreading without systemic dispersion (Extended Data Fig. 9). To assess target engagement, sections were co-stained for α-synuclein (α-Syn). Merged images showed pronounced colocalization of HA and α-Syn signals in the injected hemisphere, indicating that ND21P2F binds endogenous α-Syn in situ (Fig. 5d). Quantitative fluorescence analysis comparing the injection site with the contralateral hemisphere demonstrated a significant reduction in α-Syn signal intensity at both 24 hours (~20%) and 72 hours (~ 40%) (p < 0.01) (Fig. 5d). These results confirm that ND21P2F remains stable in the brain for at least 72 hours, retains binding specificity toward α-Syn, and exhibits local proteolytic activity sufficient to reduce endogenous α-Syn levels near the injection site. Together, these findings establish that ND21P2F possesses favorable pharmacodynamic properties in vivo , maintaining stability, tissue retention, and biochemical selectivity, and highlight its translational potential as a chemically well-defined nanobody-guided protein degrader for α-Syn-associated pathologies. Discussion This study establishes nanobody guided degraders (nanodegraders, NDs) as a modular chemical biology platform for the selective removal of pathological α-syn in human neurons. By integrating a nanobody module for molecular precision with chemoselective assembly of lysosome and proteasome recruiting signals, we demonstrate a versatile approach for programmable protein degradation. The ability to reengineer intracellular clearance pathways through site specific semisynthesis represents a conceptual advance for targeted protein degradation beyond small molecule PROTACs. Our design combines expressed protein ligation (EPL) with SuFEx (sulfur fluoride exchange) chemistry 21 to install proximity enabled covalent modules and azido lysine handles for E3 ligand conjugation, while the CMA1 motif directs cargo to lysosome. This strategy enables multi-milligram scale production and flexible incorporation of diverse chemical functionalities, yielding degraders that harness proteasomal, lysosomal, or dual proteolytic mechanisms. This flexibility bridges the gap between biologics and small molecules, creating hybrid constructs that retain the selectivity of antibodies while gaining the tunability of chemical synthesis. The resulting NDs operate through covalent proximity capture rather than transient binding, ensuring high target residence time and reducing off target proteolysis. The dual proteolytic route designs revealed pathway competition rather than additive synergy, defining practical constraints on simultaneous engagement of the proteasome and lysosome in neurons. These findings highlight the importance of pathway specific modularity for achieving efficient and predictable degradation kinetics toward neurons. Mechanistically, α-Syn A53T aggregates arise primarily from soluble oligomeric intermediates that disrupt calcium signaling, generate oxidative stress, and impair synaptic vesicle trafficking 39 . Since our NDs were developed from Syn2 nanobody which is specifically bind to soluble α-Syn but not its insoluble forms 37,40 , NDs did not efficiently eliminate total α-Syn in A53T neurons (Fig. S9) with pronounced α-Syn aggregates, but selectively reduced these toxic soluble species, which are the most neurotoxic forms, leading to marked functional recovery in A53T neurons. This selective activity parallels the natural hierarchy of α-Syn pathogenicity and suggests that complete depletion of α-Syn is unnecessary for therapeutic benefit. In our A35T human neuron model, lysosomal failures at neurites potentially leads to α‑syn aggregate buildup and local synaptotoxicity. This unevenly distributed of lysosomal functions within neurons contributes to the spatial efficacy of lysosome targeting NDs. Lysosomal-directing ND mediated α-Syn clearance occurred primarily in somatic regions, with limited neuritic α‑syn aggregate degradation. Covalently complexes of SuFEx-functionalized, lysosome-directing NDs and α-Syn potentially enhancing lysosomal access to distal compartments could improve both degradation and synaptotoxic rescue efficacies in this A53T model. Proteasome represents a complementary degradation route that operates at both cytosolic and synaptic sites. A membrane associated neuronal proteasome (NMP) has been implicated in peptide release and synaptic regulation 41 . Redirecting α-Syn toward proteasomal clearance could therefore simultaneously reduce toxic aggregates and normalize local proteostasis at synapses. Our proteasome recruiting NDs demonstrate the feasibility of coupling E3 ligase recognition through thalidomide or AHPC conjugation with nanobody precision, establishing a foundation for targeted modulation of neuronal proteasome activity. Future work should explore how NDs interact with NMPs and whether proteasome directed degradation can influence synaptic plasticity and neurotransmission. While our study focused on α-Syn, the same framework could be adapted to other misfolded or aggregation prone proteins implicated in neurodegenerative and systemic diseases. By swapping the nanobody module or degradation signal, the platform can be reprogrammed for distinct intracellular environments or disease contexts. Importantly, our in vivo experiments show that optimized NDs maintain stability and target selectivity within brain tissue for at least 72 hours, supporting their translational potential. Several challenges remain. Failures in protein homeostasis of misfolded protein disease models, particularly the A53T PD neuronal model limit uniform ND distribution and degradation efficacy. Improving pharmacokinetics, enhancing binding efficacy of ND to the target, proved by SuFEx-functionalized covalent NDs, will be critical for advancing NDs toward therapeutic use. It will also be valuable to test NDs efficacy in PD models that recapitulate α-Syn propagation and synaptic transmission in vivo to evaluate whether local degradation can halt or reverse disease progression. In summary, we demonstrate a modular chemical strategy for constructing nanobody based degraders that reprogram proteolytic systems to selectively dismantle neurotoxic α-Syn species. By integrating lysosomal, proteasomal, and covalent crosslinking mechanisms within a unified protein semisynthetic framework, this study establishes fundamental design principles for pathway specific degradation in human neurons. These findings open new directions for chemical biology approaches to restore disease-associated proteostasis and neuronal health in Parkinson’s disease and related proteinopathies. List of abbreviations α-Syn: α-Synuclein BCA: bicinchoninic acid assay CHX: cycloheximide CM: maintaining medium CMA: chaperon mediated autophagy CPP: cell penetrating peptide EPL: expressedfas protein ligation IM: induction medium iPSC: induced pluripotent stem cells Map2: Microtubule-associated protein 2 ND: nanobody-based protein degrader NGN2: neurogenin-2 NMDA: N-methyl-D-aspartate POI: protein of interestf PSD-95: Postsynaptic density protein 95 ROI: region of interest SD: standard deviation TPD: targeted protein degradation tIPSCs: transgenic induced pluripotent stem cells Tuj1: class III beta-tubulin Ub: Ubiquitin WT: wild type Declarations Author contributions NL and NC formulated the research plan, designed the nanodegraders (NDs) and interpreted experimental results with assistance from ON, UN, NA. NC, UN and NA generated NDs, purified proteins, performed protein semisynthesis and analyzed biochemical experiments. NL established human iPSC-derived neuron models with assistance from ON on iPSCs differentiations. NDs treatments on cell lines and neurons were performed and analyzed by NA, NL and ON. Mouse studies were performed by ON, RN and JN and analyzed by ON and NL. NC and NL wrote the manuscript with contributions from ON and NA. All authors edited and approved the manuscript. NC and NL jointly supervised the project. Acknowledgments We thank Dr. Michael Ward at National Institute of Neurological Disorders and Stroke (NINDS/NIH) for the K4-PB-TO-hNGN2 and K13-EF1a vectors, and Dr. William C. Skarnes at The Jackson Laboratory for Genomic Medicine for the human IPSCs lines. We also thank Dr. Dehua Pei for LC/MS analysis and Dr. Monica Venere for using cryostat. We acknowledge resources from the Campus Microscopy and Imaging Facility (CMIF) and The OSU Comprehensive Cancer Center (OSUCCC) Microcopy Shared Resource (MSR), The Ohio State University (RRID:SCR_025078). This facility is supported in part by grant P30 CA016058, National Cancer Institute, Bethesda, MD. We thank the OSUCCC's Preclinical Therapeutics Mouse Modeling Shared Resource for technical and instrumental support. The facility is supported by the OSU Comprehensive Cancer Center (OSUCCC) and the National Institute of Health under grant number P30 CA016058. The content is the authors' sole responsibility and does not necessarily represent the official views of the NIH. NC was supported by the OSUCCC Startup fund and NIH grant K22CA241105. NC, NL, NA, UN and ON were supported by NIH grant R35GM151124. NA was supported by the Pelotonia Graduate Fellowship. NL was supported by a Warren Alpert Distinguished Scholar Award. The project was supported by the OSU President’s Research Excellent Accelerator Award (NC and NL), OSU NRI Seed Grant Award (NL) and the Sanofi iDEA-Tech Award (NC and NL). Methods Chemicals, antibodies and reagents: Sodium 2-mercaptoethanesulfonate (MESNA) was purchased from Millipore Sigma (USA). Chitin resin was purchased from New England Biolabs (USA). Thalidomide was purchased from BroadPharm (California, USA). 4-(Acetylamino)phenyl]imidodisulfuryl difluoride (AISF) was purchased Chem-Impex International (USA). Rink amide resin, amino acids, and HATU were purchased from P3 BioSystems (USA). Solvents (DMF, DCM, DIEA, TFA) were purchased from Sigma-Aldrich (USA). IPTG, Dithiothreitol and ProBlock™ Protease Inhibitor Cocktail were purchased from GoldBio (USA). See Extended Data Table 1 for information of antibodies used for this study. Plasmids and cloning All nanobody expression vectors were constructed in the pTXB1 vector. The nanobodies ALFA Nb (Addgene #136626) and α-Syn Nb2 37 (gene synthesis by GenScript) were PCR-amplified to create a megaprimer and subcloned into pTXB1 vector (NEB IMPACT™ system), which encodes an Mxe GyrA intein-chitin-binding domain (CBD) fusion at the vector C-terminus. DNA oligos were designed and purchased from Integrated DNA Technologies (IDT). An N-terminal HA epitope tag was fused upstream of each nanobody open reading frame. The CMA motif was inserted in-frame at the C-terminus of each nanobody, ensuring continuous reading frame into the pTXB1 Mxe GyrA intein-CBD vector. Plasmids and clones were subsequently transformed into E. coli and verified by Sanger and whole-plasmid sequencing. Peptide synthesis: Peptides corresponding to Cys-Gly-Gly-Gly-Ser-Lys-Gly-Gly-Gly-Ser were synthesized on the automated PurePep Chorus peptide synthesizer (Protein Technologies) using Fmoc-Gly, Fmoc-Ser(tBu), Fmoc-Lys(Alloc), Boc-Cys(TrT) and Rink-Amide resin (0.05 mmol). In particular, 4 eq. of amino acid, 3.8 eq. of HATU, 8 eq. of NMM in DMF were double coupled for 1.5 h, and Fmoc groups were removed by 20% piperidine in DMF over two 10 min cycles. To synthesize peptides bearing either K-FSY or K-PEG 2 -FSY, the allyloxycarbonyl (Alloc) protecting group of N-ε-Alloc-lysine was orthogonally removed using 0.1 eq. Pd(PPh 3 ) 4 and 25 eq. phenysilane in dry DCM for 30 minutes under argon atmosphere. The resin was subsequently washed twice with 0.5 M DIEA in DMF. Next, 5 eq. of 4-hydroxybenzoic acid (Sigma), 4.75 eq. of HATU, and 5 eq. of DIEA were dissolved in DMF, added to the resin, and rotated for 1.5 hours to afford N-ε-(4-hydroxybenzoyl)-L-Lysine peptide. The resin was washed thoroughly with DMF, DCM and methanol. Finally, the sulfurylation of N-ε-(4-hydroxybenzoyl)-L-Lysine was carried out by adding a mixture of 1.2 eq. of AISF (Chem Impex, 36191), 2.2 eq. of DBU (Sigma, 139009) in dry DCM to the resin for 1.5 hours. This reaction was repeated once to ensure completion. For the K-PEG 2 -FSY peptide, prior to the coupling of 4-hydroxy benzoic acid, Fmoc-NH 2 -PEG 2 -COOH was double coupled to the lysine using the standard Fmoc strategy described above. To synthesize peptide containing thalidomide, Cys-(PEG 6 ) 2 - Gly-Gly-Gly-Ser-Lys-Gly-Gly-Gly-Ser was synthesized on the automated PurePep Chorus peptide synthesizer using Fmoc-Gly, Fmoc-Ser(tBu), Fmoc-Lys(Alloc), Fmoc-PEG 6 -COOH, Boc-Cys(TrT), and Rink-Amide resin (0.05 mmol). The Alloc protecting group of N-ε-Alloc-lysine was orthogonally removed as described above. Next, 1.2 eq. of Thalidomide-PEG 4 -NHS ester (BroadPharm) and 2.4 eq. of DIEA in dry DMF was added to the resin, and the coupling reaction was left overnight. Peptide containing TAMRA, 5-isomer and biotin was synthesized automatedly and manually. The target peptide sequence Cys-Gly-Gly-Gly-Ser-Lys(Biotin)-Gly-Gly-Gly-Ser-Lys(ivDde)-Gly-Gly-Gly-Ser was synthesized using Fmoc-Gly, Fmoc-Ser(tBu), Fmoc-Lys(ivDde), Fmoc-Lys(Biotin), Boc-Cys(TrT), and Rink-Amide resin (0.05 mmol). Fmoc-Lys(ivDde) and Fmoc-Lys(ivDde) were coupled on resin manually. The ivDde protection group of N-ε-ivDde-lysine was orthogonally removed with 5% hydrazine in DMF for 5 minutes (3 x 5 min), followed by extensive rinsing with DMF. Next, 1 eq. of TAMRA carboxylic acid, 5-isomer (Lumiprobe, 67190), 1 eq. of HATU and 4 eq. of DIEA in DMF was added to the resin, and the coupling reaction was carried out overnight. Maleimide cyclic R10 peptide was synthesized using Fmoc-L-Arg (R), Fmoc-D-Arg (r), Fmoc-Lys(Alloc), Fmoc-Glu(OAll), Fmoc-PEG 2 -COOH on Rink-Amide resin (0.05 mmol) as a linear peptide of the sequence PEG 2 -PEG 2 -K(Alloc)-RrRrRrRrRr-E(OAll) as previously described 25 . The Alloc and OAll protection groups were orthogonally removed using 0.1 eq. of Pd(PPh 3 ) 4 and 25 eq. of phenysilane in dry DCM for 30 minutes under argon atmosphere. The resin next was washed with 0.5 M DIEA in DMF to remove Pd. The cyclization of the peptide was carried out using 1 eq. of HATU and 2 eq. of DIEA in DMF for 2h at room temperature. Subsequently, the maleimide cR10 peptide was obtained by coupling 2 eq. of 2-Maleimidoacetic acid (Ambeed Inc, A110455) with 2 eq. of HATU and 4 eq. of DIEA in DMF for 1 h at room temperature. Cyclic R10 peptide bearing a C-terminal Sortase A recognition motif (LPETG) was synthesized using the same solid-phase and cyclization strategy described above. Briefly, the linear peptide K(Alloc)-RrRrRrRrRr-E(OAll)-(PEG2) 2 -SKYLELPETG was assembled on Rink-Amide resin (0.05 mmol) using standard Fmoc chemistry. Orthogonal removal of Alloc and OAll protecting groups, intramolecular cyclization, and global deprotection were performed as described for the maleimide cR10 peptide, yielding the cR10-(PEG2) 2 -LPETG product. All peptides were deprotected and cleaved from the resin with trifluoroacetic acid: water: triisopropylsilane (95:2.5:2.5, v/v/v) for 3 hours, then precipitated with chilled diethyl ether, washed twice with chilled diethyl ether, and the crude peptides were dried by nitrogen gas flow. The crude peptides were purified using preparative reverse-phase C18 HPLC column (Vydac) using a gradient of water:acetonitrile containing 0.05% trifluoroacetic acid. Pure fractions were combined, concentrated on a rotavap and then lyophilized. Peptide structures were confirmed using MALDI mass spectrometry or LC-MS/MS and peptide concentrations were determined by amino acid analysis. Protein expression and purification: SHuffle T-7 Express E. coli (NEB) bearing pTXB1 plasmid for nanobodies C-terminally fused with CMA1, Mxe GyrA intein and chitin-binding domain (CBD) was cultured in 0.5 L of LB media to reach OD 600 0.6-0.8 and then induced with 0.5 mM IPTG for 18 hrs at 16 o C. Cells were harvested by centrifugation at 5,000 rpm for 15 minutes at 4°C, and resuspended in lysis buffer (50 mM HEPES, 250 mM NaCl, 0.1% Triton X-100, 10% glycerol, pH 7.5) with one tablet of protease inhibitors (Thermo Scientific) and 1 mM PMSF. Cells were lysed using a French Press and centrifuged at 22,000 x g for 35 minutes at 4°C to collect the supernatant. The supernatant passed twice to 3 mL of chitin resin (NEB) pre-equilibrated with lysis buffer to capture the Syn Nb2-CMA1-intein-CBD. The resin was then washed with 150 mL washing buffer (25 mM HEPES pH 7.5, 500 mM NaCl, 0.1% Triton X-100) and incubated overnight in cleavage buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, pH 7.5) containing 100 mM DTT and 0.5 mM PMSF at room temperature. The cleavage buffer containing nanobody (2-3 mgs) was collected and purified using size exclusion chromatography with Superdex 75 10/300 GL column and phosphate-buffered saline (PBS) buffer, pH 7.4, 3 mM DTT. SDS-PAGE was used to assess yield and purity. Protein semisynthesis approach: Nanobody (Nb)-CMA1-intein-CBD was purified from T-7 SHuffle Express E. coli using 3 mL of chitin resin as described above. After washing, 3 mL of cleavage buffer containing 200 mM MESNA (sodium mercaptoethylsulfonate, Sigma) and 0.5 mM PMSF was added to the chitin column, incubated overnight at room temperature. The cleavage buffer containing 2-3 mgs of Nb-CMA1-thioester was collected, concentrated by ultrafiltration using an Amicon 10 kDa MWCO filter (Sigma Millipore) to 0.5 mL, exchanged into 200 mM Sodium phosphate pH 6.5 using a 2-mL zeba desalting column (Thermo). Next, Nb-CMA1-thioester was immediately reacted with 4 mM of the synthetic N-Cys containing peptides at room temperature for 3 hours, followed by overnight incubation at 4°C. The ligation products were assessed by Coomassie SDS-PAGE and were purified using size exclusion chromatography with Superdex 75 10/300 GL column (Cytiva) and PBS buffer. The pure fractions were combined, concentrated to ~1 mg/mL, aliquoted and stored at -80 o C. cR10 labeling: Semisynthetic nanobodies were conjugated with cyclic R10 (cR10) peptides using either maleimide-based thiol coupling or Sortase A–mediated ligation. For maleimide labeling, the semisynthetic nanobodies were incubated with maleimide-cR10 peptide at a molar ratio 1:7 in 1x PBS pH 7.4 for one hour at room temperature, followed by an overnight incubation at 4°C. The reaction was monitored using 15% SDS-PAGE, and the Zeba desalting columns (Thermo Fisher) were used to remove the excess cR10 peptide from cR10-NDs efficiently. For Sortase A-mediated ligation, semisynthetic nanobodies bearing an N-terminal triglycine motif were ligated to cR10-PEG2-PEG2-LPETG using GST–Sortase A. Reactions were performed at room temperature for 2 hours in ligation buffer 50 mM HEPES (pH 7.5) and 5 mM CaCl₂, using 50 µM nanobody and 500 µM cR10-PEG2-PEG2-LPETG. Upon completion, GST–Sortase A was removed by incubation with glutathione agarose beads, and excess cR10-PEG2-PEG2-LPETG was removed using Zeba desalting columns (Thermo Fisher). Ligation efficiency was assessed by SDS-PAGE. All semisynthetic nanobodies bearing cR10 are hereafter referred to as nanodegraders . All ND constructs were validated by mass spectrometry using a MALDI-TOF-TOF mass spectrometer (Bruker-Daltonics UltrafleXtreme). Spectra were obtained in both negative and positive reflectron ion modes. Cell line cultures: HeLa cells were obtained from ATCC and cultured in DMEM (Gibco, 11965118) with high glucose and 5 mM Glutamine containing 10% FBS. Cells were grown at 37 °C and 5% CO2 up to 80-90% confluency then passed on at 1 in 10 and grown for up to a week. The medium was changed every 3-4 days. Human IPSC cultures Both A53T mutant and the control WT KOLF 2.1 iPSC lines were obtained from Jackson Laboratory. IPSC lines were used for a maximum of 10 passages to avoid chromosomal and genetic aberrations that may appear during long-term passages. Accordingly, we periodically checked the human iPSC cultures to ensure they did not possess abnormal karyotype. The human iPSC lines were cultured in mTeSR™ medium (STEMCELL Technologies, 85850) in a Matrigel (BD Matrigel™, hESC-qualified Matrix, 354277) coated plate. Enzyme-free passaging reagents, ReLeSR™ (STEMCELL Technologies, 100-1438) were used for routine passaging of cells as cell clumps. The media of the iPSC cultures were changed daily for optimal growth. Neuronal differentiation Neuronal differentiation was carried out according to the published procedure 32,33,42 . Briefly, we generated stable iPSC lines expressing the transcription factor neurogenin-2 (NGN2 through Lipofectamine Stem-mediated transfection of DNA plasmids. For NGN2-expressing lines, we used the K4-PB-TO-hNGN2 and K13-EF1a vectors, kindly provided by Dr. Michael Ward at NINDS/NIH. Puromycin 1-3 μg/ml was used for selection and enrichment of transgenic IPSC cell (tIPSCs) population. Cortical neuron differentiations were performed using the established procedure 32,33 . Briefly, tIPSCs were cultured as single cells in neuronal induction medium (IM) with doxycycline (Sigma D9891) 2 μg/ml on Matrigel coated plates for 3 days. Cortical neurons differentiation was then induced with the appropriate medium (Table S2). The different days after differentiation are labelled numerically (D1 for day 1). At D4, cells were finally re-plated onto dishes coated with poly-L-ornithine (PLO) for neuronal maturation. Rho-associated protein kinase (ROCK) inhibitor Y-27632 (Tocris Bioscience, 1254) 10 μM was used in the cultures on D1 and D4 for splitting. Based on the type of interrogation the cells were either plated at low-density for immunofluorescent imaging or at high-density for preparation of cell lysates. Cells were split using Accutase (Gibco, A1110501). Following D4 final plating, neurons were cultured in neuronal maintaining medium (CM) for 7, 14, 21, 28, and 35 days. Half of the medium was changed every day with fresh pre-warm CM. At experimental endpoints, neurons were either fixed for immunofluorescence microscopy or lysed to make protein lysates. Nanodegrader treatments: Hela cell cultures after 3 days of transfections for expressing ALFA-tagged EGFP proteins were used to treat nanodegraders. Pre-treatment with cycloheximide (CHX) for 4 hours, then nano-dergraders were added into the culture medium at 500nM concentration designed timepoints. Tet-on ALFA-EGFP engineered cells were pretreated with Doxycycline 2 μg/ml to induce ALFA-EGFP expression for 2-3 days before nanodegraders were added into new culture medium at designed concentration and time points. Human iPSC-derived neurons at day 35 of differentiation were used for the treatments. Nanodegraders similarly were added into the conditional neuronal maintaining media at final concentration of 500 nM, 100 nM for 24 hours. Cell lysates and coverslip of cultures were collected for further immunoassay and biochemical analysis. Western blot (WB) Cell and neuron lysates were collected from culture in RIPA buffer or IP buffer. The total protein content of samples was measured by bicinchoninic acid assay (BCA) (Pierce, 23225). Equal amounts of protein lysate were mixed with appropriate volume of loading dye and heated for 5 min at 99°C prior to separation on Tris-Glycine SDS-PAGE gels. The proteins were then transferred onto Immobilon PVDF membranes (Immobilon-P, Millipore) and the membranes were blocked for 30 min with 5% BSA in TBST (Tris-buffered saline, 0.1% Tween 20) [1X] buffer and then incubated with primary antibodies overnight at 4°C (See Extended Data table 1 for details). Antigen-antibody complexes were detected using fluorescent goat anti-mouse IRDye 800 (green) or 680 (red) and goat anti-rabbit IRDye 680 (red) secondary antibodies, respectively, and visualized with the LI-COR Odyssey Classic Infrared Imaging System. Immunoprecipitation assay Antibody-bead conjugates were prepared by adding 2 μg of anti-HA or- α-Syn antibodies to 500 μl of Pierce IP Lysis Buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) (Thermo Fisher Scientific, 87788) in a 1.5 ml microcentrifuge tube, together with 30 μl of Dynabeads protein A/G (Invitrogen). Tubes were rotated for 2 hours at 4°C followed by two washes with IP Lysis Buffer to remove unbound antibodies. The washed antibody-bead mixture was incubated with cell lysate prepared as described below. Neuron cultures were washed in ice-cold 1x PBS, and the cells were scraped from the dishes in IP buffer supplemented with protease inhibitor (Millipore Sigma, P8340). The lysates were then sheared by repeated passage through Tuberculin syringes with 25G needle and clarified by centrifugation at 5000 rpm for 5 min. The protein concentration of the supernatants was quantified by the BCA assay. We incubated 0.5 ml at 2.0 mg/ml of lysate with the washed antibody-bead mixture for 2 hours at 4°C in a rotator at 10 rpm speed. The unbound fractions (flowthrough) were collected, and beads were subsequently washed three times with 1.0 ml of IP lysis buffer. SDS-sample buffer was then added to the beads. Equal portions of the supernatants were then separated by SDS-PAGE and immunoblotted for HA-tagged proteins and α-Syn. Immunocytochemistry staining Cells grown on coverslips were washed with PBS and fixed with 4% paraformaldehyde, then blocked in blocking buffer (1% BSA in PBS) for 30 minutes. In case of intracellular proteins detection by staining, permeabilization step with Triton-X100 0.1% for 5 minutes was performed before the blocking step. Fluorescence staining was performed using the primary and secondary antibodies listed in Table S1. Primary antibodies were made up in 1% blocking buffer and PBS. After incubation, the cells were washed with PBS; secondary antibodies were incubated in 1% blocking buffer. Finally, cells were washed 5 times, 5 min/time with PBS, and counterstained with DAPI (Invitrogen, D1306) to reveal nuclei, then mounted in Vectashield Mounting Medium (Vector Laboratories, H-1900). Coverslips were mounted on glass slides and stored at 4°C before confocal fluorescence microscopy analysis. Fluorescence in at least 10 random fields per condition was acquired on an Olympus FV 3000 confocal system, and the intensity of regions of interest (ROI) was quantified using ImageJ. Fluorescence intensity of region of interest (ROI) was measured using the ImageJ Software. Quantification experiments were carried out independently at least three times. Individual differences were assessed using individual student’s t-tests in GraphPad Prism software. Data are shown as mean ± standard deviation (SD). Dendritic spine quantification The number of dendritic spines in neuron cultures was quantified by staining with phalloidin to assess synaptotoxicity through visualization of spine morphology and quantification of spine numbers, as previously described 33,43-45 . Phalloidin staining specifically enriches for F-actin in dendritic spines of IPSC-derived neuron staining 33 . Neurons cultured on coverslips were fixed in 4% paraformaldehyde and stained with rhodamine-phalloidin to visualize dendritic spines. Images were acquired using an Olympus FV 3000 confocal microscope with a 63x objective (N.A. = 1.4). The number of dendritic spines was determined using ImageJ software. Briefly, 4-5 isolated dendritic segments were chosen from each image, and the images adjusted using a threshold that had been optimized to include the outline of the spines but not non-specific signals 46 . The number of spines was normalized to the measured length of the dendritic segment to give the number of spines/μm. For each experiment, 15-24 neurons from 3 to 4 individual experiments were imaged and quantified. Calcium Imaging iPSC-derived WT and A53T mutant neurons were differentiated for 35 days in clear-bottom, black-walled plates. A stock solution of 5mM of the calcium indicator Fluo-4 AM (Thermo Fisher, F14201) was made in DMSO. Next, neurons were loaded with 10 µM Fluo-4 AM in serum-free culture media for an hour at 37°C. After incubation, cells were washed three times with DPBS (free of Ca²⁺ and Mg²⁺) to eliminate non-specifically bound dye. To ensure complete de-esterification of the intracellular AM ester, cells were subsequently incubated in FluoroBright DMEM for 30 minutes at 37°C. On the other hand, mutant neurons were treated with our nanodegrader 24 hours before dye loading. Live-cell imaging was performed, capturing images every 30 seconds for 5 minutes. An inverted point-scanning confocal microscope equipped with 60x oil 60x oil PlanApo N SC, 1.40 N.A., 0.15 mm W.D. and immersion objective for glass bottom plate. The maximum change in fluorescence intensity over time was calculated to assess calcium dynamics in both WT and A53T neurons. For calculation of the intensity, the acquired images sequence is opened in image J software. Then, the somas of the neuronal cells are defined using the ROI measurement tool to define the ROIs at different time points. Fluorescence intensity for each cell is generated and normalized as follows: basal fluorescence intensity is used to normalize recording data at each time point. Basal level is considered as the intensity during the first minute of imaging (t= 0 second). An increase in the ratio exceeding fluctuation of the basal level has been defined based on the profile of each cell and considered as neuronal spontaneous activity. The experimental group and control group are maintained under the same conditions except the experimental treatment. The maximum change in fluorescence intensity over time was calculated to assess calcium dynamics in both WT and A53T neurons. Lysosomes labeling in neuronal cells Two distinct dyes were employed to label and assess the functionality of lysosomes in A53T neurons. For labeling, a cell-permeable red fluorescent dye, LysoTracker Deep Red (Thermo Fisher cat. #L12492), was utilized. In addition, a fluorogenic substrate for proteases, DQ Red BSA (Dye Quenched Bovine Serum Albumin, Thermo Fisher, cat. # D12051), was used to evaluate the functional status of the lysosomes within the cells. Briefly, neurons were differentiated on cover glasses, and on day 35 post-differentiation, each dye was added to the cell culture medium. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 4 hours. Following this incubation, the culture medium was discarded, and the cells were rinsed with 1x PBS. Subsequently, the cells were fixed using 4% paraformaldehyde (PFA) prepared in 1x DPBS for 15 minutes at room temperature. After fixation, the cells underwent additional washes, and their nuclei were stained with DAPI for 10 minutes. The cells were then washed three times with 1x PBS, and the coverslips were mounted onto glass slides. For imaging, a confocal microscope equipped with a 60x oil immersion Plan Apo N SC objective lens (1.40 N.A., 0.15 mm working distance) was used. Image analysis was conducted using ImageJ software (NIH). In vivo studies: Animals . Male and female wild-type C57BL/6NJ (PTMMSR-bred; original breeders from Jax, strain# 005304) mice were housed in a pathogen-free facility (the Ohio State University Comprehensive Cancer Center (OSUCCC), USA). Mice were maintained under controlled housing conditions for temperature (22°C) and light (12-hour light/dark cycle), with access to food and water. All experimental protocols were approved by the local Animal Ethical Committee (IACUC protocol # 2013A00000141-R3). Stereotaxic injection . At 17.5 weeks old, the animals were subjected to stereotaxic Intracranial injection. Briefly, mice were anesthetized with an intraperitoneal injection of ketamine/xylazine (100/20 mg/kg). After shaving and disinfecting the skull, the skull is immobilized in a stereotaxic frame. A midline incision was made to expose the skull, followed by a small craniotomy on the right side. The stereotaxic coordinates for injection were Anterior-Posterior (AP) 1 mm, Medial-Lateral (ML) -2 mm, and Dorsal-Ventral (DV) 3 mm. A Hamilton syringe was loaded with the nanodegrader (5 µM in PBS) and lowered to the target coordinates. A total of 2 µL of nanodegrader was injected unilaterally at 0.25 µL/min. For the control, the mice had an identical surgical procedure, but with PBS injection. Following injection, the skin was sutured and disinfected with betadine, and mice were monitored postoperatively until sacrifice at 24 and 72 hours. Immunofluorescence Intensity Quantification ImageJ (FIJI) was employed to measure fluorescence intensity within manually defined regions of interest (ROIs), targeting specific subcellular structures like dendritic spines, dendrites, and soma. Uniform image processing was applied, incorporating background subtraction and threshold adjustments to exclude non-specific signals. For each ROI, the mean fluorescence per pixel was obtained by dividing the total integrated density by the area measured. To minimize variability among experimental groups, fluorescence values were normalized relative to the mean intensity of the controls. Sampling included quantification from a minimum of five randomly selected fields per experimental condition, analyzing between 15-24 neurons per condition across 2-3 independent culture preparations, ensuring statistical robustness. Tissue Preparation and Immunofluorescence Tissue Preparation. Mice were deeply anesthetized via active carbon dioxide (CO₂) exposure and euthanized by cervical dislocation. Brains were quickly dissected and rinsed in ice-cold 1x PBS before immersion in cold 4% paraformaldehyde (PFA). Post-fixation was performed at 4°C for 48 hours. Subsequently, brains were cryoprotected by immersion in 30% sucrose in PBS solution for 48 hours at 4°C, then frozen and stored at -80°C until sectioning. Immunofluorescence Staining. 35 µm Coronal sections were obtained using a cryostat and processed as free-floating sections. The sections were permeabilized for 30 minutes by incubating in 1x PBS containing 0.2% Triton X-100 and blocked in Mouse-on-Mouse blocking serum (Vector MKB2213; 1:100 dilution) for an hour at room temperature (RT). Sections were then incubated overnight at 4°C under gentle agitation with primary antibodies diluted in blocking solution. The primary antibodies used were our monoclonal mouse anti-α-Syn (Syn 211; 1:500, Invitrogen #32-8100) and monoclonal rabbit anti-HA tag (1:500, Cell Signaling Technology, Cat. # 3724). The following day, sections were incubated for one hour at RT with secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 568 (1:500, Life Technologies). Nuclei were counterstained with DAPI (1:5000, Thermo Scientific #D21490), and an autofluorescence quenching step was performed using an autofluorescence eliminator reagent (Millipore, #2160). Finally, sections were mounted with Dako Fluorescent Mounting Medium. Imaging was performed using an Olympus FV3000 confocal microscope. The fluorescence intensity of HA positive and α-Syn positive cells was quantified at the injection site using image J software. The level of α-Syn between the ipsi-and contralateral sites was analyzed to determine any degradation effect. Immunofluorescence-stained brain sections were analyzed by nuclear-based single-cell segmentation to quantify per-cell fluorescence intensities of α-Syn ( a ) and ND21P2F ( b ), normalized to contralateral site controls. A relative per-cell degradation proxy was calculated as (1 − a )/( b + ε), where ε = 2.71828 was used solely as a stabilization constant at low ND21P2F signal. Quantification and statistical analysis All reported data were typically presented as mean ± S.E.M. and unpaired comparisons were analyzed by two-sided Student’s t-test unless otherwise mentioned. Multiple comparisons were analyzed by ordinary one-way ANOVA. 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Alpha-synuclein stepwise aggregation reveals features of an early onset mutation in Parkinson's disease. Commun Biol 2 , 374 (2019). Guilliams, T. et al. Nanobodies raised against monomeric alpha-synuclein distinguish between fibrils at different maturation stages. J Mol Biol 425 , 2397-411 (2013). Tamaki, Y. et al. Elimination of TDP-43 inclusions linked to amyotrophic lateral sclerosis by a misfolding-specific intrabody with dual proteolytic signals. Sci Rep 8 , 6030 (2018). Choi, M.L. et al. Pathological structural conversion of alpha-synuclein at the mitochondria induces neuronal toxicity. Nat Neurosci 25 , 1134-1148 (2022). Butler, Y.R. et al. alpha-Synuclein fibril-specific nanobody reduces prion-like alpha-synuclein spreading in mice. Nat Commun 13 , 4060 (2022). Ramachandran, K.V. & Margolis, S.S. A mammalian nervous-system-specific plasma membrane proteasome complex that modulates neuronal function. Nat Struct Mol Biol 24 , 419-430 (2017). Wang, C. et al. Scalable Production of iPSC-Derived Human Neurons to Identify Tau-Lowering Compounds by High-Content Screening. Stem Cell Reports 9 , 1221-1233 (2017). Fang, C., Imberdis, T., Garza, M.C., Wille, H. & Harris, D.A. A Neuronal Culture System to Detect Prion Synaptotoxicity. PLoS Pathog 12 , e1005623 (2016). Fang, C. et al. Prions activate a p38 MAPK synaptotoxic signaling pathway. PLoS Pathog 14 , e1007283 (2018). Mercer, R.C.C. et al. Sigma Receptor Ligands Are Potent Antiprion Compounds that Act Independently of Sigma Receptor Binding. ACS Chem Neurosci 15 , 2265-2282 (2024). Srivastava, D.P., Woolfrey, K.M. & Penzes, P. Analysis of dendritic spine morphology in cultured CNS neurons. J Vis Exp , e2794 (2011). Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations Yes there is potential Competing Interest. N. C. and N.L. are co-inventors on a patent application related to the nanobody-guided protein degradation platform described in this work. The other authors declare no competing interests. Supplementary Files SupplementaryinformationListofantibodiesandchemicals.docx Supplementary information- List of antibodies, reagents and specific chemicals ExtendedDataFiguresandLegends.pdf Extended Data Figures Table1.pdf Table 1: Summary of nanodegraders (NDs) generated in this study and their degradation activities. NDs were constructed by using EPL strategy to incorporate lysosomal targeting (CMA motif), proteasomal recruitment (E3 ligase ligands conjugation), and/or proximity-enabled covalent binding via SuFEx chemistry. Their α-Syn degradation efficiency and neuronal functional rescued via dendritic spines restored were included. Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8652640","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":591187826,"identity":"1fc85a72-a9f4-4836-ac46-ea02ee0697b6","order_by":0,"name":"Nam Chu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAr0lEQVRIiWNgGAWjYDACCSD+UPFPDsJjI1IL44wzB4xJ08LM2XYgsYFoLfyzewyYGc7cSe/nP2PA8KHsMBGW3DljwFxQ8Sx35owcA8YZ54jQYiCRY8A84wxz7oYbPAbMvG3EauFtY043OA+07i8JWg4nGBwAMhiJ0SJxI63g4IwzaYYzZwAZPefSCWvhn5G88cGHCht5fv7DGx/8KLMmrAUEDmAwRsEoGAWjYBRQCADoRztE8tedqgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-9717-5007","institution":"The Ohio State University","correspondingAuthor":true,"prefix":"","firstName":"Nam","middleName":"","lastName":"Chu","suffix":""},{"id":591187827,"identity":"fae7ae60-cc89-474d-8057-8507183032ca","order_by":1,"name":"Nhat Le","email":"","orcid":"","institution":"The Ohio State University","correspondingAuthor":false,"prefix":"","firstName":"Nhat","middleName":"","lastName":"Le","suffix":""},{"id":591187828,"identity":"024bc30f-eb04-4370-aa2d-15859a046394","order_by":2,"name":"Niyi Adelakun","email":"","orcid":"","institution":"The Ohio State University","correspondingAuthor":false,"prefix":"","firstName":"Niyi","middleName":"","lastName":"Adelakun","suffix":""},{"id":591187829,"identity":"3007e093-4c74-4562-914d-1710a185b714","order_by":3,"name":"Ouada Nebie","email":"","orcid":"","institution":"The Ohio State University","correspondingAuthor":false,"prefix":"","firstName":"Ouada","middleName":"","lastName":"Nebie","suffix":""},{"id":591187830,"identity":"f69e25aa-2822-435d-a289-9052b27b9a94","order_by":4,"name":"Upendra Nayek","email":"","orcid":"","institution":"The Ohio State University","correspondingAuthor":false,"prefix":"","firstName":"Upendra","middleName":"","lastName":"Nayek","suffix":""},{"id":591187831,"identity":"1000e8ff-dc90-47a8-a4cc-81a7fd294a27","order_by":5,"name":"Ryejun Na","email":"","orcid":"","institution":"The Ohio State University","correspondingAuthor":false,"prefix":"","firstName":"Ryejun","middleName":"","lastName":"Na","suffix":""},{"id":591187832,"identity":"8abc8acf-c8ce-4c76-8a35-04d8210ac2b2","order_by":6,"name":"Julian Meza","email":"","orcid":"","institution":"The Ohio State University","correspondingAuthor":false,"prefix":"","firstName":"Julian","middleName":"","lastName":"Meza","suffix":""},{"id":591187833,"identity":"d926ca48-e202-4b06-80f5-03d3453118a0","order_by":7,"name":"Reena Shakya","email":"","orcid":"","institution":"The Ohio State University","correspondingAuthor":false,"prefix":"","firstName":"Reena","middleName":"","lastName":"Shakya","suffix":""},{"id":591187834,"identity":"229fb987-efae-464a-be56-870e814e6479","order_by":8,"name":"Marie Butts","email":"","orcid":"","institution":"The Ohio State University","correspondingAuthor":false,"prefix":"","firstName":"Marie","middleName":"","lastName":"Butts","suffix":""}],"badges":[],"createdAt":"2026-01-20 19:59:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8652640/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8652640/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104405366,"identity":"0d080cab-867a-4137-af8f-a46dbb2e2128","added_by":"auto","created_at":"2026-03-11 12:22:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10478085,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesign and validation of the \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eALFA\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eND and nanodegrader\u003c/strong\u003e, \u003cstrong\u003eand efficient \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eALFA\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eND internalization in cells\u003c/strong\u003e. (\u003cstrong\u003ea\u003c/strong\u003e) Schematic of the expressed protein ligation (EPL) strategy to generate ALFA-Nb-CMA1-TMR and its subsequent conjugation to the cell-penetrating peptide, cR10. (\u003cstrong\u003eb\u003c/strong\u003e) Coomassie-stained SDS-PAGE gel showing the purification of ALFA-Nb-CMA1-intein (lane 1) and the successful ligation product, ALFA-Nb-CMA1-TMR (lane 2). (\u003cstrong\u003ec\u003c/strong\u003e) Coomassie-stained SDS-PAGE gel showing the conjugation of cR10 to ALFA-Nb-CMA1-TMR. (\u003cstrong\u003ed\u003c/strong\u003e) MALDI-TOF mass spectra of the final \u003csup\u003eALFA\u003c/sup\u003eND construct (ALFA-Nb-CMA1-TMR-cR10). (\u003cstrong\u003ee\u003c/strong\u003e) Live imaging of HeLa cells showing the rapid cellular uptake of TMR-labeled \u003csup\u003eALFA\u003c/sup\u003eND within 10 minutes post-treatment. Additionally, using a lysosome-specific tracker dye alongside immunofluorescence staining for ALFA-eGFP and HA-tagged \u003csup\u003eALFA\u003c/sup\u003eND, we confirmed their colocalization in the lysosome.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ef-g\u003c/strong\u003e) Immunofluorescence staining of untreated (Mock) and \u003csup\u003eALFA\u003c/sup\u003eND treated-human iPSC-derived neurons. After two hours post-treatment, the ND is localized at the dendritic regions (f). After 16 hours of ND treatment,\u003csup\u003e ALFA\u003c/sup\u003eND significantly reduced ALFA-eGFP degradation in human neurons (\u003cstrong\u003eg\u003c/strong\u003e). (\u003cstrong\u003eh\u003c/strong\u003e) Quantification of ALFA-eGFP degradation in human neurons following 16-hour treatment with 0.5 µM \u003csup\u003eALFA\u003c/sup\u003e ND. Data is presented as mean ± SD. Statistical comparisons were performed using unpaired t-tests. Significance: ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8652640/v1/104eaebdfceeba58f558bc72.png"},{"id":104328437,"identity":"670a6c71-39ed-484c-8092-f28e483b10d3","added_by":"auto","created_at":"2026-03-10 14:36:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8509668,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of iPSC-derived cortical neurons modeling A53T α-Syn pathology.\u003c/strong\u003e To establish a PD model, Wild-type (WT) and A53T neurons were differentiated for 35 days, then the level of α-Syn and some pre-, post-synaptic proteins was assessed. (\u003cstrong\u003ea\u003c/strong\u003e) Immunofluorescence images showing increased α-Synuclein aggregation in A53T-mutant neurons compared to WT neurons. (\u003cstrong\u003eb-c\u003c/strong\u003e) Confocal images of WT and A53T neurons stained with p-α-Syn (\u003cstrong\u003eb\u003c/strong\u003e), and quantification of total α-Syn and phospho-α-Syn (p-α-Syn) (\u003cstrong\u003ec\u003c/strong\u003e) levels from immunofluorescence images. The results clearly show significant upregulation of both α-Syn and p-α-Syn in A53T neurons. (\u003cstrong\u003ed\u003c/strong\u003e) Immunofluorescence images of WT and A53T neurons stained with phalloidin for dendritic spine labeling and the presynaptic marker synaptophysin (green). (\u003cstrong\u003ee\u003c/strong\u003e) Quantification of dendritic spine density shows a significant reduction in A53T neurons. Similarly, the expression of synaptophysin is significantly decreased in A53T neurons compared to WT neurons. Data are presented as mean ± SD. Statistical comparisons were performed using unpaired t-tests. Significance: ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8652640/v1/2d9d97429bf5f573df9a8c33.png"},{"id":104328441,"identity":"3298e8d3-d23e-409c-800e-b5f6e6021460","added_by":"auto","created_at":"2026-03-10 14:36:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":23425022,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNanodegrader internalization and targeting of A53T α-synuclein, with rescue of synaptic deficits in A53T neurons.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ability of ND21, ND21F and ND21P2F to target and degrade α-Syn was evaluated in A53T neurons at 35 days of culture. Fluorescence images of Mock and treated neurons were acquired and quantified. (\u003cstrong\u003ea\u003c/strong\u003e) Representative immunofluorescence images of A53T neurons treated with HA-tagged NDs. Staining for HA (green, marking NDs) and α-Syn (red) highlighted a successful internalization and degradation of the ND to neuronal soma and dendrites.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) High-magnification images demonstrate strong co-localization of covalent-binding of ND21F and ND21P2F with α-Syn aggregates compared to non-covalent controls. (\u003cstrong\u003ec\u003c/strong\u003e) Quantification of the Manders' co-localization coefficient between HA (NDs) and α-Syn signals, confirming significantly higher binding efficiency for covalent NDs. The \u003csup\u003eALFA\u003c/sup\u003eND does not bind α-Synuclein. (\u003cstrong\u003ed\u003c/strong\u003e) Representative confocal images of dendritic segments in human neurons, stained with phalloidin (red). Neurons expressing A53T α-Syn show reduced spine density, which is rescued by treatment with ND21P2F and ND21. (\u003cstrong\u003ee\u003c/strong\u003e) Quantification of dendritic spine density (spines/µm) from experiments in figure D.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8652640/v1/a3c968f851dd7b25616bf50b.png"},{"id":104328438,"identity":"b44c8673-2cc5-450c-9699-c905935512e1","added_by":"auto","created_at":"2026-03-10 14:36:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":21411984,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDual-proteolytic α-Syn nanodegraders eliminate A53T α-Synuclein in human neurons\u003c/strong\u003e. Nanodegarders that employ both the proteasome, and the lysosome pathways were tested in A53T mutant neurons. \u0026nbsp;The neurons were treated with 100 nM concentration of ND2Tha, ND21Tha, ND21P2FTha and ND21P2FVHL for 24 hours. The expression of α-Syn and dendritic spines density was evaluated by fluorescence staining. (\u003cstrong\u003ea\u003c/strong\u003e) Representative immunofluorescence images of A53T α-Syn-expressing human neurons after NDs treatment. (\u003cstrong\u003eb\u003c/strong\u003e) Quantification of α-Syn level from immunofluorescence images as shown in panel A. (\u003cstrong\u003ec\u003c/strong\u003e) Dendritic spine number count in cell treated with NDs (\u003cstrong\u003ed\u003c/strong\u003e) High resolution images showing the effect of NDs treatment on dendritic spine architecture. Compared to untreated control, we could observe a reduction in α-Syn immunoreactivity in neurons treated with nanodegraders and partial recovery of the synapses. α-Syn was labeled in green and the nanobodies were detected by HA staining (red).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8652640/v1/3aa442c68fdbec560570698a.png"},{"id":104328440,"identity":"d919e52d-46b2-46b7-ac4e-f24e300dfd03","added_by":"auto","created_at":"2026-03-10 14:36:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":20347025,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eND21P2F treatment restores neuronal calcium dynamics and demonstrates\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e in vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e stability.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Images illustrating changes in intracellular free Ca\u003csup\u003e2+\u003c/sup\u003e in WT, A53T untreated and treated neurons, monitored with fluo-4, AM. (\u003cstrong\u003eb\u003c/strong\u003e) Quantification of intracellular calcium showing a significant rescue effect in neurons treated with ND21P2F. (\u003cstrong\u003ec\u003c/strong\u003e) Schematic representation of the mouse brain, showing both coronal and sagittal slices, with a focus on the injection site (red dot) within the brain. Representative confocal images (4X) of coronal brain sections at 24- and 72-hours post-injection. Staining for the HA tag (green, marking ND) shows localization at the injection site, with some diffusion to adjacent areas. (\u003cstrong\u003ed\u003c/strong\u003e) High-magnification micrographs showing reduced endogenous α-synuclein (red) signal in the nanodegrader-injected hemisphere compared with the contralateral (control) hemisphere. (\u003cstrong\u003ee and f\u003c/strong\u003e) Quantification of α-Syn fluorescence intensity at the injection site compared to the contralateral hemisphere at 24 and 72 hours. ND treatment leads to a significant decrease in endogenous α-Syn levels (e). Violin plots show single-cell degradation efficiency at 24 hours and 72 hours post-treatment, reflecting the relative loss of α-Syn protein normalized to local ND signal. Efficiency decreases over time, indicating progressive α-Syn depletion in treated cells (f). Data are presented as mean ± SD; *p\u0026lt; 0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001 and ****p\u0026lt;0.0001 (Unpaired Student T-test).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8652640/v1/b31ff39b403c62d6e05e05f1.png"},{"id":104416365,"identity":"fc4f0aff-3929-46ce-9899-a9a69de193dc","added_by":"auto","created_at":"2026-03-11 13:15:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":78539292,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8652640/v1/06b04bb5-d259-4f75-b7f9-3043369babe3.pdf"},{"id":104328434,"identity":"b8825388-5e8f-42b3-a16a-f0dbf115d59e","added_by":"auto","created_at":"2026-03-10 14:36:38","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":31366,"visible":true,"origin":"","legend":"Supplementary information- List of antibodies, reagents and specific chemicals","description":"","filename":"SupplementaryinformationListofantibodiesandchemicals.docx","url":"https://assets-eu.researchsquare.com/files/rs-8652640/v1/765f3354f871e11fcc272c10.docx"},{"id":104328436,"identity":"07435e4e-d235-470c-8e59-6a978be24a69","added_by":"auto","created_at":"2026-03-10 14:36:38","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2530861,"visible":true,"origin":"","legend":"Extended Data Figures","description":"","filename":"ExtendedDataFiguresandLegends.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8652640/v1/2c21dc8b6a97fb865e2dcfc7.pdf"},{"id":104328435,"identity":"f7e141be-e0da-41cf-ab09-6b693299c65e","added_by":"auto","created_at":"2026-03-10 14:36:38","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":73142,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1: Summary of nanodegraders (NDs) generated in this study and their degradation activities. \u003c/strong\u003eNDs were constructed by using EPL strategy to incorporate lysosomal targeting (CMA motif), proteasomal recruitment (E3 ligase ligands conjugation), and/or proximity-enabled covalent binding via SuFEx chemistry. Their α-Syn degradation efficiency and neuronal functional rescued via dendritic spines restored were included.\u003c/p\u003e","description":"","filename":"Table1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8652640/v1/0f0ffb150eee8230f9cb1d64.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nN. C. and N.L. are co-inventors on a patent application related to the nanobody-guided protein degradation platform described in this work. The other authors declare no competing interests.","formattedTitle":"Chemoselective Semisynthesis of Covalent Nanobody-Guided Protein Degraders in Neurons","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe accumulation of misfolded and aggregation-prone proteins is a defining feature of many neurodegenerative disorders, including Parkinson\u0026rsquo;s, Alzheimer\u0026rsquo;s, Huntington\u0026rsquo;s and prion diseases. Aberrant protein aggregation disrupts proteostasis, impairs synaptic function, and drives neuronal death\u003csup\u003e1\u003c/sup\u003e. While pharmacological strategies suppress the expression or activity of individual proteins, these approaches rarely eliminate the existing pathological protein species that drive neurodegeneration\u003csup\u003e2,3\u003c/sup\u003e. Developing chemical biology strategies that enable the selective clearance of these toxic species therefore remains a major unmet challenge in neurodegenerative disease research\u003csup\u003e4,5\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTargeted protein degradation (TPD) has emerged as a powerful paradigm for removing specific proteins by co-opting the cellular degradation machinery. Proteolysis-targeting chimeras (PROTACs) \u003csup\u003e6\u003c/sup\u003e exemplify this concept by linking a ligand for the protein of interest to an E3 ligase recruiting moiety, promoting ubiquitination and proteasomal degradation. Related approaches, including molecular glues\u003csup\u003e7,8\u003c/sup\u003e, dTAG (degradation tag)\u003csup\u003e9\u003c/sup\u003e, or TRIM-Away\u003csup\u003e10\u003c/sup\u003e, similarly rely on proximity-inducing or adaptor-mediated mechanisms to recruit cytosolic E3 ligases and trigger proteasomal clearance of target proteins. More recently, TPD strategies have expanded beyond the proteasome to engage lysosomal degradation pathways. Lysosome-targeting chimeras (LYTACs)\u003csup\u003e11\u003c/sup\u003e and KineTACs\u003csup\u003e11\u003c/sup\u003e exploit receptor-mediated endocytosis to direct extracellular or membrane-associated proteins to lysosomes, while proteolysis-targeting antibodies such as AbTACs\u003csup\u003e12\u003c/sup\u003e and ProTabs\u003csup\u003e13\u003c/sup\u003e induce lysosomal elimination of cell-surface targets. Despite their success in oncology and cell signaling biology, current TPD modalities remain constrained by the availability of small-molecule binders or extracellular accessibility, and show limited applicability to undruggable proteins \u003csup\u003e14\u003c/sup\u003e, including the intrinsically disordered and aggregation-prone species central to neurodegeneration. Such proteins often lack stable tertiary structure or well-defined binding pockets, rendering them inaccessible to traditional small-molecule ligands and poorly recognized by proteasomal systems\u003csup\u003e15,16\u003c/sup\u003e. In addition, the tendency of misfolded proteins to form dense and compact aggregates can physically exclude traditional PROTACs that depend exclusively on proteasomal degradation\u003csup\u003e6\u003c/sup\u003e, underscoring the need for strategies that engage lysosomal pathways for aggregate clearance. Thus, there is an urgent need for advanced molecular platforms that can recognize, engage, and dismantle misfolded proteins within the complex intracellular environment of neurons.\u003c/p\u003e\n\u003cp\u003eRecent progress in Nanobody\u0026reg; technology provides a promising foundation for this goal. Nanobodies are small, stable antibody fragments that retain full antigen-binding capability while highly soluble and structurally compact (~15 kDa, ~2.5nm x 4 nm). They exhibit exceptional specificity and can recognize conformational epitopes that are inaccessible to small molecules, making them powerful tools for targeting misfolded or aggregated proteins inside cells\u003csup\u003e17\u003c/sup\u003e. Chemical strategies to convert nanobodies into degraders have begun to emerge. Notably, the GlueTAC platform introduced proximity-enabled SuFEx chemistry to covalently lock nanobodies to membrane proteins, enhancing target retention during endocytosis and promoting lysosome-mediated degradation\u003csup\u003e18,19\u003c/sup\u003e. While these works established the feasibility of covalent nanobody chimeras, existing approaches rely on genetic encoding of reactive amino acids and are largely restricted to membrane-associated targets and single degradation pathways. An advanced strategy is needed to improve GlueTACs by enhancing cell penetration while combining lysosomal targeting with proteasome-based degradation for broader and more efficient protein clearance.\u003c/p\u003e\n\u003cp\u003eTo address these limitations, we developed a chemoselective protein semisynthesis platform for constructing nanobody-guided degraders (nanodegraders, NDs) that merge nanobody precision with chemically programmable degradation. Our strategy combines expressed protein ligation (EPL)\u003csup\u003e20\u003c/sup\u003e with SuFEx proximity-enabled crosslinking\u003csup\u003e21\u003c/sup\u003e to introduce covalent stabilizing modules and azido-lysine handles for click conjugation of diverse E3 ligase ligands, including thalidomide (CRBN)\u003csup\u003e22\u003c/sup\u003e and AHPC (Von Hippel-Lindau, VHL)\u003csup\u003e23\u003c/sup\u003e. In parallel, incorporation of a chaperone-mediated autophagy (CMA) targeting motif provides a sequence-encoded route for lysosomal engagement\u003csup\u003e24\u003c/sup\u003e. Subsequent cell-penetrating peptide (CPP)\u003csup\u003e25\u003c/sup\u003e attachment enables efficient intracellular delivery, generating nanodegraders capable of recruiting both proteasomal and lysosomal degradation pathways in a programmable manner.\u003c/p\u003e\n\u003cp\u003eAs a disease-relevant application, we applied this platform to target alpha-synuclein (\u0026alpha;-Syn), a presynaptic protein whose pathological aggregation underlies Parkinson\u0026rsquo;s disease\u003csup\u003e26\u003c/sup\u003e. Mutations such as A53T promote \u0026alpha;-Syn misfolding, aggregation, and neurotoxicity\u003csup\u003e27\u003c/sup\u003e. Using human iPSC-derived neurons carrying the A53T mutation, we show that NDs internalize, selectively covalently engage A53T \u0026alpha;-Syn, and promote their degradation, thereby restoring synaptic integrity. Extending these findings \u003cem\u003ein vivo\u003c/em\u003e, optimized NDs mediated \u0026alpha;-Syn degradation up to 72 hours post-injection in mice, indicating favorable stability and translational potential. Together, these findings establish a generalizable chemical biology framework for reprogramming protein degradation pathways through modular nanobody semisynthesis, offering a new route to degrade pathogenic protein species in neurodegenerative disease.\u003c/p\u003e\n\u003cp\u003eNanobody\u0026reg; is a registered trademark of Sanofi or an affiliate.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. Design of nanobody-guided protein degraders\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;To establish a versatile and modular platform for constructing nanobody-guided degraders (nanodegraders, NDs), we employed a protein semisynthesis approach called expressed protein ligation (EPL)\u003csup\u003e20\u003c/sup\u003e . In this strategy, a recombinant protein fragment is fused to an intein domain and treated with a reactive thiol, such as MESNA, to generate a C-terminal thioester intermediate. This intermediate can then be chemoselectively ligated to a synthetic peptide bearing an N-terminal cysteine, forming a native amide bond through native chemical ligation. The N-terminal cysteine also supports Cys-Maleimide conjugation of the cR10 cell-penetrating peptide\u003csup\u003e25,28\u003c/sup\u003e, enabling efficient intracellular delivery. The EPL method provides precise control over nanobody modification and enables the incorporation of synthetic peptides with defined chemical functionalities.\u003c/p\u003e\n\u003cp\u003eTo test and optimize our strategy, we selected the ALFA tag nanobody\u003csup\u003e29\u003c/sup\u003e (\u003csup\u003eALFA\u003c/sup\u003eNb) as a model scaffold, which binds the ALFA epitope with low-picomolar affinity and high selectivity. We generated various \u003csup\u003eALFA\u003c/sup\u003eND constructs (Fig. 1a) designed to degrade ALFA-tagged enhanced green fluorescent protein (ALFA-eGFP) inducibly expressed in HeLa, as well as in human iPSC-derived neurons.\u003c/p\u003e\n\u003cp\u003eTo direct \u003csup\u003eALFA\u003c/sup\u003eNb-bound eGFP to the lysosomal degradation pathway, a peptide motif that promotes chaperone-mediated autophagy\u003csup\u003e24,30\u003c/sup\u003e (CMA1) was C-terminally fused to the nanobody. The CMA1 motif was rationally designed based on conserved lysosome-targeting sequences identified in known CMA substrates, including RNase A, \u0026alpha;-synuclein, and Tau, and corresponds to the amino acid sequence \u003cu\u003eK\u003c/u\u003eFER\u003cu\u003eQ\u003c/u\u003eVKKD\u003cu\u003eQK\u003c/u\u003eDRV\u003cu\u003eQ\u003c/u\u003e. This sequence contains the canonical \u003cu\u003eK\u003c/u\u003eFER\u003cu\u003eQ\u003c/u\u003e-like motif recognized by the Hsc70-LAMP2A machinery, enabling selective recruitment to the lysosomal compartment\u003csup\u003e24,30\u003c/sup\u003e. The resulting \u003csup\u003eALFA\u003c/sup\u003eNb-CMA1 fusion protein, which also contained an HA tag for detection, was recombinantly expressed and purified (Fig. 1b). For visualization and biochemical tracking, we synthesized a tetramethylrhodamine (TMR)-labeled N-ter Cysteine peptide and ligated it to the intein-mediated \u003csup\u003eALFA\u003c/sup\u003eNb\u0026ndash;CMA1 thioester, yielding fluorescently labeled \u003csup\u003eALFA\u003c/sup\u003eNb through EPL (Fig. 1b).\u003c/p\u003e\n\u003cp\u003eWe next equipped the nanobody with a cell-penetrating peptide (CPP) to facilitate cellular uptake. We used a cyclized deca-arginine peptide (cR10) that contains an N-terminal maleimide group linked through a PEG2 spacer to the cyclic sequence (KrRrRrRrRrRE)\u003csup\u003e25\u003c/sup\u003e. The cR10 peptide was synthesized by solid-phase peptide synthesis with purity greater than 95% (Extended Data Fig. 10a). Covalent conjugation of cR10 to the cysteine residue of \u003csup\u003eALFA\u003c/sup\u003eNb-CMA1 via maleimide-thiol coupling generated the final construct, cR10-\u003csup\u003eALFA\u003c/sup\u003eNb-CMA1, hereafter referred to as the ALFA nanodegrader (\u003csup\u003eALFA\u003c/sup\u003eND) (Fig. 1c-d). Importantly, the N-terminal maleimide did not interfere with nanobody structure and function (Extended Data Fig. 1a)\u003csup\u003e31\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe next examined whether \u003csup\u003eALFA\u003c/sup\u003eND could efficiently enter cells, bind ALFA-eGFP, and induce lysosomal degradation. Live-cell imaging revealed rapid uptake of \u003csup\u003eALFA\u003c/sup\u003eND, colocalize to ALFA-eGFP and then direct to lysosomes within 10 minutes after treatment (Fig. 1e). Over the following hours, HA-tag immunostaining confirmed cytoplasmic distribution of \u003csup\u003eALFA\u003c/sup\u003eND and colocalization with ALFA-eGFP in HeLa cells (Extended Data Fig. 1a) and human neurons (Fig. 1f-g). \u003csup\u003eALFA\u003c/sup\u003eND localized to lysosomal compartments together with ALFA-eGFP indicates successful targeting of the nanobody-bound substrate to the lysosomal degradation pathway. Notably, in human neurons, \u003csup\u003eALFA\u003c/sup\u003eND accumulated at dendritic spines (Fig. 1g), suggesting potential for clearing supersaturated or misfolded synaptic proteins.\u003c/p\u003e\n\u003cp\u003eFollowing 16 hours of treatment, \u003csup\u003eALFA\u003c/sup\u003eND at 0.5 \u0026mu;M induced pronounced degradation of ALFA-eGFP, reducing fluorescence intensity by approximately 90% in HeLa cells (Extended Data Fig. 1a-b) and 68% in human neurons (Fig. 1g). Time-course analysis revealed that ALFA-eGFP degradation became evident between 4 and 16 hours after \u003csup\u003eALFA\u003c/sup\u003eND administration (Extended Data Fig. 1b-c). These findings confirm that the combination of lysosome-targeting CMA1 motif and cR10-mediated delivery enables efficient cellular internalization of the nanodegrader and robust lysosomal degradation of the target protein.\u003c/p\u003e\n\u003cp\u003eTogether, these results validate the EPL-based semisynthesis approach as a powerful and modular method to design nanobody-guided degraders. The ALFA model system establishes a proof of concept for constructing programmable nanodegraders capable of achieving selective, lysosome-dependent degradation of intracellular targets.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Modeling Parkinson\u0026rsquo;s disease-associated A53T \u0026alpha;-Synuclein mutation using human iPSCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;To establish a disease-relevant neuronal system, we differentiated human induced pluripotent stem cells (hiPSCs) into cortical neurons using a rapid neurogenin-2 (NGN2)-induced protocol\u003csup\u003e32,33\u003c/sup\u003e. Stable transgenic hiPSC lines expressing doxycycline-inducible NGN2 were generated using Tet-on system, enabling efficient and scalable neuronal differentiation within 14 days (Extended Data Fig. 2a). The resulting neurons exhibited mature morphology, with dendrites and axons decorated by synaptic spines, and expressed neuronal and synaptic markers including MAP2, VGLUT1, and PSD95 (Extended Data Fig. 2a).\u003c/p\u003e\n\u003cp\u003eWe differentiated both wild-type (WT) and A53T \u0026alpha;-synuclein (\u0026alpha;-Syn) mutant hIPSC lines into cortical neurons. After 35 days of differentiation, A53T neurons displayed hallmark features of Parkinson\u0026rsquo;s disease, including elevated total and phosphorylated \u0026alpha;-Syn (pSer129)\u003csup\u003e34\u003c/sup\u003e, accumulation of higher-molecular-weight species, and punctate cytoplasmic aggregates detected by immunostaining (Fig. 2a, b). Furthermore, consistent with previous models of \u0026alpha;-Syn-induced toxicity, A53T neurons exhibited synaptic and structural impairments\u003csup\u003e35\u003c/sup\u003e. Synaptophysin levels were markedly reduced compared with WT neurons (Fig.2d, e). Phalloidin staining of F-actin, which is enriched in dendritic spine regions, was used to visualize spines and revealed a significant decrease in dendritic spine density (Fig. 2d, e). In addition, calcium imaging with Fluo-4 indicated dysregulated intracellular Ca\u0026sup2;⁺\u0026nbsp;homeostasis in A53T neurons relative to WT controls (Extended Data Fig. 2b). Finally, we assessed endo-lysosomal function using a fluorescent endocytic reporter. A53T neurons showed abnormal lysosomal activity, characterized by reduced fluorescence in dendrites compared to WT neurons (Extended Data Fig. 2d), consistent with impaired cargo degradation and lysosomal trafficking (Extended Data Fig. 2c-d).\u003c/p\u003e\n\u003cp\u003eTogether, these data demonstrate that our human iPSC-derived cortical neurons recapitulate key molecular and cellular features of A53T \u0026alpha;-Syn pathology, including aggregation, synaptic dysfunction, calcium imbalance, and dysregulated proteolysis activities, establishing a robust platform for testing nanodegrader-mediated clearance of pathogenic \u0026alpha;-Syn.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Development of lysosome-directing \u0026alpha;-Syn nanodegraders\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo demonstrate the versatility of our nanobody semisynthesis platform in a disease-relevant setting, we designed a nanodegrader targeting \u0026alpha;-Syn) a key pathogenic driver of Parkinson\u0026rsquo;s disease\u003csup\u003e36\u003c/sup\u003e. We used \u0026alpha;-Syn nanobody 2 (SynNb2)\u003csup\u003e37\u003c/sup\u003e as the targeting module and appended the previously described CMA1 lysosome-directing motif to its C terminus. To enable neuronal uptake, the construct was further equipped with the cell-penetrating peptide cR10 via cysteine-maleimide conjugation. The resulting conjugate, cR10-SynNb2-CMA1, referred to as nanodegrader 21 (ND21) (Extended Data Fig 3a), was purified by size-exclusion chromatography (Extended Data Fig 3b) and verified by MALDI-MS (Extended Data Fig 3c). ND21 represents the first lysosome-directing \u0026alpha;-Syn nanodegrader generated using this modular chemoselective assembly pipeline.\u003c/p\u003e\n\u003cp\u003eWe next assessed ND21 uptake and activity in human iPSC-derived neurons carrying the A53T \u0026alpha;-Syn mutation. Neurons were treated ND21 for 24 hours. Immunofluorescence staining for HA-taged ND21 and \u0026alpha;-Syn showed internalization and clear colocalization of ND21 with \u0026alpha;-Syn aggregates in both soma and dendritic regions (Fig. 3a). Quantitative imaging revealed degradation of \u0026alpha;-Syn within the soma, with 41.5 % reduction at 100 nM ND21, while dendritic \u0026alpha;-Syn levels remained largely unchanged compared to Mock treatment (Fig. 3a). This spatially restricted degradation pattern parallels the distribution of lysosomal activity, which is concentrated in the neuronal soma but markedly reduced in dendrites in A53T \u0026alpha;-Syn neurons (Extended Data Fig. 2c-d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo confirm that efficient ND21-guided \u0026alpha;-Syn degradation requires cell penetration, \u0026alpha;-Syn recognition and lysosomal directing abilities of ND21, we evaluated a series of nonfunctional control constructs. The \u0026alpha;-Syn Nb2-CMA1 construct, which lacks the cell-penetrating peptide cR10, failed to enter human neurons and showed no detectable \u0026alpha;-Syn binding (Extended Data Fig. 4a). In contrast, the \u003csup\u003eALFA\u003c/sup\u003eND control, which contains the CMA1 motif but does not recognize \u0026alpha;-Syn, readily internalized yet displayed diffuse cytoplasmic distribution without colocalization to \u0026alpha;-Syn aggregates (Extended Data Fig. 4a). The third construct consisting of SynNb2 equipped with cR10 peptide but lacking the CMA1 motif, together with these two control constructs showed no efficient \u0026alpha;-Syn degradation in A53T neurons (Extended Data Fig. 4b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDespite its partial spatial restriction, ND21 treatment led to robust recovering of spine loss in A53T neurons. Quantification of dendritic spines revealed ~97% recovery of spine density relative to WT \u0026alpha;-Syn neuron controls (Fig. 3d-e and Table 1), suggesting that selective reduction of A53T \u0026alpha;-Syn is sufficient to alleviate synaptic defects. Together, these results establish ND21 as a proof-of-concept lysosome-directing \u0026alpha;-Syn nanodegrader that achieves selective degradation and functional rescue in human neurons and provide a framework for designing next-generation nanodegraders that harness lysosomal pathways to eliminate pathogenic \u0026alpha;-Syn in this A53T neuronal model.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. SuFEx-enabled covalent \u0026alpha;-synuclein nanodegraders\u0026nbsp;\u003c/strong\u003eWhile ND21 efficiently engaged \u0026alpha;-Syn and promoted lysosome-dependent degradation in the neuronal soma, its noncovalent interaction with \u0026alpha;-Syn limited complex stability and degradation efficiency (~41.5%). To enhance the durability of ND-target association and enable covalent capture of \u0026alpha;-Syn, we next incorporated sulfur fluoride exchange (SuFEx) proximity-enabled crosslinking chemistry\u003csup\u003e21\u003c/sup\u003e into the ND design. The SuFEx reaction provides a proximity-enabled irreversible crosslinking of interacting proteins through selective sulfur-fluoride exchange at nucleophilic residues (Tyr, His, Lys), thereby enhancing complex stability and residence time. This approach allows formation of stable covalent linkages between the ND and its substrate upon proximity-induced SuFEx reaction, thereby increasing complex lifetime and potentially improving degradation yield. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe first validated proximity-enabled crosslinking ability of SuFEX-functionalized nanobodies and their intracellular targets in live cells using the ALFA nanobody.\u003csup\u003e\u0026nbsp;\u003c/sup\u003eIntein-mediated \u003csup\u003eALFA\u003c/sup\u003eNb-thioester was ligated by EPL to an N-terminal cysteine peptide bearing unnatural SuFEx-reactive lysine (K-FSY, where FSY denotes the aryl fluorosulfate SuFEx handle), then conjugated to cR10 via cysteine-maleimide coupling to yield cR10-\u003csup\u003eALFA\u003c/sup\u003eNb-K-FSY\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(Extended Data Fig. 5a). Live cells expressing ALFA-tagged eGFP were incubated with cR10-\u003csup\u003eALFA\u003c/sup\u003eNb-K-FSY, followed by lysis and co-IP of ALFA-eGFP. Immunoblotting detected the nanobody in the precipitate and revealed a higher-molecular-weight band corresponding to a covalent cR10-\u003csup\u003eALFA\u003c/sup\u003eNb-K-FSY::ALFA-eGFP adduct (Extended Data Fig. 5b), demonstrating proximity-enabled intracellular crosslinking between SuFEX-functionalized nanobody, hereafter termed a covalent nanobody and its targeted protein.\u003c/p\u003e\n\u003cp\u003eWe next applied this semisynthetic strategy to \u0026alpha;-synuclein nanobody 2 (SynNb2) fused to the CMA1 motif. Synthetic N-Cys peptides containing either K-FSY or K-PEG₂-FSY were ligated to SynNb2-CMA1 via EPL (Extended Data Fig. 5c), producing two covalent lysosome-directing nanodegraders, ND21F and ND21P2F, respectively. The PEG\u003csub\u003e2\u0026nbsp;\u003c/sub\u003espacer was introduced to improve solubility and provide linker flexibility of NDs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe then evaluated target engagement of covalent nanodegraders in human A53T \u0026alpha;-Syn neurons. Immunofluorescence imaging showed that HA-tagged ND21F and ND21P2F co-localized extensively with \u0026alpha;-Syn within soma and neurite locations of A53T neurons (Fig. 3c). The degree of co-localization was significantly higher than that observed with non-SuFEx NDs such as ND21 (Fig. 3c, d), indicating that FSY modification enhances nanobody-target engagement, likely by stabilizing the \u0026alpha;-Syn-ND complex. Immunoprecipitation of \u0026alpha;-Syn followed by western blot analysis further supported this observation: only the covalent-binding ND21P2F was co-immunoprecipitated with \u0026alpha;-Syn (Extended Data Fig. 5d), confirming most durable interaction with the A53T \u0026alpha;-Syn compared to other NDs. In addition, in vitro crosslinking assay between ND21F and purified A53T \u0026alpha;-Syn demonstrates that covalent binding occurred after 1 hour (Extended Data Fig. 5e). Quantitative imaging revealed increase degradation of \u0026alpha;-Syn, with 66 % and 69.5 % reduction at 100 nM ND21F and ND21P2F, respectively (Fig 3a, b and Table 1).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. Proteasome-recruiting and dual-proteolytic nanodegraders targeting \u0026alpha;-synuclein:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProteasome-recruiting \u0026alpha;-Syn nanodegraders:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo extend nanobody-guided degradation beyond lysosomal routing, we generated proteasome-recruiting \u0026alpha;-Syn nanodegraders by introducing an E3-ligase ligand into SynNb2 using the EPL strategy. SynNb2 was ligated to a synthetic N-terminal cysteine peptide containing a PEG\u003csub\u003e12\u003c/sub\u003e linker for enhanced solubility and a Thalidomide moiety conjugated to a lysine side chain via NHS-ester amidation. The ligation proceeded efficiently (\u0026gt;90 %) and yielded a purified monomeric product after size-exclusion chromatography (Extended Data\u0026nbsp;Fig.6a-b). The cR10 cell-penetrating peptide was subsequently coupled to generate the final construct ND2Tha, which was confirmed by MALDI-MS analysis (Extended Data Fig. 6c).\u003c/p\u003e\n\u003cp\u003eWe then treated ND2Tha with A53T neuron cultures, this proteasome-directing ND construct reduced \u0026alpha;-Syn levels by ~38 % and partially restored dendritic spine density (~52 % of wild-type) at 100 nM (Fig. 4a, b and Table 1). These results demonstrate that EPL-based installation of an E3-ligase ligand, Thalidomide-CRBN\u003csup\u003e22\u003c/sup\u003e, via a flexible PEG\u003csub\u003e12\u003c/sub\u003e linker can redirect nanobody-guided degraders toward the ubiquitin-proteasome pathway in neurons, though with moderate efficacy relative to lysosomal designs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDual-proteolytic \u0026alpha;-Syn nanodegraders\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDual engagement of lysosomal and proteasomal systems has been shown to improve clearance of misfolded or aggregation-prone proteins\u003csup\u003e38\u003c/sup\u003e. To test whether concurrent recruitment of lysosomal and proteasomal machinery could enhance \u0026alpha;-Syn clearance, we next introduced the CMA1 lysosomal motif into the Thalidomide-bearing design, generating ND21Tha. This construct integrates lysosomal routing (CMA1), proteasome recruitment (Thalidomide-CRBN), and cR10-mediated neuronal delivery. ND21Tha reduced \u0026alpha;-Syn levels by ~41 % and restored spine density to ~47 % of wild-type levels at 100 nM (Fig. 4a, b and Table 1). Comparing with single proteolytic pathway directing constructs, proteasome-directing ND2Tha and lysosome-directing ND21, ND21Tha shows no additive effect from dual-proteolytic pathway signaling.\u003c/p\u003e\n\u003cp\u003eTo harness both dual-proteolytic directing and SuFEx proximity-enabled chemistry within a single modular framework, we designed dual-proteolytic \u0026alpha;-syn nanodegraders that integrate multiple degradation cues and orthogonal chemical handles. Building on our optimized expressed protein ligation (EPL) platform, we incorporated SuFEx chemical handle K-PEG₂-FSY together with a Lysine-azide residue in the synthetic peptide, providing orthogonal reactivity for the site-specific attachment of E3-ligase ligands. The recombinant fragment SynNb2-CMA1 was ligated to the synthetic N-terminal cysteine peptide containing both K-PEG\u003csub\u003e2\u003c/sub\u003e-FSY (SuFEx) and Lys(N₃) functionalities (Extended Data Fig. 7a). The resulting semisynthetic nanobody fragment was subsequently subjected to azide-alkyne cycloaddition with DBCO-functionalized thalidomide (for cereblon, CRBN) or AHPC (for von Hippel-Lindau, VHL) ligands, yielding ND21P2F-Tha and ND21P2F-VHL (Extended Data Fig. 7b), respectively. Each construct retained the CMA1 motif for lysosomal engagement, thereby creating nanodegraders capable of dual proteolytic targeting through both the lysosomal and ubiquitin-proteasome pathways. Although these dual-proteolytic, SuFEx-functionalized NDs were efficiently assembled and covalently stabilized with \u0026alpha;-Syn, they reduced \u0026alpha;-Syn by only ~52 % and ~47 %, with corresponding spine recovery of ~57 % and ~59 %, respectively (Fig. 4c, d and Table 1).\u003c/p\u003e\n\u003cp\u003eThe results indicate that degradation by these constructs reveals proteolytic pathway competition rather than additive synergy. Together, these findings suggest that incorporating multiple degradation signals within a single ND can introduce kinetic interference and pathway bias instead of enhanced turnover. The data define practical design constraints for multi-pathway degraders in neurons and emphasize that pathway-specific modularity is more effective than multi-route fusion for the selective elimination of pathogenic \u0026alpha;-Syn.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e6. In vitro efficacy and in vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;stability, selectivity, and distribution of ND21P2F\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe selected ND21P2F to evaluate the \u003cem\u003ein vivo\u003c/em\u003e stability, distribution, and target selectivity of the ND strategy in the mouse brain. ND21P2F shows its most efficacy in both degradation of \u0026alpha;-Syn, restore spine density and synaptic functions of A53T neuron culture \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003e(Fig. 3 and Table 1). Live-cell calcium imaging with Fluo-4 AM of WT neurons displayed strong, synchronous calcium influx upon depolarization, whereas A53T neurons showed blunted responses, indicating impaired calcium entry and reduced excitability (Extended Data Fig. 2b). ND21P2F treatment enhanced calcium transients, restoring fluorescence amplitudes to near WT levels (Fig. 5a-b and Extended Data Fig. 8a). Furthermore, the time course treatment confirmed the ability of ND21P2F to effectively degrade \u0026alpha;-syn in A53T neurons.\u0026nbsp;The treatment of young A53T neurons with ND21P2F significantly reduced \u0026alpha;-synuclein levels in a time-dependent manner (Extended Data Fig. 8d-e)\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003ein vivo\u003c/em\u003e experiments, we performed stereotaxic injections into the mouse striatum and analyzed tissues at 24 hours and 72 hours post-injection. Confocal imaging of coronal sections stained for the HA tag revealed clear ND21P2F localization at the injection site after 24 hours, with fluorescence extending into adjacent parenchyma (Fig. 5c). After 72 hours, HA signals persisted with comparable intensity, indicating high in-tissue stability and limited degradation of the injected ND over this period. Moderate diffusion into neighboring regions was also observed, suggesting controlled spreading without systemic dispersion (Extended Data Fig. 9).\u003c/p\u003e\n\u003cp\u003eTo assess target engagement, sections were co-stained for \u0026alpha;-synuclein (\u0026alpha;-Syn). Merged images showed pronounced colocalization of HA and \u0026alpha;-Syn signals in the injected hemisphere, indicating that ND21P2F binds endogenous \u0026alpha;-Syn \u003cem\u003ein situ\u003c/em\u003e (Fig. 5d). Quantitative fluorescence analysis comparing the injection site with the contralateral hemisphere demonstrated a significant reduction in \u0026alpha;-Syn signal intensity at both 24 hours (~20%) and 72 hours (~ 40%) (p \u0026lt; 0.01) (Fig. 5d). These results confirm that ND21P2F remains stable in the brain for at least 72 hours, retains binding specificity toward \u0026alpha;-Syn, and exhibits local proteolytic activity sufficient to reduce endogenous \u0026alpha;-Syn levels near the injection site.\u003c/p\u003e\n\u003cp\u003eTogether, these findings establish that ND21P2F possesses favorable pharmacodynamic properties \u003cem\u003ein vivo\u003c/em\u003e, maintaining stability, tissue retention, and biochemical selectivity, and highlight its translational potential as a chemically well-defined nanobody-guided protein degrader for \u0026alpha;-Syn-associated pathologies.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis\u0026nbsp;study establishes nanobody guided degraders (nanodegraders, NDs) as a modular chemical biology platform for the selective removal of pathological \u0026alpha;-syn in human neurons. By integrating a nanobody module for molecular precision with chemoselective assembly of lysosome and proteasome recruiting signals, we demonstrate a versatile approach for programmable protein degradation. The ability to reengineer intracellular clearance pathways through site specific semisynthesis represents a conceptual advance for targeted protein degradation beyond small molecule PROTACs.\u003c/p\u003e\n\u003cp\u003eOur design combines expressed protein ligation (EPL) with SuFEx (sulfur fluoride exchange) chemistry\u003csup\u003e21\u003c/sup\u003e to install proximity enabled covalent modules and azido lysine handles for E3 ligand conjugation, while the CMA1 motif directs cargo to lysosome. This strategy enables multi-milligram scale production and flexible incorporation of diverse chemical functionalities, yielding degraders that harness proteasomal, lysosomal, or dual proteolytic mechanisms. This flexibility bridges the gap between biologics and small molecules, creating hybrid constructs that retain the selectivity of antibodies while gaining the tunability of chemical synthesis. The resulting NDs operate through covalent proximity capture rather than transient binding, ensuring high target residence time and reducing off target proteolysis. The dual proteolytic route designs revealed pathway competition rather than additive synergy, defining practical constraints on simultaneous engagement of the proteasome and lysosome in neurons. These findings highlight the importance of pathway specific modularity for achieving efficient and predictable degradation kinetics toward neurons.\u003c/p\u003e\n\u003cp\u003eMechanistically, \u0026alpha;-Syn A53T aggregates arise primarily from soluble oligomeric intermediates that disrupt calcium signaling, generate oxidative stress, and impair synaptic vesicle trafficking\u003csup\u003e39\u003c/sup\u003e. Since our NDs were developed from Syn2 nanobody which is specifically bind to soluble \u0026alpha;-Syn but not its insoluble forms\u003csup\u003e37,40\u003c/sup\u003e, NDs did not efficiently eliminate total \u0026alpha;-Syn \u0026nbsp;in A53T neurons (Fig. S9) with pronounced \u0026alpha;-Syn aggregates, but selectively reduced these toxic soluble species, which are the most neurotoxic forms, leading to marked functional recovery in A53T neurons. This selective activity parallels the natural hierarchy of \u0026alpha;-Syn pathogenicity and suggests that complete depletion of \u0026alpha;-Syn is unnecessary for therapeutic benefit.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn our A35T human neuron model, lysosomal failures at neurites potentially leads to \u0026alpha;‑syn aggregate buildup and local synaptotoxicity. This unevenly distributed of lysosomal functions\u0026nbsp;within neurons contributes to the spatial efficacy of lysosome targeting NDs. Lysosomal-directing ND mediated \u0026alpha;-Syn clearance occurred primarily in somatic regions, with limited neuritic \u0026alpha;‑syn aggregate degradation. Covalently complexes of SuFEx-functionalized, lysosome-directing NDs and \u0026alpha;-Syn potentially enhancing lysosomal access to distal compartments could improve both degradation and synaptotoxic rescue efficacies in this A53T model.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProteasome represents a complementary degradation route that operates at both cytosolic and synaptic sites. A membrane associated neuronal proteasome (NMP) has been implicated in peptide release and synaptic regulation\u003csup\u003e41\u003c/sup\u003e. Redirecting \u0026alpha;-Syn toward proteasomal clearance could therefore simultaneously reduce toxic aggregates and normalize local proteostasis at synapses. Our proteasome recruiting NDs demonstrate the feasibility of coupling E3 ligase recognition through thalidomide or AHPC conjugation with nanobody precision, establishing a foundation for targeted modulation of neuronal proteasome activity. Future work should explore how NDs interact with NMPs and whether proteasome directed degradation can influence synaptic plasticity and neurotransmission.\u003c/p\u003e\n\u003cp\u003eWhile our study focused on \u0026alpha;-Syn, the same framework could be adapted to other misfolded or aggregation prone proteins implicated in neurodegenerative and systemic diseases. By swapping the nanobody module or degradation signal, the platform can be reprogrammed for distinct intracellular environments or disease contexts. Importantly, our \u003cem\u003ein vivo\u003c/em\u003e experiments show that optimized NDs maintain stability and target selectivity within brain tissue for at least 72 hours, supporting their translational potential.\u003c/p\u003e\n\u003cp\u003eSeveral challenges remain. Failures in protein homeostasis of misfolded protein disease models, particularly the A53T PD neuronal model limit uniform ND distribution and degradation efficacy. Improving pharmacokinetics, enhancing binding efficacy of ND to the target, proved by SuFEx-functionalized covalent NDs, will be critical for advancing NDs toward therapeutic use. It will also be valuable to test NDs efficacy in PD models that recapitulate \u0026alpha;-Syn propagation and synaptic transmission \u003cem\u003ein vivo\u003c/em\u003e to evaluate whether local degradation can halt or reverse disease progression.\u003c/p\u003e\n\u003cp\u003eIn summary, we demonstrate a modular chemical strategy for constructing nanobody based degraders that reprogram proteolytic systems to selectively dismantle neurotoxic \u0026alpha;-Syn species. By integrating lysosomal, proteasomal, and covalent crosslinking mechanisms within a unified protein semisynthetic framework, this study establishes fundamental design principles for pathway specific degradation in human neurons. These findings open new directions for chemical biology approaches to restore disease-associated proteostasis and neuronal health in Parkinson\u0026rsquo;s disease and related proteinopathies.\u003c/p\u003e"},{"header":"List of abbreviations","content":"\u003cp\u003e\u0026alpha;-Syn: \u0026alpha;-Synuclein\u003c/p\u003e\n\u003cp\u003eBCA: bicinchoninic acid assay\u003c/p\u003e\n\u003cp\u003eCHX: cycloheximide\u003c/p\u003e\n\u003cp\u003eCM: maintaining medium\u003c/p\u003e\n\u003cp\u003eCMA: chaperon mediated autophagy\u003c/p\u003e\n\u003cp\u003eCPP: cell penetrating peptide\u003c/p\u003e\n\u003cp\u003eEPL: expressedfas protein ligation\u003c/p\u003e\n\u003cp\u003eIM: induction medium\u003c/p\u003e\n\u003cp\u003eiPSC: induced pluripotent stem cells\u003c/p\u003e\n\u003cp\u003eMap2: Microtubule-associated protein 2\u003c/p\u003e\n\u003cp\u003eND: nanobody-based protein degrader\u003c/p\u003e\n\u003cp\u003eNGN2: neurogenin-2\u003c/p\u003e\n\u003cp\u003eNMDA: N-methyl-D-aspartate\u003c/p\u003e\n\u003cp\u003ePOI: protein of interestf\u003c/p\u003e\n\u003cp\u003ePSD-95: Postsynaptic density protein 95\u003c/p\u003e\n\u003cp\u003eROI: region of interest\u003c/p\u003e\n\u003cp\u003eSD: standard deviation\u003c/p\u003e\n\u003cp\u003eTPD: targeted protein degradation\u003c/p\u003e\n\u003cp\u003etIPSCs: transgenic induced pluripotent stem cells\u003c/p\u003e\n\u003cp\u003eTuj1: class III beta-tubulin\u003c/p\u003e\n\u003cp\u003eUb: Ubiquitin\u003c/p\u003e\n\u003cp\u003eWT: wild type\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNL and NC formulated the research plan, designed the nanodegraders (NDs) and interpreted experimental results with assistance from ON, UN, NA. NC, UN and NA generated NDs, purified proteins, performed protein semisynthesis and analyzed biochemical experiments. NL established human iPSC-derived neuron models with assistance from ON on iPSCs differentiations. NDs treatments on cell lines and neurons were performed and analyzed by NA, NL and ON. Mouse studies were performed by ON, RN and JN and analyzed by ON and NL. NC and NL wrote the manuscript with contributions from ON and NA. All authors edited and approved the manuscript. NC and NL jointly supervised the project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Michael Ward at National Institute of Neurological Disorders and Stroke (NINDS/NIH) for the K4-PB-TO-hNGN2 and K13-EF1a vectors, and Dr. William C. Skarnes at The Jackson Laboratory for Genomic Medicine for the human IPSCs lines. We also thank Dr. Dehua Pei for LC/MS analysis and Dr. Monica Venere for using cryostat. We acknowledge resources from the Campus Microscopy and Imaging Facility (CMIF) and The OSU Comprehensive Cancer Center (OSUCCC) Microcopy Shared Resource (MSR), The Ohio State University (RRID:SCR_025078). This facility is supported in part by grant P30 CA016058, National Cancer Institute, Bethesda, MD. We thank the OSUCCC's Preclinical Therapeutics Mouse Modeling Shared Resource for technical and instrumental support. The facility is supported by the OSU Comprehensive Cancer Center (OSUCCC) and the National Institute of Health under grant number P30 CA016058. \u0026nbsp;The content is the authors' sole responsibility and does not necessarily represent the official views of the NIH. NC was supported by the OSUCCC Startup fund and NIH grant K22CA241105. NC, NL, NA, UN and ON were supported by NIH grant R35GM151124. NA was supported by the Pelotonia Graduate Fellowship. NL was supported by a Warren Alpert Distinguished Scholar Award. The project was supported by the OSU President’s Research Excellent Accelerator Award (NC and NL), OSU NRI Seed Grant Award (NL) and the Sanofi iDEA-Tech Award (NC and NL).\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eChemicals, antibodies and reagents:\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSodium 2-mercaptoethanesulfonate (MESNA) was purchased from Millipore Sigma (USA). Chitin resin was purchased from New England Biolabs (USA). Thalidomide was purchased from BroadPharm (California, USA). 4-(Acetylamino)phenyl]imidodisulfuryl difluoride (AISF) was purchased Chem-Impex International (USA). Rink amide resin, amino acids, and HATU were \u003cem\u003epurchased\u003c/em\u003e from P3 BioSystems (USA). Solvents (DMF, DCM, DIEA, TFA) were purchased from Sigma-Aldrich (USA). IPTG, Dithiothreitol and ProBlock™ Protease Inhibitor Cocktail were purchased from GoldBio (USA). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSee Extended Data Table 1 for information of antibodies used for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePlasmids and cloning\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll nanobody expression vectors were constructed in the pTXB1 vector. The nanobodies \u003csup\u003eALFA\u003c/sup\u003eNb (Addgene #136626) and α-Syn Nb2\u003csup\u003e37\u003c/sup\u003e (gene synthesis by GenScript) were PCR-amplified to create a megaprimer and subcloned into pTXB1 vector (NEB IMPACT™ system), which encodes an Mxe GyrA intein-chitin-binding domain (CBD) fusion at the vector C-terminus. DNA oligos were designed and purchased from Integrated DNA Technologies (IDT). An N-terminal HA epitope tag was fused upstream of each nanobody open reading frame. The CMA motif was inserted in-frame at the C-terminus of each nanobody, ensuring continuous reading frame into the pTXB1 Mxe GyrA intein-CBD vector.\u003c/p\u003e\n\u003cp\u003ePlasmids and clones were subsequently transformed into E. coli and verified by Sanger and whole-plasmid sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePeptide synthesis:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePeptides corresponding to Cys-Gly-Gly-Gly-Ser-Lys-Gly-Gly-Gly-Ser were synthesized on the automated PurePep Chorus peptide synthesizer (Protein Technologies) using Fmoc-Gly, Fmoc-Ser(tBu), Fmoc-Lys(Alloc), Boc-Cys(TrT) and Rink-Amide resin (0.05 mmol). In particular, 4 eq. of amino acid, 3.8 eq. of HATU, 8 eq. of NMM in DMF were double coupled for 1.5 h, and Fmoc groups were removed by 20% piperidine in DMF over two 10 min cycles.\u003c/p\u003e\n\u003cp\u003eTo synthesize peptides bearing either K-FSY or K-PEG\u003csub\u003e2\u003c/sub\u003e-FSY, the allyloxycarbonyl (Alloc) protecting group of N-ε-Alloc-lysine was orthogonally removed using 0.1 eq. Pd(PPh\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e4\u003c/sub\u003e and 25 eq. phenysilane in dry DCM for 30 minutes under argon atmosphere. The resin was subsequently washed twice with 0.5 M DIEA in DMF. Next, 5 eq. of 4-hydroxybenzoic acid (Sigma), 4.75 eq. of HATU, and 5 eq. of DIEA were dissolved in DMF, added to the resin, and rotated for 1.5 hours to afford N-ε-(4-hydroxybenzoyl)-L-Lysine peptide. The resin was washed thoroughly with DMF, DCM and methanol. Finally, the sulfurylation of N-ε-(4-hydroxybenzoyl)-L-Lysine was carried out by adding a mixture of 1.2 eq. of AISF (Chem Impex, 36191), 2.2 eq. of DBU (Sigma, 139009) in dry DCM to the resin for 1.5 hours. This reaction was repeated once to ensure completion. For the K-PEG\u003csub\u003e2\u003c/sub\u003e-FSY peptide, prior to the coupling of 4-hydroxy benzoic acid, Fmoc-NH\u003csub\u003e2\u003c/sub\u003e-PEG\u003csub\u003e2\u003c/sub\u003e-COOH was double coupled to the lysine using the standard Fmoc strategy described above.\u003c/p\u003e\n\u003cp\u003eTo synthesize peptide containing thalidomide, Cys-(PEG\u003csub\u003e6\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e- Gly-Gly-Gly-Ser-Lys-Gly-Gly-Gly-Ser was synthesized on the automated PurePep Chorus peptide synthesizer using Fmoc-Gly, Fmoc-Ser(tBu), Fmoc-Lys(Alloc), Fmoc-PEG\u003csub\u003e6\u003c/sub\u003e-COOH, Boc-Cys(TrT), and Rink-Amide resin (0.05 mmol). The Alloc protecting group of N-ε-Alloc-lysine was orthogonally removed as described above. Next, 1.2 eq. of Thalidomide-PEG\u003csub\u003e4\u003c/sub\u003e-NHS ester (BroadPharm) and 2.4 eq. of DIEA in dry DMF was added to the resin, and the coupling reaction was left overnight.\u003c/p\u003e\n\u003cp\u003ePeptide containing TAMRA, 5-isomer and biotin was synthesized automatedly and manually. The target peptide sequence Cys-Gly-Gly-Gly-Ser-Lys(Biotin)-Gly-Gly-Gly-Ser-Lys(ivDde)-Gly-Gly-Gly-Ser was synthesized using Fmoc-Gly, Fmoc-Ser(tBu), Fmoc-Lys(ivDde), Fmoc-Lys(Biotin), Boc-Cys(TrT), and Rink-Amide resin (0.05 mmol). Fmoc-Lys(ivDde) and Fmoc-Lys(ivDde) were coupled on resin manually. The ivDde protection group of N-ε-ivDde-lysine was orthogonally removed with 5% hydrazine in DMF for 5 minutes (3 x 5 min), followed by extensive rinsing with DMF. Next, 1 eq. of TAMRA carboxylic acid, 5-isomer (Lumiprobe, 67190), 1 eq. of HATU and 4 eq. of DIEA in DMF was added to the resin, and the coupling reaction was carried out overnight.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMaleimide cyclic R10 peptide was synthesized using Fmoc-L-Arg (R), Fmoc-D-Arg (r), Fmoc-Lys(Alloc), Fmoc-Glu(OAll), Fmoc-PEG\u003csub\u003e2\u003c/sub\u003e-COOH on Rink-Amide resin (0.05 mmol) as a linear peptide of the sequence PEG\u003csub\u003e2\u003c/sub\u003e-PEG\u003csub\u003e2\u003c/sub\u003e-K(Alloc)-RrRrRrRrRr-E(OAll) as previously described\u003csup\u003e25\u003c/sup\u003e. The Alloc and OAll protection groups were orthogonally removed using 0.1 eq. of Pd(PPh\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e4\u003c/sub\u003e and 25 eq. of phenysilane in dry DCM for 30 minutes under argon atmosphere. The resin next was washed with 0.5 M DIEA in DMF to remove Pd. The cyclization of the peptide was carried out using 1 eq. of HATU and 2 eq. of DIEA in DMF for 2h at room temperature. Subsequently, the maleimide cR10 peptide was obtained by coupling 2 eq. of 2-Maleimidoacetic acid (Ambeed Inc, A110455) with 2 eq. of HATU and 4 eq. of DIEA in DMF for 1 h at room temperature.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCyclic R10 peptide bearing a C-terminal Sortase A recognition motif (LPETG) was synthesized using the same solid-phase and cyclization strategy described above. Briefly, the linear peptide K(Alloc)-RrRrRrRrRr-E(OAll)-(PEG2)\u003csub\u003e2\u003c/sub\u003e-SKYLELPETG was assembled on Rink-Amide resin (0.05 mmol) using standard Fmoc chemistry. Orthogonal removal of Alloc and OAll protecting groups, intramolecular cyclization, and global deprotection were performed as described for the maleimide cR10 peptide, yielding the cR10-(PEG2)\u003csub\u003e2\u003c/sub\u003e-LPETG product. All peptides were deprotected and cleaved from the resin\u0026nbsp;with trifluoroacetic acid: water: triisopropylsilane (95:2.5:2.5, v/v/v) for 3 hours, then precipitated with chilled diethyl ether, washed twice with chilled diethyl ether, and the crude peptides were dried by nitrogen gas flow. The crude peptides were purified using preparative reverse-phase C18 HPLC column (Vydac) using a gradient of water:acetonitrile containing 0.05% trifluoroacetic acid. Pure fractions were combined, concentrated on a rotavap and then lyophilized. Peptide structures were confirmed using MALDI mass spectrometry or LC-MS/MS and peptide concentrations were determined by amino acid analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eProtein expression and purification:\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSHuffle T-7 Express \u003cem\u003eE. coli\u003c/em\u003e (NEB) bearing pTXB1 plasmid for nanobodies C-terminally fused with CMA1, \u003cem\u003eMxe\u003c/em\u003e GyrA intein and chitin-binding domain (CBD) was cultured in 0.5 L of LB media to reach OD\u003csub\u003e600\u003c/sub\u003e 0.6-0.8 and then induced with 0.5 mM IPTG for 18 hrs at 16\u003csup\u003eo\u003c/sup\u003eC. Cells were harvested by centrifugation at 5,000 rpm for 15 minutes at 4°C, and resuspended in lysis buffer (50 mM HEPES, 250 mM NaCl, 0.1% Triton X-100, 10% glycerol, pH 7.5) with one tablet of protease inhibitors (Thermo Scientific) and 1 mM PMSF. Cells were lysed using a French Press and centrifuged at 22,000 x g for 35 minutes at 4°C to collect the supernatant. The supernatant passed twice to 3 mL of chitin resin (NEB) pre-equilibrated with lysis buffer to capture the \u003csup\u003eSyn\u003c/sup\u003eNb2-CMA1-intein-CBD. The resin was then washed with 150\u0026nbsp;mL washing buffer (25\u0026nbsp;mM HEPES pH 7.5, 500\u0026nbsp;mM NaCl, 0.1% Triton X-100) and incubated overnight in cleavage buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, pH 7.5) containing 100 mM DTT and 0.5 mM PMSF at room temperature. The cleavage buffer containing nanobody (2-3 mgs) was collected and purified using size exclusion chromatography with Superdex 75 10/300 GL column and phosphate-buffered saline (PBS) buffer, pH 7.4, 3 mM DTT. SDS-PAGE was used to assess yield and purity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eProtein semisynthesis approach:\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNanobody (Nb)-CMA1-intein-CBD was purified from T-7 SHuffle Express \u003cem\u003eE. coli\u003c/em\u003e using 3 mL of chitin resin as described above. After washing, 3 mL of cleavage buffer containing 200 mM MESNA (sodium mercaptoethylsulfonate, Sigma) and 0.5 mM PMSF was added to the chitin column, incubated overnight at room temperature. The cleavage buffer containing 2-3 mgs of Nb-CMA1-thioester was collected, concentrated by ultrafiltration using an Amicon 10 kDa MWCO filter (Sigma Millipore) to 0.5 mL, exchanged into 200 mM Sodium phosphate pH 6.5 using a 2-mL zeba desalting column (Thermo). Next, Nb-CMA1-thioester was immediately reacted with 4 mM of the synthetic N-Cys containing peptides at room temperature for 3 hours, followed by overnight incubation at 4°C. The ligation products were assessed by Coomassie SDS-PAGE and were purified using size exclusion chromatography with Superdex 75 10/300 GL column (Cytiva) and PBS buffer. The pure fractions were combined, concentrated to ~1 mg/mL, aliquoted and stored at -80\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ecR10 labeling:\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSemisynthetic nanobodies were conjugated with cyclic R10 (cR10) peptides using either maleimide-based thiol coupling or Sortase A–mediated ligation. For maleimide labeling, the semisynthetic nanobodies were incubated with maleimide-cR10 peptide at a molar ratio 1:7 in 1x PBS pH 7.4 for one hour at room temperature, followed by an overnight incubation at 4°C. The reaction was monitored using 15% SDS-PAGE, and the Zeba desalting columns (Thermo Fisher) were used to remove the excess cR10 peptide from cR10-NDs efficiently.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor Sortase A-mediated ligation, semisynthetic nanobodies bearing an N-terminal triglycine motif were ligated to cR10-PEG2-PEG2-LPETG using GST–Sortase A. Reactions were performed at room temperature for 2 hours in ligation buffer 50 mM HEPES (pH 7.5) and 5 mM CaCl₂, using 50 µM nanobody and 500 µM cR10-PEG2-PEG2-LPETG. Upon completion, GST–Sortase A was removed by incubation with glutathione agarose beads, and excess cR10-PEG2-PEG2-LPETG was removed using Zeba desalting columns (Thermo Fisher). Ligation efficiency was assessed by SDS-PAGE.\u003c/p\u003e\n\u003cp\u003eAll semisynthetic nanobodies bearing cR10 are hereafter referred to as \u003cstrong\u003enanodegraders\u003c/strong\u003e. All ND constructs were validated by mass spectrometry using a MALDI-TOF-TOF mass spectrometer (Bruker-Daltonics UltrafleXtreme). Spectra were obtained in both negative and positive reflectron ion modes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCell line cultures: \u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeLa cells were obtained from ATCC and cultured in DMEM (Gibco, 11965118) with high glucose and 5 mM Glutamine containing 10% FBS. Cells were grown at 37 °C and 5% CO2 up to 80-90% confluency then passed on at 1 in 10 and grown for up to a week. The medium was changed every 3-4 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHuman IPSC cultures\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth A53T mutant and the control WT KOLF 2.1 iPSC lines were obtained from Jackson Laboratory. IPSC lines were used for a maximum of 10 passages to avoid chromosomal and genetic aberrations that may appear during long-term passages. Accordingly, we periodically checked the human iPSC cultures to ensure they did not possess abnormal karyotype. The human iPSC lines were cultured in mTeSR™ medium (STEMCELL Technologies, 85850) in a Matrigel (BD Matrigel™, hESC-qualified Matrix, 354277) coated plate. Enzyme-free passaging reagents, ReLeSR™ (STEMCELL Technologies, 100-1438) were used for routine passaging of cells as cell clumps. The media of the iPSC cultures were changed daily for optimal growth.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eNeuronal differentiation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNeuronal differentiation was carried out according to the published procedure\u003csup\u003e32,33,42\u003c/sup\u003e. Briefly, we generated stable iPSC lines expressing the transcription factor neurogenin-2 (NGN2 through Lipofectamine Stem-mediated transfection of DNA plasmids. For NGN2-expressing lines, we used the K4-PB-TO-hNGN2 and K13-EF1a vectors, kindly provided by Dr. Michael Ward at NINDS/NIH. Puromycin 1-3 μg/ml was used for selection and enrichment of transgenic IPSC cell (tIPSCs) population. Cortical neuron differentiations were performed using the established procedure\u003csup\u003e32,33\u003c/sup\u003e. Briefly, tIPSCs were cultured as single cells in neuronal induction medium (IM) with doxycycline (Sigma D9891) 2 μg/ml on Matrigel coated plates for 3 days. Cortical neurons \u0026nbsp;differentiation was then induced with the appropriate medium (Table S2). The different days after differentiation are labelled numerically (D1 for day 1). At D4, cells were finally re-plated onto dishes coated with poly-L-ornithine (PLO) for neuronal maturation. Rho-associated protein kinase (ROCK) inhibitor Y-27632 (Tocris Bioscience, 1254) 10 μM was used in the cultures on D1 and D4 for splitting. Based on the type of interrogation the cells were either plated at low-density for immunofluorescent imaging or at high-density for preparation of cell lysates. Cells were split using Accutase (Gibco, A1110501). Following D4 final plating, neurons were cultured in neuronal maintaining medium (CM) for 7, 14, 21, 28, and 35 days. Half of the medium was changed every day with fresh pre-warm CM. At experimental endpoints, neurons were either fixed for immunofluorescence microscopy or lysed to make protein lysates.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eNanodegrader treatments:\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHela cell cultures after 3 days of transfections for expressing ALFA-tagged EGFP proteins were used to treat nanodegraders. Pre-treatment with cycloheximide (CHX) for 4 hours, then nano-dergraders were added into the culture medium at 500nM concentration designed timepoints. Tet-on ALFA-EGFP engineered cells were pretreated with Doxycycline 2 μg/ml to induce ALFA-EGFP expression for 2-3 days before nanodegraders were added into new culture medium at designed concentration and time points. Human iPSC-derived neurons at day 35 of differentiation were used for the treatments. Nanodegraders similarly were added into the conditional neuronal maintaining media at final concentration of 500 nM, 100 nM for 24 hours. Cell lysates and coverslip of cultures were collected for further immunoassay and biochemical analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eWestern blot (WB)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell and neuron lysates were collected from culture in RIPA buffer or IP buffer. The total protein content of samples was measured by bicinchoninic acid assay (BCA) (Pierce, 23225). Equal amounts of protein lysate were mixed with appropriate volume of loading dye and heated for 5 min at 99°C prior to separation on Tris-Glycine SDS-PAGE gels. The proteins were then transferred onto Immobilon PVDF membranes (Immobilon-P, Millipore) and the membranes were blocked for 30 min with 5% BSA in TBST (Tris-buffered saline, 0.1% Tween 20) [1X] buffer and then incubated with primary antibodies overnight at 4°C (See Extended Data table 1 for details). Antigen-antibody complexes were detected using fluorescent goat anti-mouse IRDye 800 (green) or 680 (red) and goat anti-rabbit IRDye 680 (red) secondary antibodies, respectively, and visualized with the LI-COR Odyssey Classic Infrared Imaging System.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunoprecipitation assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntibody-bead conjugates were prepared by adding 2 μg of anti-HA or-\u0026nbsp;α-Syn antibodies to 500 μl of Pierce IP Lysis Buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) (Thermo Fisher Scientific, 87788) in a 1.5 ml microcentrifuge tube, together with 30 μl of Dynabeads protein A/G (Invitrogen). Tubes were rotated for 2 hours at 4°C followed by two washes with IP Lysis Buffer to remove unbound antibodies. The washed antibody-bead mixture was incubated with cell lysate prepared as described below.\u003c/p\u003e\n\u003cp\u003eNeuron cultures were washed in ice-cold 1x PBS, and the cells were scraped from the dishes in IP buffer supplemented with protease inhibitor (Millipore Sigma, P8340). The lysates were then sheared by repeated passage through Tuberculin syringes with 25G needle and clarified by centrifugation at 5000 rpm for 5 min. The protein concentration of the supernatants was quantified by the BCA assay. \u0026nbsp; We incubated 0.5 ml at 2.0 mg/ml of lysate with the washed antibody-bead mixture for 2 hours at 4°C in a rotator at 10 rpm speed. The unbound fractions (flowthrough) were collected, and beads were subsequently washed three times with 1.0 ml of IP lysis buffer. SDS-sample buffer was then added to the beads. Equal portions of the supernatants were then separated by SDS-PAGE and immunoblotted for HA-tagged proteins and α-Syn.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunocytochemistry staining\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells grown on coverslips were washed with PBS and fixed with 4% paraformaldehyde, then blocked in blocking buffer (1% BSA in PBS) for 30 minutes. In case of intracellular proteins detection by staining, permeabilization step with Triton-X100 0.1% for 5 minutes was performed before the blocking step. Fluorescence staining was performed using the primary and secondary antibodies listed in Table S1. Primary antibodies were made up in 1% blocking buffer and PBS. After incubation, the cells were washed with PBS; secondary antibodies were incubated in 1% blocking buffer. Finally, cells were washed 5 times, 5 min/time with PBS, and counterstained with DAPI (Invitrogen, D1306) to reveal nuclei, then mounted in Vectashield Mounting Medium (Vector Laboratories, H-1900). Coverslips were mounted on glass slides and stored at 4°C before confocal fluorescence microscopy analysis. Fluorescence in at least 10 random fields per condition was acquired on an Olympus FV 3000 confocal system, and the intensity of regions of interest (ROI) was quantified using ImageJ. Fluorescence intensity of region of interest (ROI) was measured using the ImageJ Software. Quantification experiments were carried out independently at least three times. Individual differences were assessed using individual student’s t-tests in GraphPad Prism software. Data are shown as mean ± standard deviation (SD).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDendritic spine quantification\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe number of dendritic spines in neuron cultures was quantified by staining with phalloidin to assess synaptotoxicity through visualization of spine morphology and quantification of spine numbers, as previously described\u003csup\u003e33,43-45\u003c/sup\u003e. Phalloidin staining specifically enriches for F-actin in dendritic spines of IPSC-derived neuron staining\u003csup\u003e33\u003c/sup\u003e. Neurons cultured on coverslips were fixed in 4% paraformaldehyde and stained with rhodamine-phalloidin to visualize dendritic spines. Images were acquired using an Olympus FV 3000 confocal microscope with a 63x objective (N.A. = 1.4). The number of dendritic spines was determined using ImageJ software. Briefly, 4-5 isolated dendritic segments were chosen from each image, and the images adjusted using a threshold that had been optimized to include the outline of the spines but not non-specific signals\u003csup\u003e46\u003c/sup\u003e. The number of spines was normalized to the measured length of the dendritic segment to give the number of spines/μm. For each experiment, 15-24 neurons from 3 to 4 individual experiments were imaged and quantified.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCalcium Imaging\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eiPSC-derived WT and A53T mutant neurons were differentiated for 35 days in clear-bottom, black-walled plates. A stock solution of 5mM of the calcium indicator Fluo-4 AM (Thermo Fisher, F14201) was made in DMSO. Next, neurons were loaded with 10 µM Fluo-4 AM in serum-free culture media for an hour at 37°C. After incubation, cells were washed three times with DPBS (free of Ca²⁺\u0026nbsp;and Mg²⁺) to eliminate non-specifically bound dye. To ensure complete de-esterification of the intracellular AM ester, cells were subsequently incubated in FluoroBright DMEM for 30 minutes at 37°C. On the other hand, mutant neurons were treated with our nanodegrader 24 hours before dye loading.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLive-cell imaging was performed, capturing images every 30 seconds for 5 minutes. An inverted point-scanning confocal microscope equipped with 60x oil 60x oil PlanApo N SC, 1.40 N.A., 0.15 mm W.D. and immersion objective for glass bottom plate. The maximum change in fluorescence intensity over time was calculated to assess calcium dynamics in both WT and A53T neurons. For calculation of the intensity,\u0026nbsp;the acquired images sequence is opened in image J software. Then, the somas of the neuronal cells are defined using the ROI measurement tool to define the ROIs at different time points. Fluorescence intensity for each cell is generated and normalized as follows: basal fluorescence intensity is used to normalize recording data at each time point. Basal level is considered as the intensity during the first minute of imaging (t= 0 second). An increase in the ratio exceeding fluctuation of the basal level has been defined based on the profile of each cell and considered as neuronal spontaneous activity. The experimental group and\u0026nbsp;control group are maintained under the same conditions except the experimental treatment.\u003c/p\u003e\n\u003cp\u003eThe maximum change in fluorescence intensity over time was calculated to assess calcium dynamics in both WT and A53T neurons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLysosomes labeling\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;in neuronal cells\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo distinct dyes were employed to label and assess the functionality of lysosomes in A53T neurons. For labeling, a cell-permeable red fluorescent dye, LysoTracker Deep Red (Thermo Fisher cat. #L12492), was utilized. In addition, a fluorogenic substrate for proteases, DQ Red BSA (Dye Quenched Bovine Serum Albumin, Thermo Fisher, cat. # D12051), was used to evaluate the functional status of the lysosomes within the cells.\u003c/p\u003e\n\u003cp\u003eBriefly, neurons were differentiated on cover glasses, and on day 35 post-differentiation, each dye was added to the cell culture medium. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂\u0026nbsp;for 4 hours. Following this incubation, the culture medium was discarded, and the cells were rinsed with 1x PBS. Subsequently, the cells were fixed using 4% paraformaldehyde (PFA) prepared in 1x DPBS for 15 minutes at room temperature. After fixation, the cells underwent additional washes, and their nuclei were stained with DAPI for 10 minutes. The cells were then washed three times with 1x PBS, and the coverslips were mounted onto glass slides.\u003c/p\u003e\n\u003cp\u003eFor imaging, a confocal microscope equipped with a 60x oil immersion Plan Apo N SC objective lens (1.40 N.A., 0.15 mm working distance) was used. Image analysis was conducted using ImageJ software (NIH).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vivo studies:\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e. Male and female wild-type C57BL/6NJ (PTMMSR-bred; original breeders from Jax, strain# 005304) mice were housed in a pathogen-free facility (the Ohio State University Comprehensive Cancer Center (OSUCCC), USA). Mice were maintained under controlled housing conditions for temperature (22°C) and light (12-hour light/dark cycle), with access to food and water. All experimental protocols were approved by the local Animal Ethical Committee (IACUC protocol #\u0026nbsp;2013A00000141-R3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStereotaxic injection\u003c/strong\u003e. At 17.5 weeks old, the animals were subjected to stereotaxic Intracranial injection. Briefly, mice were anesthetized with an intraperitoneal injection of ketamine/xylazine (100/20 mg/kg). After shaving and disinfecting the skull, the skull is immobilized in a stereotaxic frame. A midline incision was made to expose the skull, followed by a small craniotomy on the right side. The stereotaxic coordinates for injection were\u0026nbsp;Anterior-Posterior (AP) 1 mm, Medial-Lateral (ML) -2 mm, and Dorsal-Ventral (DV) 3 mm. A Hamilton syringe was loaded with the nanodegrader (5 µM in PBS) and lowered to the target coordinates. A total of 2 µL of nanodegrader was injected unilaterally at 0.25 µL/min. For the control, the mice had an identical surgical procedure, but with PBS injection. Following injection, the skin was sutured and disinfected with betadine, and mice were monitored postoperatively until sacrifice at 24 and 72 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunofluorescence Intensity Quantification\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImageJ (FIJI) was employed to measure fluorescence intensity within manually defined regions of interest (ROIs), targeting specific subcellular structures like dendritic spines, dendrites, and soma. Uniform image processing was applied, incorporating background subtraction and threshold adjustments to exclude non-specific signals. For each ROI, the mean fluorescence per pixel was obtained by dividing the total integrated density by the area measured. To minimize variability among experimental groups, fluorescence values were normalized relative to the mean intensity of the controls. Sampling included quantification from a minimum of five randomly selected fields per experimental condition, analyzing between 15-24 neurons per condition across 2-3 independent culture preparations, ensuring statistical robustness.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTissue Preparation and Immunofluorescence\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTissue Preparation.\u0026nbsp;\u003c/em\u003eMice were deeply anesthetized via active carbon dioxide (CO₂) exposure and euthanized by cervical dislocation. Brains were quickly dissected and rinsed in ice-cold 1x PBS before immersion in cold 4% paraformaldehyde (PFA). Post-fixation was performed at 4°C for 48 hours. Subsequently, brains were cryoprotected by immersion in 30% sucrose in PBS solution for 48 hours at 4°C, then frozen and stored at -80°C until sectioning.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eImmunofluorescence Staining.\u0026nbsp;\u003c/em\u003e35 µm Coronal sections were obtained using a cryostat and processed as free-floating sections. The sections were permeabilized for 30 minutes by incubating in 1x PBS containing 0.2% Triton X-100 and blocked in Mouse-on-Mouse blocking serum (Vector MKB2213; 1:100 dilution) for an hour at room temperature (RT). Sections were then incubated overnight at 4°C under gentle agitation with primary antibodies diluted in blocking solution. The primary antibodies used were our monoclonal mouse anti-α-Syn (Syn 211; 1:500, Invitrogen #32-8100) and monoclonal rabbit anti-HA tag (1:500, Cell Signaling Technology, Cat. # 3724).\u003c/p\u003e\n\u003cp\u003eThe following day, sections were incubated for one hour at RT with secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 568 (1:500, Life Technologies). Nuclei were counterstained with DAPI (1:5000, Thermo Scientific #D21490), and an autofluorescence quenching step was performed using an autofluorescence eliminator reagent (Millipore, #2160). Finally, sections were mounted with Dako Fluorescent Mounting Medium. Imaging was performed using an Olympus FV3000 confocal microscope.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe fluorescence intensity of HA positive and α-Syn positive cells was quantified at the injection site using image J software. The level of α-Syn between the ipsi-and contralateral sites was analyzed to determine any degradation effect. Immunofluorescence-stained brain sections were analyzed by nuclear-based single-cell segmentation to quantify per-cell fluorescence intensities of α-Syn (\u003cem\u003ea\u003c/em\u003e) and ND21P2F (\u003cem\u003eb\u003c/em\u003e), normalized to contralateral site controls. A relative per-cell degradation proxy was calculated as (1 − \u003cem\u003ea\u003c/em\u003e)/(\u003cem\u003eb\u003c/em\u003e + ε), where ε = 2.71828 was used solely as a stabilization constant at low ND21P2F signal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eQuantification and statistical analysis\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll reported data were typically presented as mean ± S.E.M. and unpaired comparisons were analyzed by two-sided Student’s t-test unless otherwise mentioned. Multiple comparisons were analyzed by ordinary one-way ANOVA. GraphPad Prism 10 software program was used for all statistical analysis. 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Here, we present a chemoselective semisynthesis platform that uses expressed protein ligation (EPL) to generate nanobody-guided degraders (nanodegraders, NDs) incorporating SuFEx (sulfur–fluoride exchange) covalent modules for proximity-enabled crosslinking, azido-lysine handles for E3-ligand conjugation, and a chaperone-mediated autophagy (CMA1) motif for lysosomal targeting. Subsequent attachment of a cell-penetrating peptide (CPP) enables intracellular delivery, yielding α-synuclein (α-Syn) NDs that engage lysosomal, proteasomal, or dual-proteolytic degradation pathways. Dual-proteolytic NDs revealed route competition rather than synergy, defining design limits for multi-route degradation in neurons. The optimized covalent lysosome-targeting ND efficiently internalized into human iPSC-derived A53T α-Syn neurons, cleared aggregated α-Syn, restored calcium alterations and synaptotoxic effects, and remained stable and selective in vivo. This work establishes a modular chemical strategy for engineering nanobody-based degraders that dismantle neurotoxic proteins through multiple proteolytic systems.","manuscriptTitle":"Chemoselective Semisynthesis of Covalent Nanobody-Guided Protein Degraders in Neurons","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-10 14:36:33","doi":"10.21203/rs.3.rs-8652640/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0eb8ba7e-3dbb-4171-9b90-f8d233ca33b2","owner":[],"postedDate":"March 10th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":62915875,"name":"Health sciences/Diseases/Neurological disorders/Neurodegenerative diseases"},{"id":62915876,"name":"Biological sciences/Chemical biology/Chemical modification"},{"id":62915877,"name":"Biological sciences/Chemical biology/Protein folding/Protein aggregation"},{"id":62915878,"name":"Biological sciences/Chemical biology/Chemical tools"},{"id":62915879,"name":"Biological sciences/Chemical biology/Protein design"}],"tags":[],"updatedAt":"2026-03-10T14:36:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-10 14:36:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8652640","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8652640","identity":"rs-8652640","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}