Targeting neurodegenerative disease-associated protein aggregation with proximity-inducing modalities

Abstract

Neurodegenerative diseases (NDDs) are characterized by progressive neuronal dysfunction and anatomical changes caused by neuron loss and gliosis, ultimately leading to severe declines in brain function. While these disorders arise from a variety of pathological mechanisms, a common molecular feature is the accumulation of misfolded proteins, which occurs both inside and outside neurons. For example, Alzheimer’s disease (AD) is defined by extracellular β-amyloid plaques and intracellular tau neurofibrillary tangles. These pathological protein aggregates are often resistant to traditional small molecule drugs. Recent advances in proximity-inducing chimeras such as proteolysis-targeting chimeras (PROTACs), lysosome-targeting chimeras (LYTACs), autophagy-targeted chimeras (AUTOTACs), dephosphorylation-targeting chimeras (DEPTACs) and ribonuclease-targeting chimeras (RIBOTACs) offer promising strategies to eliminate pathological proteins or mRNAs through intracellular degradation pathways. These innovative approaches open avenues for developing new therapies for NDDs. In this review we summarize the regulatory mechanisms of protein aggregation, highlight the advancements in proximity-inducing modalities for NDDs, and discuss the current challenges and future directions in therapeutic development.

Introduction

Neurodegenerative diseases (NDDs) are characterized by the progressive deterioration of neuronal structure and function, causing significant impairments in cognitive abilities and daily functioning. These NDDs commonly include Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). They pose a significant threat to human health and longevity and have been ranked the “third most common cause of death” after cardiovascular diseases and cancer [1, 2]. Therefore, urgent clinically effective therapies are required for NDDs. However, multiple underlying pathogenic mechanisms complicate the diagnosis and treatment of NDDs. Studies have indicated that aging is associated with NDD onset, with their prevalence increasing as individuals age [3, 4]. The World Health Organization has estimated that in the next twenty years, NDDs will become the second leading cause of mortality worldwide.

Recently, proximity-inducing modalities have revolutionized drug development. These modalities include proteolysis-targeting chimeras (PROTACs) [5], lysosome-targeting chimeras (LYTACs) [6, 7], autophagy-targeted chimeras (AUTOTACs) [8], autophagosome-tethered compounds (ATTECs) [9], dephosphorylation-targeting chimeras (DEPTACs) [10], ribonuclease-targeting chimeras (RIBOTACs) [11], and Trim-Away [12]. These novel technologies provide new selective strategies to target previously untreatable proteins or mRNAs, effectively blocking their pathological functions [13–15]. These proximity-inducing modalities have achieved significant success, highlighting their therapeutic potential in NDD treatment. This review briefly introduces the molecular mechanisms underlying protein aggregation and their association with NDDs, summarizes recent progress of the proximity-inducing modalities in NDDs (Fig. 1 and Table 1), and discusses therapeutic interventions and the challenges associated with clinical translation.

Fig. 1. Schematic illustration of the mechanisms and applications of proximity-inducing modalities against NDDs.

PROTAC, HyT, and TRIM21-based modalities (Trim-Away, R-Nb, and RING-Bait) degraded target proteins via the UPS. Whereas AUTAC, ATTEC, and CMATAC degraded target proteins via the autophagy-lysosome system. RIBOTAC degrades target RNAs by ribonucleases, and DEPTAC modulates the dephosphorylation of target proteins by phosphatases.

Table 1.

Summary of proximity-inducing modalities.

Modality	Mechanism	Advantages	Disadvantages
PROTAC	Hijacks the UPS to induce targeted protein degradation, and the PROTAC can be recycled	Potential to target undruggable proteins and to overcome drug resistance; high selectivity; catalytic mechanism	Poor membrane impermeability and oral bioavailability; insufficient ability to degrade aggregated proteins
AUTOTAC ATTEC CMATAC	Targets the protein of interest to autophagic pathway for degradation	Potential to clear protein aggregates and damaged organelles	Potential to influence normal autophagy-related functions
RIBOTAC	Brings target RNAs and ribonuclease together to promote the cleavage of the target RNAs	Able to destruct RNA targets such as miRNAs; target proteins indirectly through mRNA degradation	Challenging in identifying suitable binders for RNAs and ribonucleases
HyT	Using hydrophobic tags to promote the degradation of denatured or misfolded proteins	Lower molecular weight; specificity for unstable and misfolded proteins independent of ubiquitination and E3 ligase	Degradation mechanism remains to be studied; limited to degrade intracellular proteins
DEPTAC	Dephosphorylating the hyperphosphorylated proteins by recruiting phosphatases	Fine-tuned regulation of the protein functions	Difficulty in regulating the specific phosphorylation sites
Trim-Away	Utilizes antibody-based approaches to induce targeted protein degradation	Compatible with commercial antibodies for acute and rapid degradation of intracellular proteins	Delivery challenges and potential immunogenicity

Aberrant protein aggregates are the pathological hallmarks of various NDDs

Several pathological factors have been associated with the onset of NDDs; however, the deposition of pathological protein aggregates is a significant hallmark across these conditions. These pathological protein aggregates may arise from different molecular mechanisms and have been linked with NDD’s pathogenesis [16, 17]. For instance, β-amyloid accumulation leads to the formation of extracellular plaques, while tau aggregation promotes neurofibrillary tangles, both of which are characteristic features of AD [18–20]. Furthermore, α-synuclein aggregate accumulation has been associated with PD [21], whereas the aggregation of mutant Huntington’s protein (mHTT) is characteristically found in HD [22]. These disorders have a common mechanism, that is, the gradual accumulation of misfolded proteins into fibrils within the neuronal intracellular and extracellular environments. Moreover, these aggregates have been observed to transition between intracellular and extracellular environments [23]. Cells’ uptake and release of protein aggregates may promote pathology in the brain. Therefore, these protein aggregates may impair neuronal membrane permeability, disrupt calcium balance, induce inflammation, and promote oxidative stress-mediated neurotoxicity, causing chronic toxicity in the neuronal system [24].

Several factors modulate protein aggregate formation, such as protein overexpression, missense gene mutations, truncated protein production, endoplasmic reticulum stress, and protein folding cofactors deficiencies. These factors can promote the exposure of hydrophobic regions, facilitating intra-protein interactions, and facilitating misfolding. Moreover, the interactions between misfolded protein’s exposed hydrophobic regions promote aggregate formation. Once established, these aggregates can recruit normal proteins and convert them into aggregated configurations [25, 26]. For instance, protein hydrolysis or abnormal RNA splicing can release the mHTT protein’s amino-terminal polyglutamine (polyQ) region, triggering aggregate formation [27, 28]. Further, in full-length huntingtin, the structure of the expanded polyQ regions influences its interactions with other proteins [29, 30]. Misfolded protein accumulation can be caused by age-related disruptions in protein synthesis, folding, and degradation [31]. β-sheet-rich oligomers, such as α-synuclein, have been observed to resist all known protein hydrolysis pathways [32]. Furthermore, gene mutations may also promote aggregation-related diseases. For example, in ALS and FTD, TAR DNA-binding protein 43 (TDP-43) and fused in sarcoma (FUS) mutations have been found to induce abnormal cytoplasmic localization and pathological aggregates formation [33].

Targeted degradation of aggregate-prone proteins with PROTACs

Eukaryotic cells employ two primary mechanisms for protein degradation: the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway [14]. The literature suggests that approximately 80%–90% of proteins are degraded via the UPS. Therefore, proximity-inducing modalities include PROTACs, which are heterobifunctional molecules that can hijack the intrinsic UPS to degrade target proteins. These chimeras consist of a warhead that targets E3 ligases connected by a flexible linker to a binder that recognizes the target protein. The E3 ligand recruits the E3 ligase, effectively hijacking the UPS to facilitate target proteins ubiquitination and breakdown [34–37] (Fig. 2a). PROTACs hold significant promise for treating NDDs. Researchers aim to establish more effective therapies to reduce cognitive decline and improve NDD patients’ life satisfaction by specifically targeting pathological proteins and employing emerging technologies with small molecule strategies.

Fig. 2. Schematic illustration of the PROTAC-mediated protein degradation mechanisms.

a The mechanism of protein degradation mediated by PROTACs. PROTACs are heterobifunctional molecules comprising a warhead that targets an E3 ligase connected by a flexible linker to a binder that recognizes the target protein. b Chemical structures of tau-targeted PROTACs, QC-01-175 and C004019. c Chemical structure of α-synuclein-targeted PROTAC, Compound 2b. d Chemical structures of Huntingtin-targeted PROTACs, including Compounds 7, 1, and 2. e Chemical structure of TDP-43-targeted PROTAC, PROTAC 2.

Targeted degradation of aggregate-prone proteins in AD with PROTACs

The most common age-related NDD is AD, characterized by a decline in cognitive abilities and gradual memory loss. It has been observed that AD arises due to the misfolding and accumulation of β-amyloid and hyperphosphorylated tau proteins, which promote the formation of amyloid plaques and neurofibrillary tangles [18, 19].

Tau are major microtubule-associated proteins that are abundant in neurons, stabilize microtubules, and regulate vesicle transport. Abnormal hyperphosphorylation of tau, primarily induced by tau kinases, mutations, and the disruption of various genetic or environmental factors, disrupts its association with microtubules. This disruption initiates the formation of protofibrils and aggregates, causing impaired neuronal function and cell death [19].

Chu et al. developed a peptide-based PROTAC, TH006, that specifically degraded tau through UPS [38]. They revealed that TH006 promoted tau degradation in both the cortex and hippocampal CA3 region of the mouse AD model while alleviating Aβ-induced toxicity. Lu et al. synthesized another peptide-based PROTAC to degrade tau by replacing the VHL-binding peptides in TH006 with KEAP1-targeting peptides [39]. However, these peptide-based PROTACs were unstable and had membrane impermeability, which limited their degradation efficacy.

Silva et al. also developed a small molecule-based degrader, QC-01-175 (Fig. 2b), which preferentially degraded different pathological forms of tau, such as Tau-P301L, Tau-A152T, and phosphorylated tau, thus rescuing tau-mediated neuronal stress vulnerability [40]. Wang et al. also introduced a small molecule degrader, C004019 (Fig. 2b), with potent in vitro and in vivo tau-degrading activity [41]. C004019 penetrated the blood-brain barrier (BBB) and promoted tau clearance in the brains of 3xTg-AD transgenic and hTau mice. Moreover, C004019 significantly improved spatial learning and memory abilities in a mouse AD model, thus increasing cognitive function and synaptic activity.

Targeted degradation of aggregate-prone proteins in PD with PROTACs

PD is a long-term, progressive movement disorder characterized by a gradual worsening of symptoms. Its hallmark feature is the formation of Lewy bodies in dopamine-producing neurons, which predominantly comprise misfolded α-synuclein [21]. In normal physiological conditions, α-synuclein is found in the nucleus and synaptic nerve endings, where it modulates vesicular transport and neurotransmitter release. However, in pathological states, α-synuclein has been observed to accumulate in various forms, such as oligomers and aggregates, therefore causing cytotoxicity, which leads to neuronal injury and cell death. Therefore, reducing α-synuclein levels may alleviate PD onset and progression.

Qu et al. synthesized a peptide-based PROTAC, TAT-PBD-PTM, which effectively degraded α-synuclein in cultured cells [42]. Furthermore, TAT-PBD-PTM inhibited α-synuclein-induced mitochondrial dysfunction and reduced cytotoxicity in cultured cells and primary neurons. However, this degradation was sustained for only 8 h because of the peptide’s instability.

Tong et al. also designed a small molecule PROTAC, compound 2b (Fig. 2c), that promoted targeted degradation of α-synuclein aggregates [43]. Quantitative proteomic analyses of compound 2b revealed that it reduced α-synuclein aggregates with high specificity. Moreover, compound 2b inhibited cell death by diminishing α-synuclein aggregates in a cell PD model as well as alleviated neurotoxicity and oxidative stress in a Caenorhabditis elegans PD model.

Targeted degradation of aggregate-prone proteins in HD with PROTACs

HD is an autosomal dominant hereditary neurodegenerative disorder that is characterized by a progressive decline in motor skills, cognitive abilities, and emotional functions. It has been indicated that HD arises from an abnormal increase in the CAG repeat sequence (more than 35 repeats) in exon 1 of the HTT gene [22], which leads to the production of aggregation-prone mHTT, characterized by an elongated N-terminal polyQ region. Currently, there are no effective treatments for HD.

Tomoshige et al. developed compounds 1 and 2 by coupling cellular inhibitor of apoptosis protein 1 (cIAP1) ligand BE04 with protein aggregation probes benzothiazole aniline (BTA) and 1,6-bis(benzothiazol-2-yl)-hexane (PDB), respectively [44] (Fig. 2d). Both the compounds effectively reduced mHTT to nearly undetectable levels in fibroblasts of HD patients. Later on, the researchers replaced BE04 with the higher-affinity MV1 ligand to synthesize HTT-degrading compound 7 (Fig. 2d). Compound 7 reduced the levels of both wild-type (wt) HTT and mHTT; however, its effectiveness was lower than that of the initial compounds, potentially because of an unsuitable linker design [45].

Targeted degradation of aggregate-prone proteins in ALS with PROTACs

ALS is a progressively advancing NDD that primarily affects adults and targets the motor system. It has been observed that ALS promotes the degeneration of motor neurons in the brainstem and spinal cord, which eventually progresses to paralysis and, ultimately, death from respiratory muscle failure, usually within 1 to 5 years after symptoms start. Most ALS cases are sporadic; however, about 10% are inherited in an autosomal dominant pattern. Studies have identified > 50 genes associated with ALS, and the most prevalent mutations were found in the chromosome 9 open reading frame 72 (C9orf72), superoxide dismutase 1 (SOD1), TDP-43, and FUS. These mutations account for over 70% of familial cases [46]. Currently, there are no effective treatments for ALS, indicating the urgent need for novel therapeutic approaches. TDP-43 is an RNA-binding protein that is crucial for RNA synthesis and splicing. The misfolding of TDP-43 is characterized by significant nuclear clearance and cytoplasmic accumulation of aggregates, which is a hallmark of ALS-related motor neuron degeneration [47].

Tseng et al. synthesized a small molecule PROTAC, PROTAC 2 (Fig. 2e), to specifically degrade TDP-43 aggregates [48]. PROTAC 2 selectively degrades aggregated C-TDP-43 while preserving endogenous TDP-43, thus improving cell survival. PROTAC 2 inhibits aggregated C-TDP-43 levels, which improves locomotor activity in transgenic Caenorhabditis elegans.

Removing misfolded proteins with a hydrophobic tag

Hydrophobic motifs on misfolded protein surfaces can be recognized and degraded by the UPS. Therefore, the hydrophobic tag (HyT) is a promising therapeutic target for reducing pathologically elevated proteins (Fig. 3a). Gao et al. developed a peptide harboring a HyT for the selective degradation of tau [49]. This peptide contained HyT sequences, tau-binding sequences, and cell-penetrating peptide (CPP) motifs. The HyT-Tau-CPP chimera significantly reduced tau protein levels in N2A cells via the ubiquitin-proteasome-dependent mechanism (Fig. 3b). Furthermore, intravenous administration of this chimera substantially alleviated tau accumulation in AD mice’ brains. Gao et al. also synthesized a hydrophobic tag-coupled peptide called D4 for the selective TDP-43 degradation [50] (Fig. 3c). This peptide comprised a hydrophobic domain, a TDP-43 binding domain, and a CPP. D4 uses CPP to facilitate cellular entry and effectively reduce TDP-43 levels, thus alleviating TDP-43 aggregates-associated cytotoxicity. In TDP-43 overexpressing Drosophila models, D4 significantly decreased TDP-43 levels.

Fig. 3. Schematic illustration of HyT-mediated protein degradation mechanisms and the representative chemicals.

a The mechanism of protein degradation mediated by HyTs. HyTs combine a protein of interest (POI) and chaperone via the POI binder and the hydrophobic tag, respectively, and hijack the proteasome to degrade the POI. b Chemical structure of HyT targeting tau (HyT-Tau-CPP). c Chemical structure of HyT targeting TDP-43 (D4).

These studies indicate the innovative use of HyTs for the selective degradation of misfolded and aggregated proteins, highlighting the significance of HyTs as a promising therapeutic target for treating NDDs. Due to the peptide’s instability and its limited BBB permeability, the development of small molecules with HyT capable of effectively crossing the BBB is critical for treating NDDs. Ongoing research on hydrophobic motifs may discover novel hydrophobic tag-coupled compounds with enhanced efficacy, bioavailability, and BBB permeability.

Trim21-based modalities selectively remove protein aggregates

TRIM21, a member of the tripartite motif (TRIM) family, is an E3 ubiquitin ligase that promotes an antiviral response by targeting intracellular pathogens for degradation [51]. TRIM21 consists of three conserved motifs typical of TRIM proteins (a RING finger, a B-box, and a coiled-coil domain) in the N-terminal region and a C-terminal PRYSPRY domain that dictates diverse TRIM functions (Fig. 4a). TRIM21’s PRYSPRY domain act as a cytoplasmic Fc receptor to bind with neutralizing antibody and transports the virus/neutralizing antibody complex to proteasomes for degradation [52]. Based on TRIM21-mediated viral particles, Trim-Away, an innovative research tool, was developed, which leverages the cytoplasmic Fc receptor and TRIM21’s RING finger E3 ligase activity to rapidly and specifically eliminate intracellular proteins targeted with an antibody [53, 54] (Fig. 4b). However, it’s in vivo application is limited by the inherent difficulty in delivering antibodies into cells.

Fig. 4. Schematic diagrams indicating the structure and mechanisms of action of the TRIM21-based modalities.

a The domains of TRIM21 and its dimerization. b The mechanism of TRIM21-Away-mediated protein degradation. The specific antibody binds to the target protein and interacts with TRIM21’s PRYSPRY, which then promotes ubiquitination of the protein and degradation via the proteasomal pathway. c R-Nb, composed of an N-terminal TRIM21 RING domain and a C-terminal anti-tau nanobody, specifically degrades tau aggregates. Tau aggregation promotes the dimerization and clustering of R-Nb and the activation of RING E3 ligase. These aggregates are degraded via the proteasomal pathway. d RING-Bait specifically degrades tau aggregates by integrating them into growing tau aggregates. RING-Bait comprises a tau variant carrying the P301S mutation, which is fused with a TRIM21 RING domain at the C-terminus. During fiber elongation, RING-Bait integrates into expanding tau aggregates, thus promoting TRIM21 RING-mediated ubiquitination and subsequent degradation of the aggregates.

TRIM21’s ubiquitination function is activated after intermolecular dimerization of the RING finger domain. Benn et al. employed this target-induced dimerization or clustering strategy to establish a RING-nanobody degrader, R-Nb, which included an N-terminal TRIM21 RING domain, a C-terminal anti-tau nanobody F8-2, and a T2A mCherry reporter [55] (Fig. 4c). In cultured cells and animal models, R-Nb effectively inhibited or reversed tau aggregation while minimally impacting monomeric tau. R-Nb degrades aggregated tau via the UPS and valosin-containing protein (VCP). Moreover, it also degraded tau oligomers in the central nervous system (CNS) in vivo via the adeno-associated virus (AAV) delivery.

Miller et al. synthesized a RING-Bait strategy using a tau variant carrying the P301S mutation fused to a TRIM21 RING domain at the C-terminus [56] (Fig. 4d). This construct integrates into expanding tau aggregates during fiber elongation to promote TRIM21 RING-mediated ubiquitination and then aggregates degradation. The Tau-RING construct reduced 95% of tau aggregates in vitro without affecting monomeric tau levels. Furthermore, when administered via AAV 9P31 under a human synaptic protein promoter to P301S mice, it significantly reduced tau aggregates in the brain and improved locomotor performance.

R-Nb and RING-Bait utilize the cluster-dependent activation of TRIM21 to selectively degrade tau aggregates while leaving monomeric tau unaffected. This approach targets and clears already-formed tau protein aggregates rather than merely preventing the formation of new ones, potentially alleviating AD symptoms without inducing toxicity.

Bifunctional PROTACs have the limitation of insolubility, membrane impermeability, and poor pharmacokinetics due to their relatively higher molecular weights. Whereas monovalent molecular glue degraders that are specific for target proteins and E3 ubiquitin ligases for degradation are more drug-like [57]. Recently, Lu et al. identified that (S)-ACE-OH, a metabolite of the antipsychotic drug acepromazine, acts as a molecular glue degrader of the nucleoporin NUP98 by promoting NUP98 and TRIM21 binding [58]. They employed (S)-ACE-OH as a ligand for TRIM21 to develop a selective multimeric PROTAC degrader, TrimTAC. TrimTAC indicated significant potential against diseases caused by pathological protein aggregation, such as autoimmunity and NDDs. The discovery of TRIM21-based molecular glues for degrading aggregate-prone proteins using the ligand pocket of TRIM21 has provided a promising direction for future research.

Targeted degradation of aggregate-prone proteins with autophagy-associated degraders

The autophagy-lysosome pathway can more effectively degrade aggregates than the UPS. Research on mammalian and yeast systems has identified three types of autophagy pathways: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Macroautophagy is the most well-known form of autophagy and begins with the formation of a double-membrane phagosome that engulfs target proteins or organelles. The phagosome then elongates to form autophagosomes and fuses with lysosomes for degradation by lysosomal hydrolases. Macroautophagy requires cargo delivery; however, CMA directly targets lysosomal membranes by cytoplasmic proteins. It has been observed that proteins containing KFERQ motifs are linked with the constitutive chaperone heat shock cognate protein 70 (HSC70). This binding promotes the interaction of HSC70 with lysosome-associated membrane protein type 2 A (LAMP2A), thus facilitating the formation of an active translocation complex that transports proteins across the lysosomal membrane. Whereas microautophagy directly engulfs cytoplasmic cargo by lysosomes [59].

Chaperone-mediated autophagy-targeting chimera (CMATAC) is a heterodimer that can recruit the target protein and HSC70 as well as transport the target protein to the lysosome for degradation via LAMP2A (Fig. 5). Fan et al. designed a CMATAC called TAT-βsynCTM to degrade α-synuclein via CMA [60]. TAT-βsynCTM stably interacts with the target protein to form a complex. Then, the connection between the CMA targeting motifs and CMA machinery directs these complexes to lysosomes for degradation, thus significantly reducing natural α-synuclein levels in primary cultured neurons. Bauer et al. synthesized a CMATAC fusion molecule, HQ, which selectively targets mHTT for degradation [61]. They observed that HQ effectively reduced polyQ aggregation in a CMA-dependent manner. Furthermore, when packaged into an adeno-associated virus and injected into HD mice’s striatum, HQ decreased polyQ aggregates by 78.4%, improving mice’s motor abilities and increasing their survival rates.

Fig. 5. Schematic illustration of the mechanisms of action of AUTOTAC, ATTEC, and CMATAC.

AUTOTAC promotes autophagic degradation of the target protein by anchoring it to the SQSTM1/p62 complex. ATTEC is a bifunctional heterodimer that can directly bind to the target protein and autophagy receptor LC3, inducing the autophagic degradation of the target protein. CMATAC is a heterodimer that directly binds to the target protein and HSC70 and transports the target protein to the lysosome for degradation by the interaction between HSC70 and LAMP2A.

The literature has indicated that AUTOTAC achieves autophagic degradation of the target protein by anchoring it to the SQSTM1/p62 complex (Fig. 5). Lee et al. designed an AUTOTAC degrader, ATC161, for degrading α-synuclein aggregates [62]. The data revealed that ATC161 effectively degraded α-synuclein aggregates, whereas there was no effect on monomeric α-synuclein, which limited the spread of α-synuclein aggregates to neighboring cells and protected mitochondrial integrity. Furthermore, ATC161 indicated good oral bioavailability and about 10% brain penetration. Moreover, its oral administration to PD mice significantly reduced α-synuclein aggregates and inhibited the accumulation of Lewy body-like inclusions. Further, ATC161 alleviated neurotoxicity related to α-synuclein aggregation, improved the glial cell’s immune response, and enhanced mice’s motor abilities, highlighting its therapeutic potential. Mei et al. designed an AUTOTAC by combining polyQ-binding peptide 1 (QBP1) with either an ATG16L1-binding peptide (ABP) or an LC3-interacting region (LIR) (termed QBP-ABP and QBP-LIR, respectively) to selectively degrade mHTT [63]. Both chimeras reduced HTT65Q-GFP aggregates via the autophagic-lysosomal pathway. Similarly, Mei et al. further developed chimeric peptides comprising QBP, LIR, and CPP (CPP-QBP-LIR), which specifically degraded HTT65Q-GFP aggregates, depending on concentration and time. Moreover, when QBP was substituted with mitochondrial targeting sequences (MTS), the chimera enhanced the degradation of damaged mitochondria, indicating its potential applications against NDDs, where mitochondrial dysfunction is a common feature.

ATTEC is a bifunctional heterodimer that can directly bind to the target protein and the autophagy receptor LC3, promoting the autophagic degradation of the target protein (Fig. 5). Li et al. employed a small molecule microarray screen to determine four ATTECs: 10O5, 8F20, AN2, and AN1, that specifically interact with LC3 and mHTT without affecting wtHTT [64]. The compounds 10O5 and 8F20 indicated a K_d of about 100 nM for both LC3 and mHTT, significantly decreasing mHTT levels in primary cortical neurons and alleviating mHTT-induced neurotoxicity. Moreover, all four compounds substantially reduced mHTT levels in Drosophila models. Their intraperitoneal administration into HD model mice showed that 10O5 and AN2 crossed the BBB and markedly reduced mHTT levels in the cortex and striatum, as well as improved the HD-associated behavior deficits in these mice.

The CMATAC, AUTOTAC, and ATTEC techniques provide several innovative strategies for targeted protein degradation via the autophagy-lysosome pathway in NDDs. Furthermore, LYTACs, autophagy-targeting nanobody chimeras (ATNC) [65], transferrin receptor-targeting chimeras (TransTACs) [66], and cytokine receptor-targeting chimeras (KineTACs) [67] have further advanced the field of targeted protein degradation, opening new avenues for basic research and drug development.

Modulating post-translational modifications with proximity-inducing chimeras

Post-translational modifications (PTMs) have been associated with protein aggregation [68]. For instance, aberrant phosphorylation and O-GlcNAc modifications of tau have been observed to promote AD progression. Furthermore, pathological phosphorylation of tau inhibits its interaction with microtubules, thus promoting self-aggregation and reducing O-GlcNAc levels in AD patient’s brains, therefore inducing tau aggregation, microtubule destabilization, and neuronal alterations [69].

Zheng et al. established a new dephosphorylation-targeted chimera (DEPTAC) to promote tau’s interaction with the Bα subunit of protein phosphatase 2 A (PP2A), the primary tau phosphatase in the brain [70] (Fig. 6a). They observed that DEPTAC effectively dephosphorylated tau at multiple sites and significantly reduced tau accumulation in both cultured cells and animal AD models. Moreover, trans-ventricular DEPTAC injection induced neural protrusion plasticity and microtubule assembly, thus enhancing AD model mice’s learning and memory abilities. Hu et al. established a small molecule dephosphorylation chimera, Tau2-8 (Fig. 6b), which reduced 65% phosphorylation at pThr231 and pThr181 [71]. Su et al. designed D16 (Fig. 6b), a peptide-based dephosphorylation chimera that significantly decreased both phosphorylated tau and total tau levels, thus mitigating neurological impairments induced by K18 tau seeds in vitro [72]. Because it can efficiently penetrate BBB when administered intravenously into 3xTg AD mice, D16 markedly reduced tau phosphorylation at several AD-related sites and enhanced cognitive abilities in the mice.

Fig. 6. Schematic illustration of the mechanisms of action of DEPTAC and the chemicals of tau DEPTACs.

a The hyperphosphorylation of tau inhibits its interaction with microtubules, promoting microtubule disassembly. The dissociated Tau forms neurofibrillary tangles. DEPTAC facilitates the interaction of Tau with phosphatase PP2A, therefore promoting tau dephosphorylation. b Chemical structures of DEPTACs targeting hyperphosphorylated tau, Tau2-8, and D16.

Regulation of post-translational modifications with proximity-inducing chimeras can specifically target and reverse harmful PTMs on proteins, restoring their normal function without protein degradation. This approach reduces the adverse cellular effects, allows specific modulation of protein function, and mitigates pathogenic effects while preserving the protein’s structural integrity.

Targeting mRNAs with RIBOTACs to inhibit protein aggregate formation

RIBOTACs are novel tools that can selectively target and degrade specific RNA molecules. They comprise three main components: a target RNA recognizing domain, a linker moiety, and a degradation signal, such as a ribonuclease binding site. These components help the intracellular degradation machinery identify and eliminate the target RNA (Fig. 7a).

Fig. 7. Schematic illustration of the mechanisms of action of RIBOTAC and the representative chemicals.

a RIBOTACs are bifunctional molecules that promote the formation of a stable ternary complex between target mRNA and RNase L to promote the degradation of the target mRNA. b Chemical structure of RIBOTAC targeting α-synuclein, Syn-RIBOTAC. c Chemical structure of RIBOTAC targeting C9orf72, RIBOTAC 7.

Tong et al. developed Syn-RIBOTAC to selectively degrade α-synuclein mRNA [73] (Fig. 7b). They performed microarray analysis to screen small molecules that bind specifically to the iron-responsive element (IRE) located within SNCA mRNA’s 5’-untranslated region. Then, synucleozid-2.0 was linked to a ribonuclease-recruiting module to form the RNA degrader Syn-RIBOTAC. In this structure, the RNA-binding module interacted with the SNCA IRE, whereas the ribonuclease-recruiting module stimulated RNase L near the target RNA, initiating its degradation. Syn-RIBOTAC (2 µM) reduced α-synuclein transcript levels by approximately 50%, resulting in a 63% ± 9% down-regulation of protein levels. Furthermore, it inhibited cell death induced by α-synuclein overexpression and restored about half of the dysregulated genes in dopamine neurons of PD mouse model.

The expanded G₄C₂ RNA repeat sequencer [r(G₄C₂)^exp] in the C9orf72 gene is the primary genetic contributor to ALS, responsible for roughly 40% of cases. Bush et al. developed RIBOTAC 7, a RIBOTAC, for targeting r(G₄C₂)^exp degradation (Fig. 7c). RIBOTAC 7 combines an optimized binding agent for r(G₄C₂)^exp with an RNase L recruiter to recruit and activate RNase L [74]. The data showed that RIBOTAC 7 reduced about 65% of r(G₄C₂)₆₆ RNA translation and r(G₄C₂)₈ cleavage. A single intracerebroventricular injection of RIBOTAC 7 into a C9orf72 BAC mouse model for three weeks reduced r(G₄C₂)^exp levels by 44% ± 22%.

Conclusion and future perspectives

The NDDs cause a progressive loss of neuronal function, which significantly impacts cognitive and motor abilities and poses a substantial decrease in global health. With the increasing aging population, the incidence of NDDs is also increasing, presenting a major challenge for future healthcare systems. Research has indicated that pathological protein aggregates are a significant etiological cause of several NDDs. However, targeting these aggregates with traditional drugs remains challenging. Proximity-inducing modalities selectively target and remove pathogenic proteins or mRNAs by harnessing the intracellular degradation mechanisms [75]. Furthermore, they selectively degrade pathological protein aggregates to mitigate neuronal toxicity and reverse functional deficits, which is crucial for NDD’s treatment.

Although proximity-inducing chimera technologies can effectively treat NDDs, they have several challenges. First, they have poor BBB permeability. Therefore, these modalities should be combined with BBB-crossing technologies, such as transferrin receptor (TfR)- and CD98 heavy chain (CD98hc)-mediated shuttling, for targeted CNS delivery. Second, the ligands for neuronal-specific E3 ligases are still lacking, and the use of ubiquitously expressed E3 ligases may cause unwanted off-tissue on-target toxicity due to PROTAC’s catalytic mechanism of action. Third, protein aggregates are complex and usually contain multiple proteins and nucleic acids, making their specific degradation challenging. TRIM21-based clustering-activated strategies that selectively degrade protein aggregates while sparing their monomeric counterparts may potentially treat NDDs. Fourth, the mechanisms of protein degradation may be dysregulated in NDDs, necessitating careful evaluation of disease context-dependent strategies to enhance the specificity and efficacy of protein degradation. In the future, novel, effective therapeutic strategies for NDDs will emerge as research continues to identify new proximity-inducing modalities.

Competing interests

The authors declare no competing interests.