Deubiquitinating enzymes at the crossroads of blood–brain barrier integrity and neurodegeneration: mechanistic insights, therapeutic targeting and future directions

ABSTRACT

The ubiquitin-proteasome system (UPS) carries immense significance concerning cellular homeostasis that encompasses both ubiquitination and deubiquitination as key facets for maintaining protein stability. The deubiquitinating enzymes (DUBs) have emerged as critical regulators of proteostasis, neuroinflammation and blood–brain barrier (BBB) integrity by controlling the fate of crucial proteins associated with barrier architectures in CNS and neurodegenerative disorders (NDs) alike. However, a concrete understanding of their specific neurodevelopmental and neuroprotective functions is yet to be discerned. This article discusses the multifaceted roles of DUBs in the maintenance of BBB integrity, neuroprotection and various NDs and also underscores the therapeutic prospects targeting the same. While DUBs like USP7, USP9X, USP27X, UCHL1, etc. participate in neural stem cell maintenance and neurogenesis, including BBB function, USP13, USP14, USP25, BRCC3 and CYLD, among others, are associated with BBB dysfunction and NDs. The mechanistic underpinning concerning their hitherto unexplored mode of action, DUB-substrate interactions and specificity would facilitate developing the therapeutic agonists and small-molecule inhibitors to prevent or reverse neuroinflammation, BBB impairment and developmental disorders. Recent innovations concerning DUB-targeting chimaeras (DUBTACs) and proteolysis-targeting chimaeras (PROTACs) can be explored further for their plausible administration via nanoparticle-based delivery approaches to alleviate the progressive neurodegeneration.

Background

Ubiquitination and deubiquitination are dynamic post-translational modifications that regulate protein stability, localization and interactions. The ubiquitin-proteasome system (UPS) has crucial implications in cellular homeostasis, and it is mediated by E1 (activating), E2 (conjugating) and E3 (ligase) enzymes to conjugate substrates with ubiquitin chains to undergo either K48-linked proteasomal degradation or K63-linked association with various cell signaling events and response to inflammation, DNA damage, stress, etc.^1,2 Deubiquitinating enzymes (DUBs) remove ubiquitin moieties and rescue the substrates from degradation or alter their functional states. Over 100 mammalian DUBs are categorized into seven families, with context-dependent roles spanning embryonic development, epigenetic regulation, maintenance and function of barrier integrity in nervous system, DNA repair and cancer.^3,4 In the central nervous system (CNS), the blood–brain barrier (BBB), as the name suggests, is the major protective barrier present at the blood–brain interface. It not only controls selective influx and efflux of agents required for brain functions but also prevents the entry of blood-borne pathogens. The microvascular endothelial cells are the basic constituents of BBB, where DUBs influence the tight junction formation and maintenance of its structural integrity.^5,6 Any insult that includes neuroinflammation, hypoxia, injury, etc., leads to its architectural impairment and contributes to various neurological disorders, such as AD, stroke, multiple sclerosis (MS), etc.^7–10 Of late, the vital role of various DUBs acting as a double-edged sword either impairing or building BBB has been realized. Hence, deciphering the vital role of various DUBs regulating BBB integrity and functions along with the underlying mechanistic-basis of their action would present a plethora of options and opportunities to undertake therapeutic interventions targeting these enzymes to treat diseases involving BBB dysfunction.

In neurodegenerative disorders (NDs) such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS), dysregulation of either UPS or other clearance systems involving authophagosome-lysosome, mitophagy and DUB activity contribute to the pathological aggregation of amyloid-β (Aβ), hyperphosphorylated Tau, α-synuclein, TDP-43, FUS, poly-glutamate, etc.^11–13 However, targeting the UPS for controlling protein aggregation and inclusion bodies that prompt neurodegeneration has yielded contrasting (both positive and negative) as well as gender-specific responses.^14–17 Moreover, the human genome encodes ~100 DUBs compared to over 600 E3 ligases. While E3 ligases’ broad spectrum functionality and genetic heterogeneity limit their therapeutic utility, smaller family of DUBs may offer better precision while reducing off-target risks. In this context, gaining a precise insight into their specific mode of action underlying various cellular functions and organismal development may be a safer and potent option to target DUBs for the prevention and progression of neurodegeneration. This article highlights the dual role of DUBs in neuronal survival and degeneration, and emphasizes the need for combinatorial approaches to restore proteostasis, mitigate neuroinflammation and repair BBB dysfunction to prevent, protect or reverse the neurodevelopmental and degenerative disorders.

DUBs and biological barrier function in CNS

The central nervous system (CNS) is protected from toxins and pathogens in blood by BBB. The functional unit of BBB is the neurovascular unit composed of vascular cells (endothelial cells, pericytes and vascular smooth muscle cells), glial cells (astrocytes, microglia and oligodendroglia) and neurons. This intricate cellular network forms a dynamic barrier that responds to physiological and pathological stimuli.⁵ The endothelial cells in brain are tightly packed together with minimal intercellular space creating tight junctions. This arrangement prevents most blood-borne substances from passively entering the brain parenchyma.¹⁰ Emerging evidence attributes BBB dysfunction as both a contributor to and consequence of neurodegenerative disorders including AD, PD, ALS, HD and MS. The tight junction proteins – Claudin5, occludins, zonula occludens (ZO-1, ZO2 and ZO3) are required to seal the endothelial cells and limit paracellular leakage.⁸ For instance, in AD, early glucose hypometabolism and Aβ accumulation are correlated with reduced expression of Claudin5 and GLUT-1.⁷ Di Pardo et al. demonstrated that the changes in the expression of TJ proteins and signs of BBB impairment precede the onset of symptoms in HD.¹⁸

Accumulating research findings hint at the crucial role of DUBs in BBB function through post-translational regulation of proteins involved in barrier formation, maintenance and repair. Incidentally, DUBs act as double-edged sword exerting profound influence on both neurodevelopment and degeneration. Figure 1 depicts the causatives underlying BBB impairment, DUBs’ contribution during the same and also in its restoration. In a recent report, Lei et al. have highlighted the involvement of USP9X in CNS vascularization and barrier maintenance mediated via Wnt signaling activation.⁴ They have elegantly demonstrated that deubiquitination of β-catenin by USP9X leads to the transcriptional activation of Axin2 and GLUT-1, the Wnt targets that are essential for angiogenesis and barrier formation. Wnt signaling also maintains the blood-retina barrier integrity by regulating Claudin-5 and prevents vascular leakage in pathological conditions like intracerebral hemorrhage and oxygen-induced retinopathy.¹⁹ Similarly, another DUB, CYLD negatively regulates Wnt signaling in glioblastoma and is also linked to inflammatory response and plausible BBB impairment mediated via activation of NF-κB pathway and necroptosis.^20,21 Wnt/β-catenin signaling pathway has also been ascribed to effectively repair damaged BBB after stroke.²² Hence, it may be interesting to ascertain whether dysregulated CYLD activity would contribute to BBB breakdown in stroke by altering Wnt-dependent endothelial repair mechanisms. Moreover, pharmacological Wnt agonists targeting both DUBs and downstream effectors could be beneficial in rescuing BBB defects.

Figure 1.

Multifaceted DUB action spanning neuroprotection, neurodegeneration, BBB impairment and restoration.

A20 (TNFAIP3), an ubiquitin-editing enzyme, plays a critical role in maintaining BBB integrity by suppressing NF-κB signaling in endothelial cells (ECs) and reducing the expression of adhesion molecules like ICAM-1 and VCAM-1 in MS. In a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), endothelial-specific A20 deletion exhibits enhanced leukocyte infiltration into the CNS.²³ A20 also provides a protective role in BBB restoration by mediating the anti-inflammatory effects of resolvin D1 in subarachnoid hemorrhage and suppressing the NLRP3 inflammasome activity.⁹ Conversely, BRCC3 activates NLRP6 inflammasome in cerebral ischemia/reperfusion aggravating pyroptosis and neuroinflammation.²⁴ It has been further shown that the knockdown of BRCC3 reduces the expression of NLRP6, cleaved caspase-1, IL-1β and GSDMD-N and thereby suppresses the neutrophil infiltration. Hence, A20 agonist or BRCC3 antagonists could be used in conjunction with anti-inflammatory agents to restore barrier function during neuroinflammation and neurodegenerative disorders.

UCHL1, a neuronal DUB, enters the bloodstream following BBB disruption. Elevated serum UCHL1 correlates with traumatic brain injury severity and impaired barrier function, as measured by albumin quotient. Its rapid detection post-injury highlights its utility in monitoring acute BBB compromise.²⁵ In addition to this, UCHL1 stabilizes Sox17, a transcription factor crucial for angiogenesis and the recovery of blood-spinal cord barrier (BSCB) post-injury.²⁶ Using conditional knockouts in mice, they have validated the role of UCHL1 in promoting angiogenesis and BSCB restoration. Hence, therapeutic strategies that target restoring BBB integrity, enhancing transport of toxic proteins out of the brain or leveraging BBB permeability for drug delivery could provide new avenues for intervention. Further research is required to understand the complex interplay between BBB dysfunction and disease-specific pathological mechanisms crucial for developing such targeted therapeutic approaches.

DUBs in neurodegeneration

DUBs regulate the fate of ubiquitinated substrates by editing ubiquitin chains or reversing ubiquitination, and thereby dictate whether proteins should degrade via the UPS or the autophagy-lysosomal pathway (ALP). In neurodegenerative disorders, the dysfunction of UPS or ALP is implicated in pathological protein aggregation and impaired BBB. For instance, Aβ and Tau in AD are ubiquitinated but resist degradation due to dysfunctional DUB activity leading to either stabilization of these proteins or failure to clear aberrant ubiquitin signals.²⁷ USP14, a proteasome-associated DUB, enhances proteasomal degradation by removing K48-linked chains; however, it increases the phosphorylation of Tau (pTau) contributing to the pathology of AD.²⁸ Similarly, the knockdown of USP13 reduces the levels of hyperphosphorylated Tau (pTau) and amyloid-β (Aβ) by enhancing proteasomal activity and promoting clearance of pTau.²⁹

Another facet of DUBs regulation is by mitophagy, which is a selective autophagy pathway that eliminates damaged mitochondria. USP30 is localized to mitochondrial membranes, and it antagonizes Parkin-mediated mitophagy by removing K6-linked ubiquitin chains from mitochondrial substrates and delaying the clearance of dysfunctional organelles.³⁰ In contrast, USP8 promotes Parkin activation by deubiquitinating K6 chains on Parkin and enhances its recruitment to depolarized mitochondria.³¹ A recent report by Mauri et al. has also demonstrated that USP8 inhibits a Parkin-independent mitophagic pathway, emphasizing the complex and context-dependent roles of DUBs.³² Although several DUBs, including USP33, USP15 and USP35, are involved in the regulation of mitochondrial homeostasis, the inhibition of USP30 has shown significant promise in modulating Parkin-mediated mitophagy and the PINK1-dependent phosphorylation of ubiquitin.³³ These multimodal DUB functionalities also unequivocally highlight the context-dependent roles of DUBs, with USP30 inhibition mitigating mitochondrial dysfunction in PD, while USP8 activation rescuing the defective mitophagy.

Additionally, there are emerging evidences pointing toward the non-catalytic DUB functions spanning DNA repair, receptor trafficking and inflammation. USP11 is known to be involved in tau acetylation and aggregation in AD models and due to its higher expression in females might contribute to gender disparities in AD incidence.¹⁷ Similarly, USP9X stabilizes pro-survival proteins like MCL1 and regulate α-synuclein mono-ubiquitination, which affects its degradation pathway. Lower USP9X levels in α-synucleinopathies, such as PD and dementia with Lewy bodies (DLBD) correlate with increased α-synuclein mono-ubiquitination and aggregation.³⁴ These diverse functions point to the challenges of unraveling DUBs’ role in protection or pathogenesis in the context of different diseases. Figure 1 depicts the multifaceted attributes of DUBs in NDs.

Counteracting strategies in ND

AD: Aβ, Tau and synaptic dysfunction

In AD, DUBs have a direct influence on Aβ and Tau proteins. USP25 deubiquitinates BACE1 and APP in the Golgi apparatus and stabilizes these proteins by preventing their lysosomal degradation. This stabilization promotes the β-cleavage of APP, increasing Aβ generation and amyloid plaque deposition in AD models.³⁵ Similarly, USP13 stabilizes pTau by removing ubiquitin signals required for degradation, and USP13 knockdown reduces both Aβ and p-Tau in AD models, suggesting potential implication in AD pathology reversal.²⁹ Conversely, UCHL1, which is a neuron-specific DUB, exhibits reduced activity in AD brains and this correlates with Aβ accumulation and synaptic loss.³⁶ UCHL1 hydrolyses polyubiquitin chains to maintain free ubiquitin pools which enable proteasomal degradation. Hence, UCHL1 dysfunction in AD drives Aβ accumulation and synaptic loss via impaired ubiquitin recycling-mediated proteasomal overload and BACE1 regulation, while its overexpression mitigates both Aβ and Tau pathology.³⁷ Hence, restoring UCHL1 might be a promising strategy to counteract ubiquitin depletion in AD.

PD: α-synuclein and mitophagy

USP8 and USP9X play crucial roles in PD pathogenesis. USP8 enhances Parkin-mediated mitophagy, a process critical for dopaminergic neuron survival.³¹ Moreover, USP8 directly interacts with α-synuclein and removes K63-linked ubiquitin chains, preventing its lysosomal degradation.³⁸ Conversely, USP9X deubiquitinates α-synuclein and promotes its degradation through autophagy and loss of USP9X in PD cortices reduces deubiquitination, increasing monoubiquitinated α-synuclein aggregation.³⁴ However, the inhibition of USP24 increases ULK1 levels and autophagy flux, promotes the clearance of toxic aggregates and damaged mitochondria,¹³ and thereby warrants a precise targeting of USP24-substrate interactions. Taken together, the therapeutic strategies must be worked out balancing the contrasting modes of actions of the stated DUBs.

HD: mutant huntingtin (mHTT) and polyQ

DUBs have emerged as critical regulators of mHTT processing and clearance in HD, with USP12 and ATXN3 playing particularly significant roles. Both of these accelerate autophagic flux, and induction of autophagy eventually protects the HD-mediated neurotoxicity.^39,40 The interaction of ATXN3 via its polyglutamine (polyQ) domain with Beclin-1 and through its DUB activity protects the latter from undergoing proteasomal degradation and enables autophagy. In fact, the identification of these DUBs as regulators of mHTT processing confers them as promising therapeutic candidates for HD reversal. In line with the same, USP12 is reported to rescue mHTT-mediated toxicity in multiple model systems that include human induced pluripotent stem cell (iPSC)-derived neurons in-vitro and both fly and rodent models of HD in-vivo.³⁹

ALS: SOD1, TDP-43, FUS and Stress granule dynamics

The disruption of ubiquitin homeostasis by SOD1, TDP-43 and FUS aggregation and formation of stress granules contributes to the pathology of ALS. These aggregates deplete the free ubiquitin pool by sequestration of ubiquitin into cytoplasmic inclusions and impair UPS function.⁴¹ USP7 is emerged as a critical player in the regulation of proteotoxicity by controlling the turnover rate of SOD1 and TDP-43 through NEDD4L-SMAD2 axis.⁴² Although the know-how concerning the role of DUBs in ALS is limited, USP5 and USP13 are reported to be critical regulators of stress granule dynamics.⁴³ The stability of stress granules is regulated by ubiquitin chains, which is affected by hydrolysis mediated by these DUBs, potentially implicating them in ALS pathology. While USP5 shows preference toward unanchored ubiquitin chains, USP13 prefers protein-conjugated ubiquitin chains in the regulation of stress granules and that reflects their substrate-specific action.⁴³

Therapeutic innovations – challenges

The major challenges that complicate therapeutic modalities of the DUBs are selectivity, BBB penetration and contextual toxicity. The structural homology and conserved catalytic cysteine among DUBs pose significant challenges for selective inhibitor design. For instance, USP30 and USP15 both regulate mitophagy but exhibit divergent substrate specificities, as discussed earlier. Hence, targeting USP30 could result in off-target inhibition of USP15 and lead to disruption of immune signaling or DNA repair pathways due to its role in NF-κB activation and BRCA1-associated repair complexes.^44,45 Most DUB inhibitors, such as IU1 (targeting USP14) and Spautin-1 (targeting USP10/USP13), modify the catalytic cysteine, which risks cross-reactivity with other cysteine-dependent enzymes like caspases.^46,47 While IU1 shows neurotoxicity at lower concentration by inhibition of mitochondrial Complex I, its derivative, IU1–47, reduces Tau accumulation in neurons.⁴⁸ While USP7 stabilizes Tau by deubiquitination, the small-molecule inhibitor selectively targeting the non-catalytic HUBL domain of USP7 has been shown to attenuate neuroinflammation and improve cognitive outcomes in PD mouse models.^49,50 This approach of targeting non-catalytic allosteric sites might resolve challenges related to the off-target effects of DUB inhibitors. Covalent chemoproteomics have identified non-catalytic allosteric sites in 57 DUBs, and this would enable the development of specific inhibitors with enhanced selectivity.⁵¹

The presence of BBB poses a challenge in CNS disorders in the delivery of drugs to the brain.⁶ However, the innovations in nanoparticle-based delivery systems might be a potential option mitigating the BBB penetration challenge. In similar line, Zhou et al. have reported that RVG29-modified PLGA nanoparticles loaded with rifampicin can show improved cognitive function in APP/PS1 mice by reducing Aβ plaques.⁵² The other possibilities could involve DUBTACs (DUB-targeting chimeras) and PROTACs (Proteolysis-targeting chimeras).⁵³ DUBTACs are bi-functional molecules comprising a DUB recruiter linked to a ligand that binds a target protein. By inducing proximity between the DUB and the target, DUBTACs remove degradative ubiquitin chains and stabilize the proteins. The ΔF508-CFTR mutation in cystic fibrosis causes misfolding and ubiquitin-mediated degradation of the CFTR chloride channel.⁵⁴ Their work suggests that a DUBTAC (e.g., NJH-2-057) linking the OTUB1 recruiter EN523 to lumacaftor (a CFTR chaperone) stabilizes ΔF508-CFTR by deubiquitinating K48 chains and enhances its cell surface expression and chloride conductance in bronchial epithelial cells. A similar approach could be explored in the case of NDs. For instance, DJ-1 loss-of- function leads to early onset in PD and OTUB1 aggregation confers neurotoxicity.^55,56 Hence, a specific DUBTAC might recruit DUBs to stabilize DJ-1 and prevent oxidative stress-induced degeneration in PD. Similarly, PINK1-targeted DUBTAC might stabilize PINK1 by counteracting its ubiquitination and restoring mitophagy in ALS/PD models.⁵⁷ Conversely, PROTACs could be used to inhibit DUBs like OTUB1 and USP25, which exacerbates Aβ production in PD and AD, respectively.⁵⁸

PD is also associated with progressive degeneration of mid-brain dopaminergic neurons, where stem cell-based approach has emerged as a potent therapeutic option.^59,60 In fact, the recent success with clinical trials has opened a brighter horizon in the regenerative medicine arena. Incidentally, DUBs also play a major role during neurogenic proceedings. While USP7 (HAUSP) and USP9X promote neurogenesis by maintaining the neural stem cell pool, USP25 adversely affects it and also contributes to cognitive impairment.^61–63 However, the overexpression of USP7 leads to enhanced dendritic arborization and development of autistic phenotype in mice.⁶⁴ Thereby hinting at the importance of a specific threshold of USP7 maintenance during neurogenesis. Similarly, USP27X has also been linked with neural differentiation via its regulation of HES1.⁶⁵ Our ongoing work also suggests differential cell fate modulations by DUBs during embryonic stem cell differentiation. Taken together, the DUBs with their varying attributes during neural development may serve as plausible therapeutic candidates for further exploration in prevention, protection and cessation of neurodegeneration via regeneration and repair. Hence, the DUB horizon has widened with a plethora of challenges and opportunities alike. Figure 2 discusses various DUB-targeted therapeutic strategies that can be undertaken to combat CNS disorders.

Figure 2.

Schematic depiction of various therapeutic strategies targeting DUBs in treating CNS disorders.

Conclusion and future perspectives

The past decade has underscored the DUBs as central regulators of proteostasis in neurodegeneration. However, the contrasting roles of DUBs spanning neural development to disease association make them a double-edged sword requiring precise manipulation concerning therapeutic intervention. As discussed, decoding the full range of DUB functions will lead to the development of novel strategies for combating BBB impairment and NDs. While innovations like DUBTACs and covalent inhibitors demonstrate promising outcomes, there are challenges concerning isoform-specificity and BBB penetration. Further research is required to understand the complex interplay between BBB dysfunction and disease-specific pathological mechanisms that will be crucial for developing such targeted therapeutic approaches. More studies integrating patient-derived organoids, single-cell omics, biomarker stratification and machine learning driven drug design are required to translate mechanistic insights into therapies. DUB-focused therapies-led clinical trials may eventually bring a paradigm shift in treating neurodevelopmental defects and NDs.

Funding Statement

NCCS intramural.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contribution

Data Collection: FM

Conceptualization, Resource generation, Project supervision: NL

Analysis, Writing, Editing and Finalization of the manuscript: FM and NL