Therapeutic potential of lysosomal cathepsins for neurodegenerative diseases

Abstract

Lysosomes are ubiquitous and dynamic organelles with a central role in degradation and recycling of damaged cell components and misfolded proteins, otherwise known as autophagy. Autophagy plays a fundamental role in the process of correcting cell homeostasis and cellular survival. Unsurprisingly, this process is essential in the central nervous system, as neurons are not able to easily eliminate altered proteins given their post-mitotic state. Thus, lysosomal function is critical in maintaining neuronal health. Interestingly, increasing evidence suggests that impaired autophagy underlies several neurodegenerative diseases. Genetic deletion of key components of the autophagy machinery results in the accumulation of protein aggregates and subsequent neuropathologies. Moreover, some genetic variants found in lysosomal storage disorders (LSDs), which can also be hallmarked by neuronal degeneration, have been implicated as risk factors for Alzheimer’s disease (AD), Parkinson’s disease (PD) and others. Specifically, deficiency in the cathepsin family of lysosomal proteases, which play a vital role in the clearance of aggregation-prone proteins, such as alpha-synuclein (αSyn), amyloid β peptide, and saposins C (SapC) and D (SapD), seems to contribute to neuropathogenesis. Hence, targeting lysosomal function represents a novel therapeutic approach for tackling neurodegeneration. The role of lysosomal cathepsins in neuronal homeostasis: There are three major pathways by which degradation cargoes are delivered to the lysosome: microautophagy, chaperone-mediated autophagy and macroautophagy. In particular, chaperone-mediated autophagy and macroautophagy are pivotal in preserving neuronal function, as impairments in these pathways accelerate the accumulation of misfolded proteins. The catabolic capacity of the lysosome depends on its acidic environment and the function of lysosomal membrane proteins as well as soluble lysosomal hydrolases, each responsible for the bulk degradation of specific substrates. Among the ~60 lysosomal enzymes currently known, the cathepsin proteases are the most abundant. Cathepsins are classified into three categories depending on the amino acid at their active site: aspartyl (cathepsins D and E), cysteine (cathepsins B, C, F, H, K, L, O, S, V, X, and W) or serine proteases (cathepsins A and G). While they exhibit the highest enzymatic activity in the lysosomal acidic environment, cathepsins are found to also work under neutral or basal pH conditions, indicating their wide range of functions both intra- and extracellularly. In contrast to other lysosomal enzymes, aspartyl cathepsin D (CTSD) and cysteine cathepsins B (CTSB) and L (CTSL) are found in abundance, particularly in the central nervous system. Supporting their important neuronal and lysosomal roles, deficiency of CTSD underlies a serious pediatric neurodegenerative disease called neuronal ceroid lipofuscinosis (NCL) type 10, which features an aberrant autophagic function and storage bodies consisting of lipofuscin, SapC and SapD. Furthermore, the lack of both CTSB and CTSL in mice represents brain atrophy and early-onset neurodegeneration, hallmarked by a deficient autophagic flux. Interestingly, CTSD, CTSB and CTSL have long been implicated in the proteolysis of aggregation-prone proteins such as tau, αSyn, amyloid β peptide, huntingtin and prion protein (Drobny et al., 2022), further underlining the importance of enzymatic activity of cathepsins in the maintenance of neuronal homeostasis. The role of lysosomal cathepsins in αSyn clearance: αSyn is a small protein that natively exists as a soluble, unfolded conformer and is found ubiquitously in the central nervous system. The exact function of αSyn is still under debate. However, its primary location in presynaptic terminals suggests an important role in neurotransmission and synaptic plasticity. Physiological αSyn can also exist as a soluble, non-toxic oligomer that, along with the monomeric conformers, enters the lysosome via macroautophagy or chaperone-mediated autophagy for degradation. Soluble αSyn can be converted into insoluble, toxic forms under pathological conditions. For example, accumulation of glycolipids like glucosylceramide due to a decreased activity of the lysosomal enzyme β-glucocerebrosidase (GCase) can facilitate αSyn toxic oligomerization, further resulting in the formation of amyloidogenic fibrils (Zunke et al., 2018). Hence, it is not surprising that mutations in the gene encoding GCase (GBA1) represent a risk factor for PD, as 5–15% of PD patients carry a mutation within this gene. Interestingly, there are different GBA1 variants: whereas the GBA1 mutation E326K is associated with the development of PD, the L444P and N370S variants are known to underlie the most common LSD Gaucher disease, but also represent an increased risk for αSyn pathology. Variants in other genes that are implicated in LSDs and confer susceptibility to PD include for instance GALC, GLA, SMPD1 and ASAH1. Insoluble αSyn forms, which are the molecular hallmark of PD, dementia with Lewy bodies and other synucleinopathies, are preferentially delivered to the lysosome by macroautophagy. Inside the lysosome, CTSD seems to be the main mediator of αSyn proteolysis, as the complete deficiency of CTSD is associated with an increase in neuronal insoluble αSyn. Vice versa, enhancing CTSD activity reduces pathogenic αSyn levels as shown in vitro in dopaminergic neurons derived from PD patients and in an in vivo mouse model (Prieto Huarcaya et al., 2022). McGlinchey and Lee (2015) demonstrated in vitro that sole CTSD activity is unable to completely degrade αSyn, and that cysteine CTSB and CTSL are necessary to avoid amyloidogenic truncations of the C-terminus. Another cysteine protease, the asparagine endopeptidase, was found to cleave αSyn, but also to promote its toxic oligomerization (Zhang et al., 2017). Later studies suggest that in particular cysteine cathepsin activity including asparagine endopeptidase is linked to the generation of pathological C-terminal truncations (McGlinchey et al., 2019). Interestingly, CTSL gains better access to the amyloid core upon cleavage of the C-terminus, which allows the degradation of amyloid fibrils (McGlinchey and Lee, 2015; Figure 1 ). Overall, this indicates that lysosomal proteases and cathepsins in particular work in a collaborative manner to efficiently clear αSyn. Figure 1 Direct and indirect effects of lysosomal cathepsins CTSD, CTSB and CTSL in αSyn degradation. Enzyme replacement therapy (ERT) is a common therapeutic strategy for lysosomal storage disorders (LSDs). Given the convergence in pathogenic pathways between LSDs and neurodegenerative diseases, ERT could be a promising approach to tackle neurodegeneration. However, the blood-brain barrier (BBB) represents the major obstacle when treating the central nervous system. Some of the strategies to bypass the BBB that could work in combination with ERT are via intracranial injection (A), nanoparticles (B), monoclonal antibody-lysosomal enzyme fusion protein (C) and focused ultrasound (D). Recently, a study showed that the administration of recombinant proCTSD by ERT promotes pathological αSyn degradation (Prieto Huarcaya et al., 2022). αSyn exists as both soluble monomers or oligomers and is catabolic substrate of CTSD and CTSB. Under pathological conditions, the accumulated glucosylceramide (GluCer) interacts with the soluble αSyn oligomers, enhancing their toxic oligomerization and subsequent conversion into amyloid fibrils (Zunke et al., 2018). While the insoluble αSyn oligomers are cleared by CTSD, CTSB and CTSL, the amyloid fibrils are preferentially targeted by CTSL (McGlinchey et al., 2019). Lysosomal cathepsins may also degrade αSyn in an indirect manner. Both CTSD and CTSB mediate the proteolytic processing of prosaposin into mature saposin C (SapC) (Kim et al., 2022), a known activator of β-glucocerebrosidase (GCase). GCase promotes the breakdown of GluCer to glucose (Glu) and ceramide, which in turn prevents the toxic oligomerization of αSyn. αSyn: Alpha-synuclein; CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L. Created with BioRender.com. Therapeutic potential of lysosomal cathepsins for neurodegeneration: As a correct lysosomal function is imperative in maintaining efficient degradation, it is not surprising that emerging therapeutic approaches for neurodegeneration are focused on enhancing autophagy. Several genetic strategies aiming to upregulate macroautophagy and small molecules that specifically target aggregation-prone substrates are currently under development (as reviewed in Drobny et al., 2022). For example, upregulation of GBA1 via adenovirus expression or reduction of GCase substrates by administration of non-inhibitory chaperones have been shown to decrease αSyn accumulation in vivo and are under study for the treatment of Gaucher disease and PD (as reviewed in Peng et al., 2019). Enzyme replacement therapy (ERT), a common approach for treating LSDs by aiming to replenish defective enzymatic function with a recombinant protein, is also being explored as a possible treatment for neurodegeneration. Intravenous ERT is the most used and successful therapeutic approach for different LSDs including Gaucher disease. Diverse recombinant analogs of GCase like Imiglucerase (Cerezyme®, Genzyme Corporation, Boston, MA, USA) have been shown to ameliorate symptoms and notably improve the patient quality of life. However, intravenous ERT is unable to treat the pathologies within the central nervous system. One major obstacle when directly targeting neurons or lysosomes is the blood-brain barrier, which heavily restricts the entry of large molecules into the brain. Emerging strategies to avoid the blood-brain barrier are being developed, one of them via intracranial injection ( Figure 1 ). Noteworthy, clinical trials (NCT01907087 and NCT02485899) for the treatment of the LSD NCL type 2 via intraventricular infusion resulted in a slower decline in motor and language skills. Other methods to bypass the blood-brain barrier are discussed below. A recent study explored the use of the precursor form of CTSD to decrease levels of αSyn via ERT as a possible treatment for PD. First, it demonstrated that the external application of a recombinant, inactive proform of CTSD (proCTSD) was successfully endocytosed by neuronal cells and underwent protein maturation, increasing basal levels of CTSD and its activity (Prieto Huarcaya et al., 2022). Moreover, enhancing CTSD levels was able to boost autophagic function and decreased pathology-associated forms of αSyn in dopaminergic neurons derived from PD patients, without disturbing neurophysiological properties. Insoluble αSyn levels were also decreased in a ctsd knockout mouse model after intracranial injections of proCTSD, significantly reducing αSyn aggregates in the brain. In a separate study, recombinant proCTSD was shown to prolong survival and improve behavioral symptoms in these mice (Marques et al., 2020). Restoration of physiological αSyn function after proCTSD treatment was also suggested, as αSyn and synaptic-1 positive vesicles, which are clustered under pathological conditions, became dispersed upon treatment with the proform of the enzyme (Prieto Huarcaya et al., 2022). Given the remarkable effects of the recombinant proCTSD on αSyn, this strategy could be implemented for the treatment of other synucleinopathies like dementia with Lewy bodies or multiple system atrophy, since they are all characterized by αSyn burden. Furthermore, different neurodegenerative diseases such as AD, Huntington or NCL could benefit from a boost in autophagic function as their accumulating proteins are known substrates of cathepsins. Di Spiezio et al. (2021) showed that intracerebral treatment with proCTSB or proCTSL is able to partially restore the autophagic flux in NCL type 10 mice. Moreover, while proCTSL induced a reduction in SapC aggregation which subsequently improved neuroinflammation, proCTSB failed to have significant effects on SapC clearance. Even though the application of cysteine cathepsins corrected deficient autophagy, there were no improvements in the body mass or lifespan in contrast to those animals to which proCTSD was administered, which also showed an overall improvement in visceral and neuronal pathology (Marques et al., 2020; Di Spiezio et al., 2021). It is noteworthy that the combined application of proCTSD, proCTSB and proCTSL led to strong proteolytic activity in vitro, more so than a single cathepsin application, suggesting that a combined therapy might be instrumental for an effective protein turnover. Conversely, treatment with lysosomal cathepsins could indirectly enhance the clearance of aggregation-prone proteins. While it is known that CTSD regulates the processing of prosaposin into the mature homologs SapA, SapB, SapC and SapD, a recent study investigated whether CTSL and CTSB could also mediate its maturation (Kim et al., 2022). For this, a cellular PD model that showed decreased SapC protein levels was used. Kim et al. (2022) demonstrated that CTSB promotes the proteolytic yield of prosaposin to SapC, which in turn increased GCase enzymatic activity, as SapC is an important activator of this enzyme. Hence, a decrease in pathological αSyn level after boosting lysosomal cathepsin function can be a direct consequence of the increasing enzymatic function, as cathepsins target specific αSyn structural forms for degradation ( Figure 1 ). However, this increased clearance of αSyn could also be an indirect consequence, as both CTSD and CTSB promote SapC function consequently enhancing GCase activity and thus decreases glucosylceramide levels, which have been shown to have a stabilizing role on pathological αSyn conformers ( Figure 1 ). Limitations and perspective: While ERT by intracranial injection avoids the major obstacle that is the blood-brain barrier, the burden of this strategy could outweigh its benefits. This costly procedure requires having an open shunt in the cranium for the weekly or monthly administration of recombinant enzymes, which might neither seem appealing nor practical for PD or AD patients. However, familial PD or AD patients might benefit from intracranial injections as the onset of the disease in these cases may start around age 30. Of note, PD patients exhibit certain non-motor features that are usually overlooked before the onset of the classical symptoms. Gastrointestinal problems such as constipation are potentially accompanied by aberrant αSyn in the peripheral nervous system. Treatment of the periphery with proCTSD could alleviate these symptoms, possibly stopping or slowing the progression of the disease also with regards to the spreading of the pathology via the gut-brain axis. However, strong diagnostic tools for better screening of neurodegenerative disease are urgently needed to administer the treatment as early as possible. Besides intracranial injections, other strategies to bypass the blood-brain barrier and enhance the efficacy of ERT and uptake of recombinant enzymes to the central nervous system are under investigation. For instance, recombinant enzymes could be genetically fused to monoclonal IgG antibodies that target specific receptors (e.g. transferrin receptor 1 and insulin-like growth factor receptor) expressed in the brain capillary endothelium ( Figure 1 ). These lysosomal enzyme fusion proteins should be carefully selected to not interfere with the delivery of endogenous ligands to the blood-brain barrier receptors. There are currently numerous lysosomal enzyme fusion proteins that are being investigated for the treatment of various LSDs including mucopolysaccharidosis type I, Tay-Sachs disease, and GM1 gangliosiosis (Pardridge, 2022). Nanoparticles are also being exploited as a therapeutic tool against AD due to their physicochemical features, ability to carry cargoes and possibility of multi-functionalization to bypass or enhance crossing through the blood-brain barrier (Ordonez-Gutierrez and Wandosell, 2020; Figure 1 ). Focused ultrasound is a non-invasive technique that produces a safe and reversible disruption of the blood-brain barrier. Coupling of focused ultrasound with the intravenous administration of microbubbles provides a temporal opening of the blood-brain barrier contained within the targeted capillaries, allowing efficient delivery of biologics (Karakatsani et al., 2019; Figure 1 ). To this end, these strategies would greatly improve ERT by giving a more controlled and precise delivery of recombinant enzymes to neuronal cells. In conclusion, targeting lysosomal cathepsins represents a promising approach for the treatment of synucleinopathies and other neurodegenerative diseases; however, strategies to efficiently target the central nervous system need further improvement. This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) (No. 125440785 – SFB877, project B11) and by the Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-Nuremberg (Jochen-Kalden funding program N8) (both to FZ).