Targeting Protein Clearance Pathways in GBA1-associated Parkinson Disease

1. Introduction

The clearance of damaged and unwanted proteins is essential for maintaining cellular homeostasis¹. The two main mechanisms for the degradation of misfolded or aggregated proteins within the cell are the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP). Dysfunction of the UPS and ALP are seen in disorders involving protein aggregation and therefore play a critical role in common neurodegenerative diseases^{2, 3}. Specifically, deficits in protein clearance lead to the accumulation of α-synuclein protein in disorders known as the synucleinopathies, that include Parkinson disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy⁴. Additionally, recent studies have linked deficiencies in protein clearance by the UPS and ALP with aging, further supporting the importance of protein clearance pathways in neurodegeneration.

Patients with PD clinically present with bradykinesia in combination with a resting tremor, and/or rigidity. However, non-motor symptoms including depression, hyposmia and cognitive decline are also common. The neuropathological hallmark of PD is the progressive loss of dopaminergic neurons within the substantia nigra pars compacta (SNpc) and the accumulation of α-synuclein inclusions known as Lewy bodies. 5–10% of PD cases have an established monogenic cause, but over 90 different risk variants have been linked to PD in genome-wide association studies (GWAS)^{5, 6}. Many of the associated genes are related to the endolysosomal system or to protein clearance^7–10.

Mutations in the gene GBA1, which encodes for the lysosomal hydrolase glucocerebrosidase (GCase), are the most common genetic risk factor for PD¹¹. Biallelic mutations in GBA1 result in the lysosomal storage disorder Gaucher disease (GD). GBA1 mutation carriers have a more than five-fold increased risk of developing PD. As a group, patients with GBA1-associated PD (GBA1-PD) have an earlier age of onset compared to idiopathic PD and a faster disease progression¹². An inverse relationship between decreased GCase activity and increased α-synuclein aggregation has been suggested¹³. The exact mechanisms of how GBA1 mutations cause PD remains unclear. A direct effect on ALP caused by GCase activity reduction and substrate accumulation in the lysosome has been proposed, but misfolded GCase has also been shown to cause ER stress, which initiates the unfolded protein response, affecting both the ALP and UPS.

Targeting lysosomal and proteasomal dysfunction within neurodegenerative disorders has been hindered by our incomplete understanding of these processes. However, new therapies that improve protein clearance mechanisms targeting the UPS and ALP to decrease levels of protein aggregation may potentially help resolve the pathogenesis of the synucleinopathies (Figure 1). While many of these concepts can also be applied also to idiopathic forms of PD, we focus here on potential therapeutic approaches for GBA1-related PD.

Figure 1: Targeting Protein Clearance Pathways in GBA1-Associated Parkinson Disease.

New strategies for targeting the Ubiquitin-Proteasome System (e.g., PROTACs) and the Autophagy-Lysosome Pathway (e.g., autophagy-inducing small molecules) aim to increase protein clearance pathways to help degrade aggregated α-synuclein.

2. Current treatments and approaches for GBA1-PD

Currently, there are no therapies that halt or reverse disease progression in PD. Most treatments aim to restore the dopamine deficit, thereby providing symptomatic relief. The GBA1-PD link spurred hope that GD treatments (enzyme replacement therapy (ERT) and substate reduction therapy (SRT) might halt disease progression in GBA1-PD. However, ERT does not cross the blood-brain barrier (BBB)¹⁴. To improve the delivery of recombinant GCase enzyme, induced disruption of the BBB via MRI-guided focused ultrasound is being tried in a small phase I trial in PD patients (ClinicalTrials.gov, NCT04370665)¹⁵. The utility of SRT treatment GBA1-PD is questionable as the clinical relevance of lipid substrate accumulation in heterozygote GBA1-carriers remains uncertain. Despite this, a BBB-penetrant SRT (venglustat) was evaluated in a phase 2 randomized multicenter study for GBA1-PD, and while reduction of glucosylceramide in the cerebral spinal fluid (CSF) and plasma was observed, motor symptoms of the trial participants worsened, leading to discontinuation of the study ¹⁶.

Small molecule chaperones aimed to assist in GCase protein folding are also in development. These can effectively penetrate the BBB, and, competitively or non-competitively, stabilize GCase to promote trafficking from the endoplasmic reticulum (ER) to the lysosome. Inhibitory chaperones (e.g., iminosugars) are well-studied but are hampered by their intrinsic dose-dependent inhibition of enzymatic activity¹⁴. Ambroxol (ABX), a common cough expectorate, binds to the active site of GCase at neutral pH facilitating cellular translocation, but less at the lysosomal acidic condition. Preclinical studies show increased levels and activity of GCase upon Ambroxol treatment, as well as activation of macroautophagy in neurons with GBA1-mutations¹⁷. An open-label, non-randomized and non-controlled trial in 17 patients with PD, with and without GBA1-mutations, demonstrated detection of ABX in CSF and enhanced GCase and total α-synuclein levels¹⁸, but the clinical effect was difficult to interpret. More extensive studies with ABX are ongoing to evaluate motor and cognitive symptoms in patients with synucleinopathies. Other non-inhibitory GCase modulators have been shown to restore GCase activity in cellular and animal models of GBA1-PD and GD¹⁹. One allosteric modulator of GCase, LTI-29, was shown to cross the BBB and was well tolerated in healthy volunteers, but efficacy in patients is still undetermined²⁰.

In recent years, the idea of regulating GCase at the transcriptional level has emerged as a potential therapeutic approach. Transcriptional modulation using antisense oligonucleotide (ASO) treatments have recently been approved for several monogenetic neurological disorders. ASOs are specifically designed to degrade unwanted transcripts or enhance the translation of selected mRNAs. Thus, ASO therapies, potentially personalized, have been proposed as a future possible therapeutic strategy for GBA1-PD. Gene therapy aimed to introduce a copy of the wildtype GBA1 gene via a single intrathecal injection is currently in early clinical trials (ClinicalTrials.gov, NCT04127578). Preclinical evidence demonstrated effective transduction of brain cells, increased GBA1 expression levels in non-human primates, and ameliorated a behavioral phenotype in a mouse model with GBA1 deficiency^{21, 22}.

3. Targeting the autophagy-lysosomal pathway in GBA1-PD

Proper lysosomal function is required to maintain cellular homeostasis, as the accumulation of material within lysosomes leads to lysosomal impairment and further cellular dysfunction. Autophagy is the process by which cytosolic substrates are transferred to the lysosome for degradation. There are three main types, macroautophagy (commonly and hereby referred to as autophagy), chaperone-mediated autophagy (CMA), and microautophagy. Dysfunction of the ALP has been implicated in an array of neurodegenerative disorders, including PD and Alzheimer disease (AD). GCase mutations can lead to glycosphingolipid accumulation, but whether this is the case in heterozygote carriers is still a matter of debate, and today there is limited evidence supporting substrate accumulation in brains from patients with GBA1-PD. Alteration of autophagic lysosomal reformation has been showed in GCase deficient cells affecting the regeneration of lysosomes from autolysosomes²³ and misfolded GCase has also been shown to affect CMA. This indicates a wider impairment of ALP than merely the loss of enzymatic activity.

a. mTOR inhibitors

One of the major negative regulators of autophagy within the cell is the mechanistic target of rapamycin (mTOR) pathway. The mTOR pathway controls the nuclear translocation of transcription factor EB (TFEB), a master transcriptional regulator of the ALP. Thus, hyperactivation of the mTOR pathway is commonly associated with neurodegeneration. Studies examining induced pluripotent stem cell (iPSC)-derived neurons have shown that GBA1 mutations in nGD lead to mTOR hyperactivation, resulting in the subsequent inhibition of autophagy²⁴. This suggests that mTOR inhibition may be a promising strategy for nGD and GBA1-associated neurodegeneration. Treatment of mutant α-synuclein mouse models with the mTOR inhibitor rapamycin reduced aggregation of α-synuclein and improved motor function²⁵. However, rapamycin treatment is unfeasible due to long-term serious side effects. Treatment of iPSC-derived nGD neurons with a second-generation ATP-competitive mTOR inhibitor, Torin1, enhanced autophagic clearance, but has not been further tested. Alternative strategies to induce autophagy with the use of mTOR-independent autophagy activators exists but have extremely limited preclinical testing.

b. mTOR-independent autophagy activators

Alternative strategies to induce autophagy include the use of mTOR-independent autophagy activators, such as trehalose, which directly activates 5’-AMP-actived protein kinase (AMPK). In one study, trehalose treatment reduced α-synuclein accumulation in A53T α-synuclein transgenic PD mouse models²⁶. Strategies aimed at activating CMA, such as the overexpression of LAMP2A, have shown promise in some cellular and PD mouse models²⁷, but also require further study in preclinical models. Recently studies demonstrated that activation of the lysosomal calcium channel, TRPML1, can promote autophagy and presents an interesting therapeutic target in treating autophagic disorders²⁸. The application of c-Abl kinase inhibitors (e.g., nilotinib, neurotinib), which have been linked to the ALP, are also ongoing in PD animal studies²⁹.

4. Targeting the ubiquitin-proteasome system in GBA1-PD

The UPS is a selective proteolytic system that regulates protein degradation in eukaryotic organisms. The covalent attachment of ubiquitin, a small 76-amino acid protein, to lysine residues of proteins through the cascade of three enzymes (E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase) targets proteins for degradation by the proteasome. Mutations in the gene encoding Parkin (PRKN), a well-studied E3 ubiquitin ligase, impair degradation of damaged mitochondria (mitophagy) result in familial forms of PD.

a. Targeted protein degradation

Targeted protein degradation is an emerging therapeutic strategy to degrade disease-causing proteins difficult to target with traditional small molecule inhibitors³⁰. The primary class of molecules in development are PROteolysis-TArgeting Chimeras (PROTACs). Briefly, PROTACs are heterobifunctional small molecules that hijack the cell’s endogenous UPS by recruiting a protein of interest (POI) and an E3 ubiquitin ligase to facilitate the ubiquitination of the POI and its subsequent degradation by the UPS.

Studies have shown that the primary mechanism for degradation of wildtype α-synuclein within cells is CMA, while α-synuclein aggregation is the result of mutant α-synuclein blocking its own degradation³¹. Specific species of α-synuclein are thought to be degraded by either the ALP or the UPS system^{32, 33}. The proteosome is involved in GCase degradation, as mutant GCase is heavily ubiquitinated and targeted for ER-associated degradation (ERAD)³⁴. Additionally, a recent study showed mutant GCase protein gets translocated to the lysosome and blocks the degradation of α-synuclein by CMA³⁵. Thus, enhancing the clearance of pathological α-synuclein and GCase with PROTAC molecules may decrease protein aggregation and restore lysosomal function within cells. Patents have been filed for PROTAC molecules targeting α-synuclein³⁶, but no PROTAC molecules are currently approved for any disease.

5. Expert opinion

Presently, there are no therapies that modify disease progression in GBA1-PD, emphasizing the need for alternative strategies to ameliorate disease progression. The proposed inverse relationship between decreased GCase activity and increased α-synuclein aggregation underscores the importance of targeting protein clearance pathways.

Studies have shown that the primary mechanisms for the degradation of mutant α-synuclein and GCase are the ALP and UPS, respectively. The enhancement of both pathways is an exciting approach to modulating protein aggregation to potentially slow disease progression. It should however be noted that the cell’s protein clearance programs are an intricate web, where enhancement of one part, may very well affect the other. The recent study by Kuo et al. showing that GBA1-mutations do not only impair macromolecular degradation specifically by lowering GCase activity, but that they also inhibit CMA degradation in general, demonstrates the wider implications of GBA1 on cellular proteostasis³⁵. Efficient treatment might require simultaneous degradation of misfolded GCase and restoration of wildtype GCase, solving both a gain- and loss-of-function. Loss-of-function leading to substrate accumulation is an attractive theory, as shifts in lipid content affects α-synuclein folding. However, to date there is limited evidence supporting substrate accumulation in brains from patients with GBA1-PD, and therefore targeting proteostasis might be a more efficient therapeutic approach.

While artificially supporting the increased clearance of proteins does not necessarily prevent the cause of GBA1-PD, it can help facilitate the resumption of cellular homeostasis by halting the cycle where α-synuclein species increasingly disrupt protein degradation and prevent secondary effects (e.g., α-synuclein aggregation is suggested to cause an immune response which is neurotoxic, leading to neurodegeneration). Furthermore, both insufficient autophagic activity and GCase deficiency have been shown to augment cell-to-cell transmission of misfolded α-synuclein in model systems^37–39. Propagation of α-synuclein is proposed to spread in a prion-like fashion and therefore, enhanced proteolysis might theoretically also limit disease progression.

Testing of autophagy-inducing small molecules to enhance lysosomal degradation of proteins in PD animal models has provided encouraging results in decreasing α-synuclein aggregation. One concern is that mTOR inhibition could lead to widespread detrimental side effects affecting different cell types, and alternative mTOR-independent autophagy-inducers have not been well-studied. However, general autophagy inducers would increase degradation of all proteins, which is a potentially beneficial feature, as Lewy bodies include more than 90 different proteins. It is unknown if protein specific treatments (e.g., PROTACs) would halt this process. Concurrently, enhancing the function of the UPS to degrade aggregated α-synuclein and mutant GCase holds potential, but the significant drawbacks to applying PROTACs to GBA1-PD include the well-characterized “hook” effect, and the challenges of degrading mutant GCase and α-synuclein in the lysosome. Additionally, PROTACs are not selective to the misfolded POI and unwanted off-target effects could lead to degradation of the cell’s native “healthy” α-synuclein. However, the development of targeted protein degradation technology is rapidly evolving, and progress with similar approaches for the ALP provides further hope for this strategy.

Further evaluations in pre-clinical models and clinical trials are needed to confirm the validity of these strategies. The protein clearance strategies described do not target the genetic origin of GBA1-PD, and even if effective through may require continuous administration. However, until more permanent therapies like gene therapy for GBA1 become widely available, they may provide an effective strategy for patients with GBA1-associated parkinsonism. It is also possible that a combination of strategies targeting both the ALP and UPS synergistically may prove advantageous for the treatment of PD and related disorders.

Article highlights.

• Protein clearance pathways play an essential role in neurodegeneration

• Current treatments developed for Gaucher disease thus far have not proved effective for GBA1-PD

• Pathways resulting in lysosomal dysfunction are implicated in Parkinson disease

• New methods are being developed to enhance proteasome function and the autophagy-lysosomal pathway

• Targeting protein clearance pathways provide potential alternative strategies to treat GBA1-associated Parkinson disease