Breakthrough study: inhibiting USP30 gene offers hope in treating Parkinson’s disease

By Vijay Kumar MalesuReviewed by Lily Ramsey, LLMNov 15 2023

In a recent study published in Nature Communications, researchers investigated whether USP30 inhibition can serve as a disease-modifying therapy for Parkinson's disease (PD) by enhancing mitophagy and reducing α-synuclein (αSyn) pathology.

Study: Knockout or inhibition of USP30 protects dopaminergic neurons in a Parkinson’s disease mouse model. Image Credit: Chinnapong/Shutterstock.com

Background 

Mitochondrial dysfunction is a key factor in PD, highlighting the importance of mitophagy, the selective degradation of dysfunctional mitochondria. PARKIN mutations in autosomal recessive PD  (AR-PD) impair mitophagy, and PARKIN, along with PINK1 viz another gene where mutations cause AR-PD, plays a critical role in the ubiquitylation of mitochondrial proteins, essential for their autophagic degradation.

Furthermore, αSyn toxicity, linked to autosomal dominant PD (AD-PD), may induce mitochondrial impairment and reactive oxygen species production, suggesting a vicious cycle of dysfunction and αSyn accumulation.

Further research is essential to comprehensively understand and validate the role of mitophagy, particularly via USP30 inhibition, in mitigating α-synuclein toxicity and its therapeutic potential in PD.

About the study 

In this study, researchers used an AAV1/2-A53T αSyn vector to induce synucleinopathy in mice, carefully selecting the sample size and experimental endpoints based on previous studies.

The treatment groups, including male and female mice, were randomly assigned and analyzed according to the National Institutes of Health (NIH’s) sex as a biological variable policy.

Ethical considerations were paramount, with all mouse studies adhering to strict regulations and institutional review board approvals. The mice, encompassing various strains like mito-QC homozygous and Usp30 knockout (KO) homozygous, were housed in controlled environments.

The Usp30 KO mice underwent extensive phenotyping and the research incorporated cell culture experiments with SH-SY5Y cells and human iPSC-derived dopaminergic neurons. These steps were crucial in exploring the potential of Usp30 KO and the MTX115325 treatment in addressing PD pathology.

Various antibodies and vectors sourced from biotechnology companies facilitated key processes like immunostaining and immunoblotting. The study's effectiveness was further ensured through strict protocols for stereotaxic vector injection, oral administration of the inhibitor MTX115325, and consistent monitoring of its blood concentration.

Behavioral assessments, histological studies, mitophagy analysis, Western blot analysis, and dopamine measurements provided comprehensive insights. Additionally, biochemical and cell-based assays, detailed pharmacokinetics, and brain Cellular Thermal Shift Assay (CETSA) analysis were conducted.

The study's findings were solidified through rigorous statistical analysis, highlighting the thoroughness of the research approach.

Study results 

The present study successfully generated KO mice by deleting exon 4 of the Usp30 gene. These mice were viable and did not exhibit any overt pathology, maintaining normal Mendelian birth rates and showing no significant health issues with aging. Interestingly, one-year-old Usp30 KO mice showed resistance to fatty liver accumulation compared to their wild-type counterparts.

The researchers further investigated the impact of Usp30 KO on mitophagy in dopaminergic neurons. By crossing Usp30 KO mice with mito- Quality Control (QC) reporter mice, they could effectively monitor mitophagy in vivo.

The study revealed a significant increase in mitophagy levels in the dopaminergic neurons of the Usp30 KO mice compared to wild-type mice, specifically in the brain's substantia nigra, cortex, and hippocampus.

The study also explored the development of αSyn pathology and associated motor deficits in these mice. Immunostaining for phosphor-S129-αSyn, a pathological form of αSyn, showed that Usp30 KO mice had significantly reduced levels of this protein in their brains.

Additionally, colocalization analysis indicated that Usp30 depletion reduced the association of pathological S129-αSyn with mitochondria, suggesting a potential mechanism by which Usp30 KO mitigates αSyn toxicity.

In this comprehensive study, the researchers investigated the protective role of KO against αSyn induced motor deficits. They used the cylinder test to assess motor function in mice post-AAV-A53T-SNCA viz Adeno-Associated Virus - Alpha-Synuclein with A53T mutation injection. The results showed that unilateral injection of AAV-Ev did not impair motor function in any mouse group.

However, AAV-A53T-SNCA injection led to motor dysfunction in wild-type (WT) and mito-QC mice, evident from decreased usage of the forelimb contralateral to the injection site. Usp30 KO significantly protected against these αSyn-induced motor deficits in both male and female mito-QC/Usp30 KO mice.

Furthermore, the study assessed the impact of Usp30 KO on αSyn-induced loss of dopaminergic neurites and terminals in the striatum. They observed a significant decrease in the density of Tyrosine Hydroxylase positive (TH+) terminals in the striatum of WT and mito-QC mice but not in Usp30 KO mice following AAV-A53T-SNCA injection.

Additionally, Usp30 KO appeared to protect against the loss of striatal dopamine and its metabolites. These findings demonstrate that Usp30 KO not only preserves the structural integrity of dopaminergic neurons but also maintains functional dopamine levels in the striatum.

The researchers also validated the effectiveness of a brain-penetrant USP30 inhibitor, MTX115325. MTX115325 exhibited good oral bioavailability, central nervous system (CNS) penetration, and selectivity against other enzymes.

It effectively inhibited USP30 in biochemical assays and cellular models, increasing the ubiquitylation of TOM20, a mitochondrial protein, and USP30 substrate. In human dopaminergic neurons, MTX115325 increased TOM20-ubiquitylation, indicating its potential therapeutic application.

Conclusion

Finally, the study explored the effects of MTX115325 in an AAV-A53T-SNCA mouse model of PD, and the results mirrored those observed in Usp30 KO mice, with MTX115325 protecting against αSyn-induced loss of TH+ neurons and preserving striatal dopamine levels.

Moreover, MTX115325 reduced phosphorylated S129-αSyn and astrocyte activation, further supporting its potential as a therapeutic agent in PD, and these comprehensive results highlight the therapeutic promise of USP30 inhibition in mitigating PD pathology.