The value of patient-based cell models and brain organoids in unpicking the complexities of neurodegenerative diseases is again highlighted in new research showing how mutations that cause a rare subtype of Parkinson's disease operate at a cellular level.

Using stem cells derived from members of families carrying mutations in the PARK7 gene that are linked to an early onset form of Parkinson's disease, the researchers at the University of Luxembourg directly observed that these defects disrupt exon splicing.

That interferes with the activity of a small RNA called U1, reducing production of the DJ-1 protein (also called Parkinsonism associated deglycase), which among other functions, is involved in mitochondrial regulation. The researchers found that mitochondria in the patient-derived cell models malfunctioned as a result.

Based on this insight, Ibrahim Boussaad and colleagues designed a combination therapy that repaired these aberrations, halting the death of dopaminergic neurons in brain organoids.

The researchers say the study, published in the latest issue of Science Translational Medicine, provides a foundation to develop therapies that could help correct the genetic cause of some forms of Parkinson's disease.

"When PARK7 was first discovered it was described as causing the substitution of an amino acid. It's only when we got patient material we could see it's not substitution. We saw it was a problem with splicing," Boussaad told BioWorld Science.

There are more than 25 PARK7 gene mutations that are implicated in Parkinson's disease. However, it previously was unclear how loss of functional DJ-1 protein causes the neurodegenerative disease, with some studies suggesting PARK7 mutations disrupt the protein's chaperone function, leading to a toxic buildup of misfolded proteins, while others suggest PARK7 mutations impair DJ-1's ability to protect cells from oxidative stress.

Boussaad said the researchers were aiming to pinpoint specific defects in the mutant DJ-1 protein generated by the patient cell models. Instead, they found there was little or no trace of the protein at all.

The possibility that the protein was unstable and had degenerated was excluded by blocking degradation pathways to show there were no metabolites of the protein.

"So we took a step back and started to look upstream. We could detect [PARK7] RNA, but it was shorter," said Boussaad. "On sequencing the products, we saw an entire section of RNA was missing."

Luckily, one of the donors was heterozygous for PARK7, and in this case it was possible to detect both normal length and shorter RNA, helping to confirm that mis-splicing was blocking DJ-1 production.

The researchers showed it was possible to correct the defective exon skipping using genetically engineered U1-snRNA (small nuclear RNA). That corrected DJ-1 expression in neuronal precursor cells and differentiated neurons.

They also demonstrated that combination treatment with the small molecules RECTAS (rectifier of aberrant splicing) and phenylbutyric acid repaired the splicing defects, restoring DJ-1 production, correcting mitochondrial function and preventing loss of dopaminergic cells in mutant PARK7 brain organoids.

Looking at sequence databases, the researchers found that similar splicing mutations appeared more frequently in the genomic data from 2,710 patients with the common sporadic form of Parkinson's disease, than in data from 5,713 controls, pointing to a wider significance of the current study.

With the literature suggesting that as many as a third of all disease-causing mutations exert their effects through faulty exon splicing, the findings could also have relevance to other conditions.

The direct observation of the cellular-level impact of one of these mutations and the possibility of therapeutic intervention, should lead to greater focus in this area, Boussaad said. "There are a few other such mutations known and described, but they have not got the attention they should have in the field of neurodegenerative diseases."

As a next step, the researchers plan to further develop the combination treatment. "We are going into animal models to see how potent it is in entire organisms. We also want to look at availability in the brain and if compounds have the ability to cross the blood-brain barrier," said Boussaad. "And of course, the other direction will be to further explore the abundance of exon splicing mutations in Parkinson's disease."

It is becoming increasingly clear that although patients with Parkinson's present with the same motor symptoms, there are subtypes of the disease which have different etiologies. "To advance treatment, we have to stratify. We have to investigate for each patient, why they have this disease," Boussaad said.

That could form the basis of customized treatments. "If we can identify patients with exon splicing mutations it will have positive benefits. If they can be corrected, it will restore the protein and be neuroprotective, rather than treating the symptoms," said Boussaad. "It could have a significant impact." (Boussaad, I. et al. Sci Transl Med 2020, 12: eaau3960).