In fixing a protein processing malfunction in cells derived from members of a single family who carried a particular mutation in this gene, a team of scientists was able to increase DJ-1 production. That treatment led to improved cell function.
Further, the researchers discovered some sporadic mutations that had been identified in a large group of Parkinson’s patients in the same gene region as those found in this family. Those findings support an alternative therapeutic approach in people with these types of mutations, the scientists said.
“This insight fundamentally changes our view of the causes of the disease and presents entirely new possibilities for treatment,” Ibrahim Boussaad, PhD, the study’s first author and a researcher at the Luxembourg Centre for Systems Biomedicine (LCSB), said in a press release.
The study, “A patient-based model of RNA mis-splicing uncovers treatment targets in Parkinson’s disease,” was published in the journal Science Translational Medicine.
The PARK7 gene encodes the instructions for DJ-1, which is necessary for the correct functioning of nerve cells. Mutations in the gene lead to a reduction or loss of the production of DJ-1 and are associated with the development of early onset Parkinson’s.
However, how this change triggers the death of nerve cells that produce the signaling molecule dopamine — the underlying cause of Parkinson’s disease — is still unclear.
To understand further, a team of researchers based at the University of Luxembourg examined cells isolated from members of a specific family affected by Parkinson’s disease, which had been caused by PARK7 mutations.
Since it is not possible to study nerve cells (neurons) directly from patients’ brains, the scientists collected skin cells (fibroblasts) and reprogrammed them to become a type of stem cell called induced pluripotent stem cells (iPSCs). These iPSCs can be prodded into becoming any type of human cell needed for therapeutic purposes. Here, they were further transformed into immature nerve cells known as small-molecule neural precursor cells (smNPCs). The iPSCs and smNPCs were then used to derive patient-specific neurons.
Skin cells were biopsied from affected family members who carry mutations in both copies of the PARK7 gene (homozygous, one from each parent) and cells from unaffected family members who carried one mutant copy (heterozygous). The team also biopsied skin cells from healthy individuals used as controls.
An analysis of the original skin cells, as well as all types of the reprogrammed cells, showed the levels of the DJ-1 protein were “markedly reduced” in cells derived from those with Parkinson’s compared with unaffected family members or the healthy controls.
To understand why there was less DJ-1 protein, the team examined PARK7 messenger RNA (mRNA) — the molecule that carries instructions from DNA to make protein. The mRNA from the patients’ cells was shorter than expected, whereas unaffected heterozygous family members had a mix of normal and short mRNA.
Further analysis revealed that a portion of the mRNA — called exon 3 — was missing in cells derived from patients, which explained the shorter mRNA.
Notably, genes consist of exons and introns, in which exons are stretches of DNA that carry instructions to make a protein, and introns are regions that do not code for protein. Introns are removed in a process known as mRNA splicing.
The PARK7 mutation in this family led to a mistake in splicing called exon skipping that resulted in shorter mRNA, causing a lack of DJ-1 protein.
To confirm this result, the team used a molecule critical in the splicing process called U1 snRNA, which was mutated to match the PARK7 mutation. Applying this altered U1 snRNA restored full-length mRNA and rescued DJ-1 protein production.
“In the patients, an essential tool for the assembly of the protein DJ-1 fails to dock properly,” said Rejko Krüger, MD, from the University of Luxembourg. “In scientific terms, we call that exon skipping. As a result of this defect, the protein doesn’t get built at all.”
Previous studies found that a lack of DJ-1 protein causes a malfunction in small cell structures known as mitochondria, which provide energy to the cell. In skin cells derived from the patients, mitochondria function was impaired compared with healthy controls.
Using two molecules that together treat U1-dependent mis-splicing (rectifier of aberrant splicing, or RECTAS, and phenylbutyric acid), there was a dose-dependent increase in DJ-1 protein that reached statistical significance at the higher doses leading to an increase in mitochondria function.
Using a more disease-related cell model, neurons generated from smNPC — or immature nerve cells — derived from participants were treated with the two molecules. This rescued the aberrant splicing observed in the patients’ neurons and led to normal DJ-1 protein production.
Additionally, midbrain-specific organoids were grown. These are tiny, self-organized tissue cultures derived from cells to mimic brain tissue. While organoids that carry the PARK7 mutation caused a marked decrease in neurons that produce dopamine, treatment with RECTAS and phenylbutyric acid rescued this loss in a dose-dependent manner.
Finally, as PARK7 mutations are rare and the particular mutation investigated in this study comes from a single-family, the team wondered if mutations that cause aberrant mRNA splicing exists in sporadic forms of Parkinson’s disease.
To find out, genes from 372 Parkinson’s patients and 161 healthy people (controls) were analyzed. The team observed that mutations in gene regions essential for splicing were higher in patients than in controls.
Additionally, genetic data from 2,710 Parkinson’s patients and 5,713 healthy controls from the Parkinson Disease Genome Sequencing Consortium (PDGSC) project were analyzed to increase this result’s statistical power. Again, there was a high prevalence of splice-site sporadic mutations in genes from those with Parkinson’s compared with the controls.
“Our findings suggest that the pathogenic [disease-causing] relevance of exonic splicing mutations has been underestimated in [Parkinson’s disease],” the researchers wrote.
“Therefore, our study suggests an alternative strategy to restore cellular abnormalities in in vitro [in the lab] models of [Parkinson’s disease] and provides a proof of concept for neuroprotection based on precision medicine strategies in [Parkinson’s disease],” they added.
Krüger said collaboration among scientists was key to the results found in the seven-year research effort.
“Only by combining numerous disciplines — from medical practice, to laboratory research, to computer science — could we understand the cause and at the same time identify active substances for a potential treatment,” Krüger said.
We are sorry that this post was not useful for you!
Let us improve this post!
Tell us how we can improve this post?