Researchers at the University of Dundee have identified an enzyme that inhibits the protein kinase product of a gene known as LRRK2, mutations in which are the common genetic cause of Parkinson’s disease (PD). The researchers’ laboratory studies showed that the inhibitory enzyme, PPM1H, counteracts LRRK2 signaling by specifically dephosphorylating Rab proteins. The researchers suggest that the findings could lead to new therapeutic and preventive strategies against Parkinson’s disease.
“Parkinson’s is like a runaway train—at present we have no way of putting the brakes on to slow it down, let alone stop it,” commented Dario Alessi, PhD, director of the Medical Research Council Protein Phosphorylation and Ubiquitylation Unit (MRC-PPU) in the School of Life Sciences. “This new enzyme we have found acts as the brakes in the pathway that causes Parkinson’s in humans.” Alessi’s team, together with collaborators at Stanford University School of Medicine, report their findings in eLife, in a paper titled, “PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins.”
LRRK2 is a large multidomain protein kinase enzyme. Mutations in the LRRK2 gene that hyperactivate the protein cause Parkinson’s disease, the authors stated. “Mutations in LRRK2 are one of the most common genetic causes of familial Parkinson’s comprising ~5% of familial Parkinson’s, and ~1% of sporadic Parkinson’s patients. In terms of clinical presentation and late age of onset, LRRK2 mediated Parkinson’s closely resembles the common sporadic form of the disease affecting the vast majority of patients.” While the G2019S mutation is the most common LRRK2 mutation there is also evidence that the LRRK2 pathways is hyperactivated in some patients with idiopathic PD.
Accumulating evidence of a role for LRRK2 signaling in Parkinson’s disease has directed one avenue for drug development. “Pharmaceutical companies have developed LRRK2 inhibitors for treatment and prevention of PD and clinical trials have commenced and/or are planned,” the team continued. Studies have shown that LRRK2 phosphorylates a subgroup of Rab proteins, and while protein phosphatase enzymes that act on LRRK2-phosphorylated Rab proteins are known to exist and appear highly active, these enzymes haven’t yet been identified, Alessi and colleagues noted. Much of what is already known about the LRRK pathway has been discovered by the Dundee team, and the investigators set up a series of siRNA screens to identify and characterize the protein phosphatase, or phosphatases, that counteract LRRK2 signaling by dephosphorylating LRRK2-modified Rab proteins.
One of the top hits in all three screens was the enzyme PPM1H, which exhibited a remarkable ability to reverse the biology triggered by LRRK2. “Repeat studies in multiple experiments confirmed that siRNA mediated depletion of PPM1H increased Rab10 phosphorylation, without affecting overall levels of Rab10 or LRRK2,” the authors wrote. Subsequent studies showed that overexpression of PPM1H inhibited LRRK2-mediated Rab protein phosphorylation in cell lines. Conversely, using CRISPR-Cas9 editing to knock out full length PPM1H enhanced Rab10 protein phosphorylation. “Basal levels of Rab10 phosphorylation was increased 2–5-fold in 10 independent knock-out cell lines that we examined,” the scientists continued.
The Dundee team had previously shown that pathogenic LRRK2 suppresses cilia formation in cell culture and in the mouse brain, through a process that requires proteins including Rab10. Through a series of experiments the researchers also demonstrated that endogenous PPM1H protein contributes to the regulation of cilia formation in cultured cells, “… and confirms a role for wild-type LRRK2 protein in this important cellular process.”
They claim that their findings provide “compelling evidence” that PPM1H acts to dephosphorylate Rab proteins in vivo, and so counteract LRRK2 signaling. “In future work, it will be important to explore whether PPM1H contributes to PD risk,” they stated. “It would be critical to explore whether increased expression or activity of PPM enzymes protects patients with LRRK2 mutations from developing PD by enhancing Rab protein dephosphorylation. Conversely, reduced expression or activity of PPM1H phosphatase would be expected to promote Rab protein phosphorylation and enhance PD risk. Targeting PPM1H to increase its activity or expression in order to promote Rab protein dephosphorylation could be explored as a therapeutic strategy for preventing and/or treating LRRK2-mediated PD.”
“We have known for many years that the LRRK2 pathway is a major driver behind Parkinson’s but the concept of developing an activator of the PPM1H system to treat the disease is completely new, Alessi sad. “This finding opens the door for a new chemical approach to the search for Parkinson’s treatments … In terms of the current approach, Plan A is to develop a drug to inhibit LRRK2 but even once this is done we don’t know how well such a drug will be tolerated in the body so we are also looking for other ways to switch off this pathway. The purpose of this research was to find an enzyme that naturally stops LRRK2 by mediating these toxic pathways.”
The development of any form of drug derived from PPM1H is still years away, but the Alessi team and collaborators have started to work with the University’s Drug Discovery Unit to search for a compound that would switch the enzyme on for the treatment of Parkinson’s. “It would appear that the PPM1H enzyme is present in all people and it is not missing in patients with Parkinson’s so if we can find a way of switching this on then it theoretically could benefit all,” Alessi noted. “It also raises another exciting question that we want to study—is PPM1H higher in the brain of certain people and, if so, is this protecting them against Parkinson’s?”
“By making this discovery, we are now in a position to work with pharmaceutical companies and the Drug Discovery Unit here at Dundee to develop compounds that would switch on this enzyme. This will be challenging work but if we can identify appropriate drug-like molecules then the next stage would be to test them in cells and in animal models to see if they do indeed switch off this pathway. If that works it would be certain to stimulate further preclinical activity and could potentially lead to a new way to treat Parkinson’s. We have a lot of obstacles to overcome before we get to that point but this is a major discovery for us.”