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Researchers Find New Enzyme That Might Aid in ‘Putting the Brakes’ on Parkinson’s Disease

new enzyme

Researchers have discovered a new enzyme that might aid in “putting the brakes” on Parkinson’s by inhibiting the LRRK2 pathway, known to play a critical role in this neurodegenerative disease.

The findings are still at an early stage, but the team is already trying to find compounds that can switch on this enzyme in the hopes of finding a new therapy that can slow down Parkinson’s disease.

The study, “PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins,” was published in the journal eLife.

In recent years, mutations in the gene coding for the leucine-rich repeat kinase 2 (LRRK2) have been identified as the most common cause of genetic Parkinson’s, accounting for 1-2% of all cases and up to 40% in some ethnic groups.

LRRK2 works as an enzyme with kinase activity. This type of proteins, called kinases, assist in the transfer of a phosphate group — a molecule made of oxygen and phosphorus — to certain proteins. Such modification is called phosphorylation and is an essential step in turning on and off many proteins inside the cell.

Mutations that increase LRRK2 kinase activity lead to toxic effects on the nervous system, believed to play a central role in the development of Parkinson’s. Thus, looking for therapies that inhibit LRRK2 is a potential path to slow the degenerative process and could have therapeutic potential for Parkinson’s.

From a prior screening, scientist Dario Alessi’s team at the University of Dundee in Scotland already knew that human cells produced some sort of enzyme that could reverse LRRK2 activity.

Together with colleagues at Stanford University, Alessi and his team tried to discover what this enzyme was. Using human cell lines cultured in the lab, they found one — called protein phosphatase 1H (PPM1H). This enzyme is naturally produced in the body and is able to counteract LRRK2 signals. Specifically, it unlocks a type of proteins called Rab, which are inappropriately blocked by LRRK2.

“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. This new enzyme we have found acts as the brakes in the pathway that causes Parkinson’s in humans,” Alessi said in a press release.

“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. This finding opens the door for a new chemical approach to the search for Parkinson’s treatments,” added Alessi, PhD, university professor and director of the MRC Protein Phosphorylation and Ubiquitylation Unit (MRC-PPU).

So far, approaches to block LRRK2 have focused on developing compounds that inhibit the LRRK2 kinase.

“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,” Alessi said.

There currently are no treatments able to slow the progression of Parkinson’s disease. “So we need to be throwing the kitchen sink at this problem,” Alessi said.

As the PPM1H enzyme appears to be present in all people, including those with Parkinson’s, Alessi said a breakthrough could be far-reaching.

“If we can find a way of switching this on then it theoretically could benefit all,” he said. “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?”

Alessi and his colleagues have already started to work with the university’s Drug Discovery Unit to search for a compound able to switch on PPM1H, which could represent a potential treatment for Parkinson’s.

“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,” Alessi said.

The research was supported by the Michael J. Fox Foundation and the UK Medical Research Council.

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LRRK2 Inhibitors May Benefit Parkinson’s Patients With and Without Genetic Mutation, Study Finds

LRRK2

Inhibiting the activity of LRRK2 kinase — an enzyme whose mutated form is one of the most common genetic causes of Parkinson’s disease — may benefit patients both with and without this disease-related mutation, a study finds.

Molecules that block the activity of the LRRK2 kinase — such as DNL201 and DNL151, both being developed by Denali Therapeutics — are currently being tested in clinical trials.

The results of this study, “LRRK2 inhibition prevents endolysosomal deficits seen in human Parkinson’s disease,” were published in Neurobiology of Disease. The research was supported by the Michael J. Fox Foundation.

Mutations in the leucine rich repeat kinase 2 (LRRK2) gene are one of the most commonly known genetic causes of Parkinson’s disease. Evidence indicates that in people with idiopathic Parkinson’s, in which the disease has no known cause, the LRRK2 protein is overly active, regardless of the patient’s mutation status — whether or not they have a mutated LRRK2. That overly active protein leads to the malfunctioning of lysosomes, the special compartments within cells that digest and recycle different types of molecules. Lysosomal dysfunction is involved in the formation of  protein aggregates, or clumps, called Lewy bodies, which contribute to Parkinson’s and, therefore, neurodegeneration.

Therapies that can inhibit, or block LRRK2 are currently being tested in human clinical trials. However, it is still unclear whether blocking LRRK2 protein activity in people with idiopathic Parkinson’s can prevent lysosomal dysfunction and consequent neurodegenerative processes.

To learn more, investigators at the University of Pittsburgh now studied post-mortem brain samples, specifically from a motor brain region called the substantia nigra, which is severely damaged in Parkinson’s. The researchers characterized lysosomal abnormalities in the surviving dopaminergic neurons — the main source of dopamine, the loss of which is a hallmark of this disease — of idiopathic Parkinson’s patients.

When compared with healthy controls, Parkinson’s patients had more abnormal lysosomes. These changes occurred during the early stages of lysosomal development, the researchers found.

The team then investigated whether these post-mortem cellular findings could be replicated in an animal model of Parkinson’s. Rats were given two distinct dose regimens of rotenone, a pesticide that inhibits mitochondria, or the “powerhouses” of cells. Blocking mitochondria leads to cellular death and the onset of parkinsonian features.

Nine to 14 daily doses of rotenone reproduced many idiopathic Parkinson’s features, including lysosomal defects. This caused neurodegeneration in the striatum and substantia nigra, two brain areas involved in motor control.

Interestingly, five daily doses of the pesticide weren’t enough to cause cell death, but did increase the accumulation of Parkinson’s-related alpha-synuclein protein and produce changes in lysosomes.

“These data demonstrate that, in rotenone-treated rats, [alpha]-synuclein protein levels rise in the dopaminergic neurons prior to the onset of frank neurodegeneration,” the researchers said.

When overactive LRRK2 was blocked in rotenone-treated rats, the protein’s activity was reduced. That, in turn, improved the overall health of lysosomes and prevented the accumulation of alpha-synuclein. These effects were observed in animals without a genetic predisposition to develop Parkinson’s, suggesting that the LRRK2 kinase inhibitors may be effective beyond LRRK2-mutated patients.

“Our work suggests that drugs that block LRRK2, some of which have entered clinical trials, will be useful for people with typical Parkinson’s disease,” J. Timothy Greenamyre, MD, PhD, the study’s lead author, said in a press release.

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PF-360 Provides Some Benefits But Does Not Improve Dopaminergic Function, Mouse Study Shows

PF-360 mouse study

Treatment with PF-360, an investigational leucine-rich repeat kinase 2 (LRRK2) inhibitor, can efficiently decrease LRRK2’s phosphorylation levels, known to be elevated in Parkinson’s patients, in the brains of a mouse model of Parkinson’s disease, a preclinical study reports.

However, despite some observed dose-dependent therapeutic effects, including gait improvement, no robust changes in dopaminergic function were observed.

Results of the study were recently presented during the Society for Neuroscience’s 2018 conference in San Diego in a poster titled “Assessment of the Anti-parkinsonian Effects of the Potent and Selective LRRK2 Kinase Inhibitor PF-360 in the AAV-A53T Mouse Model of Parkinson’s Disease.”

The study was the result of a collaboration between several institutions including Charles River Discovery, Merck, Pfizer, Atuka Inc., and The Michael J. Fox Foundation for Parkinson’s Research.

The LRRK2 gene provides instructions for making a kinase, which is a protein that regulates the function of other molecules. Mutations in this gene put the protein into an overly activated state.

Mutations in the LRRK2 gene are one of the most commonly known genetic causes of Parkinson’s disease and usually result in the malfunctioning of lysosomes — special compartments within cells that digest and recycle different types of molecules. Lysosomal dysfunction is involved in the formation of Lewy body protein aggregates and, therefore, neurodegeneration.

Scientists believe that blocking LRRK2’s activity has the potential to slow disease progression.

Using a selective LRRK2 inhibitor called PF-360, researchers studied the dose-response efficacy of the potential therapy in two different mouse models (C57BL/6J and LRRK2-G2019S) that were injected with a “biological cocktail” of an adeno-associated virus combined with a human mutated A53T alpha-synuclein (AAV-A53T) — the major component of protein clumps called Lewy bodies, a hallmark of Parkinson’s.

They used 90 C57BL/6J mice 10-12 weeks old and 105 LRRK2-G2019S mice, 75 of which were 11-12 weeks old and 30 were 5-6 months old. In mouse “time,” 12 weeks is equal to adulthood.

This induced the degeneration of dopaminergic neurons in an area of the brain called the substantia nigra and decreased dopamine and tyrosine hydroxylase — the enzyme responsible for catalyzing levels of L-DOPA, the precursor to dopamine — in the striatum, mimicking Parkinson’s disease.

Mice were treated for 42 days with a diet containing PF-360 or a placebo (control), which was begun seven days prior to AAV-A53T injections.

PF-360 inhibited LRRK2 phosphorylation in the animals’ brain cortex and lungs at a specific site of the protein called serine 935 (serine is an amino acid, or the proteins’ building block). This protein region is required for interaction of LRRK2 with other molecules.

Phosphorylation (the adding of a phosphate group) alters a protein’s structure turning it, for instance, into an activated or deactivated state. As such, phosphorylation is the most common mechanism of regulating protein function and transmitting signals throughout the cell.

Pronounced therapeutic effects were observed with increasing doses (1 mg/kg, 3 mg/kg, 10 mg/kg, 30 mg/kg, and 60 mg/kg of PF-360) in both animal strains and age groups.

AAV-A53T injection led to motor impairments such as decreased speed (longer stride duration, shorter step length), slower swing speed, and reduced hind limb protraction (forward extension).

LRRK2-G2019S mice at 11-12 weeks old recovered their hind limb protraction and retraction with 10 mg/kg of PF-360, while older animals at 5-6 weeks of age had their overall speed (stride duration and swing speeds) improved with 30 mg/kg of the treatment.

No gait changes were observed after 42 days of PF-360 treatment in C57BL/6J mice. However, there was an insignificant treatment-related trend toward increased tyrosine hydroxylase-positive cells in the substantia nigra of C57BL/6J animals.

After treatment, a significantly higher number of tyrosine hydroxylase-positive cells were observed in older LRRK2-G2019S mice.

An increase in tyrosine hydroxylase-positive cells is indicative of an increase in the number of nerve cells that can produce either L-DOPA or dopamine.

Neurochemical analysis revealed that PF-360 delivery to younger animals did not improve striatum levels of dopamine or the intermediate end products of dopamine’s metabolism (3,4-dihydroxyphenylacetic acid and homovanillic acid).

However, treatment significantly increased homovanillic acid levels in older LRRK2-G2019S mice.

Given that most evidence suggests an LRRK2 contribution to Parkinson’s disease via abnormal phosphorylation, this study shows that although PF-360 can reduce LRRK2 phosphorylation levels, both in the brain and in the periphery, it failed to show robust improvements in dopaminergic function.

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