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In the Pipeline-Parkinson's Disease Genetics
Wild-Type LRRK2 Is Implicated in PD Patients Without Family History of Disease

ARTICLE IN BRIEF

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DR. JOSHUA SHULMAN: “This study reminds us that less common Mendelian forms of PD, such as that associated with LRRK2 mutations, can pinpoint universal mechanisms, potentially making targeted therapies more broadly applicable.”

Researchers reported evidence that the activity of LRRK2 protein might be affected in many more patients with Parkinson's disease, even when the LRRK2 gene itself is not mutated.

More than a dozen years after the identification of leucine-rich repeat kinase 2 (LRRK2) mutations in patients with familial Parkinson's disease (PD), scientists at the University of Pittsburgh have discovered that wild-type LRRK2 is activated in nigrostriatal dopamine neurons in patients without a family history.

The team developed an assay to detect LRRK2 activation and conducted a series of experiments in animal models that showed there are many paths that can lead to LRRK2 activation, including environmental toxins. They found activation in postmortem brains from patients who died with idiopathic Parkinson's. They were also able to prevent this activation (in the animal models) with experimental LRRK2 kinase inhibitors.

The findings, published in the July 25 edition of Science Translational Medicine, suggest that these LRRK2 kinase inhibitors, now in early stages of clinical testing, could be successful in treating idiopathic forms of Parkinson's.

“Our proximity ligation assays allow us to measure LRRK2 activation in dopamine neurons in the substantia nigra,” said the senior study author J. Timothy Greenamyre, MD, PhD, the Love Family professor and vice-chair of neurology and director of the Pittsburgh Institute for Neurodegenerative Diseases, and chief of movement disorders at University of Pittsburgh. “LRRK2 is activated by oxidative stress. This explains how LRRK2 is activated in the brains of patients without a LRRK2 mutation.”

The researchers showed that oxidative stress activates LRRK2 kinase activity. This leads to problems in the brain's clearance mechanisms that lead to accumulations of phosphorylated alpha-synuclein in the brains of PD patients.

LRRK2 missense mutations account for about 3 percent of PD cases, the study authors noted, making it the most common cause of autosomal dominant PD. The mutations cause a toxic increase in LRRK2 kinase activity. Scientists have also found a variant of LRRK2 that puts people at risk and another variant that confers protection. These findings sparked the scientists' interest in looking at the role of LRRK2 in idiopathic PD.

STUDY METHODS, FINDINGS

LRRK2 is present in small amounts in most cells in the brain, which makes it difficult to measure, Dr. Greenamyre said. He and his colleagues developed a proximity ligation assay that allowed them to measure LRRK2 activation, cellular location, and the identification of physiological regulators of LRRK2. The assay measures Ser1292 phosphorylation, a biomarker of LRRK2 activation. A second assay measured the interaction between activated LRRK2 and 14-3-3 proteins. The 14-3-3 proteins are known to bind LRRK2 and result in reduced LRRK2 kinase activity.

The investigators used the pesticide rotenone to trigger oxidative stress in animals; rotenone exposure also causes the animals to develop pathological features of the disease. They then compared levels of wild-type LRRK2 activation in these models with controls that had no exposure to the pesticide, and reported a 10-fold increase in activation of LRRK2 in dopamine neurons in the striatum.

They also conducted the same assay studies on adeno-associated virus type 2–mediated overexpression of wild-type human alpha-synuclein (hSNCA) injected unilaterally into the substantia nigra pars compacta of rats. This causes a slow and progressive neurodegeneration. Again, they showed a 10-fold increase in phosphorylated Ser1292 proximity ligation signal compared to the other side of the animal's brain that was not touched.

Both rotenone exposure and elevated alpha-synuclein increased the formation of reactive oxygen species and both insults activated wild-type LRRK2, explained Dr. Greenamyre. An LRRK2 kinase inhibitor prevented rotenone-induced activation of nigrostriatal LRRK2 and its downstream effects in rats. Moreover, when hydrogen peroxide was used as a reactive oxygen species to activate LRRK2 in cells, the activation could be blocked by treatment with an antioxidant.

“This is very interesting considering that for at least 40 years there have been hints that oxidative stress plays an important, but hard-to-define, role in the pathogenesis of PD. Additionally, our results show that you can end up in the same place with activation of LRRK2 with pesticide exposure as you can with mutations in the LRRK2 gene,” said Dr. Greenamyre. “It suggests that a LRRK2 kinase inhibitor could be good for whatever triggers PD.”

Next, the researchers looked at LRRK2 activation in sections of substantia nigra from postmortem brain tissue from seven iPD patients and eight age-matched controls. They reported a strong signal in idiopathic PD patients, a six-fold increase in Ser1292 proximity ligation (p< 0.0002) and a four-fold increase in phosphorylation of another LRRK2 substrate, Rab10 (p< 0.0002). There was also a fivefold decrease in the 14-3-3 proximity ligation signal (p< 0.0001).

LRRK2 is also expressed in microglia, and the assay also allowed them to measure levels in nigral microglia the human brain samples. It was more than double in the idiopathic PD patient tissue compared to the control tissue.

The researchers said that “the time course of LRRK2 activation in the human brain is unknown, and the clinical effects of LRRK2 inhibitors remain to be examined.” Still, they said: “We believe that LRRK2 inhibitors may be beneficial not only for the 3 to 4 percent of people with PD who carry LRRK2 mutations but also for iPD patients who do not carry LRRK2 mutations.”

Dr. Greenamyre added that the findings “suggest that LRRK2 activation may be a crucial player in PD. Follow-up preliminary studies indicate that dopamine itself may participate in the activation of LRRK2, and LRRK2 kinase inhibitors can protect dopamine neurons and nerve terminals.”

EXPERT COMMENTARY

“In this exciting and important study, Dr. Greenamyre and colleagues provide evidence for activation of the LRRK2 enzyme in individuals with PD, but without LRRK2 mutations as a cause,” said Joshua Shulman, MD, PhD, associate professor in the departments of neurology, neuroscience, and molecular and human genetics at Baylor College of Medicine, and an investigator at the Duncan Neurological Research Institute at Texas Children's Hospital. “In addition, in two different rat PD models (rotenone treatment or alpha-synuclein expression), they highlight similar LRRK2 activation, which they show can be induced by oxidative stress, and lead to cellular derangements in lysosomal and mitochondrial function. Inhibitors of LRRK2 can block the observed enzymatic activation and attenuate the downstream cellular consequences.”

“These results highlight that LRRK2 activation may be generally associated with PD pathogenesis, beyond simply those cases in which LRRK2 gene mutations are contributing,” Dr. Shulman said. “Recently, there has been an important push in our field for embracing ‘personalized medicine’ approaches in PD diagnosis and clinical care. This has led to a conceptualization of individualized genetic etiologies for PD, and the possibility that tailored treatments will therefore be necessary. This study reminds us that less common Mendelian forms of PD, such as that associated with LRRK2 mutations, can pinpoint universal mechanisms, potentially making targeted therapies more broadly applicable.”

“This is an exciting paper; it is a wonderful advance for the field,” said Matthew Farrer, PhD, professor in the department of medical genetics and the Don Rix BC Leadership chair in genetic medicine at the University of British Columbia. “The research connects the dots between potential environmental risk factors, such as pesticide exposure (rotenone treated rats in this case), alpha-synuclein S129 phosphorylation/oligomerization, subsequent impairments in autophagy/mitochondrial function, and LRRK2 activation. They show that treatment with LRRK2 kinase inhibitors can prevent all these downstream phenotypes.”

Dr. Farrer added that the investigators also showed that LRRK2 is expressed in dopaminergic neurons and activated in fixed tissue of postmortem brains from patients with idiopathic PD, and that LRRK2 is expressed and activated in dopaminergic neurons.

“I see LRRK2 kinase inhibitors having broad application in both monogenic parkinsonism (most reminiscent of typical, late-onset disease) and idiopathic PD,” Dr. Farrer continued. “Although it has many functions in many cell types, LRRK2 regulates the molecular machinery for synaptic-endosomal trafficking/recycling, which is particularly critical to the function and survival of dopaminergic neurons. We now need to develop an in vivo readout of the neuroprotective effect of LRRK2 kinase inhibitors on dopaminergic neurons to determine the lowest possible efficacious dose to permit a lifetime of use without peripheral non-neuronal side-effects.”

LINK UP FOR MORE INFORMATION:

•. Di Maio R, Hoffman EK, Rocha EM, et al. LRRK2 activation in idiopathic Parkinson's disease http://stm.sciencemag.org/content/10/451/eaar5429.short. Sci Transl Med 2018; 10(451): pii: eaar5429.