A decade after the discovery that mutations in leucine-rich repeat kinase 2 (LRRK2) cause Parkinson’s disease, researchers are still trying to get a handle on how the giant, 51-exon, multi-catalytic protein exerts its pathogenic power. Now, researchers led by Valina and Ted Dawson at Johns Hopkins University in Baltimore report that LRRK2 phosphorylates a ribosomal protein, resulting in a boost in protein translation. Mutations that enhance the kinase activity of LRRK2 trigger a toxic surge in protein production that results in neuronal injury and death, according to their April 10 Cell paper. Other researchers in the field were cautious about the findings, but hoped they will be confirmed by others.
Valina Dawson proposes that LRRK2’s effects on the ribosome lead to a gradual disruption of proteome homeostasis, with some proteins being more affected than others. “Initially the cells can compensate,” Dawson said, “but over time you end up in that slow slide toward misregulation and disease.”
“Understanding the basic biology of how LRRK2 exerts its effects, and identifying its immediate downstream targets, is a terribly important goal in the PD research field,” Dario Alessi of the University of Dundee in Scotland told Alzforum. However, Alessi, who was not involved in the study, questioned the rigor of the biochemical findings. To his mind, they were not compelling enough to support the mechanism proposed by the authors. “It will be key for other labs to reproduce this result,” he said. “In that case, this will be seen as a major advance.”
Lost in Translation. Overexpression of a LRRK2 mutant with elevated kinase activity triggered a shortening of neurites in human dopaminergic neurons (top). Co-expression of a ribosomal protein proposed to be a LRRK2 substrate rescued this defect when rendered phosphor-deficient (bottom panel).
Dominantly inherited mutations in LRRK2 are the most common cause of familial PD, and a majority of known LRRK2 mutations occur in the kinase domain. The most common LRRK2 mutation, G2019S, ups the activity of the kinase. Intense research has tried to identify the proteins phosphorylated by LRRK2, but with limited success (see Tsika et al., 2012). Alessi estimated that a dozen potential substrates have surfaced, mostly from in-vitro studies, but most have failed to survive as bona fide substrates when mammalian cells or tissues were examined (see Kumar et al., 2010; Trancikova et al., 2012). “The question is whether the data here are any different than the previous 12,” he said.
Valina Dawson said that an enduring issue with the identification of LRRK2 substrates has been that researchers have difficulty linking them to LRRK2 pathology. “We wanted to find the pathophysiologic targets of the LRRK2-G2019S mutant,” she said.
To pluck out a genuine pathological LRRK2 substrate from the thousands of proteins within the cell, first author Ian Martin and colleagues started by pulling out a tagged version of LRRK2 from cells and looking for phosphoproteins that came with it. The researchers next separated the phosphoproteins and analyzed their peptide makeup by mass spectrometry. Using those sequences, they then cloned and purified a majority of the 161 interacting proteins they identified, and tested which of them LRRK2 phosphorylated. They found 11 proteins that seemed to be LRRK2 substrates, and 10 were ribosomal proteins.
The investigators expanded their search to other components of the ribosome, and discovered the s15 ribosomal subunit was also phosphorylated by LRRK2 at threonine-136. In vitro, the LRRK2-G2019S mutant boosted the phosphorylation of s15. However, the LRRK2 protein appeared to phosphorylate itself far more readily than s15, which led some researchers to wonder if the ribosomal subunit represents a bona fide substrate. Dawson said they did not quantitate the number of phosphorylation events on LRRK2 versus s15. However, autophosphorylation is part of the normal biology of LRRK2, she said.
To determine whether s15 mediated the toxicity of LRRK2-GS, the researchers expressed both proteins in rat cortical neurons and in human cortical and dopaminergic neurons derived from a human embryonic stem cell line. When expressed together, LRRK2-GS and wild-type s15 drove up markers of cell death as well as the shortening of neurites. However, the rise in these toxic symptoms subsided when the researchers swapped s15 threonine-136 with an alanine residue (s15-TA), blocking phosphorylation. The s15-TA variant also prevented the loss of dopaminergic neurons and rescued deficits in climbing seen in fruit flies expressing LRRK2-GS. These results suggested that LRRK2 could mediate its neurodegenerative effects through phosphorylation of s15.
The cell and fly experiments relied on overexpression of either the substrate or the kinase or both. This drew caution from some researchers, including Bingwei Lu of Stanford University in Palo Alto, California. “The reliance on overexpression in cells throughout this study could be a cause of concern,” Lu said. He added that it would be interesting to test whether altered s15 phosphorylation is responsible for the disease-relevant phenotypes observed in neurons derived from patients carrying the LRRK2 G2019S mutation. Dawson said such experiments are underway in her lab, but that the researchers had initially wanted to establish the LRRK2-s15 link in cells with a common genetic background.
To further examine the effects of the phosphorylation of s15 by LRRK2 in vivo, the researchers measured protein translation rates in flies by feeding them 35S-methionine and measuring incorporation of the labeled amino acid into proteins in the brain. Flies expressing LRRK2-GS made slightly more protein than wild-type flies. Interestingly, expression of s15-TA reversed this boost. Anisomycin, a translation inhibitor, also reversed neuronal loss and restored climbing ability in flies expressing LRRK2-GS.
The fly results hinted that the toxic effects mediated by LRRK2-GS played out in the form of elevated protein translation, triggered by phosphorylated s15. Dawson’s lab is now in the process of determining which proteins downstream of s15 could be causing pathogenic effects.
“The hypothesis that G2019S LRRK2 toxicity leads to bulk increase in protein synthesis is interesting, as it adds to the growing body of evidence linking LRRK2 and other PD-related proteins to changes in protein homeostasis in the cell,” Gaia Skibinski of the University of California, San Francisco, wrote to Alzforum. “More specifically it adds to the increasing lines of evidence linking neurodegeneration to translation misregulation.”
Many of the biochemical and neuronal effects observed in the paper were subtle, making it difficult to draw solid conclusions from the data, Alessi said. However, he noted that LRRK2 mutations do exert subtle biological effects that take decades to cause disease. “It’s possible that the effects you would observe would be incredibly small, and therefore hard to observe,” Alessi said. “This makes a study very difficult in this field.”—Jessica Shugart
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