27 June 2011. Clinical and genetic data have linked Parkinson’s and other synucleinopathies to Gaucher’s disease, a rare lysosomal storage disorder. Now, two papers offer mechanistic support for the connection. In a study published yesterday in Cell online, researchers report that depletion of glucocerebrosidase, a lysosomal enzyme defective in Gaucher’s, leads to buildup of α-synuclein, which forms the hallmark pathology of Lewy body diseases such as PD. Moreover, α-synuclein can go back and inhibit glucocerebrosidase activity in lysosomes, thus perpetuating a vicious cycle. In another study—posted online April 3 and appearing in print in this month’s Annals of Neurology—overexpression of mutant glucocerebrosidase led to increased α-synuclein levels. The reports suggest that glucocerebrosidase can influence α-synuclein processing through both gain- and loss-of-function mechanisms, and that increasing lysosomal glucocerebrosidase activity may hold promise as a therapeutic approach for synucleinopathies.
Associations between Gaucher’s disease (GD) and PD run in both directions. GD patients and their relatives have increased risk for PD, and people with PD or idiopathic parkinsonism are more likely to carry glucocerebrosidase gene (GBA) mutations that cause Gaucher’s. A recent spate of large genetic analyses in worldwide populations has established GBA1 as the leading genetic risk factor for PD (Sidransky et al., 2009; Neumann et al., 2009; Kalinderi et al., 2009; Mitsui et al., 2009; see also ARF related news story). The Cell paper tests the loss-of-function hypothesis, whereas the Annals of Neurology study addresses the gain-of-function theory behind the glucocerebrosidase and α-synuclein connection.
Senior investigator Dimitri Krainc and first author Joseph Mazzulli of Massachusetts General Hospital, Charlestown, led the research appearing in Cell. When the researchers used short-hairpin RNA (shRNA)-carrying lentiviruses to halve endogenous GBA levels in mouse cortical neurons and human neuroglioma cells, these cells accumulated the GBA substrate glucosylceramide, and had nearly twice as much α-synuclein as control cells. The synuclein piled up due to a clearance problem, as determined by proteolysis rates in the neuroglioma cells, which express inducible synuclein. The protein degradation defect was also confirmed in pulse-chase experiments in dopaminergic neurons made from induced pluripotent stem cells derived from skin fibroblasts of a GD patient.
Further in-vitro assays showed that glucosylceramide stabilizes oligomeric α-synuclein intermediates that go on to form amyloid fibrils. Excess α-synuclein also accumulated in a GD worm model, GD mouse models with loss-of-function GBA mutations (Sun et al., 2005; Xu et al., 2003), and human postmortem brain samples from GD patients.
In addition, the scientists discovered that the surplus of synuclein resulting from GBA knockdown compromises lysosomal GBA activity, in essence forming a pathogenic loop. “α-synuclein overexpression, which is something we invariably see in synucleinopathies, can feed back onto ER-Golgi trafficking and inhibit movement of GBA into lysosomes,” Mazzulli told ARF. The drop in lysosomal GBA causes a buildup of glucosylceramide, which stabilizes synuclein oligomers and further inhibits GBA ER-Golgi trafficking, getting stronger with each round.
Brian Spencer of the University of California, San Diego, called the research “an incredibly important study linking the activity of GBA with the accumulation and fibrillization of α-synuclein.” (See full comment below.) The specificity of this link was a strength of the paper, commented James Leverenz of the University of Washington, Seattle. “When they looked at other proteins like tau and huntingtin, they didn’t see a similar affect, which makes us feel more comfortable that there’s something to this, and that this is an important link in terms of pathophysiology,” he told ARF.
Laura Parkkinen, University of Oxford, U.K., praised the “impressive amount of work,” but pointed out a shortcoming of the authors’ loss-of-function model. “It does not explain why the the majority of homozygous Gaucher’s patients, and even heterozygotes, do not develop synucleinopathy or parkinsonism,” she wrote in an e-mail to ARF (see full comment below).
In the Annals of Neurology study, a team led by Michael Schlossmacher of the University of Ottawa, Canada, tested the “gain of toxic function” hypothesis for the GBA/α-synuclein connection. In their studies with the MES23.5 PD cell culture model (Crawford et al., 1992), first author Valerie Cullen of Link Medicine, Cambridge, Massachusetts, and colleagues found that overexpression of GBA mutants did not seem to affect the activity of endogenous GBA, but caused the cells to rack up α-synuclein. However, when they overexpressed wild-type GBA, enzymatic activity went up and synuclein levels dropped. Here, α-synuclein levels were sensitive to GBA activity, similar to what Krainc and colleagues showed in their Cell paper.
Instead of a knockdown approach, the researchers used conduritol B epoxide (CBE) to chemically inhibit GBA in PC12 neuronal cells expressing wild-type α-synuclein. Unlike the MGH researchers, Cullen and colleagues did not see changes in synuclein when they lowered GBA activity. However, the CBE treatment only went for a day or so, whereas the knockdown strategy inhibited endogenous GBA for a week. “Even though we got a different result, the methodology used probably accounts for that, and I believe their result with the knockdown is probably the truer result,” Cullen told ARF.
Consistent with the Cell report, Cullen and colleagues found increased α-synuclein levels in the brains of Gaucher’s model mice with a loss-of-function GBA mutation (D409V). The researchers also showed they could reduce synuclein accumulation in their cell culture models by treating with the autophagy inducer rapamycin, or with isofagomine, a compound that promotes GBA movement into lysosomes. Amicus Therapeutics, Inc. of Cranbury, New Jersey, has tested isofagomine in a six-month Phase 2 trial of adults with type 1 Gaucher’s disease. The compound increased GBA activity but failed to improve clinical measures in 18 of 19 trial participants (see company press release). The MGH team is in discussion with several companies to develop new PD compounds that target GBA to lysosomes, Krainc told ARF.
Taken together, the papers “suggest that both GBA1 gain- and loss-of-function mechanisms conspire to promote aberrant α-synuclein processing,” wrote Pablo Sardi of Genzyme Corporation, Framingham, Massachusetts in an e-mail to ARF. Both studies suggest “that increasing GBA1 activity in lysosomes would be a putative therapeutic approach for synucleinopathies,” noted Sardi, a coauthor on the Annals of Neurology study. Sardi has an upcoming PNAS paper describing a therapeutic intervention in a Gaucher’s-related synucleinopathy animal model. Genzyme has an intravenous GBA enzyme replacement therapy for Gaucher’s patients.—Esther Landhuis.
Mazzulli JR, Xu YH, Sun Y, Knight AL, McLean PJ, Caldwell GA, Sidransky E, Grabowski GA, Krainc D. Gaucher Disease glucocerebrosidase and alpha-synuclein form a bidirectional pathogenic loop in synucleinopathies. Cell. 23 June 2011. Abstract
Cullen V, Sardi SP, Ng J, Xu YH, Sun Y, Tomlinson JJ, Kolodziej P, Kahn I, Saftig P, Woulfe J, Rochet JC, Glicksman MA, Cheng SH, Grabowski GA, Shihabuddin LS, Schlossmacher MG. Acid β-glucosidase mutants linked to gaucher disease, parkinson disease, and Lewy body dementia alter a-synuclein processing. Ann Neurol. 2011 Jun;69(6):940-53. Abstract