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

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  1. This paper by the Krainc group confirms previous work by our group and others, and provides a nice insight into the possible mechanisms by which dysregulation or mutation in the glucocerebrosidase (GBA) protein can lead to synucleinopathies.

    The cell work in this paper focused mainly on knockdown of GBA, showing that loss of the protein and its enzyme activity caused a decrease in lysosomal degradation of long-lived proteins and therefore an increase in synuclein. We also tried to inhibit the enzymatic function (rather than protein levels) with conduritol B epoxide (CBE) in cells, and did not observe an increase in synuclein, but our CBE treatment was shorter than their knockdown, which probably explains that result. One other group has previously shown that CBE can cause α-synuclein accumulation.

    Our Annals of Neurology paper (Cullen et al., 2011) instead focused on cell models of expression/overexpression of mutant GBA. We showed that expression of mutant but not wild-type GBA caused an increase in synuclein levels as measured by sensitive ELISA. This occurred without any dominant-negative effect of the mutants on endogenous enzyme function. What was also very interesting was that in one cell line, the wild-type GBA had the opposite effect; i.e., it could lower synuclein levels.

    Obviously, a therapeutic implication of the above finding would be that overexpression of GBA by viral vector or otherwise might be beneficial in synucleinopathies.

    Krainc et al. in their discussion implied that another therapeutic strategy would be to more efficiently chaperone GBA to the lysosome. We found in our study that the GBA chaperone, isofagomine, did have a trend towards reducing synuclein. Perhaps a different dose or treatment time would have been even more effective. This strategy is definitely worth pursuing in further studies.

    Krainc also nicely showed that knockdown of GBA caused an inhibition of lysosomal degradation of long-lived proteins. Although they did not mention it much, this degradation typically occurs by macroautophagy. We found that the autophagy inducer rapamycin ameliorated the increase in synuclein noted with GBA overexpression, indicating that this may be a nice therapeutic target to pursue also.

    Both papers examined synuclein accumulation in animal models of GD with mutations in GBA. The findings converged nicely and showed that dysregulation of GBA is associated with an accumulation of synuclein in various brain regions. Our group also showed that the GBA mutant mice had a similar immunohistochemical pathology as another mouse model, that of the Cathepsin D knockout, which imitates a rare but informative lysosomal storage disorder, CLN10 (see also Cullen et al., 2009). Krainc’s examination of human brain specimens is a nice extension of the mouse work.

    All in all, both papers are complementary and point to a very important interplay between GBA and synuclein metabolism. Further work on this biochemical interplay will shed new light on the genetic intersection of GBA with synucleinopathy disorders, and hopefully on new and fruitful therapeutic approaches.

    References:

    . Cathepsin D expression level affects alpha-synuclein processing, aggregation, and toxicity in vivo. Mol Brain. 2009;2:5. PubMed.

  2. Nice study by Mazzuli et al. linking glucocerebrosides to α-synuclein.

    View all comments by Subhojit Roy
  3. This certainly is an impressive amount of work focusing thoroughly on the mechanistic link between glucocerebrosidase (GCase), glucocerebroside (GlcCer), and α-synuclein by robustly combining, in vitro, Gaucher's disease (GD), animal model, and human postmortem data. Mazzulli et al. have carried out a number of elegant in vitro assays to show that GlcCer selectively stabilizes the formation of soluble α-synuclein oligomeric intermediates on the pathway toward amyloid fibrils. These findings are corroborated by the GD mouse model, showing increased levels of putative oligomers by size exclusion chromatography (i.e., 120-70 Å- and 51-44 Å-sized species), whereas only monomers were found in control mice. Importantly, when analyzing human postmortem cortical samples from GD patients with atypical Parkinson’s disease or dementia with Lewy bodies (DLB), they observed increased oligomeric α-synuclein eluting above 36 Å and migrating at 22, 44, and 75 kDa by SDS-PAGE.

    An interesting finding is that GCase function is preferentially related to α-synuclein (does not affect tau). This is also supported by pathological findings from those carrying GCase gene (GBA) mutations, who tend to show “purer “LB pathology without concomitant Alzheimer’s disease pathology (Clark et al., 2009). Additionally, in contrast to some preliminary data suggesting that GBA mutation carriers may have a more augmented LB pathology (Neumann et al., 2009), we recently showed that these carriers do not have higher age- and dementia-adjusted LB densities compared to sporadic PD cases (Parkkinen et al., 2011). This is in line with the findings by Mazzulli et al. suggesting that the GBA mutations may enhance the formation of α-synuclein oligomers that could kill the cell prior to any inclusion (i.e., LB) formation.

    In regard to the authors’ in vitro findings, the assays are quite strongly focusing on GCase knockdown models, which I’m not sure are the correct models, as Cullen and colleagues (Cullen et al., 2011) have shown that overexpression of mutant GBA proteins in vitro does indeed promote the accumulation of α-synuclein, but this appears to be independent of GCase activity (not compromised). In addition, chemical inhibition of GCase in their hands did not elevate the concentration of α-synuclein, in contrast to earlier work (Manning-Bog et al., 2009).

    In addition, using the same 4L/PS-NA mouse model (in an identical time point of 12 weeks), Cullen et al. did not note any significant α-synuclein (either soluble or insoluble) accumulation at the biochemical level. Their neuropathological analysis did, however, reveal neuronal spheroids and some enhancement of cytoplasmic α-synuclein immunoreactivity in the frontal cortex, corpus callosum, and cerebellum. However, the punctate pattern Mazzulli et al. describe with immunofluorescence is novel.

    Although GBA mutations are the most common genetic risk factor for PD, most PD patients do not harbor these mutations, and Mazzulli et al. suggest a very interesting theory for how GCase links to α-synuclein levels in these patients: The accumulation of oligomeric α-synuclein could block endoplasmic reticulum-Golgi trafficking of GCase, preventing its normal maturation, then a decrease in lysosomal GCase leads to further accumulation of GlcCer and the stabilization of oligomeric α-synuclein. This positive feedback, self-propagating loop provides a potential therapeutic target for sporadic PD. The only problem with this “loss-of-function” theory is that it does not explain why the majority of homozygous Gaucher’s patients, and even heterozygotes (all showing lower levels of GCase), do not develop synucleinopathy or parkinsonism.

    References:

    . Association of glucocerebrosidase mutations with dementia with lewy bodies. Arch Neurol. 2009 May;66(5):578-83. PubMed.

    . Glucocerebrosidase mutations in clinical and pathologically proven Parkinson's disease. Brain. 2009 Jul;132(Pt 7):1783-94. PubMed.

    . Glucocerebrosidase mutations do not cause increased Lewy body pathology in Parkinson's disease. Mol Genet Metab. 2011 Aug;103(4):410-2. PubMed.

    . Acid β-glucosidase mutants linked to Gaucher disease, Parkinson disease, and Lewy body dementia alter α-synuclein processing. Ann Neurol. 2011 Jun;69(6):940-53. PubMed.

    . Alpha-synuclein-glucocerebrosidase interactions in pharmacological Gaucher models: a biological link between Gaucher disease and parkinsonism. Neurotoxicology. 2009 Nov;30(6):1127-32. PubMed.

  4. Mazzulli et al. describe an incredibly important study linking the activity of the lysosomal enzyme, glucocerebrosidase, with the accumulation and fibrilization of α-synuclein. Their study extends previous research into the correlation of glucocerebrosidase mutations observed in Gaucher’s disease patients with an increased incidence of Parkinson’s disease in these patients. Decreased localization of glucocerebrosidase to the lysosome leads to an accumulation of glucocerebroside (the substrate of glucocerebrosidase), and thus leads to an accumulation of α-synuclein. The authors showed that the accumulating glucocerebroside polymerizes into tubules that may act as a scaffold for the polymerization of α-synuclein, leading to increased fibrillization of the latter. The authors further show that expression and accumulation of α-synuclein directly affects the endogenous localization of glucocerebrosidase, thus providing a positive feedback loop for the further accumulation of α-synuclein, and finally fibrillization.

    Examination of patients with type 2 or 3 neuronopathic Gaucher’s disease showed an increased accumulation of α-synuclein in the CNS even at very young ages (The authors’ mouse model of Gaucher’s disease may be a good model of idiopathic Parkinson’s disease. Glucocerebrosidase knockout mice die shortly after birth due to defects in the epithelium. This newer glucocerebrosidase mutant developed by Grabowski’s lab (Xu et al., 2011) shows significantly reduced levels of glucocerebrosidase activity and thus delayed onset of pathology.

    The authors suggest that a method of providing more glucocerebrosidase to the lysosomes of neurons may be an effective therapeutic avenue for Parkinson’s disease, as this would theoretically reduce the accumulation of α-synuclein as well as reduce the ability of α-synuclein to polymerize. Intravenous infusion of in vitro glycosylated glucocerebrosidase is an effective therapy for Gaucher’s disease patients (e.g., Cerezyme, Genzyme Corporation); however, the enzyme is not trafficked from the blood to the CNS, due to the presence of the blood-brain barrier. We showed a novel method for the delivery of glucocerebrosidase from the periphery to the CNS and to neuronal lysosomes (Spencer and Verma, 2007). This, too, may be an attractive therapeutic approach. Additionally, some researchers have investigated the use of chemical or protein chaperones that enhance the transport of glucocerebrosidase from the ER to the lysosomes. These would appear to be attractive for PD, too.

    References:

    . Accumulation and distribution of α-synuclein and ubiquitin in the CNS of Gaucher disease mouse models. Mol Genet Metab. 2011 Apr;102(4):436-47. PubMed.

    . Targeted delivery of proteins across the blood-brain barrier. Proc Natl Acad Sci U S A. 2007 May 1;104(18):7594-9. PubMed.

  5. Unique clinical observations and elegant genetic research have established that approximately 10-12 percent of people with Parkinson’s have a mutation in one copy of a gene encoding the lysosomal enzyme glucocerebrosidase, or GBA1 (Sidransky et al., 2009). Today, mutations in GBA1 are considered the most common known genetic risk factor for the synucleinopathies, PD and DLB.

    Despite the wealth of clinical and genetic evidence supporting the association between GBA1 and α-synuclein accumulation, the underlying mechanisms by which GBA1 mutations can lead to α-synuclein misprocessing are not understood.

    A GBA1 loss-of-function hypothesis and a mutant, toxic gain-of-function hypothesis have emerged to explain the effects of GBA1 on α-synuclein processing. Importantly, these hypotheses are not mutually exclusive, and are supported by clinical and genetic evidence. (For more details see, Velayati et al., 2010).

    Recent Data: Gain of Function Versus Loss of Function?

    In recent months, several articles have shed more light into this complex interaction. In collaboration with Michael Schlossmacher at the University of Ottawa, we demonstrated that overexpression of mutant forms of GBA1 can increase α-synuclein levels in a dose- and time-dependent manner (Cullen et al., 2011). Furthermore, we showed that increasing GBA1 activity reduces the levels of α-synuclein in a cell-specific manner, suggesting the therapeutic potential of modulating this pathway in synucleinopathies.

    Now, the group led by Dimitri Krainc at Massachusetts General Hospital shows that reduction in GBA1 activity can increase α-synuclein toxicity by stabilizing oligomeric intermediates, possibly through the accumulation of a GBA1 substrate (GlcCer). Interestingly, these authors also show that elevated α-synuclein levels impair GBA1 function in the lysosome, suggesting a vicious cycle whereby α-synuclein accumulation leads to decreased GBA1 activity, which in turn incites more α-synuclein accumulation.

    The current evidence suggests that both GBA1 gain- and loss-of-function mechanisms conspire to promote aberrant α-synuclein processing. Mutations in GBA1 appear to be sufficient to initiate α-synuclein misfolding, while a decrease in GBA1 activity (regardless of mutations) seems to accelerate its misprocessing. Importantly, elevation of α-synuclein seems to prevent normal trafficking of GBA1 to the lysosome, thereby promoting more α-synuclein accumulation. Together, these mechanisms would support increasing GBA1 activity in the lysosomes as a therapeutic approach.

    In summary, GBA1-mediated pathways have emerged as attractive targets for Gaucher's-related and sporadic synucleinopathies and, possibly, for other neurodegenerative diseases. These recent reports by Cullen et al., and this paper by Mazzulli et al., are important stepping stones in unveiling the complex complot between GBA1 and α-synuclein. As the worldwide pioneers in treating Gaucher's disease, we (at Genzyme) have been actively pursuing this line of research, and we hope to provide more answers in the near future.

    References:

    . Multicenter analysis of glucocerebrosidase mutations in Parkinson's disease. N Engl J Med. 2009 Oct 22;361(17):1651-61. PubMed.

    . The role of glucocerebrosidase mutations in Parkinson disease and Lewy body disorders. Curr Neurol Neurosci Rep. 2010 May;10(3):190-8. PubMed.

    . Acid β-glucosidase mutants linked to Gaucher disease, Parkinson disease, and Lewy body dementia alter α-synuclein processing. Ann Neurol. 2011 Jun;69(6):940-53. PubMed.

References

News Citations

  1. More Than Gaucher’s—GBA Throws Its Weight Around Lewy Body Disease

Paper Citations

  1. . Multicenter analysis of glucocerebrosidase mutations in Parkinson's disease. N Engl J Med. 2009 Oct 22;361(17):1651-61. PubMed.
  2. . Glucocerebrosidase mutations in clinical and pathologically proven Parkinson's disease. Brain. 2009 Jul;132(Pt 7):1783-94. PubMed.
  3. . Complete screening for glucocerebrosidase mutations in Parkinson disease patients from Greece. Neurosci Lett. 2009 Mar 13;452(2):87-9. PubMed.
  4. . Mutations for Gaucher disease confer high susceptibility to Parkinson disease. Arch Neurol. 2009 May;66(5):571-6. PubMed.
  5. . Gaucher disease mouse models: point mutations at the acid beta-glucosidase locus combined with low-level prosaposin expression lead to disease variants. J Lipid Res. 2005 Oct;46(10):2102-13. PubMed.
  6. . Viable mouse models of acid beta-glucosidase deficiency: the defect in Gaucher disease. Am J Pathol. 2003 Nov;163(5):2093-101. PubMed.
  7. . A novel N18TG2 x mesencephalon cell hybrid expresses properties that suggest a dopaminergic cell line of substantia nigra origin. J Neurosci. 1992 Sep;12(9):3392-8. PubMed.

External Citations

  1. GBA1
  2. company press release
  3. enzyme replacement therapy

Further Reading

Papers

  1. . Dynamics of plaque formation in Alzheimer's disease. Biophys J. 1999 Mar;76(3):1330-4. PubMed.
  2. . APPSw transgenic mice develop age-related A beta deposits and neuropil abnormalities, but no neuronal loss in CA1. J Neuropathol Exp Neurol. 1997 Sep;56(9):965-73. PubMed.
  3. . Rapid appearance and local toxicity of amyloid-beta plaques in a mouse model of Alzheimer's disease. Nature. 2008 Feb 7;451(7179):720-4. PubMed.
  4. . Characterizing the appearance and growth of amyloid plaques in APP/PS1 mice. J Neurosci. 2009 Aug 26;29(34):10706-14. PubMed.
  5. . Multicenter analysis of glucocerebrosidase mutations in Parkinson's disease. N Engl J Med. 2009 Oct 22;361(17):1651-61. PubMed.

Primary Papers

  1. . Acid β-glucosidase mutants linked to Gaucher disease, Parkinson disease, and Lewy body dementia alter α-synuclein processing. Ann Neurol. 2011 Jun;69(6):940-53. PubMed.
  2. . Gaucher disease glucocerebrosidase and α-synuclein form a bidirectional pathogenic loop in synucleinopathies. Cell. 2011 Jul 8;146(1):37-52. PubMed.