title: Acid β-glucosidase mutants linked to Gaucher disease, Parkinson disease, and Lewy body dementia alter α-synuclein processing.
abstract: OBJECTIVE
  Heterozygous mutations in the GBA1 gene elevate the risk of Parkinson disease and dementia with Lewy bodies; both disorders are characterized by misprocessing of α-synuclein (SNCA). A loss in lysosomal acid-β-glucosidase enzyme (GCase) activity due to biallelic GBA1 mutations underlies Gaucher disease. We explored mechanisms for the gene's association with increased synucleinopathy risk.
  
  
  METHODS
  We analyzed the effects of wild-type (WT) and several GBA mutants on SNCA in cellular and in vivo models using biochemical and immunohistochemical protocols.
  
  
  RESULTS
  We observed that overexpression of all GBA mutants examined (N370S, L444P, D409H, D409V, E235A, and E340A) significantly raised human SNCA levels to 121 to 248% of vector control (p < 0.029) in neural MES23.5 and PC12 cells, but without altering GCase activity. Overexpression of WT GBA in neural and HEK293-SNCA cells increased GCase activity, as expected (ie, to 167% in MES-SNCA, 128% in PC12-SNCA, and 233% in HEK293-SNCA; p < 0.002), but had mixed effects on SNCA. Nevertheless, in HEK293-SNCA cells high GCase activity was associated with SNCA reduction by ≤32% (p = 0.009). Inhibition of cellular GCase activity (to 8-20% of WT; p < 0.0017) did not detectably alter SNCA levels. Mutant GBA-induced SNCA accumulation could be pharmacologically reversed in D409V-expressing PC12-SNCA cells by rapamycin, an autophagy-inducer (≤40%; 10μM; p < 0.02). Isofagomine, a GBA chaperone, showed a related trend. In mice expressing two D409Vgba knockin alleles without signs of Gaucher disease (residual GCase activity, ≥20%), we recorded an age-dependent rise of endogenous Snca in hippocampal membranes (125% vs WT at 52 weeks; p = 0.019). In young Gaucher disease mice (V394Lgba+/+//prosaposin[ps]-null//ps-transgene), which demonstrate neurological dysfunction after age 10 weeks (GCase activity, ≤10%), we recorded no significant change in endogenous Snca levels at 12 weeks of age. However, enhanced neuronal ubiquitin signals and axonal spheroid formation were already present. The latter changes were similar to those seen in three week-old cathepsin D-deficient mice.
  
  
  CONCLUSIONS
  Our results demonstrate that GBA mutants promote SNCA accumulation in a dose- and time-dependent manner, thereby identifying a biochemical link between GBA1 mutation carrier status and increased synucleinopathy risk. In cell culture models, this gain of toxic function effect can be mitigated by rapamycin. Loss in GCase activity did not immediately raise SNCA concentrations, but first led to neuronal ubiquitinopathy and axonal spheroids, a phenotype shared with other lysosomal storage disorders.
authors.affiliation: Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
authors.forename: Valerie
authors.initials: V
authors.lastname: Cullen
authors.forename: S Pablo
authors.initials: SP
authors.lastname: Sardi
authors.forename: Juliana
authors.initials: J
authors.lastname: Ng
authors.forename: You-Hai
authors.initials: YH
authors.lastname: Xu
authors.forename: Ying
authors.initials: Y
authors.lastname: Sun
authors.forename: Julianna J
authors.initials: JJ
authors.lastname: Tomlinson
authors.forename: Piotr
authors.initials: P
authors.lastname: Kolodziej
authors.forename: Ilana
authors.initials: I
authors.lastname: Kahn
authors.forename: Paul
authors.initials: P
authors.lastname: Saftig
authors.forename: John
authors.initials: J
authors.lastname: Woulfe
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Rochet
authors.forename: Marcie A
authors.initials: MA
authors.lastname: Glicksman
authors.forename: Seng H
authors.initials: SH
authors.lastname: Cheng
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Grabowski
authors.forename: Lamya S
authors.initials: LS
authors.lastname: Shihabuddin
authors.forename: Michael G
authors.initials: MG
authors.lastname: Schlossmacher
mesh_terms: D000818:Animals; D002402:Cathepsin D / Q000172:deficiency / Q000235:genetics; D002460:Cell Line; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005776:Gaucher Disease / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D005962:Glucosylceramidase / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D020961:Lewy Body Disease / Q000235:genetics*; D051379:Mice; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation / Q000235:genetics*; D010300:Parkinson Disease / Q000235:genetics*; D051381:Rats; D020123:Sirolimus / Q000494:pharmacology; D014162:Transfection; D051844:alpha-Synuclein / Q000235:genetics / Q000378:metabolism*
chemical_list: D007166:Immunosuppressive Agents; D051844:alpha-Synuclein; D049452:Green Fluorescent Proteins; D005962:Glucosylceramidase; D002402:Cathepsin D; D020123:Sirolimus