Our data indicate that deficient GCase leads to accumulation of GlcCer in neurons that in turn promotes formation of toxic α-syn oligomers. GCase depletion causes a decline in lysosomal proteolysis that preferentially affects α-syn (). It is likely that the unique inherent property of α-syn to form amyloid fibrils plays a critical role in the neurotoxicity that occurs with GCase KD, since expression of α-syn mutant lacking the 71–82 region did not effect neuronal viability in our culture model (). Interestingly, another aggregation-prone protein involved in neurodegenerative disorders, tau, did not accumulate ( and ) indicating that GCase function is preferentially related to α-syn. Importantly, similar results were observed in human iPS neurons in the presence of endogenous mutations in GCase, suggesting that GCase protein depletion or expression of loss-of-function GCase mutants exhibit comparable phenotypes.
Biochemical analysis indicated that GCase KD caused a dramatic increase in the levels of soluble oligomers (). This effect was distinct from that observed by leupeptin, which primarily resulted in elevated levels of T-insoluble α-syn without altering soluble forms. This suggests that in addition to general lysosomal inhibition, GlcCer accumulation specifically affects the conformation and solubility of α-syn, by stabilizing the levels of soluble intermediates. In vitro
studies using purified recombinant α-syn demonstrated that GlcCer has the ability to prolong the lag phase of fibril growth and stabilize oligomeric intermediates only at acidic pH ( and S4
). This pH-dependent effect is consistent with accumulation of α-syn within LAMP1-positive vesicles and subcellular fractions upon GCase KD (Figure S3h,i
). After the lag phase, GlcCer accelerated amyloid formation, and formed fibrils that appeared to extend from GlcCer lipid tubules (). It is possible that GlcCer tubules provide a scaffold or platform for oligomeric intermediates to form which, once saturated, proceed to rapid polymerization of fibrils. This ability may be a crucial step in pathogenesis, since the documentation of α-syn oligomers appears to be correlated with neurodegeneration in neuronal cultures, mouse models, and human neuronopathic GD brain.
SEC analysis of postmortem GD and PD brain demonstrated elevated levels of a previously undocumented 36–45 Å-sized soluble oligomeric α-syn species that correlated with a neurological phenotype. The oligomers prominently reacted with the mAb syn303, an antibody generated against oxidized/nitrated α-syn that preferentially detects pathological conformations of the protein that exhibit toxic properties (Tsika et al., 2010
). The pathological α-syn oligomers were also detected in infantile neuronopathic GD cases, and in a child with type III GD (), strongly suggesting that GBA1
mutations and specific alterations in the GlcCer metabolism pathway influence α-syn oligomerization that is not necessarily age dependent.
The absence of oligomeric α-syn in samples from type I GD without parkinsonism () indicates that other factors, in addition to deficiency of GCase, likely contribute to oligomerization of α-syn in neuronopathic GD. For example, oxidation and nitration of α-syn has been shown to impede clearance and stabilize α-syn oligomers in vitro
(Hodara et al., 2004
), and chaperones have also been shown to abrogate α-syn toxicity and aggregation (Auluck et al., 2002
). While our analysis of GD brain indicated increased levels of oxidized α-syn in neuronopathic forms (), further studies are required to examine how oxidation and other age-dependent processes interact with deficiency of GCase in promoting α-syn oligomerization.
Our data also demonstrate that elevated α-syn inhibits intracellular trafficking and lysosomal function of normal GCase in neurons ( and S7
). This indicates that decreased GCase activity not only contributes to toxicity in patients with GBA1
mutations, but may also affect the development of more common sporadic forms of PD and synucleinopathies that do not have mutations in the GBA1
gene. Interestingly, we show that variation of α-syn levels in healthy control subjects can also alter ER-Golgi flux of GCase, a property that may be potentiated by α-syn oligomerization. This is further suggested by normal GC activity in neurons expressing aggregation-incompetent Δ71-82-α-syn () as well as the increased immunoreactivity to syn303 in controls that contain higher levels of ER GCase (Figure S7g
). However, further studies are required to delineate the precise mechanism of α-syn mediated inhibition of GCase maturation.
Taken together, our results suggest that elevated levels of toxic α-syn species lead to depletion of lysosomal GCase and further stabilization of α-syn oligomers by GlcCer accumulation. This self-propagating positive feedback process proceeds until a pathogenic threshold is surpassed, resulting in neurodegeneration (). Therefore, specific treatments that promote targeting of GCase to lysosomes are expected to diminish the formation of toxic α-syn oligomers and break the pathogenic cycle of α-syn aggregation and toxicity in PD and other synucleinopathies.