The lysosomal storage disorder Gaucher disease (GD) is caused by autosomal recessive mutations in the glucocerebrosidase (GBA) gene. GBA encodes a lysosomal enzyme (GCase) that catalyses the metabolism of the sphingolipid glucosylceramide to ceramide and glucose. Deficiency of GCase activity results in accumulation of substrate in the lysosomes of several tissues, including brain. Mutations in GBA result in 3 clinical manifestations. Type 1 GD occurs in both children and adults and predominantly impacts on the non-neuronal organs, whereas types 2 and 3 have an onset in childhood and adolescence, respectively, and exhibit neurological deficits.1
Parkinson disease (PD) is primarily characterized by the motor symptoms of resting tremor, bradykinesia, rigidity, and postural instability. Pathological hallmarks include loss of dopaminergic neurons from the substantia nigra (SN) and the presence of cytoplasmic inclusions known as Lewy bodies in the surviving cells of affected brain regions.2
Typical parkinsonism is among the neurological complications of GD (including type 1).3, 4 The neuropathology of GD brains includes the typical hallmarks of PD, such as cortical and brainstem Lewy bodies.5 Heterozygote carriers of GBA mutations also have an increased frequency of PD, and these mutations are the most common genetic risk factor for developing the disease.6–8
Although the pathogenesis of PD is still unknown, the accumulation of α-synuclein and other ubiquitinated proteins in Lewy bodies has implicated protein mishandling as a putative cause. The proteasome and lysosomes are the 2 principal mechanisms for degrading cellular constituents. Autophagy utilizes lysosomes to degrade long-lived proteins, misfolded/aggregated proteins, and organelles such as mitochondria.9 Defective autophagy and/or lysosomal depletion have been implicated in PD.10–13 Cellular or animal models of GCase deficiency have caused α-synuclein accumulation.14–18 GCase has also been suggested to bind directly with α-synuclein in lysosomes19 and the GCase substrate glucosylceramide stabilizes soluble oligomeric α-synuclein species.18 These observations have led to the notion that GCase deficiency might contribute to the α-synuclein aggregation characteristic of PD pathology.
Despite the recognized association between GBA mutations and PD, it is unknown how heterozygous GBA mutations affect GCase activity in PD brains. In this paper, we provide the first report of the activity of GCase in several regions of PD brains from GBA mutation carriers and sporadic PD brains. GCase deficiency was greatest in the SN of PD brains with GBA mutations. This loss of activity was in part mediated by a decrease in GCase protein levels. GCase activity was also significantly decreased in the SN of sporadic PD brains.
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We report the first comprehensive biochemical analysis of the effects of GBA mutations in PD brains. There is widespread deficiency of GCase activity and protein levels in PD+GBA brains, with the most severe defect located in the SN (58%) and putamen (48%). This loss of GCase is unlikely to simply represent neurodegeneration, as there was a 47% defect in cerebellum, an area not involved in the degenerative process of PD. GCase activity was also unaffected in the amygdala of AD brains, a region associated with marked neurodegeneration. There was no decrease in GCase mRNA levels in the putamen, indicating that the reduction in protein levels, in this region at least, was not a result of downregulation of gene expression, but rather a post-translational effect on protein levels. Three markers of UPR/ERAD were increased and might play a role in reduced protein levels, although further studies are required to confirm this. Given the normal cathepsin D levels and hexosaminidase activity, the loss of GCase protein was not due to a general reduction in lysosomal content or activity. There was a trend for LC3-II levels to be increased in PD+GBA putamen, suggesting an increase in autophagosome number, a feature previously reported in PD SN and amygdala.10, 11 As autophagy flux cannot be measured in postmortem tissue, it is unclear whether this is due to an increase in macroautophagy or an inhibition of lysosomal degradation of autophagosomes.
We also report for the first time a significant deficiency of GCase activity in PD SN (33%) and cerebellum (24%), with reduced protein levels, in sporadic PD brains. Enzyme activities were comparable to control in other PD brain regions. The patterns of lysosomal proteins and UPR/ERAD markers were similar in PD and GBA+PD brains.
The SN is the site of greatest pathology in PD brain and biochemical abnormalities thought relevant to PD pathogenesis, including α-synuclein deposition, mitochondrial dysfunction, and oxidative stress. Although the loss of GCase activity in PD+GBA brains is in part related to GBA mutations, this cannot be the explanation in PD brains. Thus, we hypothesized that mitochondrial dysfunction and/or oxidative stress, or increased α-synuclein levels might cause this defect in PD and an exacerbation of the GCase deficiency in PD+GBA. Mitochondrial dysfunction/oxidative stress as a result of PINK1 KD28, 29 caused a loss of GCase activity and protein in cells. Increased α-synuclein expression also significantly decreased GCase activity and protein levels.
Recent studies have highlighted the reciprocal relation between GCase activity and α-synuclein. Decreased GCase activity caused increased α-synuclein levels in toxin and transgenic GBA mouse models, and in neuronal cultures.14–18 Accumulation of the GCase substrate glucosylceramide can stabilize soluble α-synuclein oligomers in vitro,18 and oligomeric α-synuclein has been reported in patients with homozygous or heterozygous GBA mutations with Lewy body dementia, although not in PD.18, 31 No statistical differences in Lewy body pathology were reported between sporadic PD brains and PD+GBA brains studied here.8 We also found no noticeable difference in the solubility or amount of monomeric α-synuclein between PD+GBA brains and sporadic PD. Differences in α-synuclein conformation between these groups warrants further investigation.
GCase has also been shown to bind α-synuclein at lysosomal pH, and increased α-synuclein levels in the cortex can result in the depletion of lysosomal GCase.18, 19 Our in vitro data suggest that increased sensitivity of GCase to endo-H in cells with increased α-synuclein results in the aberrant trafficking of GCase through the ER. α-Synuclein can affect ER/Golgi apparatus trafficking,32, 33 but the mechanism by which GCase transport is reduced remains unclear. LIMP-2, the protein required for GCase transport to the lysosome,30 does not bind α-synuclein. However, less GCase is bound to LIMP-2 in α-synuclein–overexpressing cells, and therefore the amount of enzyme delivered to the lysosome is decreased. LIMP-2 levels were unaffected in the SN of PD brains. The decrease in steady-state GCase mRNA levels observed in vitro needs further investigation to determine whether this is a decrease in transcription or increased degradation of mRNA, and the extent to which this contributes to GCase deficiency.
Accumulation of proteins in the ER can induce UPR/ERAD. The 2 most common heterozygote GBA mutations associated with PD (N370S and L444P)7 have been reported to undergo UPR/ERAD in cultured cells.24, 25, 34 However, 2 Gaucher mouse studies could not find evidence of UPR/ERAD in the brain.17, 35 UPR/ERAD markers were increased in both PD brains with GBA mutations and sporadic PD. Aberrant trafficking of GCase might contribute to this increase in UPR/ERAD. However, it could also be a result of perturbed calcium homeostasis, redox status, or proteostasis or mitochondrial dysfunction, all of which are linked with PD pathogenesis.26, 36
Surprisingly, GCase activity was decreased in the cerebellum of PD and PD+GBA brains, although this is not a site associated with neurodegeneration, α-synuclein accumulation,37 mitochondrial dysfunction, or oxidative stress in PD.38 It is unclear why GCase activity is decreased in this region. We speculate that deficiency in the cerebellum is caused by a mechanism separate from that occurring in recognized pathogenic areas of PD such as the SN, putamen, and amygdala.
In conclusion, our studies confirm a widespread deficiency of GCase activity in the brains of PD patients carrying GBA mutations. We also demonstrate that PD patients without GBA mutations exhibit deficiency of GCase in SN. Based upon the relation between GCase, α-synuclein, and mitochondrial function shown here and by others, we propose that PD pathology is exacerbated and accelerated, but not necessarily initiated, by GBA mutations (Fig 5). This would explain why not all GBA mutation carriers develop PD, and why those who do tend to do so at an earlier age.
Figure 5. Scheme of glucocerebrosidase enzyme (GCase) deficiency in PD pathogenesis. Mutant (Mut) GCase (purple squares) disrupts the degradation of α-synuclein and organelles such as mitochondria by the autophagy–lysosomal pathway (1). Some GCase mutants may also become trapped in the endoplasmic reticulum (ER) and activate the unfolded protein response (UPR)/endoplasmic reticulum-associated degradation (ERAD). Increased levels of α-synuclein (α-syn) impair the trafficking of wild-type (WT) GCase (blue rectangle) via the ER/Golgi apparatus (2), resulting in less WT GCase being delivered to the lysosomes by lysosomal integral membrane protein-2 (3). This will exacerbate any lysosomal/autophagic dysfunction (4). Dysfunctional mitochondria can also affect WT GCase protein levels by an unidentified mechanism (5).
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