Glucocerebrosidase mutations alter the endoplasmic reticulum and lysosomes in Lewy body disease

Abstract

Lewy body disease (LBD) development is enhanced by mutations in the GBA gene coding for glucocerebrosidase (GCase). The mechanism of this association is thought to involve an abnormal lysosomal system and we therefore sought to evaluate if lysosomal changes contribute to the pathogenesis of idiopathic LBD. Analysis of post-mortem frontal cortex tissue from 7 GBA mutation carriers with LBD, 5 GBA mutation carriers with no signs of neurological disease and human neural stem cells exposed to a GCase inhibitor was used to determine how GBA mutation contributes to LBD. GBA mutation carriers demonstrated a significantly reduced level of GCase protein and enzyme activity and retention of glucocerebrosidase isoforms within the endoplasmic reticulum (ER). This was associated with enhanced expression of the lysosomal markers LAMP1 and LAMP2, though the expression of ATP13A2 and Cathepsin D was reduced, along with the decreased activity of Cathepsin D. The ER unfolded protein response (UPR) regulator BiP/GRP78 was reduced by GBA mutation and this was a general phenomenon in LBD. Despite elevation of GRP94 in LBD, individuals with GBA mutations showed reduced GRP94 expression, suggesting an inadequate UPR. Finally, human neural stem cell cultures showed that inhibition of GCase causes acute reduction of BiP, indicating that the UPR is affected by reduced glucocerebrosidase activity. The results indicate that mutation in GBA leads to additional lysosomal abnormalities, enhanced by an impaired UPR, potentially causing α-synuclein accumulation.

Fig 1

Peptide N-glycosidase F (PNGase F)-treated glucocerebrosidase in normal and GBA mutation carriers: Brain homogenates were treated with PNGase F to show protein molecular weight and Western blotting for glucocerebrosidase used to detect glucocerebrosidase protein (a). The major isoform of glucocerebrosidase was reduced in GBA-mutation heterozygotes (GBA mutations: L444P, RecNciI, IVS2 + 1 G>A, N370S and L105R) (LBDmt, n = 5; Cmt, n = 5) compared to wild-type GBA individuals (LBDwt, n = 5; Cwt, n = 5) (b). Reductions in the major isoform were seen irrespective of the presence of Lewy body disease. Changes were seen in the levels of the short isoform of glucocerebrosidase in controls with GBA mutations, although this was not significant in LBD cases and GBA mutation carriers; overall, the short isoform was significantly reduced (c). Figures are Mean GBA/GAPDH ratio ± SD. Numbering of mutation bearing cases is according to Table 1.

Fig 2

Brain glucocerebrosidase enzyme activity is decreased in GBA mutation carriers: Samples of grey matter (LBDmt n = 7, LBDwt n = 15, Cmt n = 5, Cwt n = 10) were assayed for glucocerebrosidase enzyme activity using the specific substrate and levels of 4-methylumbelliferone (4-MU) production were determined. Comparison with tissue from individuals with wild-type GBA were used as control and showed reduced glucocerebrosidase enzyme activity in GBA mutation carriers. Glucocerebrosidase activity was reduced in both normal and Lewy body disease (LBD) cases to a similar extent. Note that there is an overlap in glucocerebrosidase activity between control and mutant samples.

Fig 3

Major lysosomal membrane proteins are elevated in GBA mutation carriers: Western blotting was used to determine the relative levels of LAMP1, LAMP2 and LIMP-2 protein in brain homogenates from individuals with and without GBA mutations (LBDmt n = 5–7, LBDwt n = 14–15, Cmt n = 5, Cwt n = 10–11)(a, c). Elevated levels of LAMP1 and LAMP2 were seen in Lewy body disease (LBD) and normal individuals in the presence of GBA mutation (b). The major protein involved in import of glucocerebrosidase into lysosomes LIMP-2 (SCARB2) was also seen to be elevated in control mutation carriers (c) but was not significantly elevated in LBD mutation carriers. Error bars are ± SD.

Fig 4

Selective alterations in lysosomal proteins in GBA mutation carriers with Lewy body disease: Brain tissue from GBA mutation carriers was screened for levels of the Kufor–Rakeb Parkinsonism protein ATP13A2 and the lysosomal protease Cathepsin D by Western blotting (LBDmt n = 7, LBDwt n = 14, Cmt n = 5, Cwt n = 11) (a) and relative levels determined against GAPDH levels. Reduced levels of ATP13A2 were seen in LBD cases carrying GBA mutations but not in normal individuals with GBA mutation (b) and similar reductions in Cathepsin D heavy chain (28 kDa) were seen associated selectively with LBD patients. Error bars are ± SD.

Fig 5

The ER Unfolded Protein Response (UPR) is abnormal in GBA mutation carriers: The levels of ER proteins BiP/GRP78 and HERP were determined in brain homogenates from individuals with Lewy body disease (LBD) and in normal control individuals with and without GBA mutations (LBDmt n = 7, LBDwt n = 13–15, Cmt n = 5, Cwt n = 10–11) (a, c). Relative levels of BiP/GRP78 were reduced in LBD cases, independent of mutation status, compared to controls (p < 0.001) by approximately 10–15% but reduced in control individuals (Cmt) carrying GBA mutation (b). HERP, a stress-induced ER protein was similarly reduced in control and LBD GBA mutation carriers (d) although there was no change between LBD and control samples overall (p = 0.3). Error bars are ± SD.

Fig 6

The chaperone GRP94 is elevated in Lewy body disease (LBD) but reduced by GBA mutation: Brain tissue from LBD and control individuals was probed for the major ER chaperone protein GRP94 by Western blotting (LBDmt n = 5, LBDwt n = 13, Cmt n = 4, Cwt n = 11) (a). Compared to controls (b) there was an elevation of GRP94 in LBD brain of approximately 15% (p < 0.001) suggesting the presence of an UPR, though this was reduced by the presence of GBA mutation in controls and to a lesser extent in LBD. Error bars are ± SD.

Fig 7

α-synuclein is elevated in frontal cortex of GBA mutation carriers: Increased α-synuclein protein expression is only observed in LBD patients with heterozygous GBA mutations and does not appear to be elevated in normal controls with GBA mutations (total brain tissue homogenates were used in the analysis) (LBDmt n = 6, LBDwt n = 11, Cmt n = 5, Cwt n = 11).

Fig 8

Effects of glucocerebrosidase inhibition in human neural cultures using CBE: Differentiated human neural stem cells were exposed to 100 μM of the specific glucocerebrosidase inhibitor conduritol Beta epoxide (CBE) for 72 h and protein extracts prepared for western blotting. CBE caused a slight elevation of steady-state levels of glucocerebrosidase (a) but caused an acute change of ER stress and the UPR as evidenced by reduced levels of BiP (b). Overall, no significant change was seen in the major lysosomal protein LAMP1 (c). Example blots are representative of three independent experiments. The data presented consist of the mean of all three independent replicates. Error bars are ± SD.