CRISPR/Cas9 Editing for Gaucher Disease Modelling

Abstract

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the acid β-glucosidase gene (GBA1). Besides causing GD, GBA1 mutations constitute the main genetic risk factor for developing Parkinson's disease. The molecular basis of neurological manifestations in GD remain elusive. However, neuroinflammation has been proposed as a key player in this process. We exploited CRISPR/Cas9 technology to edit GBA1 in the human monocytic THP-1 cell line to develop an isogenic GD model of monocytes and in glioblastoma U87 cell lines to generate an isogenic GD model of glial cells. Both edited (GBA1 mutant) cell lines presented low levels of mutant acid β-glucosidase expression, less than 1% of residual activity and massive accumulation of substrate. Moreover, U87 GBA1 mutant cells showed that the mutant enzyme was retained in the ER and subjected to proteasomal degradation, triggering unfolded protein response (UPR). U87 GBA1 mutant cells displayed an increased production of interleukin-1β, both with and without inflammosome activation, α-syn accumulation and a higher rate of cell death in comparison with wild-type cells. In conclusion, we developed reliable, isogenic, and easy-to-handle cellular models of GD obtained from commercially accessible cells to be employed in GD pathophysiology studies and high-throughput drug screenings.

Keywords: CRISPR/Cas9; Gaucher disease; acid β-glucosidase; cellular model; high-throughput drug screenings; neuroinflammation; unfolded protein response; α-synuclein.

Figure 1

Development and characterization of THP-1 GBA1 mutant cells. Schematic representation of GBA1 editing workflow (A). WB analysis of THP-1 GBA1 mutant D2, D6 and F5 clones showed lower levels of GCase expression in comparison to THP-1 wt (B). GCase enzymatic activity was significantly reduced in all three clones (C). A massive LysoGL1 accumulation was detected in THP-1 GBA1 mutant clone D2 (D). Results are expressed as mean ± SD of three independent experiments. ** p < 0.001, *** p < 0.0001.

Figure 2

Development and characterization of U87 GBA1 mutant cells. WB analysis of U87 GBA1 mutant cells showed lower levels of GCase expression in comparison to U87 wt (A). A significant reduction of GCase enzymatic activity (B), as well as LysoGL1 accumulation (C), were detected. When digested by Endo-H, U87 GBA1 mutant showed lack of mature GCase (indicated by arrows in D); the Endo-F cleavage pattern confirmed that difference in protein migration on SDS-PAGE was only due to protein glycosylation [*GCase detection was performed at differential exposure time between U87 wt and U87 GBA1 mutant] (D). A significant increase of GCase protein abundance was detected in cells treated with the proteasome inhibitor MG132 by WB (E). The quantification of GCase signals, normalized to actin, in the presence and absence of MG132 showed that 32% of the mutant protein was subjected to proteasomal degradation (F). mRNA levels of ER stress markers BiP and Chop were significantly increased in U87 GBA1 mutant cells in comparison to U87 wt (G). Results are expressed as mean ± SD of three independent experiments. * p < 0.05, ** p < 0.001, *** p < 0.0001. Abbreviations: M = marker, ND = non digested, D = digested, (↑exp) = longer exposure.

Figure 3

Inflammatory markers. Cytokines levels evaluated by capture ELISA in the supernatant of U87 GBA1 mutant cells showed an increased production of IL-1β without (-) or with (LPS + ATP) inflammosome activation (A). No changes in TNFα (B) nor IL-6 (C) were detected, with or without LPS stimuli. Results are expressed as mean ± SD of three independent experiments. * p < 0.05, ** p < 0.001.

Figure 4

Accumulation of α-synuclein and cell death. The higher α-syn abundance shown by the U87 GBA1 mutant by WB analysis (A). Quantification of α-syn signals normalized to actin (B) showed that mutant cells displayed a 56% increase in α-syn content. This increment was accompanied by an increased percentage of apoptotic cells as assessed by Annexin V positive and propidium iodide (IP) negative (C) and LDH activity release (D). Results are expressed as mean ± SD of three independent experiments. * p < 0.05, ** p < 0.001, *** p < 0.0001.

Figure 5

Schematic representation of the main cellular features and their possible correlation. Mutations in the GBA1 gene cause the synthesis of a mutant form of GCase, which is retained in the ER causing ER stress and subjected to degradation via the ubiquitin-proteasome system (UPS degradation), triggering unfolded protein response (UPR). The low enzymatic activity of the mutant GCase leads to LysoGL1 accumulation in lysosomes, which has been associated with neuroinflammation. Mutated GCase and substrate storage might lead to α-syn accumulation, which may have a direct role in the development of inflammation. Moreover, α-syn accumulation, UPR and/or inflammation may be the key triggers of apoptotic processes, eventually causing cell death.