A new glucocerebrosidase deficient neuronal cell model provides a tool to probe pathophysiology and therapeutics for Gaucher disease

Abstract

Glucocerebrosidase is a lysosomal hydrolase involved in the breakdown of glucosylceramide. Gaucher disease, a recessive lysosomal storage disorder, is caused by mutations in the gene GBA1 . Dysfunctional glucocerebrosidase leads to accumulation of glucosylceramide and glycosylsphingosine in various cell types and organs. Mutations in GBA1 are also a common genetic risk factor for Parkinson disease and related synucleinopathies. In recent years, research on the pathophysiology of Gaucher disease, the molecular link between Gaucher and Parkinson disease, and novel therapeutics, have accelerated the need for relevant cell models with GBA1 mutations. Although induced pluripotent stem cells, primary rodent neurons, and transfected neuroblastoma cell lines have been used to study the effect of glucocerebrosidase deficiency on neuronal function, these models have limitations because of challenges in culturing and propagating the cells, low yield, and the introduction of exogenous mutant GBA1 . To address some of these difficulties, we established a high yield, easy-to-culture mouse neuronal cell model with nearly complete glucocerebrosidase deficiency representative of Gaucher disease. We successfully immortalized cortical neurons from embryonic null allele gba −/− mice and the control littermate ( gba +/+ ) by infecting differentiated primary cortical neurons in culture with an EF1α-SV40T lentivirus. Immortalized gba −/− neurons lack glucocerebrosidase protein and enzyme activity, and exhibit a dramatic increase in glucosylceramide and glucosylsphingosine accumulation, enlarged lysosomes, and an impaired ATP-dependent calcium-influx response; these phenotypical characteristics were absent in gba +/+ neurons. This null allele gba −/− mouse neuronal model provides a much-needed tool to study the pathophysiology of Gaucher disease and to evaluate new therapies.

Value of Glucosylsphingosine (Lyso-Gb1) as a Biomarker in Gaucher Disease: A Systematic Literature Review

“…Of the 74 articles included, 56 articles pertained to lyso-Gb1 and GD pathology. They encompassed (in a non-mutually exclusive manner) ten clinical (autopsy) studies [ 54 , 55 , 56 , 57 , 58 , 59 , 61 , 62 , 63 , 93 ] and 48 studies reporting preclinical data (19 studies reporting in vitro data and 32 studies reporting in vivo data) [ 26 , 45 , 53 , 58 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 ,…”

“…A human in vitro study that reported normal lyso-Gb1 concentrations and lyso-Gb1 hydrolysis in human fibroblasts isolated from patients with types 1, 2, and 3 GD was rationalized by a relatively high residual activity of GBA versus that found in other cells [ 99 ]. Intracellular lyso-Gb1 also accumulated in GBA knockout mice models and in newborn neural cells from a type 3 GD mouse model in the presence of CBE [ 84 , 101 , 102 ].…”

Glucocerebrosidase haploinsufficiency in A53T α-synuclein mice impacts disease onset and course

“…15 μg of midbrain and forebrain lysate and 1.2 μM imiglucerase (Genzyme, Cambridge, MA), used as a control, were incubated with 100 nM of the GCase-specific MDW933 fluorescent probe at 37° C for 90 min and run on a 4–20% Criterion: TGX™ gel (Bio-Rad laboratories). The results were analyzed using an excitation wavelight of 488 nm and emission of 520 nm to measure the fluorescent signal in the gel [23]. …”

Type 2 Gaucher disease in an infant despite a normal maternal glucocerebrosidase gene

“…Cells were disrupted by two ten‐second sonication pulses at 50% amplitude, and cell debris was removed by centrifugation at 5,000 rpm for 5 min. Western blotting was performed and glucocerebrosidase levels were quantified as previously described (Westbroek et al, ), using β‐actin (abcam 20272, 1:4000) as the loading control.…”

“…Glucocerbrosidase activity in the mother and proband was analyzed using 4‐methylumbelliferyl‐β‐D (4‐MU) glucopyranoside substrate, as previously described (Raghavan, Topol, & Kolodny, ; Westbroek et al, ), with the following modifications. Reactions were performed in pH 4.2 citrate‐phosphate buffer and incubated for 2.5 hr at 37°C.…”