Inhibition of Mitochondrial Complex I Impairs Release of α-Galactosidase by Jurkat Cells.

Fabry disease (FD) is caused by mutations in the GLA gene that encodes lysosomal α-galactosidase-A (α-gal-A). A number of pathogenic mechanisms have been proposed and these include loss of mitochondrial respiratory chain activity. For FD, gene therapy is beginning to be applied as a treatment. In view of the loss of mitochondrial function reported in FD, we have considered here the impact of loss of mitochondrial respiratory chain activity on the ability of a GLA lentiviral vector to increase cellular α-gal-A activity and participate in cross correction. Jurkat cells were used in this study and were exposed to increasing viral copies. Intracellular and extracellular enzyme activities were then determined; this in the presence or absence of the mitochondrial complex I inhibitor, rotenone. The ability of cells to take up released enzyme was also evaluated. Increasing transgene copies was associated with increasing intracellular α-gal-A activity but this was associated with an increase in Km. Release of enzyme and cellular uptake was also demonstrated. However, in the presence of rotenone, enzyme release was inhibited by 37%. Excessive enzyme generation may result in a protein with inferior kinetic properties and a background of compromised mitochondrial function may impair the cross correction process.

Inhibition of neuronal mitochondrial complex I or lysosomal glucocerebrosidase is associated with increased dopamine and serotonin turnover.

Parkinson's disease (PD) is a neurodegenerative disorder caused by loss of dopaminergic and serotoninergic signalling. A number of pathogenic mechanisms have been implicated including loss of mitochondrial function at the level of complex I, and lysosomal metabolism at the level of lysosomal glucocerebrosidase (GBA1). In order to investigate further the potential involvement of complex I and GBA1 in PD, we assessed the impact of loss of respective enzyme activities upon dopamine and serotonin turnover. Using SH-SY5Y cells, complex I deficiency was modelled by using rotenone whilst GBA1 deficiency was modelled by the use of conduritol B epoxide (CBE). Dopamine, its principal metabolites, and the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the extracellular medium were quantified by HPLC. Inhibition of complex I significantly increased extracellular concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-HIAA. Comparable results were observed with CBE. These results suggest increased monoamine oxidase activity and provide evidence for involvement of impaired complex I or GBA1 activity in the dopamine/serotonin deficiency seen in PD. Use of extracellular media may also permit relatively rapid assessment of dopamine/serotonin metabolism and permit screening of novel therapeutic agents.

Increased glucocerebrosidase (GBA) 2 activity in GBA1 deficient mice brains and in Gaucher leucocytes.

Lysosomal glucocerebrosidase (GBA1) deficiency is causative for Gaucher disease. Not all individuals with GBA1 mutations develop neurological involvement raising the possibility that other factors may provide compensatory protection. One factor may be the activity of the non-lysosomal ß-glucosidase (GBA2) which exhibits catalytic activity towards glucosylceramide and is reported to be highly expressed in brain tissue. Here, we assessed brain GBA2 enzymatic activity in wild type, heterozygote and GBA1 deficient mice. Additionally, we determined activity in leucocytes obtained from 13 patients with Gaucher disease, 10 patients with enzymology consistent with heterozygote status and 19 controls. For wild type animals, GBA2 accounted for over 85 % of total brain GBA activity and was significantly elevated in GBA1 deficient mice when compared to heterozygote and wild types (GBA1 deficient; 92.4 ± 5.6, heterozygote; 71.5 ± 2.4, wild type 76.8 ± 5.1 nmol/h/mg protein). For the patient samples, five Gaucher patients had GBA2 leucocyte activities markedly greater than controls. No difference in GBA2 activity was apparent between the control and carrier groups. Undetectable GBA2 activity was identified in four leucocyte preparations; one in the control group, two in the carrier group and one from the Gaucher disease group. Work is now required to ascertain whether GBA2 activity is a disease modifying factor in Gaucher disease and to identify the mechanism(s) responsible for triggering increased GBA2 activity in GBA1 deficiency states.