Proceedings of The Physiological Society

Mitochondria: Form and function (London, UK) (2017) Proc Physiol Soc 38, C02

Oral Communications

GBA1 deficiency sensitises neurons to glutamate toxicity and causes calcium dyshomeostasis

N. Plotegher1, D. Perocheau3, G. Massaro2, A. Rahim2, S. Waddington3, M. Duchen1

1. Department of Cell and Developmental Biology, University College London, London, United Kingdom. 2. School of Pharmacy, University College London, London, - Non US -, United Kingdom. 3. Institute of Women's Health, University College London, London, - Non US -, United Kingdom.


Glucocerebrosidase (GBA1) is a lysosomal enzyme whose homozygous mutations cause Gaucher's disease (GD), a lysosomal storage disorder. Severity and symptoms are variable but include a severe neurodegenerative ‘neuropathic' form. Heterozygous mutations in GBA1 are the major known genetic risk factor for Parkinson's disease (PD). The deficiency of GBA1 causes impaired autophagy but was also associated with reduced mitochondrial membrane potential, mitochondrial fragmentation and respiratory chain defects (1). Since mitochondrial dysfunction can impact on free radical production and calcium homeostasis, we used fluorescence imaging microscopy and biochemical approaches to investigate these variables in neurons cultured from a GBA1 knockout (KO) mouse stimulated with a non-toxic glutamate concentration (10μM). We found that basal rates of reactive oxygen species (ROS) (measured by imaging the dye dihydroethidium) are increased in GBA1 KO neurons compared to wild-type (WT) and heterozygous (HET) neurons in basal conditions, while all the genotypes responded similarly to glutamate, showing an increase in the rates of ROS generation. The increased free radical production did not correspond to a change in the expression of the antioxidant proteins superoxide dismutases 1 and 2. The calcium responses to glutamate showed an immediate ‘early' response to 10μM glutamate stimulation which was significantly higher in HET and KO neurons compared to WT, measured by Fura2 imaging. Moreover, a higher proportion of HET and KO neurons showed a late increase in cytosolic calcium termed ‘delayed calcium deregulation (DCD)' that is usually seen only at higher glutamate concentration in WT neurons (>100μM) and leads to cell death. This was associated with dysregulation of mitochondrial calcium handling: measurements of mitochondrial calcium uptake (using mitochondrial targeted aequorin) showed a decrease in response in HET and KO compared to WT cells. This was also associated with a reduced expression of the mitochondrial calcium uniporter (MCU), which is the channel responsible for the mitochondrial calcium uptake. Expression levels of glutamate receptors (glutamate ionotropic receptor kainate type subunit 4 and glutamate ionotropic receptor NMDA type subunit 2B) were not changed at the transcript level (as probed by pQCR) showing that the increased response to glutamate in GBA1 KO cells was not due to their altered expression. These data together suggest calcium handling is dysregulated in GBA1 KO neurons, with an increased sensitivity to otherwise innocuous glutamate concentrations, a mechanism which may contribute to neurodegeneration in GD. Strikingly, HET neurons showed a behavior that was more similar to KO rather than to WT, consistent with a role of these mechanisms in the pathogenesis of PD.

Where applicable, experiments conform with Society ethical requirements