Inhibition of Calcium Uptake via the Sarco/Endoplasmic Reticulum Ca2+-ATPase in a Mouse Model of Sandhoff Disease and Prevention by Treatment with N-Butyldeoxynojirimycin

Dori Pelled,Emyr Lloyd-Evans,Christian Riebeling,Mylvaganam Jeyakumar,Frances M Platt,Anthony H Futerman

doi:10.1074/jbc.m302964200

Abstract

Gangliosides are found at high levels in neuronal tissues where they play a variety of important functions. In the gangliosidoses, gangliosides accumulate because of defective activity of the lysosomal proteins responsible for their degradation, usually resulting in a rapidly progressive neurodegenerative disease. However, the molecular mechanism(s) leading from ganglioside accumulation to neurodegeneration is not known. We now examine the effect of ganglioside GM2 accumulation in a mouse model of Sandhoff disease (one of the GM2 gangliosidoses), the Hexb-/- mouse. Microsomes from Hexb-/- mouse brain showed a significant reduction in the rate of Ca2+-uptake via the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), which was prevented by feeding Hexb-/- mice with N-butyldeoxynojirimycin (NB-DNJ), an inhibitor of glycolipid synthesis that reduces GM2 storage. Changes in SERCA activity were not due to transcriptional regulation but rather because of a decrease in Vmax. Moreover, exogenously added GM2 had a similar effect on SERCA activity. The functional significance of these findings was established by the enhanced sensitivity of neurons cultured from embryonic Hexb-/- mice to cell death induced by thapsigargin, a specific SERCA inhibitor, and by the enhanced sensitivity of Hexb-/- microsomes to calcium-induced calcium release. This study suggests a mechanistic link among GM2 accumulation, reduced SERCA activity, and neuronal cell death, which may be of significance for delineating the neuropathophysiology of Sandhoff disease.

Highlights

This study suggests a mechanistic link among GM2 accumulation, reduced sarco/ endoplasmic reticulum Ca2ϩ-ATPase (SERCA) activity, and neuronal cell death, which may be of significance for delineating the neuropathophysiology of Sandhoff disease
We demonstrate that brain microsomes and neurons cultured from these mice show dramatically reduced levels of Ca2ϩ-uptake via the sarco/ endoplasmic reticulum Ca2ϩ-ATPase (SERCA),1 which is not related to transcriptional regulation of SERCA but rather to changes in Vmax
Impairment of SERCA activity usually results in neuronal cell dysfunction and/or death [8, 9], and our observations that Ca2ϩ-uptake via SERCA is severely impaired upon GM2 accumulation may suggest a molecular mechanism to explain, at least in part, the neuropathophysiology in Sandhoff disease

Summary

EXPERIMENTAL PROCEDURES

Activator deficiency [1, 2]. The HEXA and HEXB genes code for the ␤-hexosaminidase ␣- and ␤-subunits, respectively, which dimerize to produce two forms of the enzyme, A (␣␤) and B (␤␤),. Mouse cortical microsomes (350 ␮g protein) were incubated at 37 °C in 1.5 ml of Buffer B (40 mM imidazole, pH 7.0, 100 mM KCl, 5 mM MgCl2, 5 mM NaN3, 5 mM potassium oxalate, 0.5 mM EGTA, 1 ␮M ruthenium red [21] (which blocks spontaneous Ca2ϩ-release via the ryanodine receptor [22]), 10 ␮Ci 45Ca2ϩ, and CaCl2, to yield the required final concentration of free Ca2ϩ (determined using an algorithm [23] and software available at www.stanford.edu/ϳcpatton/maxc.html). For analysis of Ca2ϩ-uptake [29], neurons were plated at a density of 1.1 ϫ 106 cells per 60-mm culture dish that contained three 24-mm polylysine-coated coverslips. Neurons were examined using a Plan 25ϫ/ 0.45 numerical aperture objective of a Zeiss Axiovert 35 microscope

RESULTS

Inhibition of SERCA in Sandhoff Disease

We next examined the functional significance of the reduced

DISCUSSION