Dori Pelled

Elevation of Intracellular Glucosylceramide Levels Results in an Increase in Endoplasmic Reticulum Density and in Functional Calcium Stores in Cultured Neurons

Gaucher disease is a glycosphingolipid storage disease caused by defects in the activity of the lysosomal hydrolase, glucocerebrosidase (GlcCerase), resulting in accumulation of glucocerebroside (glucosylceramide, GlcCer) in lysosomes. The acute neuronopathic type of the disease is characterized by severe loss of neurons in the central nervous system, suggesting that a neurotoxic agent might be responsible for cellular disruption and neuronal death. We now demonstrate that upon incubation with a chemical inhibitor of GlcCerase, conduritol-B-epoxide (CBE), cultured hippocampal neurons accumulate GlcCer. Surprisingly, increased levels of tubular endoplasmic reticulum elements, an increase in [Ca2+] i response to glutamate, and a large increase in [Ca2+] i release from the endoplasmic reticulum in response to caffeine were detected in these cells. There was a direct relationship between these effects and GlcCer accumulation since co-incubation with CBE and an inhibitor of glycosphingolipid synthesis, fumonisin B1, completely antagonized the effects of CBE. Similar effects on endoplasmic reticulum morphology and [Ca2+] i stores were observed upon incubation with a short-acyl chain, nonhydrolyzable analogue of GlcCer, C8-glucosylthioceramide. Finally, neurons with elevated GlcCer levels were much more sensitive to the neurotoxic effects of high concentrations of glutamate than control cells; moreover, this enhanced toxicity was blocked by pre-incubation with ryanodine, suggesting that [Ca2+] i release from ryanodine-sensitive intracellular stores can induce neuronal cell death, at least in neurons with elevated GlcCer levels. These results may provide a molecular mechanism to explain neuronal dysfunction and cell death in neuronopathic forms of Gaucher disease.

Glucosylceramide and Glucosylsphingosine Modulate Calcium Mobilization from Brain Microsomes via Different Mechanisms

We recently demonstrated that elevation of intracellular glucosylceramide (GlcCer) levels results in increased functional Ca2+ stores in cultured neurons, and suggested that this may be due to modulation of ryanodine receptors (RyaRs) by GlcCer (Korkotian, E., Schwarz, A., Pelled, D., Schwarzmann, G., Segal, M. and Futerman, A. H. (1999) J. Biol. Chem. 274, 21673–21678). We now systematically examine the effects of exogenously added GlcCer, other glycosphingolipids (GSLs) and their lyso-derivatives on Ca2+ release from rat brain microsomes. GlcCer had no direct effect on Ca2+ release, but rather augmented agonist-stimulated Ca2+ release via RyaRs, through a mechanism that may involve the redox sensor of the RyaR, but had no effect on Ca2+ release via inositol 1,4,5-trisphosphate receptors. Other GSLs and sphingolipids, including galactosylceramide, lactosylceramide, ceramide, sphingomyelin, sphingosine 1-phosphate, sphinganine 1-phosphate, and sphingosylphosphorylcholine had no effect on Ca2+ mobilization from rat brain microsomes, but both galactosylsphingosine (psychosine) and glucosylsphingosine stimulated Ca2+ release, although only galactosylsphingosine mediated Ca2+ release via the RyaR. Finally, we demonstrated that GlcCer levels were ∼10-fold higher in microsomes prepared from the temporal lobe of a type 2 Gaucher disease patient compared with a control, and Ca2+ release via the RyaR was significantly elevated, which may be of relevance for explaining the pathophysiology of neuronopathic forms of Gaucher disease.

Enhanced calcium release in the acute neuronopathic form of Gaucher disease.

Gaucher disease is an inherited metabolic disorder caused by defective activity of the lysosomal enzyme, glucocerebrosidase, resulting in accumulation of the lipids, glucosylceramide (GlcCer), and glucosylsphingosine (GlcSph). Little is known about the mechanism leading from lipid accumulation to disease, particularly in the acute and subacute neuronopathic forms of Gaucher disease, types 2 and 3, respectively. Recent work from our laboratory has shown, in animal models, that GlcCer enhances agonist-induced calcium release from intracellular stores via the ryanodine receptor, which results in neuronal cell death. We now test whether calcium release is altered in human brain tissue obtained post-mortem from Gaucher disease patients. Agonist-induced calcium release via the ryanodine receptor was significantly enhanced (P < 0.05) in brain microsomes from the acute neuronopathic form of Gaucher disease (type 2) (43 +/- 6% of the calcium in microsomes) compared to the subacute (type 3) (27 +/- 3%) and the non-neuronopathic (type 1) (28 +/- 6%) forms, and controls (18 +/- 3%), and correlated with levels of GlcCer accumulation. These findings suggest that defective calcium homeostasis may be a mechanism responsible for neuropathophysiology in acute neuronopathic Gaucher disease, and may potentially offer new therapeutic approaches for disease management.

Phosphatidylcholine synthesis is elevated in neuronal models of Gaucher disease due to direct activation of CTP:phosphocholine cytidylyltransferase by glucosylceramide

Glucosylceramide (GlcCer) accumulates in the inherited metabolic disorder, Gaucher disease, because of the defective activity of lysosomal glucocerebrosidase. We previously demonstrated that upon GlcCer accumulation, cultured hippocampal neurons exhibit modified growth patterns, altered endoplasmic reticulum density, and altered calcium release from intracellular stores. We here examined the relationship between GlcCer accumulation and phospholipid synthesis. After treatment of neurons with an active site‐directed inhibitor of glucocerebrosidase, or in neurons obtained from a mouse model of Gaucher disease, [14C]methyl choline incorporation into [14C]phosphatidylcholine ([14C]PC) and [14C]sphingomyelin was elevated, as were [14C]CDPcholine levels, suggesting that CTP:phosphocholine cytidylyltransferase (CCT) is activated. Indeed, CCT activity was elevated in neurons that had accumulated GlcCer. GlcCer, but not galactosylceramide (GalCer), stimulated CCT activity in rat brain homogenates, and significantly higher levels of CCT were membrane associated in cortical homogenates from a mouse model of Gaucher disease compared with wild‐type mice. Because CCT mRNA and protein levels were unaltered in either neurons or brain tissue that had accumulated GlcCer, it appeared likely that GlcCer activates CCT by a post‐translational mechanism. This was verified by examination of the effect of GlcCer on CCT purified about 1200‐fold from rat brain. GlcCer stimulated CCT activity, with stimulation observed at levels as low as 2.5 mol% and with maximal activation reached at 10 mol%. In contrast, GalCer had no effect. Together, these data demonstrate that GlcCer directly activates CCT, which results in elevated PC synthesis, which may account for some of the changes in growth rates observed upon neuronal GlcCer accumulation.

The increased sensitivity of neurons with elevated glucocerebroside to neurotoxic agents can be reversed by imiglucerase

We have recently demonstrated that incubation of cultured rat hippocampal neurons with conduritol β-epoxide (CBE), an inhibitor of glucocerebrosidase, the enzyme defective in Gaucher disease, results in changes in intracellular morphology and in functional calcium stores. Changes in levels of functional calcium stores are directly related to neuronal cell death. We now show that neurons incubated with either CBE or a non-hydrolysable analogue of GlcCer (glucosylthioceramide), are more sensitive to the toxic effects of high concentrations of glutamate and of a variety of metabolic inhibitors. A linear relationship exists between level of accumulation of GlcCer and the extent of neuronal cell death. The deleterious effects of elevated GlcCer levels can be completely reversed by addition of human glucocerebrosidase (imiglucerase) to the culture medium. Imiglucerase is internalized to lysosomes, where it presumably degrades excess GlcCer. This suggests that the limited success of enzyme replacement therapy in neuronopathic forms of Gaucher disease is not due to lack of efficacy of glucocerebroside in degrading GlcCer in neurons of the central nervous system, and adds impetus to attempts to develop ways to efficiently deliver glucocerebrosidase to the brains of neurologically compromised Gaucher disease patients.

Autoimmune Epilepsy: Some Epilepsy Patients Harbor Autoantibodies to Glutamate Receptors and dsDNA on both Sides of the Blood-brain Barrier, which may Kill Neurons and Decrease in Brain Fluids after Hemispherotomy

Purpose: Elucidating the potential contribution of specific autoantibodies (Abs) to the etiology and/or pathology of some human epilepsies. Methods: Six epilepsy patients with Rasmussens encephalitis (RE) and 71 patients with other epilepsies were tested for Abs to the –B— peptide (amino acids 372-395) of the glutamate/AMPA subtype 3 receptor (GluR3B peptide), double-stranded DNA (dsDNA), and additional autoimmune disease-associated autoantigens, and for the ability of their serum and cerebrospinal-fluid (CSF) to kill neurons. Results: Elevated anti-GluR3B Abs were found in serum and CSF of most RE patients, and in serum of 17/71 (24%) patients with other epilepsies. In two RE patients, anti-GluR3B Abs decreased drastically in CSF following functional-hemispherotomy, in association with seizure cessation and neurological improvement. Serum and CSF of two RE patients, and serum of 12/71 (17%) patients with other epilepsies, contained elevated anti-dsDNA Abs, the hallmark of systemic-lupus-erythematosus. The sera (but not the CSF) of some RE patients contained also clinically elevated levels of –classical— autoimmune Abs to glutamic-acid-decarboxylase, cardiolipin, β2-glycoprotein-I and nuclear-antigens SS-A and RNP-70. Sera and CSF of some RE patients caused substantial death of hippocampal neurons. Conclusions: Some epilepsy patients harbor Abs to GluR3 and dsDNA on both sides of the blood-brain barrier, and additional autoimmune Abs only in serum. Since all these Abs may be detrimental to the nervous system and/or peripheral organs, we recommend testing for their presence in epilepsy, and silencing their activity in Ab-positive patients.

Lyso-glycosphingolipids mobilize calcium from brain microsomes via multiple mechanisms.

Recently, we demonstrated that the GSL (glycosphingolipid), GlcCer (glucosylceramide), modulates Ca2+ release from intracellular stores and from microsomes by sensitizing the RyaR (ryanodine receptor), a major Ca2+-release channel of the endoplasmic reticulum, whereas the lyso derivative of GlcCer, namely GlcSph (glucosylsphingosine), induced Ca2+ release via a mechanism independent of the RyaR [Lloyd-Evans, Pelled, Riebeling, Bodennec, de-Morgan, Waller, Schiffmann and Futerman (2003) J. Biol. Chem. 278, 23594-23599]. We now systematically examine the mechanism by which GlcSph and other lyso-GSLs modulate Ca2+ mobilization from rat brain cortical and cerebellar microsomes. GlcSph, lactosylsphingosine and galactosylsphingosine all mobilized Ca2+, but at significantly higher concentrations than those required for GlcCer-mediated sensitization of the RyaR. GlcSph-induced Ca2+ mobilization was partially blocked by heparin, an inhibitor of the Ins(1,4,5) P3 receptor, and also partially blocked by thapsigargin or ADP, inhibitors of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), but completely blocked when both acted together. In contrast, neither lactosylsphingosine nor galactosylsphingosine had any effect on Ca2+ release via either the Ins(1,4,5) P3 receptor or SERCA, but acted as agonists of the RyaR. Finally, and surprisingly, all three lyso-GSLs reversed inhibition of SERCA by thapsigargin. We conclude that different lyso-GSLs modulate Ca2+ mobilization via different mechanisms, and discuss the relevance of these findings to the GSL storage diseases in which lyso-GSLs accumulate.

Reduced rates of axonal and dendritic growth in embryonic hippocampal neurones cultured from a mouse model of Sandhoff disease.

Sandhoff disease is a lysosomal storage disease in which ganglioside GM2 accumulates because of a defective β‐subunit of β‐hexosaminidase. This disease is characterized by neurological manifestations, although the pathogenic mechanisms leading from GM2 accumulation to neuropathology are largely unknown. We now examine the viability, development and rates of neurite growth of embryonic hippocampal neurones cultured from a mouse model of Sandhoff disease, the Hexb–/– mouse. GM2 was detected by metabolic labelling at low levels in wild type (Hexb+/+) neurones, and increased by approximately three‐fold in Hexb–/– neurones. Hexb–/– hippocampal neurones were as viable as their wild type counterparts and, moreover, their developmental programme was unaltered because the formation of axons and of the minor processes which eventually become dendrites was similar in Hexb–/– and Hexb+/+ neurones. In contrast, once formed, a striking difference in the rate of axonal and minor process growth was observed, with changes becoming apparent after 3 days in culture and highly significant after 5 days in culture. Analysis of various parameters of axonal growth suggested that a key reason for the decreased rate of axonal growth was because of a decrease in the formation of collateral axonal branches, the major mechanism by which hippocampal axons elongate in culture. Thus, although the developmental programme with respect to axon and minor process formation and the viability of hippocampal neurones are unaltered, a significant decrease occurs in the rate of axonal and minor process growth in Hexb–/– neurones. These results appear to be in contrast to dorsal root ganglion neurones cultured from 1‐month‐old Sandhoff mice, in which cell survival is impaired but normal outgrowth of neurones occurs. The possible reasons for these differences are discussed.