Neuronopathic Gaucher Disease Grant
The funds to support this research have been raised by the family of Ellie Carter who died on 9 February 2004 aged seven months. Ellie had Type 2 Gaucher disease In memory of Ellie her
parents Jill and Ian, set up ELF, the Eleanor Lily Foundation, to raise funds to go towards research into understanding more about Type 2 Gaucher disease.
In accepting the research grant Prof. Mia Horowitz said “I was delighted to realise that I was the recipient of this award from the UK Gaucher association, which will support our efforts toward understanding the molecular mechanism underlying brain cell death in neuronopathic Gaucher disease. As part of this project, we intend to study what are the consequences of the endoplasmic reticulum associated degradation of the mutant glucocerebrosidase forms.
We believe that the accumulation of mutant glucocerebrosidase forms in the endoplasmic reticulum of Gaucher patients with neuronopathic forms of the disease lead to unfolding protein response and cell death. We would like to test this hypothesis. During these processes mutant glucocerebrosidase is supposed to interact with several proteins, which we would like to unravel."
See Year 1 report - STUDIES TOWARD UNDERSTANSING THE MOLECULAR MECHANISM UNDERLYING CELL DEATH IN NEURONOPATHIC GAUCHER DISEASE
PURPOSE OF THE PROJECT
The aims of the presented project were:
A. To test the correlation between endo-H sensitivity in different cell types and neuronopathic Gaucher disease severity.
B. To identify interactors of mutant glucocerebrosidase variants, that participate in its Endoplasmic Reticulum Associated Degradation (ERAD)
C. To test whether Endoplasmic Reticulum (ER) stress caused to brain cells in patients with type 2 or 3 Gaucher disease lead to their death through apoptosis or autophagy.
In Gaucher disease low or no activity of the lysosomal enzyme glucocerebrosidase leads to accumulation of non-degraded glucosyl-ceramide(s). We believed that this accumulation, which affects mostly cells of the reticuloendothelial system, leads to enlarged spleen and liver, anemia, thrombocytopenia, bone fractures, necrosis and pains and in some cases, to neurological symptoms.
Lately it has become clear that mutant lysosomal variants are misfolded in the ER and are subjected to ER associated degradation (ERAD) – therefore they do not travel to the lysosome. Our data indicated that mutant glucocerebrosidase variants are misfolded in the ER and undergo ER associated degradation in the proteasomes - htpp://en.wikipedia.org/wiki/Proteasome. Our data strongly indicated that in Gaucher disease there is a direct correlation between the level of ER-retained mutant glucocerebrosidase, which is endoglycosidase H (endo-H) sensitive and severity of Gaucher disease. Moreover, our data strongly suggest that in the neuronopathic forms of Gaucher disease there is very little mature enzyme and most of it undergoes ERAD – therefore it does not get to the lysosome and break down the accumulated substrate. It has already been documented that in response to the accumulation of unfolded proteins in the ER, the rate of general translation initiation is attenuated (weakens), the expression of ER resident protein chaperones and protein foldases is induced, the ER compartment proliferates (becomes rapid), and ER-associated degradation (ERAD) is activated to eliminate the irreparably misfolded proteins. When the prosurvival efforts are exhausted, ER-stress related apoptosis (a form of programmed cell death) commences. We hypothesize that in neurons or microglia (a type of glial cell that acts as the first and main form of active immune defense in the central nervous system) of neuronopathic Gaucher patients the extensive ERAD leads to ER stress and cell death. We propose to test this hypothesis. We would also like to isolate proteins, interacting with glucocerebrosidase, which participate in its ERAD process.
RESULTS
Establishment of a correlation between endo-H sensitivity of glucocerebrosidase in different cell types and neuronopathic Gaucher disease severity
We wished to establish a biochemical test for severity of the neuronopathic forms of Gaucher disease. As has been described, we have shown a good correlation between Gaucher disease severity and sensitivity of mutant glucocerebrosidase to digestion with the enzyme endoglycosidase H (endo-H). During the last year we have found that in cell lines (skin fibroblasts or lymphoblastoid cell lines) from patients with neuronopathic GD there is a significant reduction in the amount of glucocerebrosidase. Most mutant glucocerebrosidase is retained in the ER and therefore is endo-H sensitive (see attached note re: Endo –H). The mutant glucocerebrosidase variants undergo ER associated degradation in the proteasome. Therefore, there is very little mutant enzyme that reaches the lysosomes. The severity of the disease is determined by the fraction that reaches the lysosomes and has some activity there.
Test whether ER stress caused to brain cells in patients with type 2/3 Gaucher disease lead to their death through apoptosis or autophagy
Mutant glucocerebrosidase variants undergo ERAD due to their inability to properly fold. Ample evidence strongly indicates that when the amount of unfolded protein exceeds the capacity of the ERAD, ER-stress related apoptosis commences. We could show that the constant accumulation of misfolded glucocerebrosidase variants in the ER induces the unfolded protein response in cells that derived from patients with Gaucher disease. We also tested whether the chronic accumulation of misfolded glucocerebrosidase in the ER causes a calcium depletion, which may also contribute to the unfolded protein response. Our results show that this is the case, namely in Gaucher cells the ER calcium stores are smaller than those in normal cells.
We also showed that elavated intracellular cholesterol levels limit the ability of the ER quality control system to refold misfolded proteins and therefore more mutant glucocerebrosidase is destined for degradation instead of trafficking to the lysosomes.
We hypothesized that in the neuronopathic forms of Gaucher disease, the degree of unfolded mutant glucocerebrosidase in the ER of neuronal cells or microglial cells, which are derived from monocytes and therefore resemble macrophages, exceeds its capacity to degrade it and therefore the cells are destined to cell death. As explained in the project proposal, we aimed at creating a system model in which we will be able to test our model. We used neuronal dopaminergic cells, which we stably transfected with glucocerebrosidase cDNA carrying the N370S mutation and the L444P mutation, which are associated with the chronic and the neuronopathic types of Gaucher disease, respectively. We use this model system to test whether there is abnormal interaction of mutant glucocerebrosidase with proteins that are present in these cells, which may lead to their enhanced death.