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Results of a Three Year Study On Understanding the Molecular Mechanism Underlying Cell Death In NGD

In 2007, the Association awarded Prof Mia Horowitz of the Department for Cell Research and Immunology at Tel Aviv University a three-year grant to understand the molecular mechanism underlying brain cell death in neuronopathic Gaucher disease. The funds to support this research had been raised by the family of Ellie Carter who died in 2004 aged seven months; Ellie had Type 2 Gaucher disease. This research project completed in August, Professor Mia Horowitz outlines her research findings and possible ideas for further study"

Research objectives:

1. To establish a correlation between endo-H sensitivity of glucocerebrosidase (Gcase) in different cell types and nGD severity.

2. To identify interactors of mutant glucocerebrosidase variants that participate in its Endoplasmic Reticulum Associated Degradation (ERAD).

3. To test whether Endoplasmic Reticulum (ER) stress caused to brain cells Type 2 or 3 patients leads to death through apoptosis (cell death) or autophagy (degradation of a cell's own components).

The Results:

1. Our results strongly indicate that the L444P mutant GCase variant, which is associated, in homozygocity, with Type 3 Gaucher disease is retained in the Endoplasmic Reticulum (ER) and undergoes Endoplasmic Reticulum Associated Degradation (ERAD). In this process mutant molecules that enter the ER are recognized as misfolded and after several attempts to refold them they are degraded. Therefore, there is little mutant enzyme that reaches the lysosomes and has some activity there.

Unfortunately, enzyme replacement therapy (ERT) cannot reverse or alleviate neurological signs since the administered enzyme does not cross the blood brain barrier. To overcome this, enzyme enhancement therapy (EET) has been developed. EET uses small molecules, known as pharmacological chaperones (PCs), to stabilize the native conformation of a mutant enzyme as it folds in the ER, allowing more functional molecules to form and evade the ERAD pathway by instead being passed on to the ER transport machinery, resulting in increased amounts in the lysosome.

We found that Ambroxol, a known expectorant, lately described as a chaperone for mutant GCase, removes the L444P mutant GCase variant from the ER and transports it to the lysosomes, where it has some activity.

2. An association has been established between Gaucher disease (GD) and Parkinson's disease (PD). Namely, the prevalence of PD was found to be higher among GD patients than in the general population. It was also documented that the prevalence of GCase mutations among individuals suffering from Parkinsonism is higher than in the general population, suggesting that the presence of mutant GCase contributes to a vulnerability to Parkinsonism.
Parkin, mutated in autosomal recessive, juvenile form of PD, is an enzyme that plays a role in tagging proteins for degradation. As GCase mutant variants undergo different degrees of ERAD and are eliminated in the proteasome, we tested the possibility that the concurrence of GD and PD reflects an association between parkin and misfolded mutant GCase variants.

Tests indicated that mutant GCase forms were less stable in the presence of parkin, indicating that parkin mediates degradation of mutant GCase. Mutant GCase variants underwent "Degradation tagging" and proteasomal degradation in the presence of parkin.

We hypothesize that, due to its occupation with mutant GCase variants, parkin is unable to efficiently degrade its natural substrates in cells in the mid brain, involved in movement. This leads to accumulation of these substrates and death of these cells, namely, development of PD.

3 Mutant glucocerebrosidase variants undergo ERAD due to their inability to properly fold. Ample evidence indicates that when the amount of unfolded protein exceeds the capacity of the ERAD, ER-stress related apoptosis commences. ERAD, as well as the productive folding mechanism, is induced in response to ER stress, an imbalance between the load of unfolded proteins that enter the ER and the capacity of the cellular machinery that handles this load sets. These are two processes, regulated by a transcriptional program termed the Unfolded Protein Response (UPR), leading to degradation of unfolded proteins and accelerated refolding. Since UPR leads to changes in gene expression, it can be followed by testing the RNA levels of several key genes in this process.

We chose 12 different GD derived cells in tissue culture to test the level of UPR in  neuronopathic versus non-neuronopathic GD. Surprisingly; we found that in a major part of the cells studied there was UPR. However, there was no direct correlation between the level of UPR and GD severity. We still have to examine whether growth rate of the cells in tissue culture or the number of passages influence the level of UPR.

Editors Note: The Association as delighted to learn that a poster giving the details of this work was given an award at the 9th EWGGD meeting in June 2010.