No evidence for activation of the unfolded protein response in neuronopathic models of Gaucher disease.

Gaucher disease (GD), the most common lysosomal storage disorder (LSD), is caused by defects in the activity of the lysosomal enzyme, glucocerebrosidase, resulting in intracellular accumulation of glucosylceramide (GlcCer). Neuronopathic forms, which comprise only a small percent of GD patients, are characterized by neurological impairment and neuronal cell death. Little is known about the pathways leading from GlcCer accumulation to neuronal death or dysfunction but defective calcium homeostasis appears to be one of the pathways involved. Recently, endoplasmic reticulum stress together with activation of the unfolded protein response (UPR) has been suggested to play a key role in cell death in neuronopathic forms of GD, and moreover, the UPR was proposed to be a common mediator of apoptosis in LSDs (Wei et al. (2008) Hum. Mol. Genet. 17, 469-477). We now systematically examine whether the UPR is activated in neuronal forms of GD using a selection of neuronal disease models and a combination of western blotting and semi-quantitative and quantitative real-time polymerase chain reaction. We do not find any changes in either protein or mRNA levels of a number of typical UPR markers including BiP, CHOP, XBP1, Herp and GRP58, in either cultured Gaucher neurons or astrocytes, or in brain regions from mouse models, even at late symptomatic stages. We conclude that the proposition that the UPR is a common mediator for apoptosis in all neurodegenerative LSDs needs to be re-evaluated.

Successful low-risk hematopoietic cell therapy in a mouse model of type 1 Gaucher disease.

Hematopoietic stem cell-based gene therapy offers the possibility of permanent correction for genetic disorders of the hematopoietic system. However, optimization of present protocols is required before gene therapy can be safely applied as general treatment of genetic diseases. In this study we have used a mouse model of type 1 Gaucher disease (GD) to demonstrate the feasibility of a low-risk conditioning regimen instead of standard radiation, which is associated with severe adverse effects. We first wanted to establish what level of engraftment and glucosylceramidase (GCase) activity is required to correct the pathology of the type 1 GD mouse. Our results demonstrate that a median wild-type (WT) cell engraftment of 7%, corresponding to GCase activity levels above 10 nmoles/hour and mg protein, was sufficient to reverse pathology in bone marrow and spleen in the GD mouse. Moreover, we applied nonmyeloablative doses of busulfan as a pretransplant conditioning regimen and show that even WT cell engraftment in the range of 1%-10% can confer a beneficial therapeutical outcome in this disease model. Taken together, our data provide encouraging evidence for the possibility of developing safe and efficient conditioning protocols for diseases that require only a low level of normal or gene-corrected cells for a permanent and beneficial therapeutic outcome.

Murine models of acute neuronopathic Gaucher disease.

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the glucosidase, beta, acid (GBA) gene that encodes the lysosomal enzyme glucosylceramidase (GCase). GCase deficiency leads to characteristic visceral pathology and, in some patients, lethal neurological manifestations. Here, we report the generation of mouse models with the severe neuronopathic form of GD. To circumvent the lethal skin phenotype observed in several of the previous GCase-deficient animals, we genetically engineered a mouse model with strong reduction in GCase activity in all tissues except the skin. These mice exhibit rapid motor dysfunction associated with severe neurodegeneration and apoptotic cell death within the brain, reminiscent of neuronopathic GD. In addition, we have created a second mouse model, in which GCase deficiency is restricted to neural and glial cell progenitors and progeny. These mice develop similar pathology as the first mouse model, but with a delayed onset and slower disease progression, which indicates that GCase deficiency within microglial cells that are of hematopoietic origin is not the primary determinant of the CNS pathology. These findings also demonstrate that normal microglial cells cannot rescue this neurodegenerative disease. These mouse models have significant implications for the development of therapy for patients with neuronopathic GD.

Effective cell and gene therapy in a murine model of Gaucher disease.

Gaucher disease (GD) is a lysosomal storage disorder due to an inherited deficiency in the enzyme glucosylceramidase (GCase) that causes hepatosplenomegaly, cytopenias, and bone disease as key clinical symptoms. Previous mouse models with GCase deficiency have been lethal in the perinatal period or viable without displaying the clinical features of GD. We have generated viable mice with characteristic clinical symptoms of type 1 GD by conditionally deleting GCase exons 9-11 upon postnatal induction. Both transplantation of WT bone marrow (BM) and gene therapy through retroviral transduction of BM from GD mice prevented development of disease and corrected an already established GD phenotype. The gene therapy approach generated considerably higher GCase activity than transplantation of WT BM. Strikingly, both therapeutic modalities normalized glucosylceramide levels and practically no infiltration of Gaucher cells could be observed in BM, spleen, and liver, demonstrating correction at 5-6 months after treatment. The findings demonstrate the feasibility of gene therapy for type 1 GD in vivo. Our type 1 GD mice will serve as an excellent tool in the continued efforts toward development of safe and efficient cell and gene therapy for type 1 GD.