Understanding the skeletal phenotype of Gaucher disease

描述（由申请人提供）：戈谢病是一种使人衰弱的溶酶体贮积症，其特征是显著的内脏肿大和致残性骨折的高风险。它是由葡萄糖脑苷脂酶（GBA 1）基因突变引起的，该突变损害了鞘脂催化剂所需的β-糖苷酶。虽然酶替代疗法（伊米苷酶）是有效的，但其对骨折风险的影响尚未完全了解。为了确定非神经元病性1型GD（GD 1）的新治疗靶点，已经尝试在小鼠中敲入突变并删除Gba 1。我们已经成功地删除Gba 1在造血和间充质细胞谱系的细胞使用Mx 1启动子。我们的Mx 1-Cre：GD 1小鼠表型几乎完全模仿人GD 1，表现出严重的肝脾肿大、血细胞减少和骨质疏松症。小鼠还显示Th 1和Th 2高细胞因子血症和免疫细胞缺陷，这可能不仅导致淋巴增生性恶性肿瘤的风险增加，而且还导致骨疾病。我们的数据进一步表明，骨质疏松症是由于成骨细胞骨形成的缺陷，而不是骨吸收。我们发现，Mx 1-Cre：GD 1小鼠基质细胞培养物中观察到的成骨细胞活力降低，通过暴露于鞘氨醇（一种在GD 1中积累的鞘脂）而重现。我们假设，尽管免疫细胞功能障碍可能会影响骨，但Mx 1-Cre：GD 1小鼠中观察到的骨质减少主要是由于鞘氨醇对成骨细胞的直接作用，从而降低骨形成。因此，在特定目标1中，我们将确定骨形成缺陷是否是自主的，如果是，哪种骨细胞-成骨细胞，骨细胞或破骨细胞-驱动它。为此，我们将分别使用Col2.3-Cre，Dmp 1-Cre和CathK-Cre小鼠删除三种细胞类型中的Gba 1。在具体目标2中，我们将通过抑制或删除葡萄糖神经酰胺合酶（Gcs）（Gba 1上游的一种酶）来降低鞘脂水平。为此，我们将向Mx 1-Cre：GD 1小鼠注射一种Gcs抑制剂--酒石酸依格列司他，并在相同细胞中平行产生缺乏Gba 1和Gcs的小鼠。值得注意的是，我们发现酒石酸依利司他降低了血清GL-1，并逆转了GD 1患者的内脏肿大和血细胞减少。最后，在特定目标3中，为了研究导致成骨细胞抑制的特定脂质，我们将降低鞘氨醇，但不降低LysoGL-1水平。我们假设，由于溶酶体外酶Gba 2将LysoGL-1转化为鞘氨醇，Gba 2缺失或AMP-DNM对其的抑制应逆转Mx 1-Cre：GD 1小鼠的骨质减少。为了进一步研究鞘氨醇和其他鞘脂对成骨细胞的作用，我们将在体外研究分化，细胞周期和凋亡。我们的研究不仅要确定GD 1中骨质减少的靶细胞和负责分子，还要确定新的治疗靶点，包括Gba 1的上游（Gcs）和下游（Gba 2），用于GD 1相关和其他常见类型的骨质疏松症。

DESCRIPTION (provided by applicant): Gaucher Disease is a debilitating lysosomal storage disorder characterized by striking visceral enlargement and a high risk of crippling fractures. It s caused by mutations in the glucocerebrosidase (GBA1) gene that impair -glycosidase, an enzyme required for sphingolipid catabolism. While enzyme replacement therapy (imiglucerase) is effective, its effects on fracture risk are not fully understood. To identify new therapeutic targets for non-neuronopathic type 1 GD (GD1), attempts have been made to knock in mutations and delete Gba1 in mice. We have successfully deleted Gba1 in cells of the hematopoietic and mesenchymal cell lineage using an Mx1 promoter. Our Mx1-Cre:GD1 mouse phenocopies human GD1 almost in its entirety, displaying severe hepatosplenomegaly, cytopenia, and osteoporosis. The mouse also displays Th1 and Th2 hypercytokinemia and immune cell defects, which might contribute not only to the increased risk of lymphoproliferative malignancy, but also to the bone disease. Our data further show that the osteoporosis is due to a defect in osteoblastic bone formation, not osteoclastic bone resorption. We find that reduced osteoblast viability noted in stromal cell cultures from Mx1-Cre:GD1 mice is recapitulated by exposure to sphingosine, a sphingolipid that accumulates in GD1. We hypothesize that, despite the immune cell dysfunction that may affect bone, the osteopenia noted in Mx1-Cre:GD1 mice arises mainly from the direct action of sphingosine on the osteoblast, thus lowering bone formation. Therefore, in Specific Aim 1, we will determine whether the bone formation defect is autonomous, and if so, which bone cell - osteoblast, osteocyte, or osteoclast - drives it. For this we will delete Gba1 in the three cell types, respectively, using Col2.3-Cre, Dmp1-Cre and CathK-Cre mice. In Specific Aim 2, we will lower sphingolipid levels by inhibiting or deleting glucosylceramide synthase (Gcs), an enzyme upstream of Gba1. For this, we will inject Mx1-Cre:GD1 mice with eliglustat tartrate, a Gcs inhibitor, and, in parallel, generate mice lacking Gba1 and Gcs in the same cells. Of note, we find that eliglustat tartrate lowers serum GL-1 and reverses the visceromegaly and cytopenia in GD1 patients. Finally, in Specific Aim 3, to hone in on the specific lipid that causes osteoblast inhibition, we will lower sphingosine, but not LysoGL-1 levels. We hypothesize that, as the extralysosomal enzyme Gba2 converts LysoGL-1 to sphingosine, Gba2 deletion or its inhibition by AMP-DNM should reverse the osteopenia in Mx1-Cre:GD1 mice. To further examine the action of sphingosine and other sphingolipids on the osteoblast, we will study differentiation, cell cycling and apoptosis in vitro. Our investigations should not only define the target cell and responsible molecule for the osteopenia in GD1, but also identify new therapeutic targets, both upstream (Gcs) and downstream (Gba2) of Gba1, for GD1-associated and other common types of osteoporosis.