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性疾病，其特征是显著的内脏增大和致残性骨折的高风险。它是由葡萄糖脑苷酶（GBA1）基因突变引起的，该基因会损害-糖苷酶，而-糖苷酶是鞘脂分解代谢所必需的酶。虽然酶替代疗法（imiglucerase）是有效的，但其对骨折风险的影响尚不完全清楚。为了确定非神经性1型GD （GD1）的新治疗靶点，已经尝试在小鼠中敲入突变并删除Gba1。我们使用Mx1启动子成功地删除了造血细胞和间充质细胞谱系中的Gba1。我们的Mx1-Cre:GD1小鼠几乎完全复制了人类GD1，表现出严重的肝脾肿大、细胞减少和骨质疏松症。小鼠还表现出Th1和Th2高细胞分裂血症和免疫细胞缺陷，这不仅可能导致淋巴增生性恶性肿瘤的风险增加，而且可能导致骨病。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，而不是破骨细胞骨吸收。我们发现，Mx1-Cre:GD1小鼠基质细胞培养中发现的成骨细胞活力降低，可以通过暴露于鞘脂（一种在GD1中积累的鞘脂）来重现。我们推测，尽管免疫细胞功能障碍可能影响骨骼，但Mx1-Cre:GD1小鼠的骨质减少主要是由于鞘氨醇对成骨细胞的直接作用，从而降低了骨形成。因此，在Specific Aim 1中，我们将确定骨形成缺陷是否是自主的，如果是，是哪种骨细胞——成骨细胞、骨细胞还是破骨细胞——驱动它。为此，我们将在Col2.3-Cre， Dmp1-Cre和CathK-Cre小鼠中分别删除三种细胞类型中的Gba1。在Specific Aim 2中，我们将通过抑制或删除葡萄糖神经酰胺合成酶（Gcs）来降低鞘脂水平，Gcs是Gba1上游的一种酶。为此，我们将给Mx1-Cre:GD1小鼠注射Gcs抑制剂——酒石酸依利司他，同时在相同的细胞中产生缺乏Gba1和Gcs的小鼠。值得注意的是，我们发现酒石酸依利司他降低血清GL-1，逆转GD1患者的内脏肿大和细胞减少。最后，在Specific Aim 3中，为了深入研究导致成骨细胞抑制的特定脂质，我们将降低鞘氨醇水平，但不降低LysoGL-1水平。我们推测，由于溶酶体外酶Gba2将LysoGL-1转化为鞘氨醇，Gba2的缺失或AMP-DNM对其的抑制应能逆转Mx1-Cre:GD1小鼠的骨质减少。为了进一步研究鞘磷脂和其他鞘脂对成骨细胞的作用，我们将在体外研究成骨细胞的分化、细胞周期和凋亡。我们的研究不仅要确定GD1中骨质减少的靶细胞和相关分子，还要确定Gba1的上游（Gcs）和下游（Gba2）治疗GD1相关骨质疏松症和其他常见骨质疏松症的新靶点。

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.