Understanding the skeletal phenotype of Gaucher disease

描述(申请人提供)：高谢病是一种衰弱的溶酶体储存障碍，其特征是显著的内脏肿大和严重骨折的高风险。S是由葡萄糖脑苷酶基因突变引起的，该基因损害了神经鞘糖脂分解代谢所需的糖苷酶。尽管酶替代疗法(亚硫糖苷酶)是有效的，但其对骨折风险的影响尚不完全清楚。为了确定非神经病1型GD(GD1)的新治疗靶点，已经尝试在小鼠中敲入突变和删除Gba1。我们已经成功地利用Mx1启动子在造血细胞和间充质细胞系的细胞中删除了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。小鼠还表现出Th1和Th2高细胞分裂素血症和免疫细胞缺陷，这不仅可能导致淋巴增殖性恶性肿瘤风险增加，而且还可能导致骨骼疾病。我们的数据进一步表明，骨质疏松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，在Mx1-Cre：Gd1小鼠的基质细胞培养中，成骨细胞活力的降低可以通过暴露于鞘氨醇(一种在Gd1中积累的鞘磷脂)来再现。我们推测，尽管免疫细胞功能障碍可能会影响骨骼，但在Mx1-Cre：Gd1小鼠中注意到的骨量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，在特定的目标1中，我们将确定骨形成缺陷是否是自主的，如果是的话，哪个骨细胞-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、Dmp1-Cre和KatK-Cre小鼠来删除这三种细胞类型中的Gba1。在具体目标2中，我们将通过抑制或删除Gba1上游的葡萄糖神经酰胺合成酶(GCS)来降低鞘磷脂水平。为此，我们将给Mx1-Cre：Gd1小鼠注射GCS抑制剂伊立卢斯特酒石酸盐，同时在同一细胞中产生缺乏Gba1和GCS的小鼠。值得注意的是，我们发现酒石酸立格列他可降低血清GL-1，并逆转GD1患者的内脏肿大和细胞减少。最后，在特定的目标3中，为了磨练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，当溶酶外酶Gba2将LysoGL-1转化为鞘氨醇时，Gba2的缺失或AMP-DNM的抑制应能逆转Mx1-Cre：Gd1小鼠的骨量减少。为了进一步研究鞘氨醇和其他鞘磷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骨量减少的靶细胞和负责分子，而且要确定新的治疗靶点，包括Gba1上游(Gcs)和下游(Gba2)，用于Gd1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.