CNS in Congenital DM1: Pathogenesis and Therapeutic Opportunities

强直性肌营养不良1型（DM 1）是一种复杂的疾病，影响许多器官，包括骨骼肌， 中枢神经系统（CNS）。在DM 1领域的大多数机制研究都集中在肌肉和非常少的是已知的DM 1中枢神经系统功能障碍的分子发病机制。DM 1是由扩展的CUG重复序列引起的，其错误调节两个家族的RNA结合蛋白MBNL和CUGBP（CELF）。这两个家族的蛋白质在许多组织中表达，包括CNS。CUGBP 1活性受GSK 3-cyclin D3-CDK 4依赖性磷酸化调节。在DM 1中，CUG重复序列通过增加GSK 3激酶减少CUGBP 1在S302处的磷酸化，从而将活性CUGBP 1转化为无活性的un-pS302-CUGBP 1（称为CUGBP 1阻遏物或CUGBP 1 REP），因为它阻断应激颗粒中mRNA的翻译。我们发现GSK 3在先天性DM 1（CDM 1）小鼠（DMSXL模型）的脑中增加。CUGBP 1 REP在DMSXL小鼠的大脑中也升高。用GSK 3抑制剂tideglusib（TG）对DMSXL小鼠进行产前治疗，提高了发育不全的DMSXL小鼠的存活率，改善了它们的生长和力量，并减轻了它们的焦虑。这些发现表明，GSK 3抑制剂是通过纠正脑中GSK 3-CUGBP 1 REP通路来减少CDM 1中CNS缺陷的良好候选药物。为了支持这一假设，最近针对成人CDM 1的IIa期临床试验表明，TG对CDM 1患者的认知功能具有有益作用。由于GSK 3在脑内有多种底物，GSK 3的抑制剂可能影响多种途径。该项目将确定GSK 3-CUGBP 1 REP通路在CDM 1 CNS发病机制中的作用（目的1）。我们将通过分析与新小鼠模型杂交的DMSXL小鼠来解决这个问题，在新小鼠模型中，CUGBP 1（S302）的GSK 3-细胞周期蛋白D3-CDK 4位点突变（S302 A敲入模型）。将DMSXL小鼠脑中被突变体DMPK mRNA破坏的分子途径与S302 A小鼠脑中被CUGBP 1 REP破坏的分子途径进行比较。我们发现，GSK 3的抑制剂TG减少了来自CDM 1患者的骨骼肌细胞前体中的突变DMPK mRNA，纠正了成肌细胞中的剪接事件。为了确定TG在CNS中是否具有与CDM 1相同的功能，我们将测试TG是否减少DMSXL小鼠脑中的突变体DMPK mRNA，使脑特异性mRNA的剪接正常化（Aim 2）。用TG对DMSXL小鼠进行产前治疗的积极长期效果表明，早期纠正CDM 1中的GSK 3对于改善CDM 1患者的发育至关重要。该项目的目的3将测试用TG进行的短期产后治疗是否对DMSXL小鼠的表型具有长期的积极影响。还将在DMSXL小鼠中检查TG的治疗效果，在幼年和成年时进行治疗。所产生的知识对于开发基于GSK 3的CDM 1 CNS功能障碍疗法至关重要。

Myotonic Dystrophy type 1 (DM1) is a complex disease affecting many organs including skeletal muscle and the Central Nervous System (CNS). The majority of mechanistic studies in the DM1 field have been focused on muscle and very little is known about the molecular pathogenesis of CNS dysfunction in DM1. DM1 is caused by the expanded CUG repeats which misregulate RNA-binding proteins of two families, MBNL and CUGBP (CELF). Proteins of both families are expressed in many tissues, including the CNS. CUGBP1 activity is regulated by GSK3-cyclin D3-CDK4 dependent phosphorylation. In DM1, CUG repeats reduce phosphorylation of CUGBP1 at S302 via increase of GSK3 kinase, thereby converting active CUGBP1 into inactive un-pS302-CUGBP1 (termed CUGBP1 repressor or CUGBP1REP) because it blocks translation of mRNAs in Stress Granules. We found that GSK3 is increased in the brains of congenital DM1 (CDM1) mice (DMSXL model). CUGBP1REP is also elevated in the brains of DMSXL mice. The prenatal treatment of DMSXL mice with the inhibitor of GSK3, tideglusib (TG), increases survival rate of underdeveloped DMSXL mice, improves their growth and strength and reduces their anxiety. These findings suggest that the inhibitors of GSK3 are good candidate drugs to reduce CNS defects in CDM1 via correction of GSK3-CUGBP1REP pathway in brain. In support of this hypothesis, recent Phase IIa clinical trial for adult CDM1 showed that TG has beneficial effect on cognitive function in patients with CDM1. Since GSK3 has many substrates in brain, the inhibitors of GSK3 might affect many pathways. This project will determine the contribution of the GSK3-CUGBP1REP pathway in CNS pathogenesis of CDM1 (Aim 1). We will address this question by analysis of DMSXL mice crossed with a new mouse model in which the GSK3-cyclin D3-CDK4 site of CUGBP1 (S302) is mutated (S302A knock in model). The molecular pathways disrupted by the mutant DMPK mRNA in the brains of DMSXL mice will be compared to those disrupted by CUGBP1REP in the brains of S302A mice. We found that the inhibitor of GSK3, TG, reduces the mutant DMPK mRNA in skeletal muscle cell precursors from patients with CDM1, correcting splicing events in myoblasts. To determine if TG has the same function in CNS of CDM1, we will test if TG reduces the mutant DMPK mRNA in the brains of DMSXL mice, normalizing splicing of brain-specific mRNAs (Aim 2). Positive long-term effect of the prenatal treatment of DMSXL mice with TG suggests that early correction of GSK3 in CDM1 is critical for the improved development of CDM1 patients. Aim 3 of the project will test if short postnatal treatments with TG have a long-term positive effect on phenotype of DMSXL mice. The therapeutic effect of TG will be also examined in DMSXL mice, treated at young age and in adulthood. The knowledge generated is critical for the development of the GSK3 based therapies of CNS dysfunction in CDM1.