Validation of Novel Pathogenic Post-Translational Modifications of Huntingtin, and of Modifying Enzymes as Therapeutic Targets for Huntington's Disease

亨廷顿蛋白的新型致病性翻译后修饰以及修饰酶作为亨廷顿病治疗靶点的验证

基本信息

批准号：
10599877
负责人：
Christopher A Ross
金额：
$ 57.38万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-04-01 至 2025-01-31
项目状态：
未结题

项目摘要

Validation of Novel Pathogenic Post-Translational Modifications of Huntingtin, and of Modifying Enzymes as Therapeutic Targets for Huntington's Disease HD is a progressive neurodegenerative disorder caused by a single gene mutation, the CAG repeat expansion in Huntingtin (Htt). The best-validated therapeutic target in HD remains Htt itself, and RNAi approaches are under development to lower mutant Htt in HD patients. However, these approaches have daunting obstacles of delivery large molecules to the CNS. Small molecule therapeutics remains an important alternative. We have identified near 40 PTMs (phosphorylation and acetylation) on endogenous Htt from human and mouse brain using quantitative proteomics. We find that the PTMs are arranged in clusters, and we have initial evidence for crosstalk among certain sites in these clusters.. Furthermore, we find that alteration of the residues for several of the PTMs abrogates mutant Htt neuronal toxicity, showing that PTMs are modulators of mutant Htt toxicity. These data indicate that identification of the enzymes catalyzing these PTMs will have potential for yielding defined molecular targets for disease-modifying therapy for HD. We now propose the next series of steps in these studies, in order to identify additional PTMs, and to identify enzymes that catalyze modifications, as potential defined molecular targets for rational therapeutics for HD. We will take advantage now of our newly developed HD iPSC-derived immortalized striatal precursor cell lines. In Aim 1, we will identify novel PTMs on Htt using our novel striatal precursor cell lines (SPNs) derived from HD iPS cells. These can be differentiated into neurons with a medium-spiny neuron phenotype. We will generate PTM-specific antibodies to high-priority PTMs, in order to evaluate localization in cellular compartments using immunofluorescence, and cell fractionation followed by Western blot, in SPNs and human brain. In Aim 2 we will identify kinases and other modifying enzymes. In Aim 3 we will confirm the role of PTMs and kinases in HD cellular pathogenesis and their validation as therapeutic targets. We will perform CRISPR/Cas9 PTM alterations for highly-ranked functional PTMs, as well as CRISPR/Cas9 knock-out of most relevant kinases in striatal precursor neuron model in collaboration with Gene Edit BioLab and Xiao and Shihua Li, who are expert at gene editing in relation to HD. Taken together, these studies will further elucidate the basic biology of mutant Htt. The identification of modifying enzymes has the potential to provide defined molecular targets for preclinical therapeutic studies.

亨廷顿的新型致病后翻译后修饰的验证，并修改酶为亨廷顿氏病的治疗靶点 HD是由单个基因突变引起的进行性神经退行性疾病，CAG重复亨廷顿（HTT）的扩张。 HD中最佳验证的治疗靶标保持HTT本身，RNAi 正在开发方法，以降低HD患者的突变HTT。但是，这些方法有向中枢神经系统的大分子递送的巨大障碍物。小分子疗法仍然是重要的选择。我们已经在人类的内源性HTT上确定了接近40个PTM（磷酸化和乙酰化）使用定量蛋白质组学和小鼠脑。我们发现PTM是在集群中排列的，我们有这些集群中某些地点之间串扰的最初证据。此外，我们发现几个PTM的残留物消除了突变的HTT神经元毒性，表明PTMS是调节剂突变的HTT毒性。这些数据表明，催化这些PTM的酶的鉴定将具有产生定义的HD疾病修饰治疗的分子靶标的潜力。我们现在提出下一个这些研究中的一系列步骤，以识别其他PTM，并确定催化的酶修改，作为HD理性疗法的潜在定义的分子靶标。我们将利用现在，我们新开发的HD IPSC衍生的永生纹状体前体细胞系的成绩。在AIM 1中，我们将确定 HTT上的新型PTM使用我们的新型纹状体前体细胞系（SPN）衍生自HD IPS细胞。这些可以用中棘神经元表型分化为神经元。我们将生成PTM特异性抗体高优先级PTM，以评估使用免疫荧光和细胞分馏，然后在SPN和人脑中进行蛋白质印迹。在AIM 2中，我们将确定激酶和其他修饰酶。在AIM 3中，我们将确认PTM和激酶在HD细胞发病机理及其中的作用验证作为治疗靶标。我们将执行CRISPR/CAS9 PTM更改，以实现高度排名的功能 PTM以及最相关激酶在纹状体前体神经元模型中的CRISPR/CAS9敲除与Gene Edit Biolab和Xiao和Shihua Li合作，他们是基因编辑的专家高清。综上所述，这些研究将进一步阐明突变体HTT的基本生物学。识别修饰酶有可能为临床前治疗研究提供定义的分子靶标。

项目成果

期刊论文数量（17）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

pS421 huntingtin modulates mitochondrial phenotypes and confers neuroprotection in an HD hiPSC model.

DOI：
10.1038/s41419-020-02983-z
发表时间：
2020-09-25
期刊：
Cell death & disease
影响因子：
9
作者：
Xu X;Ng B;Sim B;Radulescu CI;Yusof NABM;Goh WI;Lin S;Lim JSY;Cha Y;Kusko R;Kay C;Ratovitski T;Ross C;Hayden MR;Wright G;Pouladi MA
通讯作者：
Pouladi MA

Quantitative Proteomic Analysis Reveals Similarities between Huntington's Disease (HD) and Huntington's Disease-Like 2 (HDL2) Human Brains.

DOI：
10.1021/acs.jproteome.6b00448
发表时间：
2016-09-02
期刊：
Journal of proteome research
影响因子：
4.4
作者：
Ratovitski T;Chaerkady R;Kammers K;Stewart JC;Zavala A;Pletnikova O;Troncoso JC;Rudnicki DD;Margolis RL;Cole RN;Ross CA
通讯作者：
Ross CA

Elevated arteriolar cerebral blood volume in prodromal Huntington's disease.

DOI：
10.1002/mds.25591
发表时间：
2014-03
期刊：
MOVEMENT DISORDERS
影响因子：
8.6
作者：
Hua, Jun;Unschuld, Paul G.;Margolis, Russell L.;van Zijl, Peter C. M.;Ross, Christopher A.
通讯作者：
Ross, Christopher A.

Nuclear retention of full-length HTT RNA is mediated by splicing factors MBNL1 and U2AF65.

DOI：
10.1038/srep12521
发表时间：
2015-07-28
期刊：
Scientific reports
影响因子：
4.6
作者：
Sun X;Li PP;Zhu S;Cohen R;Marque LO;Ross CA;Pulst SM;Chan HY;Margolis RL;Rudnicki DD
通讯作者：
Rudnicki DD

Transgenic mouse model expressing the caspase 6 fragment of mutant huntingtin.

DOI：
10.1523/jneurosci.1305-11.2012
发表时间：
2012-01-04
期刊：
The Journal of neuroscience : the official journal of the Society for Neuroscience
影响因子：
0
作者：
Waldron-Roby E;Ratovitski T;Wang X;Jiang M;Watkin E;Arbez N;Graham RK;Hayden MR;Hou Z;Mori S;Swing D;Pletnikov M;Duan W;Tessarollo L;Ross CA
通讯作者：
Ross CA