Dysregulated phosphorylation of Rab GTPases by LRRK2 induces neurodegeneration.
Dysregulated phosphorylation of Rab GTPases by LRRK2 induces neurodegeneration.
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DOI：
10.1186/s13024-018-0240-1
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发表时间：
2018-02-13
影响因子：
15.1
通讯作者：
Lee BD
中科院分区：
文献类型：
--
作者：
Jeong GR;Jang EH;Bae JR;Jun S;Kang HC;Park CH;Shin JH;Yamamoto Y;Tanaka-Yamamoto K;Dawson VL;Dawson TM;Hur EM;Lee BD
富含亮氨酸重复序列激酶2（LRRK 2）的突变是家族性和散发性帕金森病（PD）的最常见原因。激酶活性升高与LRRK 2毒性相关，但介导神经退行性变的底物仍不明确。鉴于越来越多的证据表明LRRK 2在膜和囊泡运输中的作用，在这里，我们系统地筛选了Rab GTP酶，囊泡动力学的核心调节因子，作为LRRK 2的潜在底物，并研究了这种磷酸化在细胞和体内的功能后果。用45种纯化的人Rab GTP酶进行体外LRRK 2激酶测定以鉴定Rab家族蛋白作为LRRK 2的底物。我们通过串联质谱鉴定了磷酸化位点，并通过体外LRRK 2激酶测定和细胞中的磷酸化评估进行了确认。Rab磷酸化对神经变性的影响在原代培养物中进行了研究，并通过颅内注射表达野生型或磷酸突变体Rab 35的腺相关病毒载体（AAV）在体内进行了研究。我们的筛选揭示了LRRK 2在开关II区域中的保守苏氨酸残基处磷酸化几种Rab GTP酶，并且通过使用激酶失活的LRRK 2-D1994 A和致病性LRRK 2-G2019 S沿着与其中LRRK 2位点突变的Rab蛋白，我们验证了Rab蛋白的子集，包括Rab 35，在体外和细胞中都是LRRK 2的真实底物。我们还发现，磷酸化的Rab调节GDP/GTP结合的细胞特性。此外，在原代皮层神经元中，LRRK 2位点的突变在几个Rabs引起神经毒性，这是最严重的诱导磷酸化Rab 35。此外，将AAV-Rab 35-T72 A或AAV-Rab 35-T72 D颅内注射到黑质中实质上诱导了体内多巴胺能神经元的变性。在这里，我们表明，一个子集的Rab GTP酶是真实的基板LRRK 2在体外和细胞。我们还提供了证据表明，Rab磷酸化在LRRK 2网站的失调诱导原代神经元的神经毒性和体内多巴胺能神经元的变性。我们的研究表明，Rab GTP酶可能介导LRRK 2毒性在PD的进展。本文的在线版本（10.1186/s13024-018-0240-1）包含补充材料，可供授权用户使用。
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson’s disease (PD). Elevated kinase activity is associated with LRRK2 toxicity, but the substrates that mediate neurodegeneration remain poorly defined. Given the increasing evidence suggesting a role of LRRK2 in membrane and vesicle trafficking, here we systemically screened Rab GTPases, core regulators of vesicular dynamics, as potential substrates of LRRK2 and investigated the functional consequence of such phosphorylation in cells and in vivo. In vitro LRRK2 kinase assay with forty-five purified human Rab GTPases was performed to identify Rab family proteins as substrates of LRRK2. We identified the phosphorylation site by tandem mass-spectrometry and confirmed it by assessing phosphorylation in the in vitro LRRK2 kinase assay and in cells. Effects of Rab phosphorylation on neurodegeneration were examined in primary cultures and in vivo by intracranial injection of adeno-associated viral vectors (AAV) expressing wild-type or phosphomutants of Rab35. Our screening revealed that LRRK2 phosphorylated several Rab GTPases at a conserved threonine residue in the switch II region, and by using the kinase-inactive LRRK2-D1994A and the pathogenic LRRK2-G2019S along with Rab proteins in which the LRRK2 site was mutated, we verified that a subset of Rab proteins, including Rab35, were authentic substrates of LRRK2 both in vitro and in cells. We also showed that phosphorylation of Rab regulated GDP/GTP-binding property in cells. Moreover, in primary cortical neurons, mutation of the LRRK2 site in several Rabs caused neurotoxicity, which was most severely induced by phosphomutants of Rab35. Furthermore, intracranial injection of the AAV-Rab35 -T72A or AAV-Rab35-T72D into the substantia nigra substantially induced degeneration of dopaminergic neurons in vivo. Here we show that a subset of Rab GTPases are authentic substrates of LRRK2 both in vitro and in cells. We also provide evidence that dysregulation of Rab phosphorylation in the LRRK2 site induces neurotoxicity in primary neurons and degeneration of dopaminergic neurons in vivo. Our study suggests that Rab GTPases might mediate LRRK2 toxicity in the progression of PD. The online version of this article (10.1186/s13024-018-0240-1) contains supplementary material, which is available to authorized users.
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DOI：
10.1212/nxg.0000000000000009
发表时间：
2015-06
期刊：
Neurology. Genetics
影响因子：
--
作者：
Lesage S;Bras J;Cormier-Dequaire F;Condroyer C;Nicolas A;Darwent L;Guerreiro R;Majounie E;Federoff M;Heutink P;Wood NW;Gasser T;Hardy J;Tison F;Singleton A;Brice A;French Parkinson's Disease Genetics Study Group (PDG) and the International Parkinson's Disease Genomics Consortium (IPDGC)
通讯作者：
French Parkinson's Disease Genetics Study Group (PDG) and the International Parkinson's Disease Genomics Consortium (IPDGC)
DOI：
10.15252/embj.201591593
发表时间：
2015-11-12
期刊：
The EMBO journal
影响因子：
--
作者：
Lai YC;Kondapalli C;Lehneck R;Procter JB;Dill BD;Woodroof HI;Gourlay R;Peggie M;Macartney TJ;Corti O;Corvol JC;Campbell DG;Itzen A;Trost M;Muqit MM
通讯作者：
Muqit MM
DOI：
10.1073/pnas.1318306111
发表时间：
2014-02-18
影响因子：
11.1
作者：
Beilina, Alexandria;Rudenko, Iakov N.;Cookson, Mark R.
通讯作者：
Cookson, Mark R.
影响因子：
11.2
作者：
Biskup, Saskia;Moore, Darren J.;Dawson, Valina L.
通讯作者：
Dawson, Valina L.
影响因子：
3.5
作者：
Chiariello, M;Bruni, CB;Bucci, C
通讯作者：
Bucci, C