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Parkinson's disease (PD) is the second most common neurodegenerative disease in the world, with a prevalence of about 2% in middle-aged and elderly people over 65 years old α. The vast majority of patients with PD have sporadic PD, and about 10% of patients with PD are due to genetic mutations. Mutations in the PINK1 and Parkin genes can lead to early-onset autosomal recessive PD due to loss of gene function. PINK1 is a protein kinase, and Parkin is an E3 ligase. Based on a large number of in vitro experiments, the traditional theory is that PINK1 kinase can phosphorylation at Ser65 to activate Parkin, and the two together remove damaged mitochondria and protect nerve cells through mitophagy. Although there are more than 3,300 papers related to PINK1 and Parkin, none of them have reported evidence that PINK1 can phosphorylate and activate Parkin in mammals in physiological states to participate in mitophagy. Moreover, the mouse and porcine models with PINK1/Parkin knockout could not simulate the important pathological features of neuronal cell degeneration and death in the brain of PD patients, which limited the feasibility of studying the functions of PINK1 and PARKIN in animals.
Considering that the non-human primate model is closer to humans, Xiaojiang Li's team established the world's first PINK1 gene target monkey model in 2019 after six years and demonstrated that PINK1 deletion can lead to nerve cell death in the primate brain (Yang et al., Cell Res 2019). Subsequently, Yang Weili from the team further discovered that PINK1 protein is specifically expressed in the primate brain in physiological states and maintains the survival of primate nerve cells through other important kinase functions independent of mitophagy (Yang et al., Protein Cell 2022, Chen et al., Zool Res, 2024), suggesting that the expression and function of the same disease protein in different species are different. However, the function of Parkin, the classic substrate of PINK1, in the primate brain is still unclear and needs to be explored urgently.
On October 15, 2024, the team of Weili Yang, Shihua Li and Xiaojiang Li from the Guangdong-Hong Kong-Macao Institute of Central Nervous System Regeneration of Jinan University published a paper titled "Deficiency of Parkin causes neurodegeneration and accumulation of pathological α-synuclein in" in the Journal of Clinical Investigation Research paper by monkey models. In this study, CRISPR/Cas9 technology was used to establish a model of Parkin-deficient monkeys of different ages, and it was found that Parkin deletion had no significant effect on the survival of nerve cells in the early development of the monkey brain, but with the increase of age, it would cause obvious degeneration and death of dopamine nerve cells in the substantia nigra of the monkey midbrain, decreased dopamine synthesis in the striatum and pathological pS129-α-syn aggregation (Figs. 1 and Fig. 2), which successfully simulated the important pathological features in the brain of PD patients. This is in stark contrast to the important pathology of neuronal cell degeneration and death in the brain of PD patients in the mouse model and pig model of Parkin knockout, suggesting the importance and necessity of using non-human primate models to study Parkin's function.
图1. （A）猴脑注射AAV病毒载体表达CRISPR/Cas9敲除Parkin基因。 （B， C）免疫组化染色证明在Parkin基因打靶后猴脑黑质区多巴胺神经细胞（TH）出现明显退变死亡，且在年老（25岁）猴中的神经细胞死亡更为严重。 （D）电镜结果显示，Parkin敲除可造成猴中脑黑质神经细胞的退变死亡及髓鞘退化。 （E）FDOPA PET结果显示敲低猴脑黑质Parkin可显著减少纹状体部位多巴胺的水平。 （Credit: Journal of Clinical Investigation）
图2. Parkin敲除可造成猴中脑黑质出现病理性pS129-α-syn聚集。 （A）免疫组化染色显示Parkin敲除可在猴黑质中造成pS129-α-syn表达增加，且随着衰老这种蛋白聚集更为显著。 （B）高倍镜图显示毒性α-synuclein蛋白形成路易小体。 （C）对A图的聚集体进行统计。 （D）在老年（25岁）猴脑黑质神经细胞的胞内及胞外均有病理性pS129-α-syn聚集体形成。 （Credit: Journal of Clinical Investigation）
It has been reported that Parkin Ser65 mutations can also cause PD. A large number of in vitro studies have found that PINK1 can activate Parkin by phosphorylation at Ser65, but there is no in vivo evidence that PINK1 mediates phosphorylation of Parkin in physiological states due to low expression in small animal brains and common cell lines. In this study, it was further found that knocking out the PINK1 gene significantly reduced the expression of phosphorylated Parkin (pS65-Parkin) and was accompanied by the appearance of Lewy bodies formed by the aggregation of pS129-α-syn protein, which revealed for the first time that PINK1 can phospho-Parkin in animals in physiological states The decrease in phosphorylation is associated with aggregation of pS129-α-syn protein. This provides a new molecular mechanism for PD patients with PINK1 and Parkin mutations to have similar phenotypes in clinical practice.
In order to further validate the important function of PINK1-mediated Parkin phosphorylation in monkey brain, this study mutated the Ser65 site (S65A) of wild-type Parkin protein so that it could not be phosphorylated by PINK1. The results showed that both overexpression of wild-type PINK1 and Parkin could reduce the aggregation of pathological α-synuclein protein in the brain of elderly monkeys by increasing the phosphorylation level of Parkin, thereby exerting neuroprotective effects. However, overexpression of the Parkin mutant (S65A), which cannot be activated by PINK1 phosphorylation, does not effectively clear the toxic effects of α-synuclein protein. In addition, the study also verified the phenomenon in monkey brain tissue of patients with sporadic PD, and found that the expression of phosphorylated Parkin in the brain of patients with sporadic PD was down-regulated, and the expression of soluble Parkin phosphorylated protein in the brain of normal aging monkey brain was also significantly down-regulated, suggesting that aging may be an important factor in the down-regulation of phosphorylated Parkin function and thus mediating abnormal brain function (Fig. 3). In summary, this study used primate models to reveal the important protective role of PINK1 kinase-mediated phosphorylation of Parkin protein in senescence and PD caused by mutations in PINK1 and Parkin genes, providing a new potential target for the clinical treatment of PD.
图3. 磷酸化Parkin在PD病理中的重要机制模型。 衰老、PINK1和Parkin基因突变均可导致磷酸化Parkin的减少, 从而导致包括毒性α-synuclein蛋白的累积及神经细胞死亡（Credit: Journal of Clinical Investigation）
Notably, unlike the PINK1-deficient monkey model, Parkin deletion did not have a significant effect on neuronal cell function in early brain development in monkeys, which may be due to the fact that PINK1 is also involved in the regulation of other important substrates as an upstream kinase. For example, the team also used fresh wild-type monkey brain tissue to isolate different subcellular organelles by sucrose density gradient centrifugation, and found that PINK1 and Parkin proteins have different subcellular localization (Liu et al, Neural Regen Res 2024), suggesting that PINK1 and Parkin proteins may have independent functions in addition to their functions through Parkin phosphorylation.
bibliography
1. Chen XS, Han R, Liu YT, Huang W, Wang Q, Xiong X, Zhang Y, Zhao JG, Li SH, Li XJ, Yang WL. Comparative analysis of primate and pig cells reveals primate-specific PINK1 expression and phosphorylation. Zool Res. 2024 Mar 18; 45(2):242-252.2. Liu Y, Huang W, Wen J, Xiong X, Xu T, Wang Q, Chen X, Zhao X, Li S, Li X, Yang W. Differential distribution of PINK1 and Parkin in the primate brain implies distinct roles. Neural Regen Res. 2025 Apr 1; 20(4):1124-1134.3. Yang W, Liu Y, Tu Z, Xiao C, Yan S, Ma X, Guo X, Chen X, Yin P, Yang Z, Yang S, Jiang T, Li S, Qin C, Li XJ. CRISPR/Cas9-mediated PINK1 deletion leads to neurodegeneration in rhesus monkeys. Cell Res. 2019 Apr; 29(4):334-336.4. Yang W, Guo X, Tu Z, Chen X, Han R, Liu Y, Yan S, Wang Q, Wang Z, Zhao X, Zhang Y, Xiong X, Yang H, Yin P, Wan H, Chen X, Guo J, Yan XX, Liao L, Li S, Li XJ. PINK1 kinase dysfunction triggers neurodegeneration in the primate brain without impacting mitochondrial homeostasis. Protein Cell. 2022 Jan; 13(1):26-46.5. Han R, Wang Q, Xiong X, Chen XS, Tu ZC, Li B, Zhang F, Chen CY, Pan MT, Xu T, Chen LQ, Wang ZF, Liu YT, He DJ, Guo XY, He F, Wu P, Yin P, Liu YB, Yan XX, Li SH, Li X-J, Yang W. Deficiency of Parkin causes neurodegeneration and accumulation of pathological α-synuclein in monkey models. J Clin Invest. 2024.https://www.jci.org/articles/view/179633
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