​Experts comment on Protein & Cell overturning the classic theory, and Li Xiaojiang's team reveals a new mechanism for hereditary Parkinson's disease

Comments | Lu Boxun (Fudan University), Chen Shengdi (Shanghai Jiaotong University), Deng Hanxiang (Northwestern University), Tang Beisha (Central South University), editor in charge | Parkinson's Disease (PD) is the second only The second largest neurodegenerative disease of Alzheimer's disease, the global incidence rate is as high as 1-2% in people over 65 years old
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Among them, PINK1 gene mutation can cause juvenile Parkinson's disease due to loss of function
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For a long time, Parkinson's disease has been thought to be related to damage to the organelles (mitochondria) that supply energy in cells.
PINK1, as a mitochondrial membrane protein, maintains normal mitochondria and protects nerve cells
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This classic theory is based on the fact that damaged mitochondria in in vitro cell culture experiments can recruit full-length PINK1 protein to activate Parkin and ubiquitinase to form a complex that binds to damaged mitochondria so that they can be cleared by lysosomes in time (Figure 1)
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This process is defined as mitochondrial autophagy to maintain mitochondrial homeostasis and normal function, and to protect nerve cells under different pathological conditions
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Therefore, the expression and function of PINK1 have long been considered to be closely related to mitochondrial autophagy
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Figure 1.
The classic theory that PINK1 is involved in mitochondrial autophagy
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On November 20, 2021, the Li Xiaojiang team of the Guangdong-Hong Kong-Macao Central Nervous Regeneration Institute of Jinan University published a research article (Research Article) PINK1 kinase dysfunction triggers neurodegeneration in the primate brain without impacting mitochondrial homeostasis in Protein & Cell.
The dysfunction of PINK1 kinase in the brain-like brain can cause neurodegeneration without affecting mitochondrial homeostasis)
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This study uses gene-edited monkey models and dead human brain tissue to deeply study the expression and function of Parkinson’s disease-causing gene PINK1, overturning the long-standing classic theory based on in vitro and small animal experiments, and providing a treatment for Parkinson’s disease.
New ideas and basis have been established
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Animal models are important tools for research on disease mechanism and clinical translation
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However, none of the reported Pink1 knockout mouse models showed pathological features and mitochondrial damage similar to the obvious neurodegeneration in the patient's brain
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The traditional theory believes that under normal physiological conditions, the expression level of PINK1 is extremely low.
Only under specific mitochondrial stress, the endogenous PINK1 expression level increases and is mainly located in the outer mitochondrial membrane
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The Li Xiaojiang team of the Guangdong-Hong Kong-Macao Central Nervous Regeneration Institute of Jinan University, together with Professor Qin Chuan and Professor Liu Yunbo of the Institute of Medical Laboratory Animals of the Chinese Academy of Medical Sciences, established the world's first PINK1 mutant monkey model in 2019 using embryonic CRISPR/Cas9 gene editing technology.
It was found that PINK1 mutation can cause severe neuronal degeneration and death in monkey brains (Yang et al.
, Cell Res.
29:334-336, 2019), which is consistent with the Pink1 knockout mouse model without obvious neuronal death in old age The features formed a sharp contrast
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Why does knockout of the same gene in different species make such a huge difference? After in-depth research, Li Xiaojiang's team found that PINK1 protein is abundantly expressed in primate brains, but it is difficult to detect in mice (Figure 2)
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Figure 2.
The expression of PINK1 protein in mice, monkeys and humans.
Further studies have found that PINK1 protein is not enriched in mitochondria in the primate brain, but mainly exists in the cytoplasm as a kinase
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Therefore, the survival of primate nerve cells does not depend on the regulation of PINK1 on mitochondria as described in the classic theory, but PINK1 acts as a kinase to maintain the survival of nerve cells by phosphorylating proteins related to nerve cell function
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This main function is completely different from the PINK1-mediated mitochondrial autophagy observed through mitochondrial stress in a large number of in vitro cell culture experiments (Figure 3)
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Figure 3.
PINK1 in primate brain tissue mainly acts as a kinase phosphorylation protein to maintain the survival of nerve cells, while PINK1 in cultured cells in vitro mainly exerts mitochondrial autophagy function after mitochondrial stress
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This discovery overturns the traditional theory based on a large number of in vitro and small animal experiments for a long time, revealing that the main function of PINK1 in primate brain tissue is to phosphorylate key neuroproteins, rather than the mitochondrial self-autonomy in the classical theory.
Phagocytosis provides new ideas and basis for the treatment of Parkinson's disease
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Professor Junying Yuan, a member of the American Academy of Sciences, wrote an article in the journal at the same time commenting that the study challenges the theory that PINK1 maintains mitochondrial function and provides sufficient evidence that it is necessary to explore the non-mitochondrial function of PINK1 in Parkinson's disease
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In recent years, Li Xiaojiang’s team discovered pigs (Yan et al.
, Cell, 173(4):989-1002, 2018) and monkey models (Yang et al.
, Cell Res.
29:334-336, 2019; Yin et al.
, Acta Neuropathol, 137(6):919-937, 2019) can better simulate the typical pathological changes of neurodegenerative diseases.
This study further confirms that the use of large animal models can more accurately reveal the disease mechanism and find effective Treatment methods
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The first author and co-corresponding author of the paper is researcher Yang Weili from Jinan University
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Associate researcher Guo Xiangyu, researcher Tu Zhuchi and Professor Li Shihua of Jinan University also participated in the research of this project
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The team of Professor Lujian Liao from East China Normal University has made important contributions to the analysis of protein phosphorylation
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Professor Li Xiaojiang is the co-corresponding author
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Original link: https://doi.
org/10.
1007/s13238-021-00888-x https://link.
springer.
com/article/10.
1007/s13238-021-00889-w Expert comment on Lu Boxun: (Life of Fudan University Professor of the Academy of Sciences, neurodegenerative disease research expert): Good research work often solves an important scientific problem, while epoch-making research work may change the concept of scientists in one field or even multiple fields and open up new research fields
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This research paper recently published by Professor Li Xiaojiang's team in Protein & Cell undoubtedly has the potential to become an epoch-making research work
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At present, there are about 120,000 papers on Parkinson’s disease, about 6,000 papers on mitochondrial autophagy, about 3,000 papers on PINK1, and up to 2 million papers on mouse models.
This paper may have ideas about scientists in the above fields.
All have an impact
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Speaking of PINK1 and Parkinson’s disease, the first thing scientists think of is that it participates in the function of mitochondrial autophagy on the mitochondria, thereby participating in the occurrence and development of Parkinson’s disease; the paper reveals that it mainly plays a role in the cytoplasm through phosphorylation of various proteins.
It completely overturns the above viewpoints, which may have a profound impact on the three research areas of Parkinson's disease, mitochondrial autophagy, and PINK1 function
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What's important is that such findings were made in primate models, and such research is basically impossible in mouse models
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Since the early 1990s, the mouse model has become the most important animal model for biological research, and there are indeed many milestone discoveries derived from mouse model experiments
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In recent years, with the advancement of technology, large animal models including primate models have begun to emerge, and it is believed that they may be able to better simulate human diseases and physiology
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However, at the molecular level, mouse models are generally considered sufficiently conservative to be used to study the core molecular mechanisms of physiological processes or important diseases
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However, studies on PINK1 mutant monkey models revealed that the effects of primate PINK1 mutations are not only very different from mouse Pink1 in terms of tissue phenotype (Yang et al.
, Cell Res.
29:334-336, 2019), even in The molecular mechanism and intracellular localization may be fundamentally different from mouse Pink1
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This is worth thinking about every scientist who uses mouse models
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Of course, to become truly epoch-making research requires more accumulation and time testing
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This paper opens a new field to study the function, mechanism and pathological significance of PINK1 phosphorylation of key neuroproteins in the cytoplasm
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There are many unsolved scientific questions here, such as which phosphorylated substrates are the most important? How to be regulated by PINK1? What is its pathological significance? In addition, why is there such a fundamental difference between primate PINK1 and mouse Pink1? How is its structure different? Or is there any difference in its regulation, post-translational modification, and interacting proteins that caused such a difference? Solving the above problems may guide us to really figure out when we can use mouse models and when we need to use primates or other large animal models, so as to better construct and use animal models
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I look forward to more breakthroughs in the related fields of Professor Li Xiaojiang's team
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Chen Shengdi (Professor of Ruijin Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Parkinson’s Disease Research Expert): A large number of in vitro studies have found that PINK1 protein can participate in mitochondrial autophagy and the regulation of mitochondrial function with Parkin, another pathogenic gene of Parkinson’s disease (PD) However, this traditional theory lacks evidence from in vivo experiments
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The Pink1 knockout mouse model does not show the pathological features of neuronal cell death in the brain of PD patients, and it is difficult to detect endogenous Pink1 in the brain of the mouse
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In 2018, another study reported that Pink1 knockout mice did not affect mitochondrial autophagy under physiological conditions (McWilliams et al.
, Cell Metab.
2018 27(2):439-449), which led to a key question: PINK1 What is the main function? Does it mainly act on mitochondrial autophagy and maintain mitochondrial homeostasis? To solve this key question, an animal model that can simulate the neuropathological characteristics of patients with PINK1 mutations is urgently needed
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Li Xiaojiang’s team has been focusing on using large animal models to study neurodegenerative diseases in the past ten years.
In 2019, they established a PINK1 mutant monkey model through embryonic CRISPR/Cas9 targeting (Yang et.
al.
, Cell Res.
29(4) ):334-336, 2019), it was found that loss of PINK1 can cause severe neurodegenerative death in the monkey brain
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The team’s article published in Protein & Cell is an important result of their further research on the function of PINK1 in the primate brain
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The study first found that PINK1 is expressed abundantly in primate brains in the form of kinases, but it is difficult to detect its expression in mouse models
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At the same time, endogenous PINK1 kinase in monkey brain is mainly expressed in the cytoplasm without significant co-localization with mitochondria, and the death of nerve cells caused by PINK1 deletion is not accompanied by obvious mitochondrial dysfunction
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More importantly, the loss of PINK1 caused a large amount of neuronal cell protein phosphorylation to decrease and lead to neuronal cell death
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It should be said that the results of this research challenge the traditional theory that PINK1 regulates mitochondrial autophagy discovered in a large number of in vitro experiments
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Small animal models have played a very important role in life science and disease research, elucidating a series of major biological laws and common pathways for molecular regulation
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In recent years, the continuous development of technologies such as CRISPR/Cas9 has provided us with a good tool for studying the occurrence and development of diseases and biological laws using species that are closer to humans in evolution
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It is not easy to use monkeys to build disease models.
The time period is too long, the price is expensive, and the workload is huge.
However, the research of Li Xiaojiang's team also makes us think: Some disease-causing genes have different functions in human and small animal models
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Obviously, the study of the same pathogenic gene PINK1 causing such significant pathological differences in mouse and monkey models will provide us with a good reference for studying other important pathogenic genes: that is, mice cannot simulate the pathological characteristics of patients.
Disease genes may need to consider species differences and require in-depth research using large animal models
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Han-Xiang Deng (Professor of Northwestern University School of Medicine, Parkinson’s Disease Researcher): Both PINK1 and Parkin gene mutations can cause Parkinson’s disease, and these two proteins are believed to maintain mitochondria through mitochondrial autophagy Homeostasis protects nerve cells
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However, this classic theory is mainly based on the results of experiments with damaged mitochondria in cultured cells in vitro
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The reported PINK1 knockout mouse model does not have obvious characteristics of nerve cell death, nor does it affect the basic function of mitochondrial autophagy (McWilliams et al.
, Cell Metab.
2018 27(2):439-449)
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Therefore, the in vivo function of PINK1 is still unclear
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The study published in Protein & Cell used a non-human primate model to reveal the non-mitochondrial function of PINK1 for the first time
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The results of this research have at least two meanings: one is that non-human primate models can reveal important pathological changes that cannot be simulated by dyskinetic models; the other is that the same disease-causing gene can have different functions and cause different pathological changes in different species
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Therefore, the research results also have important reference significance for the study of other important diseases
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 Since the Parkin-deficient mouse model has no obvious nerve cell death, the next important question is whether Parkin loss can also cause pathological changes similar to those in the patient's brain in non-human primates
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Tang Beisha (Professor of Xiangya Hospital of Central South University, Parkinson's disease research expert): Parkinson's disease (PD) is a common neurodegenerative disease, its etiology and pathogenesis are still unclear, genetic and environmental factors It is closely related to its onset
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Has reported that more than 20 genes that cause familial PD, such as Parkin, PINK1, DJ-1 and so on
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As a mitochondrial kinase, PINK1 protein can participate in mitochondrial autophagy and mitochondrial quality regulation, and it can also regulate cell functions through phosphorylation of a variety of proteins
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PINK1 gene is one of the common autosomal recessive PD pathogenic genes.
Its mutation can lead to loss of PINK1 protein function and participate in the occurrence of PD.
The mutation forms include missense mutations, nonsense mutations, frameshift mutations and exon deletions, etc.
The age of onset of PD patients with PINK1 gene mutations is mostly 20-40 years old, and some patients’ age of onset is earlier than 20 years old.
Neuropathological examinations confirmed that PD patients with PINK1 gene mutations have lost dopaminergic neurons in the substantia nigra of the midbrain, but Louie was small.
Body formation is very rare
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Since the discovery that PINK1 gene mutation is associated with the pathogenesis of PD, researchers have been exploring its specific pathogenic mechanism
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In 2019, Xiaojiang Li's team used CRISPR/Cas9 technology to establish a non-human primate monkey model with a mutation in the PINK1 gene (Yang et.
al.
, Cell Res.
2019), and found that the loss of PINK1 in the embryonic stage can lead to the appearance of monkey brains Severe neuronal degeneration and death, and Pink1 knockout mice did not show obvious neuronal degeneration and death even in old age
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Recently, Li Xiaojiang’s laboratory published a study in Protein & Cell (Yang et.
al, Protein & Cell, 2021), which proved for the first time in a non-human primate monkey model that PINK1 deletion does not affect mitochondrial autophagy, but it can affect protein Phosphorylation causes neuron death.
This finding is in sharp contrast to the traditional theory that PINK1 regulates mitochondrial autophagy
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The reported PINK1 knockout pig model (Zhou et al.
, Cell Mol Life Sci.
2015; Wang et al.
, Sci Rep, 2016) also did not show obvious neuronal disease, so the results of this study imply that the PINK1 protein It may be a specific functional protein in the primate brain, which is of great significance for revealing the function of PINK1 protein
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In addition, the transgenic monkey model used in this study is a large fragment deletion mutation of the PINK1 gene.
The neuron death caused by the deletion mutation indicates that the PINK1 protein plays a vital role in neuron survival.
Of course, considering the PINK1 gene Point mutations are more common in PD patients.
In follow-up work, for example, a non-human primate monkey model with point mutations in the PINK1 gene can be established to explore whether point mutations in the PINK1 gene can cause neuronal death, and to further study the PINK1 gene The molecular mechanism of neuronal death caused by point mutations will provide more theoretical basis for clinical diagnosis and precise treatment
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