Mechanism of removal of microglial debris by Nat Comms

A debris removal of microglia is an important and neglected biological process

Microglia are important immune cells in the central nervous system (CNS) and play a key role
in multiple physiological and pathological processes such as CNS development, homeostasis maintenance, and neurodegeneration.
As specialized phagocytes within the CNS, microglia perform functions such as removing dead cell debris, dendritic spines, extracellular matrix, and invading pathogens through phagocytosis1-6
.
When cell debris is not removed in time, it will cause CNS dysfunction, including an increase in the level of inflammation7,8
.
Therefore, the efficient phagocytosis of microglia is critical for maintaining CNS homeostasis and ^{function1-3,6}
.

However, unlike nerve cells, which are maintained throughout adulthood, microglia undergo continuous turnover throughout the
body's life cycle.
During this process, senescent microglial death is accompanied by the regeneration of new microglia9-13
.
Microglia regenerate through self-renewal and are coupled to the death of senescent cells, thereby maintaining a stable number of microglia14
.
In a steady-state scenario, approximately 30% of microglia are replaced each year in humans and ^mice13,15
.
In other words, nearly one-third of the microglia in the normal brain die
each year.
In disease states, microglia are replaced more ^quickly15,16
.
If the debris produced by dead microglia is not removed in time, it will accumulate in the brain, causing dysfunction of the CNS and accelerating the pathological process of neurodegeneration7,8
.
In addition, in recent years, it has been found that more than 99% of microglia in the brain can be quickly killed in a short period of time by inhibiting the colony-stimulating factor 1 receptor (CSF1R
).
However, this process is not accompanied by a large accumulation of cellular debris, and inflammation is not ^{detectable17-20}
.
It can be seen that there is an efficient mechanism
for removing dead microglia debris in the body.
Therefore, studying how microglial debris is cleared is critical
to understanding the homeostasis maintenance of the CNS and the mechanism of neurodegeneration.

Research on the pathways and mechanisms of microglial debris removal in the field has been neglected for a long time, mainly for three reasons: (1) Microglia themselves assume the role of professional phagocytes, and researchers usually focus on how microglia engulf the debris produced after the death of other types of cells, while ignoring how the debris after the death of microglia itself is removed
。 (2) If the cell debris generated by the replacement of microglia cannot be removed, a large amount of debris will accumulate
over a long period of time.
However, in steady-state conditions, the real-time replacement rate of microglia is relatively slow, with only about 0.
1% of microglia dying every ^day13,15
.
The low real-time death rate of microglia results in less microglial debris in the brain, making it inconvenient to screen and study
cell types that engulf microglial debris.
(3) It has been speculated that the fragments of microglia may be cleaned
up by phagocytosis of microglia.
However, microglial debris carries molecular markers of normal microglia (such as IBA1 and CX3CR1), and it is difficult to distinguish the same type of cell fragments
engulfed in microglia by traditional in vivo experimental methods.
For these three main reasons, the study of this important biological process of microglial debris removal has long been neglected
.

The C4b-mediated pathway of microglial debris is engulfed by astrocytes and degraded by the non-classical autophagy pathway

Peng Bo's team at Fudan University has long focused on the research of
microglia replacement.
The team's previous series of research work elucidated the origin and migration mechanism of neomicroglia during the replacement of microglia (Figure 1) (Nature Neuroscience, 2018 and Cell Discovery, 2018)17,20, and developed three different microglial replacement strategies based on this (Figure 1) (Cell Reports, 2020)21.
It provides new treatment ideas and clinically achievable solutions
for neurological diseases.
In addition, the team further found that NeuroD1 mediates microglia apoptosis through the BCL2 pathway, suggesting that this pathway can be used as a switch-off control to enhance the safety of microglia replacement therapy (Neuron, 2021) (Figure 1)22
.
Based on its previous research, the team used a variety of research methods to further explore the mechanism
of removal of dead cell debris during the replacement of microglia 。 The research results were published in Nature as Microglial debris is cleared by astrocytes via C4b-facilitated phagocytosis and degraded via RUBICON-dependent noncanonical autophagy in mice Communications (Figure 1-2) (Nature Communications, 2022) 23
.

Fig.
1 The academic achievements
of Peng Bo's team around microglia replacement in recent years.

Figure 2 The first page of the paper

Due to the slower turnover rate of microglia under physiological conditions, very little microglial debris is produced, which is not conducive to screening and observation
of cell types that perform debris phagocytosis.
Therefore, Peng's team first killed microglia in the brain by CSF1R inhibitor PLX5622, which produced a large number of cell debris in a short period of time to the main cell types in the central nervous system (including astrocytes, Müller glial cells, oligodendrocytes and their precursor cells, pericytes, smooth muscle cells, neuronal cells, neural stem cells, CNS macrophages) for observation and screening
.
The team found that only astrocytes (brain, retina and spinal cord) and Müller glial cells (a unique class of glial cells within the retina) exhibited the ability
to engulf microglial debris at this time 。 Furthermore, by observing the phagocytosis of astrocytes on microglial fragments produced under natural replacement in homeostasis and disease states, the researchers found that their phagocyte rate was positively correlated with the microglial turnover rate in different regions, which proved that astrocytes have important physiological and pathological significance
for the phagocytosis of microglial fragments.

In the next step, the research team sparsely labeled P2Y12-CreER-GFP::Ai14 and CX3CR1-CreER::Ai14 with low-concentration tamoxifen as a way to explore whether microglia, as specialized phagocytes in the brain, can remove cell debris
produced by the death of the same type of cells 。 The team found that under in vivo conditions, microglia could not engulf the debris produced by dead microglia (either in homeostasis or artificially induced microglial debris).

Subsequently, through in vitro experiments, the team found that microglia have the ability to
engulf the same type of cell debris.
However, due to distance-dependent competition under in vivo conditions, this process does not occur
under in vivo conditions.
Thus, the team demonstrated that microglial debris in the brain is cleared by astrocytes (Figure 2).

Further, the team explored the mechanism by which astrocytes engulf and degrade microglial debris, and found that complement C4b promotes astrocyte phagocytosis of microglial debris
.
IN ASTROCYTES, MICROGLIAL DEBRIS IS DEGRADED BY RUBICON-DEPENDENT NONCLASSICAL AUTOPHAGY PATHWAYS, ALSO KNOWN AS LC3-ASSOCIATED ENDOCYTOSIS (FIGURE 2).

Fig.
2 Schematic diagram
of the removal mechanism of microglial debris.

Fig.
3 Astrocytes and Müller glial cells eating hot pot (microglial fragments).

Professor Peng Bo of Fudan University is the corresponding author of this paper, and Dr.
Zhou Tian is the first author
of the team.
This research was supported by Academician Duan Shumin of Fudan University/Zhejiang University, Academician Gao Tianming of Southern Medical University, Dean Mao Ying of Huashan Hospital, Professor Rao Yanxia of Fudan University, Professor Li Dali of East China Normal University, Professor Yuan Lifei of the Affiliated Mental Health Center of Shanghai Jiaotong University, Professor Peng Jiyun of Nanchang University, Professor Mei Xifan of Jinzhou Medical University, Professor Ji Shengjian of SUSTech, and Professor Shu Yousheng, Professor Guo Fanwei and Professor Lu Wei of Fudan University
。 The paper was completed with the support of the China Brain Program Major Project, the National Natural Science Foundation of China, the Science and Technology Leading Talent Team of the Ministry of Education, the Shanghai Basic Research Academic Special Zone, and the Shanghai Outstanding Academic Leader.

The full text of the paper style="font-size: 16px;line-height: 18.
4px" _istranslated="1">
.

Peng Bo's research group currently has young researchers (professors), young associate researchers (associate professors) and postdoctoral fellows, and welcomes researchers/students who are passionate about scientific research and have academic ideals to join
.
Applicants should send a detailed and complete resume (both in Chinese and English) to peng@fudan.
edu.
cn by email, indicating the name + position
applied for.
Young associate researchers (associate professors) and postdoctoral applicants should provide the contact information
of 2~3 referees.

Corresponding author Professor Peng Bo introduced:

National Youqing, Chief Scientist of the Major Project of Science and Technology Innovation 2030 Brain Science and Brain-like Research (China Brain Program), Head of the Science and Technology Leading Talent Team of the Ministry of Education, Shanghai Excellent Academic Leader (Youth), Doctoral Supervisor, Assistant
to the President of the Institute of Brain Science Translation.
From 2004 to 2008, he studied at Huazhong University of Science and Technology and obtained a bachelor's degree
in biotechnology.
From 2008 to 2011, he studied neurobiology
at the Institute of Neuroscience, Chinese Academy of Sciences.
He graduated from the University of Hong Kong with a Ph.
D.
degree in 2015.

In December 2015, he established an independent neuroimmunology laboratory at the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, and served as the leader of the research group (PI).

He joined Fudan University
in September 2019.

Peng's research group focuses on the replacement of microglia in the central nervous system (including cell aging, death, and regeneration), cell reprogramming, and new strategies for intervention and treatment with the idea of microglial replacement/transplantation in a variety of neurodegenerative diseases.

In addition, the research group focused on the related mechanism of microglia in the maintenance of central nervous system
homeostasis.
Since the establishment of the research group, the main results have been published as corresponding authors in important journals such as Nature Neuroscience, Neuron, Cell Reports, Nature Communications, eLife and Cell Discovery
.
The research work of Peng's group was selected for the 2018 Year In Review: Neuroimmunology 2018 by Nature Reviews Immunology, a global total 5 jobs were selected
.
Since 2020, Peng Bo has been continuously rated as a highly cited scholar
in China by ELSEVIER.
In addition, Peng Bo has served as a guest reviewer
for several important academic journals, including Nature Neuroscience and Nature Aging.

Attached: Representative papers published by Peng Bo's research group in the past four years

(1) Rao Y.
*, Du S.
, Yang B.
, Wang Y.
, Li Y.
, Li R.
, Zhou T.
, Du X.
, He Y.
, Wang Y.
, Zhou X.
, Yuan T.
-F.
*, Mao Y.
* and Peng B.
* (2021) NeuroD1 induces microglial apoptosis and cannot induce microglia-to-neuron cross-lineage reprogramming, Neuron, 109.

(2) Huang Y.
^#, Xu, Z.
^# , Xiong S.
, Sun F.
, Qin G.
, Hu G.
, Wang J.
, Zhao L.
, Liang Y.
-X.
, Wu T.
, Lu Z.
, Humayun M.
S.
, So K.
-F.
, Pan Y.
, Li N.
, Yuan T.
-F.
*, Rao Y.
* and Peng B.
* (2018).
Repopulated microglia are solely derived from the proliferation of residual microglia after acute depletion.
Nature Neuroscience 21, 530-540.

(3) Xu Z.
^#, Rao Y.
^#, Huang Y.
, Zhou T.
, Feng R.
, Xiong S.
, Yuan T.
F.
, Qin S.
, Lu Y.
, Zhou X.
, Li X.
, Qin B.
, Mao Y.
, and Peng B.
* (2020).
Efficient strategies for microglia replacement in the central nervous system.
 Cell Reports 32, 108041.

(4) Huang Y.
^#, Xu Z.
^#, Xiong S.
, Qin G.
, Sun F.
, Yang J.
, Yuan T.
F.
, Zhao L.
, Wang K.
, Liang Y.
X.
, Fu L.
, Wu T.
, Lu Z.
, So K.
F.
, Rao Y.
* and Peng B.
* (2018) Dual origins of retinal microglia in the model of microglia repopulation.
 Cell Discovery 4, 9.

(5) Zhou T.
, Li Y.
, Li X.
, Zeng F.
, Rao Y.
, He Y.
, Wang Y.
, Liu M.
, Li D.
, Xu Z.
, Zhou X.
, Du S.
, Niu F.
, Peng J.
, Mei X.
, Ji S.
-J.
, Shu Y.
, Lu W.
, Guo F.
, Wu T.
, Yuan T.
-F.
, Mao Y.
and Peng B.
* (2022) Microglial debris is cleared by astrocytes via C4b-facilitated phagocytosis and degraded via RUBICON-dependent noncanonical autophagy in mice.
Nature Communications.

(6) Niu F.
, Han P.
, Zhang J.
, She Y.
, Yang L.
, Yu J.
, Zhuang M.
, Tang K.
, Shi Y.
, Yang B.
, Liu C.
, Peng B.
* and Ji S.
-J.
* (2022) The m6A reader YTHDF2 is a negative regulator for dendrite development and maintenance of retinal ganglion cells.
eLife 11, e75827.

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