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Written by Sucre, edited by Sucre, Wang Sizhen α-synuclein (α-syn) is a small molecule protein with a molecular weight of 14kD encoded by the SNCA gene.
It is highly expressed in the central nervous system and is mainly distributed in the presynapses Nerve endings
.

Under physiological conditions, α-syn is a soluble protein with no specific structure in aqueous solution [1]
.

Under pathological conditions, α-syn oligomerizes and aggregates to form Lew bodies (LB) and Lewy neurites (LN).
The formation of LB and LN is a typical pathology of a variety of neurodegenerative diseases.
Including Parkinson Disease (PD), Dementia with Lewy bodies (DLB) and Multiple System Atrophy (MSA) [2]
.

In addition, SNCA gene mutation or overexpression of α-syn can cause pathological α-syn aggregation and lead to loss of dopaminergic neuron death and movement disorders [3]
.

 A number of studies have shown that under in vivo and in vitro conditions, pathological α-syn can spread between cells and promote the development of diseases [4-5]
.

Therefore, improving the clearance efficiency of pathological α-syn and reducing the accumulation of pathological α-syn may be an effective way to treat synucleinopathies such as PD
.

 Microglia, as the main immune cells of the central nervous system, play an important role in mediating inflammation, maintaining brain homeostasis, removing cell debris, and providing neurotrophic factors [6]
.

At the same time, studies have found that in the brains of patients with a variety of synucleinopathies, pathological α-syn aggregation is often accompanied by the activation of microglia, indicating that microglia are involved in the pathological process of α-syn [7]
.

At present, although people have done a lot of research on the involvement of microglia in the removal of α-syn, unfortunately, it is still unclear about the mechanism of action of microglia in the removal of α-syn
.

 On September 22, 2021, the Michael T.
Heneka team from the University of Bonn Medical Center in Germany, the German Neurodegenerative Disease Center and the University of Massachusetts Medical School published a titled "Microglia jointly degrade fibrillar alpha-synuclein cargo by distribution" on Cell.
"through tunneling nanotubes" is the latest research paper
.

The study found that through the formation of F-actin (F-actin)-dependent cell connection network between microglia, pathological α-syn transfer between microglia, and promote pathological α-syn degradation and Clear
.

In this article, the author first studied the ability of microglia to phagocytose (ie, clear) pathological α-syn and the effect of pathological α-syn on the transcriptome of microglia
.

To this end, the authors confirmed through cellular immunochemistry and flow cytometry that microglia can take up fluorescently labeled α-syn fibrils, and this process can be significantly inhibited by the phagocytosis inhibitor CytD
.

Next, the authors analyzed the effect of α-syn fibrils on the transcription level of microglia and found that compared with untreated cells, there were 2189 differentially expressed genes in microglia treated with α-syn fibrils, of which 687 were differentially expressed genes.
Gene expression was up-regulated, and 1502 genes were down-regulated
.

Furthermore, the author found that differentially expressed genes are closely related to inflammation and programmed cell death through GO (gene ontology) analysis and BINGO (Biological Network Gene Ontology tool) analysis tools
.

Therefore, these results indicate that α-syn fibrils can be phagocytosed by microglia and cause the up-regulation of microglia inflammation-related genes and apoptosis-related factors (Figure 1)
.

Figure 1 Phagocytosis of α-syn fibrils causes microglia inflammation and apoptosis pathway-related gene expression (picture quoted from: Scheiblich H, et al.
Cell.
2021) Next, the author studied the uptake of α-syn by microglia After the degradation mechanism
.

First of all, the author found through cellular immunochemistry, flow cytometric sorting and western blotting that after 24 hours of treatment, 40%-50% of α-syn fibrils are still located in microglia and have not yet been degraded
.

Furthermore, the results of cellular immunochemistry showed that F-actin-positive cellular connection networks formed between microglia.
Although these structures are of different lengths and thicknesses, they all contain α-syn fibrils
.

Furthermore, the results of electron microscopy showed that the cell membranes of adjacent microglia were in contact with each other, accompanied by the participation of a variety of organelles
.

In order to observe this process in real time, the author adopted a time-lapse shooting method and found that the transfer of α-syn fibrils between microglia can be achieved through two different types of transport mechanisms.
One: short and thick connections are formed between cells Structure, the transfer of large α-syn fibrils between cells can be realized within 40-60 minutes; the second: the formation of long and thin connection structures between cells, the transfer of small α-syn fibrils between cells can be realized in about 3 minutes
.

It is worth mentioning that α-syn-treated microglia formed significantly more cell connection networks than untreated microglia
.

These results indicate that the transfer of α-syn fibrils from cell to cell is realized through the formation of a connecting network between microglia (Figure 2)
.

Figure 2 α-syn fibrils promote the formation of the connection network between microglia (picture quoted from: Scheiblich H, et al.
Cell.
2021) Subsequently, the author conducted an in-depth study of the transport of α-syn fibrils between microglia Molecular mechanism
.

To this end, the author co-cultured donor cells containing fluorescently labeled α-syn and recipient cells labeled with CellTracer.
After 5 hours of culture, it was found that an average of 8% of the recipient cells that formed direct contact with the donor cells It becomes α-syn positive, and the donor cell α-syn fibrils decrease, indicating that α-syn fibrils can be transferred between adjacent microglia and cause α-syn fibrils to redistribute
.

Next, the author analyzed the cytoskeleton of the recipient cell and the donor cell and found that the two had similar changes, which were specifically manifested in the increase and growth of cell branches
.

At the same time, through GO analysis, it was found that the Rho signal transduction level of α-syn-treated microglia increased
.

A large number of evidences have shown that Rho kinase ROCK participates in regulating the occurrence of cytoskeleton by regulating downstream actomyosin complex
.

Next, the authors used ROCK selective inhibitor Y-27632 to treat the cells and found that Y-27632 significantly promoted the transfer of α-syn fibrils from donor cells to recipient cells.
At the same time, the cells were treated with the kinase inhibitor Blebbistatin.
It also significantly promotes the transfer of α-syn fibrils, and is accompanied by an increase in the formation of cell connection networks
.

The difference is that the use of F-actin turnover inhibitor CytD significantly inhibits the transfer of α-syn fibrils between cells, and the formation of cell connection networks is obstructed
.

The results of cellular immunohistochemistry showed that the network connection formed between donor cells and recipient cells was positive for cytoskeleton marker proteins, that is, positive for myosin II and F-actin
.

Finally, through knockout experiments, the authors found that knocking out ROCK1 does not affect the transfer of α-syn fibrils between microglia, while knocking out ROCK2 significantly promotes the transfer of α-syn fibrils between microglia
.

These combined results indicate that after microglia uptake of α-syn fibrils, they activate the Rho signaling pathway to regulate the formation of downstream actomyosin complexes and promote the transfer of α-syn fibrils between cells (Figure 3)
.

Figure 3 The transfer of α-syn fibrils between microglia depends on F-actin (picture quoted from: Scheiblich H, et al.
Cell.
2021) Next, the author studied intercellular α-syn fibrils The effect of metastasis on gene expression of microglia
.

First, the author co-cultured donor cells containing fluorescently labeled α-syn and recipient cells labeled with CellTracer, and then performed flow cytometric sorting and RNA sequencing at different time points, and calculated the Pearson coefficients ρ and As a basis for comparison of transcriptome similarity
.

It was found that as the co-cultivation time was extended, the transcriptome similarity of the two gradually approached, that is, the transcription profile of the donor cell gradually approached the transcription profile of the recipient cell.

.

At the same time, the author compared the transcription profiles of the donor cells and recipient cells before and after co-cultivation, and found that the donor cells originally expressed abundantly expressed differentially expressed genes, including inflammation-related genes and apoptosis-related genes, as the co-cultivation time prolonged , The expression levels of the above-mentioned genes were gradually down-regulated; while the recipient cells had no significant difference in transcription profiles before and after co-cultivation
.

These results indicate that the transfer of α-syn fibrils between cells can down-regulate the expression of inflammation-related genes in microglia (Figure 4)
.

Figure 4 Intercellular transfer of α-syn fibrils down-regulates the expression of microglial inflammation-related genes (picture quoted from: Scheiblich H, et al.
Cell.
2021) Further, the author studied the effect of intercellular α-syn fibril transfer on small cells.
The effect of glial cell function
.

First, the authors discovered that the treatment of microglia with α-syn fibrils disrupts the integrity of cell membranes and leads to cell death
.

Interestingly, the co-culture of donor cells and ligand cells greatly protects the integrity of the donor cell membrane and effectively prevents the penetration of SYTOX into the cells without affecting the integrity of the recipient cell membrane
.

At the same time, the authors found that the number of concentrated mitochondria in donor cells increased and the mitochondrial network structure was decomposed, which in turn led to an increase in the production of reactive oxygen species (ROS)
.

Next, the author explored the role of ROS in the redistribution of α-syn fibrils
.

The results showed that the use of ROS scavenger NAC (N-Acetylcystein, N-acetylcysteine) can significantly inhibit the transfer of α-syn fibrils between cells, on the contrary, the treatment of cells with H2O2 can effectively reverse the above phenomenon
.

These results indicate that ROS may affect the transfer of α-syn fibrils between cells
.

 Next, the author used MitoTracker to label the mitochondria of recipient cells and record their dynamic changes over time during the co-culture process
.

The results showed that the number of MitoTracker-positive donor cells increased significantly after the cells were co-cultured for 5 hours.
The cell immunochemical results also confirmed that MitoTracker-positive mitochondria and α-syn fibrils appeared in the network formed between cells
.

Similarly, the mitochondria of the donor cell can also be transferred to the recipient
.

In addition, transcriptome analysis revealed that the donor cells' inherent apoptosis signaling pathway-related genes (abnormal mitochondrial function) expression were down-regulated after co-culture
.

These results indicate that the transfer of α-syn fibrils between microglia is accompanied by mitochondrial exchange
.

 Existing studies have shown that the G2019S mutation of the LRRK2 gene is the most common genetic factor leading to PD, and the LRRK2 gene is closely related to abnormal mitochondrial function
.

Therefore, next, the authors used WT mice and LRRK2 G2019S mutant mice to compare the differences in oxygen consumption and ROS production of microglia in response to the challenge of α-syn fibrils
.

The results showed that the oxygen consumption, mitochondrial circulation rate and ROS production of LRRK2 G2019S microglia mitochondria were significantly lower than those of wild-type (WT) microglia mitochondria
.

At the same time, the transfer of α-syn between LRRK2 G2019S microglia is blocked
.

Next, the author tried to use WT microglia to redistribute functional mitochondria to save the function of LRRK2 G2019S microglia
.

To this end, the author co-cultured WT and LRRK2 G2019S microglia through a combination of different forms
.

The results showed that WT microglia as recipient cells can effectively reduce the ROS production of donor cells (WT or LRRK2 G2019S microglia) and accompanied by effective exchange of mitochondria, while LRRK2 G2019S microglia as recipient cells cannot effectively reduce ROS production.
Donor cells (WT or LRRK2 G2019S microglia) ROS production is accompanied by mitochondrial exchange barriers
.

These results indicate that the LRRK2 G2019S mutation may cause familial PD by hindering pathological α-syn metastasis and mitochondrial exchange (Figure 5)
.

Figure 5 The transfer of α-syn fibrils between cells is accompanied by mitochondrial exchange.
Functional mitochondrial transfer reduces ROS levels and helps cells avoid cytotoxicity and death (picture quoted from: Scheiblich H, et al.
Cell.
2021) To further clarify the transfer mechanism of the above-mentioned α-syn, the author injected CellTracer-labeled primary microglia containing α-syn into organotypic cultured brain slices.
After 24 hours of co-cultivation, the injected microglia and brain slices The microglia in the cells form a variety of connections
.

More interestingly, the authors found that the microglia in the brain slices contain α-syn fibrils, which proves that the transfer of α-syn occurs between microglia
.

In addition, the author injected CellTracer-labeled primary microglia containing α-syn into the cerebral cortex of Cx3xr1GFP+/- mice, and used a two-photon microscope for in vivo imaging
.

It was found that near the injection site, a variety of cell connections were formed between the injected microglia and GFP-positive microglia.
More importantly, the author found through time-lapse imaging that the cells containing α-syn branched towards Neighboring cells extend and form connections
.

These results indicate that α-syn fibrils can also be transferred between microglia under in vivo conditions (Figure 6)
.

Figure 6 Under in vivo conditions, α-syn fibrils transfer between microglia (picture quoted from: Scheiblich H, et al.
Cell.
2021) At the end of the article, the author found that after death brain tissue samples from DLB and MSA patients There are aggregate α-syn in some microglia.
It is worth mentioning that many of these cells form connections with cells containing aggregate α-syn
.

This result prompted the author to speculate that pathological α-syn may spread between the patient’s microglia
.

To this end, the author induced and verified the differentiation of isolated peripheral blood mononuclear cells (PBSCs) into macrophages/microglia
.

Next, the authors found that compared with the microglia derived from the healthy control group and induced to differentiate, the microglia derived from DLB patients and induced to differentiate had a significantly lower ability to transfer α-syn fibrils
.

These results indicate that pathological α-syn can spread between human microglia, and the ability of microglia in DLB patients to metastasize pathological α-syn is significantly reduced (Figure 7)
.

Figure 7 In human tissues, α-syn fibrils transfer between human microglia (picture quoted from: Scheiblich H, et al.
Cell.
2021) Figure 8 Summary: Microglia responds to pathological α-syn (Picture quoted from: Scheiblich H, et al.
Cell.
2021) Conclusion and discussion of the article, inspiration and prospects As an important immune cell of the central nervous system, microglia through pattern recognition receptors (pattern recognition receptor) Respond to misfolded proteins in cells and mediate inflammation [8, 9]
.

However, the molecular mechanism by which microglia degrades pathological α-syn has always been unclear
.

The emergence of this research work has provided us with new insights
.

 First of all, through the formation of F-actin-dependent functional cell connection network between microglia, pathological α-syn transfer between microglia can be realized, on the one hand, it can reduce the pathological α-syn of affected cells.
On the other hand, it can accelerate the degradation of pathological α-syn and promote cell survival
.

In addition, the article found that WT microglia can exchange mitochondria at the same time, and can effectively alleviate the cytotoxicity caused by ROS
.

The difference is that pathological α-syn transfer and mitochondrial exchange between LRRK2 gene G2019S mutant microglia are significantly affected, and LRRK2 G2019S mutation is the most common genetic factor of familial PD [10]
.

Therefore, it can be speculated that the LRRK2 G2019S mutation may cause familial PD by hindering pathological α-syn metastasis and mitochondrial exchange
.

 In this article, the author thoroughly studied the process of pathological α-syn transfer between microglia and confirmed that this phenomenon is common in vitro, in vivo, and in patients with DLB
.

So, do other types of misfolded proteins, such as pathological tau, also transfer between microglia? In addition, studies have shown that pathological α-syn mainly exists in neurons, and can spread between neurons and promote disease development
.

Then, is there a similar pathological α-syn transport phenomenon and mechanism in this article between microglia and neurons? They are all issues worthy of further study
.

 Finally, the author used synthetic α-syn fibrils to indicate pathological α-syn, and studied the process of metastasis after being phagocytosed by microglia
.

While praising the novelty of this work, there are also points worthy of further discussion.
First: Although α-syn fibrils are aggregates of α-syn monomers, previous studies have shown that clinical patients have a high degree of pathological α-syn.
Heterogeneous [11, 12], therefore, whether synthetic α-syn fibrils have the ability to represent pathological α-syn is debatable
.

Second: The author uses fluorescently labeled α-syn fibrils to study the phagocytosis and metastasis of microglia.
However, considering that α-syn fibrils may attach to the surface of microglia, it may lead to results Some deviations
.

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