PNAS︱Chun Han's group reveals a new mechanism of neuronal degeneration caused by external phagocytosis

Written by ︱ Ji Hui, edited by Han Chun ︱ Wang Sizhen Physical injury usually leads to the loss of function of neuron axons and dendrites
.

Damaged neurites far from the neuronal cell body undergo a series of changes known as Wallerian degeneration (WD) [1-3], during which the damaged nerve breaks apart and is eventually cleared by surrounding phagocytes [4]
.

Immediate clearance of neuronal debris can hinder neurite regeneration and lead to neuroinflammation and other secondary injuries [5, 6]
.

 Although Wallerian degeneration of axons and dendrites is generally considered to be an endogenous "self-destruction" process in neurons, and phagocytes only play a role in cleaning up after neurite fragmentation [7], whether phagocytes are actively involved in neural The process of sudden fragmentation is still unknown
.

 Nicotinamide mononucleotide adenyltransferase (Nmnat) is an important member of the WD pathway
.

Nmnat is responsible for the synthesis of nicotinamide adenine dinucleotide (NAD+) in vivo and has a protective effect on neurons[8, 9]
.

The decrease of NAD+ level caused by Nmnat deletion can stimulate the NAD+ hydrolase activity of the downstream protein Sarm, further reduce the NAD+ level and trigger the "self-destruction program"[10-15]
.

In contrast, phagocytosis of neurites is accomplished through the recognition of "eat-me" signals on the surface of damaged neurons by phagocytic receptors on phagocytes [7, 16-18]
.

The author's previous study found that serine phospholipid (phosphatidylserine, PS) eversion is an "eat-me" signal on neurons that triggers phagocytosis [18]
.

This work aimed to investigate whether the WD pathway also regulates PS eversion and whether phagocytosis is actively involved in neurite fragmentation
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 On January 25, 2022, Han Chun's research group from Cornell University published a paper entitled "Phagocytosis and self" in the Proceedings of the National Academy of Sciences of the United States of America (PNAS).
-destruction break down dendrites of Drosophila sensory neurons at distinct steps of Wallerian degeneration" research paper
.

The authors found that neural dendrites display an "eat-me" signal on the cell surface when damaged or when Nmnat is mutated
.

This signal triggers peripheral phagocytes to actively attack dendrites, leading to dendrite fragmentation, and the neuron's "self-destruction program" is then initiated to ensure the effective removal of dendrites
.

This article enriches our understanding of the mechanism of neuronal degeneration at the cellular level, proposing for the first time an active role of phagocytosis in the clearance of damaged neurons
.

 To investigate whether the WD pathway triggers phagocytosis, the authors used Drosophila larval sensory neurons (dendritic arborization neurons, da neurons) as a model, combined in vivo imaging and specific in-tissue gene editing technology (clustered regularly interspaced short palindromic repeats (CRISPR)-mediated tissue-restricted mutagenesis, CRISPR-TRiM) carried out the following studies
.

 First, the authors observed the degeneration of neurons after knocking down Nmnat (Fig.
1 AB, GH), and confirmed that the degeneration depends on the decrease of NAD+ level (Fig.
1 CD, GH) and the activation of Sarm (Fig.
1 EF, GH)
.

Figure 1 Nmnat knockout triggers neuronal degeneration, which is dependent on Sarm activity (Credit: Ji, H.
, Sapar, M.
, et al.
, PNAS, 2022) Next, the authors used genetically encoded probes to observe To PS everted on the neuronal membrane after Nmnat knockout (Fig.
2 AC)
.

Both inhibition of PS eversion or inhibition of phagocytosis effectively prevented the degeneration of Nmnat-knockout neurons (Fig.
2 DL)
.

This result demonstrates that PS-mediated phagocytosis is the main reason for the degeneration of Nmnat-knockout neurons
.

Figure 2 PS-mediated phagocytosis is the main reason for the degeneration of Nmnat-knockout neurons (Source: Ji, H.
, Sapar, M.
, et al.
, PNAS, 2022) Previous studies have shown that damaged degenerated dendrites Also accompanied by PS valgus phenomenon [18]
.

Here, in order to explore whether PS valgus is regulated by the WD pathway, the authors used high-intensity lasers to damage dendrites of Sarm knockout neurons and Nmnat fusion protein WldS-overexpressing neurons, respectively, and found that PS valgus on damaged dendrites Both phenomena and debris-like changes were suppressed (Fig.
3)
.

This result suggests that Nmnat and Sarm upstream regulate PS eversion on damaged dendrites
.

Figure 3 WldS overexpression and Sarm knockout inhibited PS valgus and fragmentation of damaged dendrites (Source: Ji, H.
, Sapar, M.
, et al.
, PNAS, 2022) Next, the authors conjectured that, PS eversion-mediated phagocytosis can lead to fragmentation of damaged dendrites
.

To verify its adequacy, the authors genetically modulated PS transporters to ectopic PS in Sarm knockout neurons and WldS-overexpressing neurons
.

Injured Sarm knockout neurons with PS eversion, as well as WldS-overexpressing neurons, both exhibited fragmentation-like changes that were close to those of injured wild-type neurons (Fig.
4)
.

This result revealed that PS-mediated phagocytosis plays an important role in the degeneration of damaged neurons
.

Figure 4 The introduction of PS eversion resulted in normal dendrite fragmentation of damaged WldS-overexpressing neurons and Sarm knockout neurons (Source: Ji, H.
, Sapar, M.
, et al.
, PNAS, 2022) At the same time, The authors found that the degeneration of damaged dendrites did not use phagocytosis as the sole driving force, in contrast to the degeneration triggered by Nmnat knockdown that was entirely dependent on phagocytosis (Figure 2 EL)
.

When WD progressed to a more severe degree than PS eversion, inhibition of phagocytosis failed to completely inhibit the degeneration of damaged neurons and Sarm overactivated neurons (Fig.
5), indicating that the "self-destruction" program of neurons at this time has become A major driver of neuronal degeneration following phagocytosis
.

Figure 5 Injured neurons and Sarm overactivated neurons undergo phagocytosis-independent degeneration (Credit: Ji, H.
, Sapar, M.
, et al.
, PNAS, 2022) To explore other molecules in the WD pathway For neuronal degeneration, the authors knocked out the Axondead (Axed) gene, which has been shown to have an integrated role in axonal degeneration studies, and damaged dendrites
.

The results showed that Axed did not completely control the degeneration of damaged neurites, nor did it regulate PS eversion, but synergized with phagocytosis to promote the degeneration of damaged dendrites (Fig.
6)
.

Figure 6 Axed-mediated "self-destruction" program cooperates with phagocytosis to promote the degeneration of damaged dendrites (Credit: Ji, H.
, Sapar, M.
, et al.
, PNAS, 2022) For further study of damaged trees To investigate the mechanism of dendritic degeneration, the authors compared the fragmentation characteristics of dendritic cell membranes in the case of Nmnat knockout and in the damaged condition
.

The authors created a genetically encoded probe based on split green fluorescent protein (split GFP) to facilitate the observation of fine cell membrane fragmentation in live imaging (Fig.
7A)
.

The results showed that only damaged dendrites would undergo membrane fragmentation during degeneration, while dendrites of Nmnat knockout neurons did not undergo this process (Fig.
7 BF), further suggesting that Nmnat knockout induced Dendritic degeneration is mainly accomplished by phagocytosis, whereas the degeneration of damaged dendrites is more severe
.

Figure 7 Damaged dendrites undergo a process of cell membrane fragmentation (Credit: Ji, H.
, Sapar, M.
, et al.
, PNAS, 2022) Distinctive calcium ion dynamics within lesional dendrites
.

After injury, calcium levels undergo a rapid and repeated up-and-down phase-resting-second-rising phase until dendrite fragmentation (Fig.
8 AD)
.

Both WldS and Axed regulate this calcium dynamics (Figure EL), suggesting that the regularity of calcium changes may be a factor regulating the degeneration of damaged dendrites
.

Figure 8 The unique calcium dynamics in damaged dendrites (Image source: Ji, H.
, Sapar, M.
, et al.
, PNAS, 2022) Conclusion and discussion, inspiration and prospect In vivo imaging and genetic manipulation techniques have found that the Wallerian degeneration (WD) pathway regulates neuronal serine phospholipid (PS) eversion, and triggers the phagocytosis of dendrites by surrounding phagocytes, promoting dendrite degeneration.
When WD progresses to the next During the stage, the "self-destruction" program within the dendrite is initiated, which further ensures that dendrite degeneration occurs in an orderly manner
.

In addition, the study revealed the membrane fragmentation process experienced by damaged dendrites and the unique calcium dynamics within damaged dendrites
.

 There are also unresolved questions in this study, such as whether the "self-destruction program" and phagocytosis triggered by the damaged WD pathway covers all the mechanisms that lead to the degeneration of damaged neurons? Are the phenomena the authors found in dendrites also present in axons? In conclusion, this study is the first to discover the promoting effect of phagocytes around neurons on neuronal degeneration, and also suggests a potential mechanism for the occurrence of neurodegenerative diseases
.

Link to the original text: https:// Group photo of the laboratory: first author Ji Hui (front row, third from left), first author Maria Sapar (front row, first from left), corresponding author Han Chun (first from the left in the back row) (Photo provided by: Han Chun's laboratory) For more details of Han Chun's laboratory work, please refer to: https://han.
wicmb.
cornell.
edu/ In addition, Han Chun's laboratory is looking for different disciplines Postdoctoral fellows and technicians with backgrounds are paid favorably, and aspiring young people who are interested in neuroscience are welcome to join
.

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Waller, A.
, Experiments on the Section of the Glossopharyngeal and Hypoglossal Nerves of the Frog, and Observations of the Alterations Produced Thereby in the Structure of Their Primitive Fibres.
Philosophical Transactions of the Royal Society of London, 1850.
140: p.
423-429.
2.
Coleman , MP and MR Freeman, Wallerian degeneration, wld(s), and nmnat.
Annu Rev Neurosci, 2010.
33: p.
245-67.
3.
Tao, J.
and MM