PNAS︱ He Shujun’s research group reveals a new mechanism for glial cells to provide time signals to regulate neuron development

Written by Yang Mengying, edited by Wang Honglei, Wang Sizhen The central nervous system is composed of neurons and glial cells differentiated from neural stem cells
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In the past, the inherent cognition believed that neurons are the dominant cells of the nervous system, and glial cells play a supporting role
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However, in recent years, more and more studies have shown that glial cells, as an important part of the nervous system, actively regulate the development and function of the nervous system, including neuronal axon extension, synapse formation and neural signal transmission [1 】
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In addition, the excessive division of glial cells will lead to the occurrence of glioma disease, which is harmful to human health [2]
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These findings indicate that glial cells have an indispensable role in the nervous system; however, many other new functions of glial cells have not yet been discovered and are waiting to be examined
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 The development process of neurons is carefully and tightly regulated
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The Mushroom Body (MB) of Drosophila melanogaster is the center that regulates learning and memory, and its function is equivalent to the hippocampus of the human brain
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Different types of mushroom body neurons are regulated by temporal and spatial factors, exhibiting diversity that is vital to biological functions, regulating smell, learning and memory, sleep, exercise and other behavioral abilities [3]; some mechanisms are known to depend on nerves The role of autonomic regulation within the cell, and the pathways and molecules that mediate the development of non-cell-autonomous mushroom body neurons are still poorly understood
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 On June 8, 2021, the He Shujun research group of the School of Life Science and Technology, ShanghaiTech University published a study titled "Glia-derived temporal signals orchestrate neurogenesis in the Drosophila mushroom body" in the Proceedings of the National Academy of Sciences (PNAS) The paper proposes that maintaining protein homeostasis in glial cells is an important mechanism for regulating the normal development of neurons
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Yang Mengying and Wang Honglei are the co-first authors of the paper
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In this study, the authors found that glial cell ubiquitination ligase dSmurf regulates the protein stability of Hedgehog (Hh) signaling pathway receptor Patched (Ptc) and cell adhesion factor Fasciclin II (FasII), thereby regulating the brain's learning and memory centers The development process of the mushroom body
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The previous work of the research team showed that ubiquitination modification is the main pathway that affects the differentiation and proliferation of glial cells.
Once the function is lost, it will lead to abnormal development of the nervous system [4]
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On the basis of these early stages, this research deeply explores the regulation mechanism of glial cell ubiquitination pathway and protein homeostasis on neuron development, clarifies the glial cell-neuron information transmission, and promotes the pathogenesis of neurodevelopmental diseases.
Understanding of the mechanism
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The author first observed that Drosophila systemically lack of dSmurf and the mushroom body nerve axons that selectively inhibit the expression of glial cells dSmurf are abnormal.
In 3 different types of mushroom body neurons (α/β, α'/β', g ) Only α/β neurons are affected: when the expression level of dSmurf in glial cells changes, the thickness of α/β neuron axon bundles will change accordingly, and the number of neurons will decrease, while the number of the other two neuron types will follow.
The shape remains normal
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This phenomenon can be repaired by re-expression of dSmurf (Figure 1A-E)
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 Experimental results show that the regulation of α/β neurons by glial cells dSmurf begins with the division and proliferation of Drosophila mushroom neural stem cells (MB NBs): 4 MB NBs divide and proliferate normally in the early stages of development, until the pupal stage α/β When neurons start to divide, the physiological level of glial cells dSmurf changes, which regulates the division and proliferation of MB NBs at this stage (that is, the pupal stage), thereby regulating the number of α/β neurons and their axon morphology (Figure 1F-I )
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 Figure 1 Glia dSmurf regulates the proliferation of MB neuroblasts and the number of α/β cells
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(Image source: He Shujun laboratory) It is known that dSmurf regulates the development of different organs through multiple signal pathways, including Hedgehog (Hh), BMP, and Hippo [5-9]
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However, the regulation mechanism of dSmurf on neurodevelopment is still blank
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After a series of explorations, the author identified the downstream pathway regulated by glial cell dSmurf and found that glial cell dSmurf activates glial cell Hh signal by regulating the protein stability of the Hh signaling pathway transmembrane receptor Ptc
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By monitoring the expression of the reporter gene Ptc-LacZ of Hh signal, and marking glial cells with Drosophila UAS/GAL4 expression system (repo-GAL4>UAS-IVS-Syn21-GFP-p10), using β-galactosidase (β -galactosidase, β-gal) antibody immunostains the brain, and then detects the level of LacZ and determines the co-positive cells of GFP and β-gal to quantify the glial Hh signal (Figure 2A, D)
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When the Hh receptor Smoothened (smo) (inhibited by Ptc), ptc-RNAi, or dSmurf is expressed in glial cells, the β-gal intensity in glial cells is found to increase, indicating that the Hh signal level is increased, and MB α/β The number of pH3-positive cells displayed in the neuron cell body area was significantly reduced (pH3 marked the division and proliferation ability of MB NBs), and the division and proliferation ability of MB NBs was impaired (Figure 2C)
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These results indicate that glial cell dSmurf activates glial cell Hh signal, thereby inhibiting the division and proliferation of MB NBs
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 According to existing studies, Hh signals can transmit signals to neighboring cells through non-cell autonomous propagation [10]
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Therefore, the author further proposed a conjecture: glial cell dSmurf activates glial cell Hh signal, and activates neuronal Hh signal through non-cell-autonomous propagation, thereby inhibiting the division and proliferation of MB NBs
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In order to verify this conjecture, the author used the reporter gene Ptc-LacZ to monitor the Hh signal in α/β neuronal cells while regulating the expression of dSmurf in glial cells (Figure 2B, E, F); The expression of dSmurf, smo or ptc-RNAi in the cells all lead to a significant increase in the intensity of β-gal in α/β neuronal cells, that is, the Hh signal of α/β cells is activated; at the same time, these results also indicate that the Hh signal of neurons is in the gel Plasma cell Hh signal is also up-regulated under the condition that it is activated
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Therefore, glial cell dSmurf regulates the division and proliferation of MB NBs by activating the Hh signal transmission from glial cells to neurons
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Figure 2 Glial cell dSmurf activates glial cell and neuron Hh signal
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(Image source: He Shujun laboratory) In addition to identifying the mechanism by which glial cell dSmurf regulates mushroom body neuron development through Hh, the author also revealed another Hh-independent signaling pathway, that is, by regulating the protein stability of the cell adhesion molecule FasII Sex, thereby affecting the axon morphology of mushroom body neurons
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 The author’s previous study found that inhibiting the expression of dSmurf in glial cells resulted in an increase in the expression of the cell adhesion molecule FasII, suggesting that FasII may act downstream of dSmurf
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Based on this, the author first proved that the FasII subtype FasIIB expressed in glial cells has a binding effect with dSmurf and may form the same complex (Figure 3A)
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Cell experiment results revealed that dSmurf stabilizes FasIIB and increases its half-life, while dSmurf lacking the WW domain cannot bind to FasIIB and accelerate the degradation of FasIIB (Figure 3B-E).
It is known that the WW domain can be enriched by proline.
Contains sequence recognition substrate 
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These results suggest that FasIIB may act on the downstream pathway of dSmurf to regulate mushroom body neuron development
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 Furthermore, in view of these findings, the authors established a dual system for simultaneously manipulating gene expression in Drosophila neurons and glial cells to detect the influence of glial cell FasIIB-mediated cell adhesion on the axon morphology of mushroom body neurons
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The authors found that the expression of glial cell FasIIB or neuronal FasII subtype FasIIA-PEST alone induced severe MB α lobe defects (30% and 72% of the analyzed brains showed abnormal lobes, respectively), while the two types of FasII The co-expression of subtypes will repair their respective α/β leaf defects, and more than 73% of the leaves are morphologically normal; as a control, only 17% of Drosophila brain expressions when FasII-RNAi is expressed in α/β neuronal cells A normal α axon lobe (Figure 3F, G)
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These results indicate that FasII mediates the adhesion between glial cells and MB α/β neurons, thereby regulating α/β axon morphology
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In addition, the authors also found that dSmurf can stabilize the expression and distribution of FasIIB on the glial cell membrane, and regulate the binding of FasIIB to FasIIA on MB α/β neuron axons, which in turn leads to the weakening of the homophilic FasIIA affinity between MB axon bundles.
Disorders with axonal fascification
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 Figure 3 The homophilic FasII molecular interaction between glial cells and MB axons regulates axon integrity
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(Picture source: He Shujun laboratory) At the end of the article, considering that Drosophila MB is the center for regulating learning, memory and a variety of behaviors, the author tested the effects of Drosophila lacking glial cells dSmurf in exercise, learning and memory, and anti-epileptic paralysis.
Important role, as shown in Figure 4, selective regulation of the expression of dSmurf in glial cells leads to dysmotility and learning and memory impairment in Drosophila, and produces anti-epileptic paralytic behavior (Figure 4A-C)
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In the memory experiment, Drosophila's choice of smell was normal, indicating that the sense of smell was not affected, but learning and memory were impaired (Figure 4D, E)
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Figure 4 The dysregulation of dSmurf expression in glial cells leads to MB-related behavioral defects in Drosophila
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(Image source: He Shujun laboratory) Figure 5 Summary of the article: glial cells provide time signals to regulate mushroom body neuron development
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(Image source: He Shujun laboratory) The conclusion and discussion of the article shows that in the early stage of pupal development in Drosophila, the imbalance of expression of dSmurf in glial cells will increase the internalization and conversion efficiency of Ptc protein in glial cells.
The inhibition of Smo, another Hh receptor, is weakened, thereby activating the Hh signal of glial cells
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Through non-cell-autonomous transmission, neuronal Hh signals are activated, inhibiting the proliferation of MB neuroblasts, resulting in a decrease in the number of α/β nerve cells and abnormal leaf formation
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In addition, glial cell dSmurf stabilizes the expression of FasIIB on the cell membrane, and regulates the axon morphology of neurons through its autoadhesion with α/β neuron FasIIA
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This study identified the mechanism by which the glial cell ubiquitination ligase dSmurf regulates the development of MB neurons (Figure 5)
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 Moreover, because MB is the information integration center in the Drosophila nervous system and regulates a variety of behaviors at the same time, its developmental abnormalities can cause severe motor, learning and memory disorders
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Therefore, revealing the regulation mechanism of glial cell protein homeostasis on its neurodevelopment and mediating behavior has important research significance
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Yang Mengying (back row, second from right) and Wang Honglei (front row, second from right) are the co-first authors of the paper, and Professor Shujun He (front row, second from left) is the corresponding author (picture source: He Shujun laboratory) Original link: https://doi.
org/10.
1073/pnas.
2020098118 Selected articles from previous issues [1] PNAS︱ The network balance mechanism formed by the rich cognitive functions of the brain [2] Cell︱ breakthrough! The new mechanism of human brain spatial navigation and memory: hippocampus and entorhinal cortex phases Precession [3] PNAS︱ new hope for visual restoration? CXCR4/CXCL12 signal-mediated new mechanism of damaged optic nerve regeneration [4] Sci Transl Med︱ A new mechanism to improve the pathology of Alzheimer’s disease [5] Alzheimer's Dementia︱ The latest scientific hypothesis! JNK is a therapeutic target for neurodegenerative diseases [6] Nat Neurosci ︱ for the first time! Neuron ApoE is a new mechanism that affects immune response genes to cause AD pathology.
Recommended high-quality scientific research training courses [1] "Scientific research image processing and mapping" offline : June 26-27, Shanghai; July 10-11, Beijing [2] Patch clamp and optogenetic and calcium imaging technology seminar (June 26-27, two days and one night) [3] Single cell sequencing Data Analysis and Project Design Network Practice Class (July 24-25) References (swipe up and down to view) [1] Allen, NJ & Lyons, DA Glia as architects of central nervous system formation and function.
Science 362, 181- 185, doi:10.
1126/science.
aat0473 (2018).
【2】Molinaro, AM, Taylor, JW, Wiencke, JK & Wrensch, MR Genetic and molecular epidemiology of adult diffuse glioma.
Nat Rev Neurol 15, 405-417, doi :10.
1038/s41582-019-0220-2 (2019).
[3] Lee, T.
, Lee, A.