The abnormal timing of Cereb Cortex®MET tyrosine kinase signal transduction is a key mechanism affecting the development and behavior of normal cortical neural circuits in mice

Written by Ma Xiaokuang, edited by Ma Xiaokuang, Wang Sizhen In the development of cerebral cortical neural circuits, the formation of normal neuronal synaptic connections is essential, including regulating neuron growth, balancing the formation and depletion of excitability and inhibitory synapses, and Control maturity and plasticity at the right time [1]
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Receptor tyrosine kinase (receptor tyrosine kinase) mediated molecular signals significantly affect these processes [2]
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Disorders of these processes are the hallmark pathological features of many neurodevelopmental and neuropsychiatric disorders, including autism spectrum disorder (ASD) [3]
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 The human MET gene encodes a receptor tyrosine kinase that was initially identified as a high-confidence ASD risk gene in a family-based genetic study
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The rs1858830'C' allele in the MET proximal promoter reduces gene transcription level by 50% [4]
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In the cortical samples of ASD patients, the expression of the MET gene is reduced by about 50%, which is almost absent in patients with Rett syndrome [5]
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It is worth noting that although MET is not a pathogenic gene for ASD, human functional neuroimaging studies have shown that the'C' allele is highly correlated with changes in cortical functional connectivity [6]
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A large number of neurodevelopmental studies have shown that the MET protein regulates the dendritic branching, dendritic spine morphology, excitatory synaptic maturation and cortical plasticity of excitatory pyramidal neurons by participating in multiple intracellular molecular pathways, as well as mediating developmental Signal transduction in the cortex and hippocampus [7, 8]
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The expression of MET is only relatively high in early cortical development, and it is rapidly down-regulated three weeks after the mouse is born
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However, people's understanding of the functional significance of the regulation of the occurrence and termination of MET developmental signals is still insufficient
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 On September 1, 2021, Professor Shenfeng Qiu’s team from The University of Arizona College of Medicine-Phoenix published an online publication on Cerebral Cortex entitled "Disrupted Timing of MET Signaling Derails the Developmental Maturation of Cortical Circuits and Leads to Altered Behavior in Mice" research paper
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Dr.
Ma Xiaokuang is the first author of the paper
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The research team used controllable Met gene overexpression transgenic mice to overexpress MET by extending the duration of the MET signal, and found that extending the time course of MET conduction can affect the development and maturity of the cerebral cortex neural circuit in mice and lead to mice.
Behavior changes reveal that MET signal is a key mechanism that controls the development of cortical neural circuits and normal behavior in mice
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Previous studies have shown that MET signals are strictly time-regulated during the development of cerebral cortical circuits
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Although MET signal has regional and cell type specificity in time regulation, in general, the expression of MET protein in multiple cortical regions such as prefrontal cortex (PFC), hippocampus (HPC) and primary visual cortex (VC) It is strongest in the first 2-3 weeks
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What remains unchanged is that compared with the expression level in the first week after birth, the MET protein and signal are drastically down-regulated on the 21st day after birth (P21) in all cortical areas [9]
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Therefore, the researchers hypothesized that the timely termination of MET signaling is a key molecular transformation, which is necessary for the subsequent development of cortical neural circuits and typical behaviors in mice
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 In this study, in order to determine the functional significance of the down-regulation of MET signaling, the authors used a controllable transgenic overexpression Met (cto-MET) mouse line.
The cto-MET mouse allows for the expression of MET.
Precise time control, by feeding mice with doxycycline (doxycycline, DOX) feed to inhibit the transmission of MET signal in cto-MET in transgenic mice
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On the contrary, after removing the DOX-containing feed, the MET transgenic signal can be retransmitted (Figure 1A-B)
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When the mice were born, they started to feed the mothers/pups with DOX-containing feed, and the DOX-containing feed was removed on P14 days.
Western blotting results showed that the expression of PFC and HPC in cto-MET mice was obvious on P25-28 days.
Higher than the control group (Figure 1C)
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These data indicate that the cto-MET mouse model can significantly increase the total MET protein, and the cto-MET signal can be precisely controlled by DOX in time
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Figure 1.
Using the cto-MET mouse model to extend MET signaling in the developing cortex (picture quoted from: Ma X, et al.
, Cerebral Cortex, 2021, bhab323) The author found in previous studies that it is in development In the visual cortex, cto-MET enhanced the activities of small GTPase Cdc42 and Rac1
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Small GTPases, also known as small G-proteins, are a class of hydrolases that can bind and hydrolyze guanosine triphosphate (GTP)
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So is this a general mechanism that exists in different cortical regions? The author found through western blotting that high expression of MET protein can also significantly enhance the activity of Cdc42 and Rac1 in the prefrontal PFC and the expression of P-Ser3 (that is, phosphorylation of cofilin) ​​during development (Figure 2A) ), the author further tested in PFC how cto-MET affects the level of synaptic proteins related to synapse occurrence and synaptic function
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These data indicate that MET signaling activates the small GTPase-mediated signaling pathway in the developing PFC, which has been shown to extensively affect actin dynamics
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The authors hypothesized that MET controls dendritic spines morphogenesis and synaptic molecular composition (Figure 2C)
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In summary, these results indicate that the MET signal in PFC is involved in the mechanism related to dendritic spine morphogenesis and synaptic maturation
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Figure 2.
Cto-MET leads to small GTPase activation and synaptic protein changes in PFC (picture quoted from: Ma X, et al.
, Cerebral Cortex, 2021, bhab323) Based on the above research results, the author guessed that the prefrontal projection neurons Does the normal development of morphology need to down-regulate the expression of MET protein in time
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The author injected biotin into the neurons projecting to the striatum (CSt) of the fifth layer of the PFC to obtain the cell morphology of the L5 neurons, and then used the Neurolucida system to three-dimensionally reconstruct the L5 neuron morphology (Figure 3B) and use The Imaris system reconstructs the morphology of the dendritic spines of L5 neurons (Figure 3A)
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The results showed that the dendritic length distribution and the number of dendritic branches in cto-MET neurons increased significantly.
In addition, the density and length distribution of dendritic ridges in cto-MET neurons increased significantly, and the number of immature dendritic ridges increased significantly.
As it increases, the number of mature dendritic ridges decreases significantly (Figure 3C)
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These results indicate that the overexpression of MET protein significantly affects the state of dendritic branching and the mature state of dendritic spines of PFC L5 neurons
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 It is worth noting that the increase in dendritic ridge density induced by cto-MET and the inhibitory phosphorylation of the actin-binding protein cofilin indicate that prolonged MET signal may lead to the destruction of dendritic spine dynamics pruning
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Therefore, the researchers used two-photon in vivo imaging technology (Figure 3D) to determine whether the increase in dendritic spine density may be the result of increased dendritic spine production or decreased dendritic spine pruning
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The author crossed cto-MET mice with Thy1-GFP mice to obtain cto-MET:Thy1-GFP triple transgenic mice.
The two-photon imaging was repeated twice in the primary visual cortex by thinning the skull (interval of 7 days).
The formation and reduction of dendritic spines during this cycle
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The results showed that the dendritic spine formation rate and elimination rate of cto-MET were significantly increased (Figure 3E)
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These in vivo imaging data reveal that the MET signal can affect the pruning and dynamics of dendritic spines
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Figure 3.
Continuous expression of MET increases dendritic branching and dendritic spine density of CSt neurons in PFC (picture quoted from: Ma X, et al.
, Cerebral Cortex, 2021) Next, the researchers used laser scanning light stimulation ( Laser-scanning photostimulation (LSPS) combined with glutamate uncaging technology to explore whether MET overexpression can change the interlayer and local synaptic connections of PFC L5 neurons
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PFC L5 neurons receive the main synaptic input from neurons in the L2/3 layer, as well as weak synaptic input from other layers and other types of neurons [10]
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The author uses LSPS to quantify the synaptic input to PFC L5 neurons [11]
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Interestingly, it was found that overexpression of MET significantly affected the excitatory synaptic input of the L2/3 layer, but did not affect the inhibitory synaptic input of the L2/3 layer (Figure 4)
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These results suggest that the continuous enhancement of the MET signal will lead to changes in the local synaptic connections of PFC L5 neurons, which may disrupt the integration of the different input and output sources of these projection neurons
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Figure 4.
Cto-MET leads to changes in PFC local neural circuit connections (picture quoted from: Ma X, et al.
, Cerebral Cortex, 2021, bhab323).
In summary, these data indicate that after the endogenous MET signal is down-regulated, cto -MET maintains the continuous expression of MET protein for two weeks, which will lead to changes in the biochemical characteristics, cell morphology and electrophysiological characteristics of PFC L5 neurons
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These changes can explain the changes in synaptic activity and cortical neural circuits
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The time-controlled characteristics of MET signals in cortical neurons can regulate the plasticity and function of cortical neural circuits, and ultimately may affect animal behavior
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Therefore, the researchers continued to express MET at P21-35 after weaning, and then tested the open field, elevated plus maze, and three-chamber social interactions in the 16-20 weeks.
interaction) (Figure 5)
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The results show that continuous expression of MET during adolescence can lead to behavioral changes including repetitive stereotyped behaviors and social behaviors in adult mice
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Figure 5.
Cto-MET can cause behavioral changes in adult mice during development (picture quoted from: Ma X, et al.
, Cerebral Cortex, 2021, bhab323) Conclusion and discussion of the article, inspiration and prospects for the use of this research A new genetically modified mouse with adjustable ability, combined with interdisciplinary methods, to determine the signal mechanism of excitatory synaptic connections and functional maturity in the cerebral cortex
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This study proved through experiments that the normal temporal regulation of MET signals is a key factor in the maturation of the PFC neural circuit in mice
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The data shows that after the normal endogenous MET signal is down-regulated, cto-MET transgenic mice can artificially continue to express MET for two weeks, which can change the dendritic structure of cortical neurons, the morphology of dendritic spines, the maturation of synapses, and cortical nerves.
The maturity of the circuit and changes in behavior and social behavior involving the PFC circuit
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 This study proved that although cto-MET does not affect the basic exercise and anxiety behaviors of mice, it increases a large number of repetitive/stereotyped behaviors
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Most notably, these mice showed impaired social skills and reduced social novelty (Figure 5).
The current work supports an emerging view that neurodevelopmental disorders, such as ASD, may be to some extent The above is caused by untimely signal transduction during the critical and sensitive period of neural circuit development [12]
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The effects of MET on the developing brain may be multifaceted, long-term, and far-reaching
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There is increasing evidence that the critical timing of molecular signaling events during development may have long-term effects on brain function
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The cells, neural circuits, and behavioral phenotypes described in this study illustrate how molecular regulatory mechanisms can lead to specific network level (PFC) dysfunctions.
Even if these are not the main causes of specific neurodevelopmental disorders, they are still important for research.
Disease has a powerful effect
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Original link: https://academic.
oup.
com/cercor/advance-article-abstract/doi/10.
1093/cercor/bhab323/6362003?redirectedFrom=fulltext laboratory member (from right to left): Shenfeng Qiu (corresponding author) , Pat Levitt, Jing Wei, Ma Xiaokuang (first author), Chen Ke (picture source: Qiu Lab) Selected previous articles [1] Nat Biomed Eng︱Ye Yuru's team develops whole-brain gene editing-mediated treatment of Alz The new strategy of Heimer's disease [2] Luo Liqun Science heavy review System interpretation ︱ Neural circuit structure-the system that makes the brain "computer" run at high speed [3] Sci Adv ︱ important discovery! The calcium homeostasis regulatory protein Calhm2 regulates the activation of microglia and participates in the process of Alzheimer's disease [4] EMBO J︱ new discovery! AGHGAP11B promotes the expansion of the neocortex into adulthood and improves cognitive ability [5] Cell Death Differ︱ Qi Yitao/Wu Hongmei and others cooperate to reveal the molecular mechanism of SUMO modification regulating neurogenesis in adult mice [6] Cereb Cortex︱A2A receptor antagonist can Reversing the sequence learning impairment induced by abnormal aggregation of α-Syn [7] Neuron︱Nicotine promotes the new discovery of anxiety—the important role of inhibiting the ventral tegmental area-amygdala dopamine pathway [8] Int J Mol Sci︱ Frontier review Interpretation: Pathophysiological response and role of astrocytes in traumatic brain injury [9] Cereb Cortex | Wang Lang's research group reveals that astrocytes have experience-dependent steady-state plasticity [10] New discovery in Nature︱! References on social communication of oxytocin neurons causing maternal behavior (slide up and down to view) 【1】Reh, RK, et al.
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【2】Park, H.
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