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.2014 Dec 3;34(49):16166-79.
doi: 10.1523/JNEUROSCI.2580-14.2014.

MET receptor tyrosine kinase controls dendritic complexity, spine morphogenesis, and glutamatergic synapse maturation in the hippocampus

Affiliations

MET receptor tyrosine kinase controls dendritic complexity, spine morphogenesis, and glutamatergic synapse maturation in the hippocampus

Shenfeng Qiu et al. J Neurosci..

Abstract

The MET receptor tyrosine kinase (RTK), implicated in risk for autism spectrum disorder (ASD) and in functional and structural circuit integrity in humans, is a temporally and spatially regulated receptor enriched in dorsal pallial-derived structures during mouse forebrain development. Here we report that loss or gain of function of MET in vitro or in vivo leads to changes, opposite in nature, in dendritic complexity, spine morphogenesis, and the timing of glutamatergic synapse maturation onto hippocampus CA1 neurons. Consistent with the morphological and biochemical changes, deletion of Met in mutant mice results in precocious maturation of excitatory synapse, as indicated by a reduction of the proportion of silent synapses, a faster GluN2A subunit switch, and an enhanced acquisition of AMPA receptors at synaptic sites. Thus, MET-mediated signaling appears to serve as a mechanism for controlling the timing of neuronal growth and functional maturation. These studies suggest that mistimed maturation of glutamatergic synapses leads to the aberrant neural circuits that may be associated with ASD risk.

Keywords: MET receptor tyrosine kinase; autism; glutamatergic circuit; hippocampus; mouse model; synaptogenesis.

Copyright © 2014 the authors 0270-6474/14/3416166-14$15.00/0.

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Figures

Figure 1.
Figure 1.
MET signaling affects growth and morphological development of hippocampal neuronsin vitro.A, MET signaling competency in live P3 hippocampus slices and DIV 7 cultured hippocampal neuron. HGF stimulation leads to tyrosine (Y1234/1235) phosphorylation of MET.B, Cultured hippocampal neurons were transfected with GFP to reveal morphology. HGF treatment enhances the growth of both developing dendrites and axons.C, Quantification of neurite growth as shown inB (*p < 0.05; ***p < 0.001).D, Test of efficacy of three 19-nt RNAi sequences by cotransfection with MET cDNA in HEK293 cells. RNAi sequences were cloned into PLVTHM vector, which has bicistronic expression of GFP. RNAi #1 sequence is most efficient in reducing MET expression.E, Test of knockdown efficiency of RNAi sequence #1 on endogenous MET expression in cultured hippocampal neurons. This RNAi sequence was cloned into pSuper vector for faster expression and designated as ‘RNAi’ hereafter.F, MET OE and RNAi affect dendritic protrusions of cultured hippocampal neurons during the second week in culture.G, Quantification ofF. MET OE significantly increased (**p < 0.01), while RNAi significantly decreased (**p < 0.01), the density of dendritic protrusions.H–K, MET OE or RNAi alters dendritic spine density and morphology (H). MET OE increases (***p < 0.001), while RNAi decreases (*p < 0.05), the density of dendritic spines (I). A scrambled sequence of RNAi was without effect. MET OE decreases, while RNAi increases, spine head area (J) (p < 0.01, for both effects). MET OE also significantly increases dendritic spine length (K) (*p < 0.05). Scale bars:F,H, 10 μm.
Figure 2.
Figure 2.
Altered MET signaling affects morphological development of hippocampal neuronsin vivo.A, Schematic illustration of deliveringMet cDNA and RNAi to developing CA1 neurons using IUEP. Offspring were harvested at P22–P25 for morphological analysis.B, Individual CA1 neurons labeled with IUEP were subjected to morphological analysis. Dendritic arbor of CA1 neurons were traced and reconstructed with Neurolucida. High-resolutionZ-stack images were then collected, and dendritic spines were reconstructed with Imaris.C, Representative neurons labeled by IUEP with control GFP, MET, RNAi, and scr RNA, and their reconstructed 3D dendritic arborization and dendritic spines.D, Sholl analysis of CA1 neurons revealed that MET OE increases, while RNAi decreases, the number of dendritic intersections at distinct compartments of the apical dendritic tree (*p < 0.05,#p < 0.05).E,F, MET OE significantly increases total apical dendritic length (E, *p < 0.05), the number of apical dendritic branches (F, *p < 0.05), whereas RNAi significantly decreases total apical dendritic length (**p < 0.01) and the number of apical dendritic branches (*p < 0.05).G,H, MET OE significantly decreases dendritic spine head volume (G, **p < 0.01) and increased spine density (H, *p < 0.05), whereas RNAi significantly enlarges dendritic spine head volume (**p < 0.01) and decreased spine density (**p < 0.01).I, Altered MET signaling changes the categorical spine classification based on the morphology. MET OE significantly decreased (p < 0.05), whereas RNAi increased (p < 0.05) the proportion of the more mature, mushroom-type spines.
Figure 3.
Figure 3.
Disrupted MET signalingin vivo alters CA1 neuron synaptic function.A, Photomicrograph illustrates labeling of neurons in hippocampal slices prepared from mice that had undergone IUEP. CA1 neurons transfected with GFP, MET, and RNAi were selected for patch-clamp recording.B, Quantification of A:N ratio. MET OE leads to reduced A:N ratio (*p < 0.05), whereas RNAi increased A:N ratio (*p < 0.05).C, Representative sample mEPSC traces recorded in CA1 neurons transfected with GFP, MET, and RNAi by IUEP. Average mEPSC from a 1 min epoch are displayed on the right.D, Plot of the cumulative fraction of mEPSC amplitude distribution. MET OE leads to significantly reduced size mEPSC amplitude (p < 0.01), whereas RNAi treatment results in significantly increased mEPSC amplitudes (p < 0.05).E, MET OE in CA1 neuron results in decreased mEPSC frequency (*p < 0.05). RNAi did not result in statistically significant changes in frequency.
Figure 4.
Figure 4.
Ablation of MET signaling affects glutamatergic synapse development.A, Photomicrograph illustration of double labeling of GluN1 and GluA1, or PSD-95 and synapsin I. Colocalized synaptic puncta were used to represent putative functional synapses. Cultures were prepared from individualMetfx/fx;emx1cre embryos and theirMetfx/fx littermate controls.B, Quantification of GluN1 and GluA1 immunolabeling. No statistical difference was found between the two genotypes for density of individual GluN1+ or GluA+ puncta. However, a significant increase in the proportion of putative functional synapse (defined by colabeling of GluN1 and GluA1 profiles) was found inMetfx/fx;emx1cre neurons (*p < 0.05).C, Quantification of PSD-95 and synapsin I immunoreactivity. No overall difference was found for density of individual PSD-95+ or synapsin I+ puncta. A significant increase in the proportion of putative functional synapse (colabeled PSD-95 and synapsin I puncta) was found inMetfx/fx;emx1cre neurons (*p < 0.05).D, Sample mEPSC recording traces obtained fromMetfx/fx andMetfx/fx;emx1cre cultures.Metfx/fx;emx1cre neurons showed significantly enlarged mEPSC amplitude (p < 0.02). No change of mEPSC frequency was seen.E, Representative image on Neurolucida and Imaris reconstructions of single CA1 neuronal dendrites and spines from P21–P22Metfx/fx mice that had undergone IUEP with CAG-GFP or CAG-GFP/cre plasmids.F–I, Quantification of single neuron knock-out ofMet by IEUP. CAG-GFP/cre electroporated neurons displayed significantly reduced total dendritic length in both basal and apical compartments (F, *p < 0.05, **p < 0.01), and decreased number of apical dendrites (G, *p < 0.05). Dendritic spines from CAG-GFP/cre electroporated neurons show a significantly reduced density (H, **p < 0.01) and enlargement of head volume (I, **p < 0.01) compared with that from CAG-GFP electroporated neurons.
Figure 5.
Figure 5.
MET loss of function leads to accelerated glutamatergic circuit maturation.A, Analysis of mEPSC in P12–P14 acute hippocampal slices revealed an enlarged mEPSC amplitude inMetfx/fx;emx1cre neurons (p < 0.02).B,Metfx/fx;emx1cre CA1 neurons in P6–P7 and P14 slices show significantly increased A:N ratio (*p < 0.05).C,Metfx/fx;emx1cre CA1 neurons in P7–P9 and P14 slices show significantly decreased sensitivity to ifenprodil (*p < 0.05; **p < 0.01), as measured by reduction of NMDAR current after ifenprodil (3 μm).D, No change of paired pulse ratio was found at various interpulse intervals at both P7–P8 and P14.E, Illustration of isolation of whole CA1 tissue lysate (1), total synaptosome protein (2), and synaptic surface protein (3), and quantification protocol.F, Representative Western blot of different glutamate receptor subunit proteins, postsynaptic scaffold proteins, and signaling molecules in whole CA1 lysates.G, Representative blots and quantification of glutamate receptor subunits in total synaptosome fraction and synaptic membrane surface compartments. Quantitative analysis of synaptic surface proteins revealed increased GluA1, GluN2A, and decreased GluN2B levels of immunoreactive protein (*p < 0.05). No significant change of total synaptosome protein was seen (quantification not shown).
Figure 6.
Figure 6.
MET loss of function reduces silent synapses and enhances AMPAR quantal transmission.A, Representative recording probes the proportion of silent synapses in P12–P14 slices of bothMetfx/fx andMetfx/fx;emx1cre mice.B,C, Quantification indicates thatMetfx/fx;emx1cre neurons had significantly lower proportion of silent synapses (*p < 0.05), as indicated by a smaller difference of failure rate at −70 mV and 40 mV.D, Nonstationary fluctuation analysis was applied to analyze the quantal parameters of minimum stimulation-induced EPSCAMPA. Traces from successful trails are displayed on top, with the trace averages highlighted. Two average traces from both genotypes are peak scaled to indicate no change of kinetics. Calculated variances (dotted lines) of these traces following peak scaling were displayed above. EPSC variance was plotted against binned mean EPSC amplitude, averaged AMPAR numbers that open at the peak of mean EPSC, and single-channel conductance was calculated by curve fitting from 7 or 8 experiments.
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