Exp Neurobiol 2020; 29(2): 138-149
Published online April 30, 2020
© The Korean Society for Brain and Neural Sciences
Ting-Wei Mi1,2†, Xiao-Wen Sun1,3†, Zhi-Meng Wang1,3†, Ying-Ying Wang1,3†, Xuan-Cheng He1,2†, Cong Liu1,3, Shuang-Feng Zhang1,4, Hong-Zhen Du1,2, Chang-Mei Liu1,2,3* and Zhao-Qian Teng1,2,3*
1State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, 2Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, 3Savaid Medical School, University of Chinese Academy of Sciences, Beijing 100049, 4School of Life Sciences, University of Science and Technology of China, Hefei 230026, China
Correspondence to: *To whom correspondence should be addressed.
Zhao-Qian Teng, TEL: 86-10-82619699, FAX: 86-10-64807099
Chang-Mei Liu, TEL: 86-10-82619690, FAX: 86-10-64807316
†These authors equally contributed to this work.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License
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Neuropsychiatric disorders are the leading cause of mental and intellectual disabilities worldwide. Current therapies against neuropsychiatric disorders are very limited, and very little is known about the onset and development of these diseases, and their most effective treatments.
Neuropsychiatric disorders, including schizophrenia, autism, depression, and anxiety, are very common all around the world . The limited effectiveness of current therapies against neuropsychiatric disorders and neurological disorders highlights the urgent need for understanding their pathological mechanisms and for developing new approaches to prevent or retard the disease progression .
Genome-wide association studies (GWAS) have identified
Neural activity depends on electric signals that are transmitted from the presynaptic neuron to the postsynaptic cell via chemical signaling. The positive or negative change in membrane potential of the postsynaptic neuron is caused by the activation of postsynaptic receptors, which are ion channels whose activation alters permeability for specific ions . Although genetic variation in genes coding for ion channels increases risk for psychiatric disorders [14-17], little is known about the function of miR-137 on ion channels in neurons.
In this study, we provide the first evidence that loss of miR-137 results in impaired homeostasis of potassium in neurons, both
All experiments involving animals were performed in accordance with the animal protocol approved by the Institutional Animal Care and Use Committee at the Institute of Zoology, Chinese Academy of Sciences. Mice were housed in groups of 3~5 animals under a 12 h light/12 h dark cycle, and were fed ad libitum on a standard mouse diet. The miR-137f/f mice were generated as previously described . The Emx1-Cre transgenic mice were bought from Jackson Laboratory (Stock No. 005628). The miR-137 conditional knockout mice were generated by breeding miR-137f/f mice with Emx1-Cre transgenic mice, as described previously .
Hippocampal neurons were isolated from P0
Approximately 300 base pairs around the predicted target site from the KCC2 3’UTR was cloned into the pIS2 vector using the XhoI and NotI restriction sites in the multiple cloning region downstream of the luciferase reporter gene. Mutagenesis of the binding site on KCC2 3’UTR was performed using the QuickChange II Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol. All plasmid clones were then verified by sequencing.
Dual luciferase transfection assays were performed as previously described [20, 21]. In brief, HEK293 cells in 24-well plates were transfected with sh-miR-137 (pCR2.1 TOPO vector) and pIS2-3’UTR or mutated pIS2-3’UTR using Lipofectamine 2000 (Invitrogen). Meanwhile, pIS2 vector with no 3’UTR was cotranfected with U6-neg-shRNA (pCR2.1 TOPO vector) or sh-miR-137 to set up as a control. All Luciferase readings were recorded using Dual-Luciferase Reporter 1000 System (Promega) following manufacturer's instructions.
Whole-cell patch-clamp recordings were carried out using an Axopatch 700B amplifier (Axon Instruments, Union City, CA). The pClamp10.6 software was used for data acquisition and analysis. Patch pipettes (6~10 MΩ) were pulled from borosilicate glass capillaries with a micropipette puller (Sutter instrument, USA). The internal pipette solution contained (in mM): 135 K-gluconate, 10 HEPES, 2 MgCl2, 10 EGTA, 0.3 MgGTP, and 0.5 Na2ATP (pH 7.3 with KOH). The membrane potential was held at -65 mV. Series resistances and cell capacitance compensation were carried out prior to recording. The recordings were included only in those with high resistance seal (>1 GΩ) and a series resistance <25 MΩ.
Total RNA was extracted from hippocampus tissue or cultured neurons using TRIzol reagent (Invitrogen). Two micrograms of total RNA were reverse transcribed with either oligo (dT) primers or specific primers by a Transcriptor First Strand cDNA Synthesis Kit (Roche). For qRT-PCR analysis, 25 ng of cDNA and 0.5 µM primers were used in a final volume of 20 µl according to the manufacturer’s instructions (SYBR Green Master, Roche). Each reaction was run in triplicate and analyzed following the △△CT method using U6 or GAPDH as a normalization control. The following primers are used: KCC2 (forward: 5’-GGGCAGAGAGTACGATGGC-3’; reverse: 5’-TGGGGTAGGTTGGTGTAGTTG-3’), GAPDH (forward: 5’-AAGGTCATCCCAGAGCTGAA-3’; reverse: 5’-AGGAGACAACCTGGTCCTCA-3’).
Hippocampal tissues or cultured neurons were lysed in a buffer containing 25 mM HEPES at pH7.9, 150 mM NaCl, 1 mM PMSF, 20 mM NaF, 1 mM DTT, 0.1% NP40, and proteinase inhibitor cocktails (Roche). Protein concentrations were determined by Folin phenol method with bovine serum albumin as protein standard. Twenty micrograms of protein were separated on 8~12% SDS-PAGE gels (Bio-Rad) and transferred to PVDF membranes (Millipore). The membranes were then blocked in 5% BSA in TBS-T with 0.05% Tween-20, and incubated with primary antibodies at 4°C overnights. Dilutions of primary antibodies were 1:1,000 for KCC2 (Millipore, #07–432), and 1:10,000 for β-actin antibody (Sigma). As for the secondary antibodies, we used HRP-linked goat anti-rabbit at 1:500. Enhanced chemo luminescence (ECL, Pierce) was used for detection. Quantifications of Western blots were determined using Quantity One V4.4.0 (BioRad).
Mice were anesthetized and transcardially perfused with cold PBS, followed by 4% PFA in PBS (pH 7.4). Brain tissue was dissected out, equilibrated in 30% sucrose, and sectioned into 40 µm-thick sections. The brain sections were washed in PBS for 15 min three times, and then blocked in a blocking solution (3% BSA, 0.3%Triton X-100, 0.2% sodium azide) at room temperature for 1 h. The primary antibodies we used were as follows: anti-KCC2 (1:1,000, Millipore, #07–432), anti-Map2 (1:1,000, Millipore, Mab3418). After overnight incubation with primary antibody at 4°C, the brain sections were washed with TBS for 30 min three times and then incubated with the secondary antibodies conjugated with Alexa Fluor 488 or 594 (1:500). Sections were finally stained with DAPI and mounted on glass-slides using adhesion anti-fade medium.
KCC2 shRNA sequence (CUACGAGAA GACAUUAGUA)  was inserted in the U6-shRNA lentiviral construct. Lenti-sh-KCC2 and lenti-sh-Neg (negative control) viruses were produced with titers at a range around 1×109 TU/ml as described previously [23, 24]. Lentivirus was grafted stereotaxically into the hippocampus of 8-week-old male
Mice were kept in groups of 4~5 animals on a 12:12 h light:dark cycle. The open field test and the light-dark preference test were performed during the light phase at week 3 after lentiviral injection as previously described . Videos were recorded and analyzed by the software Smart V3.0.03 (Panlab, Barcelona, Spain).
Either unpaired Student’s two-tailed t tests or ANOVA with Tukey’s post hoc tests were conducted using IBM SPSS Statistics V26 software. Samples sizes were provided in each figure legend. All data were presented as mean±SEM. Differences were considered statistically significant when p<0.05.
We originally generated miR-137 conditional knockout mice that displayed dysregulated synaptic plasticity, repetitive behavior, anxiety-like behavior, and impaired learning and social behavior . Since neurological and neuropsychological diseases are pathophysiologically linked to potassium channel dysfunction [26, 27], we speculated that loss of miR-137 may play a role in regulating K+ currents and thus result in neurodysfunction.
To examine the electrophysiological properties of primary hippocampal neurons isolated from
To get more insight into the molecular mechanism underlying the effect of miR-137 loss-of-function on voltage-gated K+ current, we performed a combined computational and experimental study to identify the downstream targets of miR-137 in the brain. We first used the TargetScan program to predict mRNA targets . TargetScan analysis identified 15 potassium channel associated targets predicted to be responsive to miR-137 and conserved among species. Among these candidate targets, the K+–Cl- cotransporter 2 (aka: KCC2 and SLC12A5) is known to play pivotal roles in the physiology of neurons, and its malfunction has been linked to multiple neurological diseases including seizures, epilepsy, and schizophrenia [29-33]. Indeed, there is a highly conserved binding site of miR-137 on the 3’-UTR sequence of mouse
Next, we examined the expression levels of KCC2 in
To further explore the role of KCC2 in the electrophysiological properties of neurons, we used a pharmacological agent, the KCC2 antagonist VU0240551, to intervene the function of KCC2 by preincubating neurons with the agent for 1 hour (10 µM), and performed the whole-cell patch-clamp recordings of primary hippocampal neurons at DIV 14. All primary hippocampal neurons elicited multiple action potentials upon the injection of depolarizing currents (Fig. 4A) and large fast-inactivating inward currents followed by outward potassium currents when evoked by a series of voltage steps (Fig. 4B). We then assessed membrane properties of hippocampal neurons that threated with VU0240551 by recording voltage-dependent currents in voltage-clamp mode. We found that maximum peak outward potassium amplitude of VU0240551-treated
Recently, we found that mice with forebrain-specific miR-137 loss-of-function can survive to adulthood, but exhibit anxiety-like behavior . To examine whether knockdown of KCC2 might be beneficial to ameliorate anxiety-like behavior in
In the open field test, sh-KCC2 significantly ameliorates the anxiety-like behavior in
Dysfunction of miR-137 has been linked with the pathogenesis of schizophrenia , bipolar disorder , anxiety and depression , and autism spectrum disorders [5, 10]. Fine-tuning the expression of miR-137 is critical in regulating neural development and synaptic plasticity [9, 20, 34]. Overexpression of miR-137 results in changes in synaptic vesicle pool distribution, impaired induction of mossy fiber long-term potentiation and deficits in hippocampus-dependent learning and memory . He et al.  then confirmed these observed changes in synaptic transmission upon miR-137 overexpression. Although selective synaptic vesicle docking defects were not obtained, miR-137 overexpression had remarkable effects on docking, active zone length and total vesicle number . Syt1, complexin-1 and neuroligin-3 are known miR-137 targets involved in synapse development [9, 34]. In contrast, a complete knockout of
KCC2 mutations or dysfunctions have been identified as a critical component in the development of autism spectrum disorder , schizophrenia, epilepsy and seizures [38-42], neuropathic pain . KCC2 has been well-known for its role in maintaining a low intracellular Cl- concentration ([Cl-]i) essential for hyperpolarizing inhibition mediated by GABAA receptors , its loss-of-function results in enhanced [Cl-]i which thereby represses the inhibitory strength of GABA and glycine, whose cognate receptors are ligand-gated ion channels permeable to Cl- and HCO3- ions [45-47]. Besides its central role in hyperpolarizing inhibitory signaling based on chloride currents which are mediated by GABA- or glycine-gated receptor channels, KCC2 also acts a structural protein crucially involved in the maturation and regulation of excitatory glutamatergic synapses [48-51]. KCC2 is required for neuronal maturation by rendering GABA hyperpolarizing [49,52-54], and overexpression of KCC2 enhances dendritic spines in the adult nervous system in mice . Therefore, we speculate that the elevated expression of KCC2 may also contribute to dysregulated synaptic plasticity and altered behaviors in
Potassium is essential for the proper function of all cells . In neurons, the sodium-potassium flux generates the electrical potential that aids the conduction of nerve impulses . Potassium channels participate in cell ionic balance and serve the fundamental function of supporting action potentials and electrical signal propagation along the neurons and their myelinated axons [57-59]. The increased outward K+ currents in
Surprisingly, increased K+ currents in
In summary, we show here that miR-137 is a crucial player in the homeostasis of potassium by directly targeting KCC2, and treatment with the KCC2 antagonist can maintain potassium homeostasis in
This work was supported by grants from the National Key Research and Development Program of China Project (2018YFA0108001), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16010300), the National Science Foundation of China (91753140), the Beijing Natural Science Foundation (7182107), and the Open Project Program of State Key Laboratory of Stem Cell and Reproductive Biology.
MiR-137 deficiency impairs K+ efflux of potassium in primary mouse hippocampal neurons. (A) Phase-contrast images of primary mouse hippocampal neurons during patch clamp recordings at DIV 14. (B) Representative traces of action potentials in response to step current injections in primary mouse hippocampal neurons at DIV 14. Membrane potential was maintained at approximately -40 mV. Step currents were injected from -50 pA to +250 pA in 50 pA increments (middle panel). All neurons elicited multiple action potentials upon the injection of depolarizing currents. (C) Representative traces of whole-cell currents in voltage-clamp mode. Primary neurons from
KCC2 is a direct target of miR-137. (A) Sequence alignment of miR-137 and the KCC2 3’-UTR, which contains a predicted conserved miR-137-biding site. The seed-recognizing site in the KCC2 3’-UTR is indicated in red, while the mutant KCC2 3’-UTR site is denoted in green. (B) KCC2 WT 3’-UTR-dependent expression of a Renilla luciferase was reduced, while mutation of the miR-137 binding site in the KCC2 3’-UTR did not affect the Renilla luciferase activity. (C) KCC2 mRNA expression levels were upregulated both in the hippocampus and in the prefrontal cortex of
KCC2 is upregulated upon the loss of miR-137 both
KCC2 antagonist maintains potassium homeostasis in mouse
KCC2 knockdown rescues anxiety-like behaviors in