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MicroRNA-128 Governs Neuronal Excitability and Motor Behavior in Mice

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Science  06 Dec 2013:
Vol. 342, Issue 6163, pp. 1254-1258
DOI: 10.1126/science.1244193

Not Too Much, Not Too Little

The microRNA miR128 is expressed in brain neurons of the mouse. Lek Tan et al. (p. 1254) now find that miR128 is crucial to stable brain function. Mice deficient in miR128 developed hyperactivity and were susceptible to fatal seizures, whereas overexpression of miR128 correlated with reduced motor activity and reduced susceptibility to proconvulsive drugs. Experiments using ex vivo–isolated adult brain tissues suggested that miR-128 controlled motor activity by governing the signaling network that determines the intrinsic excitability and signal responsiveness of neurons.

Abstract

The control of motor behavior in animals and humans requires constant adaptation of neuronal networks to signals of various types and strengths. We found that microRNA-128 (miR-128), which is expressed in adult neurons, regulates motor behavior by modulating neuronal signaling networks and excitability. miR-128 governs motor activity by suppressing the expression of various ion channels and signaling components of the extracellular signal–regulated kinase ERK2 network that regulate neuronal excitability. In mice, a reduction of miR-128 expression in postnatal neurons causes increased motor activity and fatal epilepsy. Overexpression of miR-128 attenuates neuronal responsiveness, suppresses motor activity, and alleviates motor abnormalities associated with Parkinson’s–like disease and seizures in mice. These data suggest a therapeutic potential for miR-128 in the treatment of epilepsy and movement disorders.

MicroRNA-128 (miR-128) is one of the most abundant and highest enriched miRNAs in the adult mouse and human brain (fig. S1A) (1, 2). The expression of miR-128 in the mouse brain increases gradually during postnatal development and peaks in adulthood (fig. S1B) (3, 4). miR-128’s expression in diverse brain regions (fig. S1C) suggests an important role for this miRNA in processes that are common to many neuronal cell types.

The indication of a potent regulatory role for miR-128 in brain function came from our observation of early-onset fatal epilepsy in mice deficient in miR-128 (Fig. 1A). miR-128 is encoded by two separate genes, miR-128-1 and miR-128-2, on mouse chromosomes 1 and 9 (fig. S2, A and B) or human chromosomes 2 and 3, respectively. In mice, germline miR-128-2 deficiency results in an 80% reduction of miR-128 expression in the forebrain, whereas ablation of the miR-128-1 gene eliminates only 20% of miR-128 (fig. S2, A and B). The profound decline in miR-128 expression levels in miR-128-2−/− but not miR-128-1−/− mice is associated with the development of hyperactivity and increased exploration at 4 weeks of age (Fig. 1A and fig. S2, C and D). The juvenile hyperactivity in miR-128-2−/− mice progresses quickly to severe seizures and death at 2 to 3 months of age (Fig. 1, A and B, and movie S1). The lethal impact of miR-128 deficiency in mice can be prevented by treatment with the anticonvulsant drug valproic acid (Fig. 1C), thus demonstrating the causal role of seizures in the animals’ death.

Fig. 1 miR-128 controls motor behavior in mice.

(A) Deficiency in miR-128-2 causes hyperactivity and premature death in mice. (Left) Motor activity was determined by measuring total horizontal distance in a 60-min open field assay (n = 23 and 12 mice). (Right) The life spans of miR-128-2−/− mice and littermate controls are shown (n = 20 and 46 mice). (B and C) miR-128 deficiency causes fatal seizures that can be prevented with anticonvulsant treatment. (B) Representative display of spontaneous tonic-clonic seizure episodes in miR-128-2−/− (black) or Camk2a-cre; miR-128-2fl/fl mice (red) during a 22-day observation period. (C) The life spans of control miR-128-2−/− [dotted line, as shown in (A)] or sodium valproate-treated (red, n = 11 mice) miR-128-2−/− mice are shown. (D) Deficiency in miR-128 in postnatal neurons causes hyperactivity and fatal epilepsy. Motor activity and survival rates of Camk2a-cre; miR-128-2fl/fl mice (n = 21 and 25 mice) and littermates (n = 8 and 47 mice) are shown. (E) Ectopic expression of miR-128 normalizes hyperlocomotion and prevents death of Camk2a-cre; miR-128-2fl/fl mice. Motor activity in Camk2a-cre; miR-128-2fl/fl; Rosa-miR-128 (blue, n = 4 mice) and control mice (gray, n = 10 mice) are shown. The life spans of Camk2a-cre; miR-128-2fl/fl mice in the presence (blue, n = 4 mice) or absence (black, n = 9 mice) of ectopic miR-128 expression are shown. (F) miR-128 deficiency in D1-neurons causes hyperactivity and fatal epilepsy. Motor activity (n = 26 and 42 mice) and life spans (n = 16 and 28 mice) of mice with a D1-neuron–specific miR-128 deficiency or control mice are shown. Error bars show SEM, Welch’s t test, nonsignificant (ns), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Kaplan-Meier graph shows survival curves of mutant and littermate control mice; *P ≤ 0.05, ***P ≤ 0.001, log rank tests.

The hyperactivity and fatal epilepsy in miR-128-2–deficient mice reflects the ability of miR-128 to control the excitability of postnatal neurons. Selective inactivation of the miR-128-2 gene in forebrain neurons (Camk2a-cre; miR-128-2fl/fl) leads to a reduction of miR-128 expression, followed by early onset hyperactivity, seizures, and death, as observed in miR-128-2−/− mice (Fig. 1, B and D, and fig. S3A). Moreover, correction of miR-128 deficiency by ectopic miR-128-2 expression in neurons normalizes motor activity and prevents the seizure-induced death (Fig. 1E and fig. S4, A and C).

To gain an understanding of the mechanism that mediates miR-128–dependent control of motor activity, and to avoid interference between phenotypes caused by the loss of miR-128 in diverse neuronal cell types, we restricted the miR-128-2 deficiency to dopamine-responsive neurons that regulate motor behavior in mice and humans. There are two major dopamine-responsive Camk2a-expressing neuron types in the mouse forebrain, which have distinct contributions to motor activity (5). Although activation of the dopamine 1 receptor–expressing neurons (D1-neurons) increases locomotion, activation of dopamine 2 receptor–expressing neurons (D2-neurons) reduces locomotion in mice (6). We found that miR-128 deficiency in D1-neurons (Drd1a-cre;miR-128-2fl/fl), but not in D2-neurons (A2a-cre;miR-128-2fl/fl), leads to juvenile hyperactivity, followed by lethal seizures at ~5 months of age (Fig. 1F and fig. S3, B and C).

To identify miR-128 targets that are responsible for the abnormal motor activity, we analyzed mRNAs associated with the RNA-induced silencing complex (RISC) in adult neurons in vivo. The RISC-bound mRNAs represent the pool of cellular mRNAs that become a subject of miRNA-mediated suppression (7). We used mice that express the epitope-tagged RISC component Argonaute 2 (Ago2) (8) in Camk2a-neurons (fig. S5A). Immunoprecipitation of Ago2 [high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) (9)] from the forebrain of these mice yielded the neuron-specific RISC-associated mRNAs (fig. S5, A and B). The perfect base pairing of at least six nucleotides between the miRNA seed sequence and the 3′ untranslated region (3′UTR) of the RISC-associated mRNAs (10) was considered to be the minimal requirement for any potential miRNA-mediated mRNA suppression (fig. S5, B and C). Using these criteria, we found that the miR-128 seed target sequence (ACUGUG) is the most represented hexamer among all RISC-associated mRNAs (fig. S5C and table S1) and identified a total of 1061 potential miR-128 target mRNAs in adult neurons (table S2).

We investigated these miR-128 target genes by analyzing their expression in neurons deficient for miR-128. We expected that mRNA transcripts that are targeted directly by miR-128 in neurons would show an increase in mRNA expression and subsequent ribosome association in the miR-128–deficient cells. We reasoned that the relative homogeneity of the D1-neuron population might provide the most accurate assessment of miR-128–dependent target genes that are responsible for controlling motor activity. The impact of miR-128 deficiency on mRNA expression was evaluated by D1 cell-type–specific translating ribosome affinity purification (TRAP) in mice (11). The TRAP approach allows a direct comparison between ribosome-associated mRNAs from wild-type and miR-128–deficient D1-neurons in vivo (fig. S6A). Using Sylamer analysis (12), we confirmed the expected enrichment of potential miR-128 binding sites among the most up-regulated genes in miR-128–deficient D1-neurons (fig. S6B). We found that the deficiency of miR-128 in D1-neurons results in a significant up-regulation of 154 of the predicted RISC-associated miR-128 target genes (Fig. 2A and table S3). The fact that only ~15% of the potential RISC-associated miR-128 targets display increased expression is likely to reflect the known redundancy among miRNAs. Many mRNAs are regulated by more than one miRNA (13, 14), thus limiting the actual impact of individual miRNA deficiency on the expression of miRNA targets in vivo.

Fig. 2 miR-128 controls signaling protein expression and activation of the ERK signaling network in neurons.

(A) Venn diagram shows the RISC-associated mRNA targets of miR-128 (red) and mRNAs that are up-regulated in miR-128–deficient D1-neurons (blue). The overlapping 154 mRNAs are considered as direct miR-128 targets. (B) (Left) Gene ontology annotations of the 154 miR-128 target genes are shown with pathway enrichment presented as –log10 (P value). The dotted orange line indicates P = 0.05. (Right) The components of the ERK1/2 network (P = 10−46, right-tailed Fisher’s exact test) that are directly targeted by miR-128 are indicated in solid gray. (C) Expression levels of miR-128–targeted ERK regulators in the striatum of Drd1a-cre; miR-128-2fl/fl and littermate controls were analyzed by means of Western blotting (n = 4 mice each). (D) Increased ERK2 phosphorylation in the striatum of mice with D1-neuron–specific miR-128 deficiency. Representative Western blot analysis of ERK1/2 phosphorylation in the striatum of control and Drd1a-cre; miR-128-2fl/fl mice is shown; bar graphs display phospho-ERK/ERK protein ratios (n = 4 mice). Error bars show SEM, Welch's t test, *P ≤ 0.05, **P ≤ 0.01.

Bioinformatic network and pathway analyses of the miR-128 target genes indicate the ability of miR-128 to affect molecular processes that are intrinsically linked to the regulation of neuronal excitability and motor behavior in mice and humans (Fig. 2B). In particular, miR-128 regulates the expression of numerous ion channels and transporters, as well as genes that contribute to neurotransmitter-driven neuronal excitability and motor activity (Fig. 2B and tables S3 and S4). Several of these genes are linked to epilepsy in humans, some of which—including the neurotransmitter γ-aminobutyric acid transporter Slc6a1, the high-affinity glutamate receptor Slc1a1, the voltage-gated sodium channels Scn2b and Scn4b, the voltage-dependent calcium channels Cacna2d3 and Cagn2, as well as the carbonic anhydrase Car7—are potential targets of clinically approved anti-seizure drugs (tables S3 and S4) (15). The high abundance of extracellular signal–regulated kinase (ERK1/2) signaling network components among the miR-128 targets underscores the potential of this miRNA to control signaling processes associated with neuronal excitability (Fig. 2B). Moreover, many of the neuronal signaling proteins and channels that we identified as direct miR-128 target genes are involved in the regulation of upstream signaling events, which can affect ERK activity (tables S3 and S4). Although ERK1 and ERK2 are not directly targeted by miR-128, the ERK network appears to be at the center of the miR-128–controlled signaling circuit in neurons. The protein expression levels of potent ERK network regulators, which are directly targeted by miR-128—such as Pea15a (16), D4Ertd22e/Szrd1 (17), and the TARPP protein that is encoded by the long splice variant of the miR-128-2 host gene Arpp21 (18, 19)—are increased in the striatum of mice with a D1-neuron–specific deficiency in miR-128 (Fig. 2C and fig. S7). Furthermore, mice with a D1-neuron–specific deficiency of miR-128-2 display an increase in ERK2 activation as compared with that of their littermate controls (Fig. 2D). Only ERK2, but not ERK1, displays increased phosphorylation (Fig. 2D). Deficiency of miR-128 in D1-neurons appears to specifically activate ERK2 phosphorylation, without affecting the activation of other MAP kinase pathways components, such as the stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK) or protein kinase B (AKT) (fig. S8).

Electrophysiological studies in striatal slices from Drd1a-cre; miR-128-2fl/fl mice revealed an increase in D1-neuron excitability. The miR-128–deficient D1-neurons show normal membrane excitability at the soma (fig. S9A) but display enhanced dendritic excitability (Fig. 3A) as well as a ~20% increase of functional dendritic spines (Fig. 3B and fig. S9B). These findings are consistent with a critical role of the ERK2 network in neuronal excitability and synaptic plasticity (20, 21).

Fig. 3 miR-128 controls D1-neuron excitability and responsiveness to dopamine.

(A and B) miR-128 regulates D1-neuron dendritic excitability and number of spines. (A) Single action potentials were generated in the soma, and action potential invasion was calculated by dividing the distal calcium signal by the maximum proximal calcium signal per cell (n = 4 cells, 11 to 21 shafts per group). Mann-Whitney nonparametric test, ***P ≤ 0.001. (B) Representative maximum intensity projection images of distal dendrites in control and mutant D1-neurons are shown. Boxplots display population spine densities (n = 10 to 11 cells per group). Mann-Whitney nonparametric test, error bars show 90th percentile interval, *P ≤ 0.05. (C to E) miR-128 regulates motor response, ERK2 phosphorylation, and immediate early gene (IEG) induction upon dopamine D1 receptor (Drd1) activation in D1-neurons. (C) Motor activity of Drd1a-cre; miR-128-2fl/fl and control mice (n = 25 and 30 mice) was evaluated in an open-field chamber. Saline and 3 mg/kg Drd1 agonist SKF81297 were injected intraperitoneally at 10 and 20 min, respectively. (D) ERK2 phosphorylation was quantified by means of Western blotting of striatal lysates derived from Drd1a-cre; miR-128-2fl/fl and control mice that received saline or D1-agonist SKF81297 injection (n = 5 mice each). Bar graph displays the ratio of phospho-ERK2 to total ERK2 expression. (E) IEG and D1-neuron–expressed Darpp32 gene expression levels were measured by means of quantitative reverse transcription polymerase chain reaction of D1-neuron–specific polyribosome-associated mRNAs purified from saline or SKF81297-treated Drd1a-TRAP; Drd1a-cre; miR-128-2fl/fl and control mice (n = 5 mice each). Error bars display SEM, Welch's t test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Enhanced ERK2 activation is linked to increased motor activity and seizures in mice (2224). The hyperactivation of ERK2 and concomitant increase in D1-neuron sensitivity to dopamine occurs also during Parkinson’s-like disease in mice caused by chemically induced depletion of dopamine in the mouse striatum (2527). The reduced levels of dopamine and concurrent increase of D1-neuron sensitivity result in hyperresponsiveness to the motor activity–inducing effects of dopamine (2628). In humans, the D1-neuron hyperresponsiveness is one of the major causes of dyskinesia, a side effect of l-DOPA treatment in Parkinson’s disease (2527).

We found that miR-128 deficiency in striatal D1-neurons mimics the hypersensitivity of D1-neurons in mice suffering from Parkinson’s-like syndrome. The deficiency of miR-128 in D1-neurons enhances motor activity in response to Drd1-specific agonist treatment in mice (Fig. 3C). The D1-neuron hyperresponsiveness to the Drd1 agonist is also associated with an increase in ERK2 phosphorylation in the striatum of Drd1a-cre; miR-128-2fl/fl mice (Fig. 3D). The increase in dopamine sensitivity and enhanced ERK2 activation in mice with Parkinson’s-like disease are accompanied by increased expression of dopamine-induced immediate early genes (IEGs) in D1-neurons (2527). Similarly, Drd1 agonist treatment enhances IEG expression in miR-128–deficient D1-neurons as compared with the D1-neurons of control mice (Fig. 3E). The increased locomotor activity characteristic of Drd1a-cre; miR128-2fl/fl mice was normalized by means of pharmacological inhibition of the mitogen-activated protein kinase kinase MEK1, a major activator of ERK2 in neurons. In vivo–administered MEK1-specific inhibitor SL327 does not affect motor activity in wild-type mice (22) but does normalize ERK2 phosphorylation and motor activity in the mutant mice (Fig. 4A). In turn, overexpression of miR-128 in Camk2a-neurons is associated with reduced ERK2 activation (fig. S10A) and decreased motor activity (fig. S4B) in mice. The effect of increased miR-128 expression in adult neurons protects mice against abnormal motor activities associated with chemically induced Parkinson’s disease (Fig. 4B and fig. S10B) and seizures (Fig. 4C).

Fig. 4 Abnormal motor activity caused by miR-128 deficiency is corrected by pharmacological ERK inhibition or ectopic miR-128 expression.

(A) Drd1a-cre; miR-128-2fl/fl and littermate control mice were injected intraperitoneally with either vehicle or 12 mg/kg of the MEK1 inhibitor SL327 (n = 5 mice per group). Western blot analysis of ERK2 phosphorylation at 30 min after drug injection (left) and motor activity after vehicle or SL327 injection (right) are shown. Two-way analysis of variance followed by Bonferroni post-test. Error bars show SEM, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. (B) Overexpression of miR-128 suppresses D1-neuron hyperresponsiveness in the dopamine-depleted striatum. The number of contralateral rotations at baseline and in response to cocaine (10 mg/kg) or D1-agonist SKF81297 (5 mg/kg) in unilateral 6-hydroxydopamine–lesioned Camk2a-cre; Rosa-miR-128 or control mice (n = 11 mice per group) are shown. Error bars show SEM,Welch’s t test, **P ≤ 0.01. (C) miR-128 reduces the susceptibility to chemically induced seizures in mice. The numbers of Camk2a-cre; Rosa-miR-128 or littermate control mice (n = 12 mice per group) that exhibit tonic-clonic seizures 60 min after intraperitoneal injection of proconvulsive drugs kainic acid (30 mg/kg, P = 0.005) or picrotoxin (3 mg/kg, P = 0.04) are shown. P values were calculated by means of Fisher’s exact test.

We have identified miR-128 as a modulator of signaling pathways that control neuronal excitability and motor activity in mice. The human miR-128-2 gene on chromosome 3p lies within a region that has been linked to idiopathic generalized epilepsy (29, 30). It is tempting to speculate that changes in miR-128 or miR-128 target gene expression could be a potential cause of increased neuronal excitability and epilepsy in humans. Our understanding of miR-128’s role in neuronal signaling could prove advantageous in the design of novel therapeutics for epilepsy and motor disorders.

Supplementary Materials

www.sciencemag.org/content/342/6163/1254/suppl/DC1

Materials and Methods

Figures S1 to S10

Tables S1 to S4

References (3139)

Movie S1

References and Notes

  1. Acknowledgments: We thank K. Rajewsky, G. Schuetz, and N. Heintz for mice and reagents. We thank C. Bargman and A. Tarakhovsky for valuable comments and P. Zamore for advice and critical reading of the manuscript. This work was supported by the NIH 1DP2MH100012-01 and DA025962 (A.S.), P50MH090963 (P.G., A.S., and D.J.S.), DA10044 (P.G.), NS34696 (D.J.S.), the Seaver Autism Foundation (A.S.), the USAMRAA W81XWH-090100402 and JPB Foundation (P.G.), the Lundbeck Foundation and Center for Integrative Sequencing at Aarhus University (M.T.V. and J.K), and the CHDI Foundation (D.J.S.). The authors declare no competing financial interests. The microarray data reported in this paper are archived at the Gene Expression Omnibus (GEO) Repository (www.ncbi.nlm.nih.gov/geo, accession no. GSE48813). Correspondence and requests for materials should be addressed to A.S. via anne.schaefer@mssm.edu. Mount Sinai and The Rockefeller University filed a United State provisional patent application (61/896,463 and 61/898,952) that covers the treatment of epilepsy using miR-128.
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