MicroRNA-219 modulates NMDA receptor-mediated neurobehavioral dysfunction

Abstract

N-methyl-D-aspartate (NMDA) glutamate receptors are regulators of fast neurotransmission and synaptic plasticity in the brain. Disruption of NMDA-mediated glutamate signaling has been linked to behavioral deficits displayed in psychiatric disorders such as schizophrenia. Recently, noncoding RNA molecules such as microRNAs (miRNAs) have emerged as critical regulators of neuronal functions. Here we show that pharmacological (dizocilpine) or genetic (NR1 hypomorphism) disruption of NMDA receptor signaling reduces levels of a brain-specific miRNA, miR-219, in the prefrontal cortex (PFC) of mice. Consistent with a role for miR-219 in NMDA receptor signaling, we identify calcium/calmodulin-dependent protein kinase II gamma subunit (CaMKIIgamma), a component of the NMDA receptor signaling cascade, as a target of miR-219. In vivo inhibition of miR-219 by specific antimiR in the murine brain significantly modulated behavioral responses associated with disrupted NMDA receptor transmission. Furthermore, pretreatment with the antipsychotic drugs haloperidol and clozapine prevented dizocilpine-induced effects on miR-219. Taken together, these data support an integral role for miR-219 in the expression of behavioral aberrations associated with NMDA receptor hypofunction.

Fig. 1.

NMDA-R hypofunction is associated with decreased miR-219 expression in prefrontal cortex. (A) miR-219 expression was quantified by RT-PCR from the PFC, hippocampus, and cerebellum of adult male C57BL/6 mice injected with saline (n = 9) or dizocilpine (MK-801) (0.5 mg/kg; i.p. injection; n = 9) for 15 min. MiR-219 was down-regulated significantly (P < 0.05) 15 min after dizocilpine injection in the PFC but not in the hippocampus or cerebellum. (B) Expression of miR-219 was also significantly (P < 0.05) down-regulated in the PFC of NR1 hypomorphic mice compared to wild-type (WT) controls (n = 5–8 per group). (C) C57BL/6J mice were chronically treated with dizocilpine (0.2 mg/kg per hour) for 5 days by minipump (n = 9 for wild-type controls and n = 10 for dizocilpine-treated animals). After 5 days of dizocilpine treatment, the brains were removed and the prefrontal cortex and hippocampus were dissected for microRNA analysis. (D) Another group of mice were chronically treated with dizocilpine by i.p. injection (0.5 mg/kg once per day) for 14 days. After 14 days, the PFC and hippocampus were harvested and analyzed for miR-219 expression. (C and D) Chronic treatment with dizocilpine had no significant effect on miR-219 levels with either minipumps (C) or daily injections (D) in any of the brain regions examined.

Fig. 2.

Antipsychotic treatment reverses dizocilpine-mediated miR-219 regulation. (A) Distance traveled over a 2-h time period after pretreatment of mice for 30 min with haloperidol or clozapine (0.1 mg/kg haloperidol, 0.75 mg/kg clozapine; i.p.) (B) Pretreatment of mice for 30 min with haloperidol or clozapine (0.1 mg/kg haloperidol, 0.75 mg/kg clozapine; i.p.; n = 5–8 per group) reversed the effects of dizocilpine administration on miR-219 expression (P < 0.05), compared with saline treatment.

Fig. 3.

Identification of CaMKIIγ as an mRNA target of miR-219. (A) miR-219 was subcellularly localized in mouse P19 cells by using an LNA-modified ISH probe. In the P19 cells, miR-219 was concentrated around the nucleus of the neurons, as depicted by an overlay of the green Alexa Fluor 488 signal derived from the LNA-miR-219 probe with the blue nuclear DAPI stain. (B) CaMKIIγ was identified as an in vitro target of miR-219. Wild-type and mutant luciferase reporter constructs containing the miR-219 target site were generated and subsequently transfected (n = 6 for all treatments) into P19 cells. There was significant down-regulation of luciferase signal from the wild-type construct compared to the mutant control, suggesting that endogenous miR-219 indeed represses translation of CaMKIIγ. Additionally, luciferase signal from the wild-type construct could be partially derepressed in cells, where miR-219 activity was inhibited with an LNA-antimiR molecule complementary to the mature microRNA sequence compared to mismatch (MM) LNA control. One of the primary functions of miRNAs is translational arrest. Hence, protein levels of CaMKIIγ were subsequently analyzed from P19 cells displaying overexpression (O/E) with miR219 RNA. Western blot analyses revealed a repression of CaMKIIγ protein levels in miR-219 O/E cells, further validating CaMKIIγ as a target of this microRNA. (C) To examine the cortical regulation of CaMKIIγ by miR-219, cortical cells derived from C57BL/6 mice were overexpressed with miR-219 and protein was isolated 48 h after transfection (n = 4 each for miR-219 overexpression and scrambled control). Western blot analysis revealed that overexpression of miR-219 significantly reduced the protein levels of the kinase. (D) CaMKIIγ protein expression was assessed in vivo in LNA-antimiR treated mice. Animals were killed after 7 days of osmotic minipump administration of saline (control), mismatch LNA, or LNA-antimiR-219, and the PFC was subsequently harvested from the brains for protein analysis. Western blot analysis revealed up-regulation of CaMKIIγ protein in the PFC of LNA-antimiR-219 treated animals compared to mismatch or controls. *, P < 0.05 for wild-type versus mutant groups; #, P < 0.05 for wild-type construct groups.

Fig. 4.

Antagonism of miR-219 in vivo by LNA-antimiR alters dizocilpine-induced behavior. The effects of in vivo antagonism of miR-219 in the brain on dizocilpine induced behavior were examined in mice. Adult male C57BL/6 mice were administered saline (n = 5), a 2-nt mismatch LNA control (n = 5; 0.24 mg per day), or LNA-antimiR complementary to miR-219 (n = 5; 0.24 mg per day) for 5 days by osmotic minipump. After 5 days of saline (surgical controls) or oligonucleotide administration, mice were habituated in an Open-Field box before acute administration of dizocilpine. Data were recorded for total distance traveled and stereotypy during the 2-h pretreatment habituation and for 90 min after dizocilpine (Dz) or saline (Sal) administration (the 30- to 90-min interval is shown here for the posttreatment data). Hyperlocomotion (measured as distance traveled) (A and B), as well as stereotypy (C and D), were all significantly attenuated after Dz challenge in the LNA-antimiR-219 treated mice when compared to the mismatch or Dz-treated controls. *, P < 0.05 compared with Sal treated controls; #, P < 0.05 for Dz controls compared with Dz-treated groups.

Fig. 5.

Schematic of miR-219 regulation in NMDA signaling. Outlined in the schematic, we propose that miR-219 negatively regulates the function of NMDA receptors. The silencing of miR-219 releases the translational repression of the microRNA on the 3′UTR of the CaMKIIγ mRNA, thus providing a compensatory mechanism to maintain NMDA-R function during acute antagonism of the receptor.