Microrna-134 Activity in Somatostatin Interneurons Regulates H-Ras Localization by Repressing the Palmitoylation Enzyme, DHHC9

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MicroRNA-134 activity in somatostatin interneurons regulates H-Ras localization by repressing the palmitoylation enzyme, DHHC9

Sunghee Chai1,2, Xiaolu A. Cambronne1, Stephen W. Eichhorn3, and Richard H. Goodman2

Vollum Institute, Oregon Health and Science University, Portland, OR 97239

Contributed by Richard H. Goodman, September 18, 2013 (sent for review July 30, 2013) MicroRNA-134 (miR-134) serves as a widely accepted model for characterizing the expression patterns of microRNAs within a microRNA function in synaptic plasticity. In this model, synaptic heterogeneous population of neurons requires approaches that activity stimulates miR-134 expression, which then regulates den- monitor microRNA function at the single-cell level. drite growth and spine formation. By using a ratiometric microRNA In previous studies, we used a ratiometric microRNA sensor to sensor, we found, unexpectedly, that miR-134 activity in cortical monitor another activity-regulated microRNA, miR-132 (12). This neurons was restricted to interneurons. Using an assay designed allowed us to quantify miR-132 function in individual cells by to trap microRNA–mRNA complexes, we determined that miR-134 comparing the ratios of GFP (fused to a sequence containing the interacted directly with the mRNA encoding the palmitoylation microRNA binding site) to an internal control, DsRed, driven by enzyme, DHHC9. This enzyme is known to palmitoylate H-Ras, a shared promoter. Using a similar sensor designed to monitor fi fi a modi cation required for proper membrane traf cking. Treat- miR-134, we found that unlike miR-132, miR-134 was not active in ment with bicuculline, a GABAA receptor antagonist, decreased the majority of cortical neurons. Rather, activity was detected in DHHC9 expression in somatostatin-positive interneurons and cells that also stained for neuropeptides somatostatin (SST) or membrane localization of an H-Ras reporter in a manner that calretinin. This indicated that miR-134 activity was largely limited depended on miR-134. Thus, although miR-134 has been proposed to inhibitory GABAergic interneurons. The finding that func- to affect all types of neurons, we showed that functionally active tionally active miR-134 was produced in only a limited population miR-134 is produced in only a selected population of neurons where it influences the expression of targets, such as DHHC9, that of neurons must be considered in analysis of potential mRNA regulate membrane targeting of critical signaling molecules. targets. Using the RNA-induced silencing complex (RISC)-trap method, designed to characterize microRNA–mRNA interactions fi palmitoyltransferase | RISC-trap biochemically (13), we identi ed the transcript encoding the palmitoylation enzyme, DHHC9, as a putative miR-134 target. Pal- mitoylation is a lipid modification critical for proper trafficking of onsiderable progress has been made in understanding the membrane proteins (14). In most instances, the relationship bet- contributions of microRNAs to neuronal development, but C ween a particular palmitoylation enzyme and its targets is poorly microRNAs are also found in mature neurons, where their lo- fi calization in dendrites, and possibly axons, has been proposed to de ned, in that each enzyme has multiple targets and each target contribute to synaptic plasticity (1–5). This is an appealing con- can be palmitoylated by multiple enzymes. This does not appear to cept because local regulation of protein translation is essential for plasticity and microRNAs can regulate dendritic growth, Significance spine formation, and growth cone guidance, properties generally believed to underlie plasticity. Activity-regulated microRNAs are Most brain regions comprise numerous neural cell types that are particularly well suited to participate in these mechanisms. One distinct in function and molecular characteristics. We examined of the best-studied activity-dependent microRNAs in brain is microRNA-134 (miR-134) activity in cortical cultures using microRNA-134 (miR-134), which has been shown to regulate a microRNA sensor and discovered that miR-134 was induced in dendritic spine morphogenesis through effects on LIM domain response to neuronal activity in somatostatin- and calretinin- kinase 1 (Limk1) (1) and dendrite growth through the translational expressing interneurons but not in pyramidal neurons. We repressor, pumilio 2 (Pum2) (5). Mutations in LimK1 have been identified the palmitoylation enzyme DHHC9 as a direct target linked to a human mental retardation syndrome (6), and Pum2- of miR-134 and showed that it contributed to the regulation of deficient mice showed behavioral abnormalities (7). Recent studies H-Ras in somatostatin interneurons. H-Ras is a signaling mole- have demonstrated involvement of miR-134 in long-term poten- cule crucial for neuronal development and function. This study tiation (LTP) via regulation of sirtuin 1 (SIRT 1) and in epi- uncovered an activity-dependent miR-134 regulatory pathway leptogenesis (8, 9). Like most studies involving brain microRNAs, in cortical interneurons that can potentially affect membrane these reports make the implicit assumption that miR-134 is localization of multiple signaling molecules. expressed uniformly in excitatory neurons. It is well known, however, that brain neurons are highly heterogeneous, and in Author contributions: S.C., X.A.C., and R.H.G. designed research; S.C., X.A.C., and S.W.E. many regions, excitatory (largely glutamatergic) and inhibitory performed research; S.C., X.A.C., and S.W.E. analyzed data; and S.C., X.A.C., and R.H.G. (largely GABAergic) interneurons form complex networks that wrote the paper. fl balance opposing actions (10). Although many microRNAs are Con ict of interest: X.A.C. was involved in the commercial licensing of the RISC-trap technology. This did not influence the experimental design, data collection, or analysis probably expressed in both excitatory and inhibitory neurons, of the project data. some are likely to be restricted to one or the other subclass. 1S.C. and X.A.C. contributed equally to this work. Indeed, analysis of Argonaute (Ago)2 complexes in individual 2 fi fi To whom correspondence may be addressed. E-mail: [email protected] or chaisu@ neuronal subtypes within the mouse brain identi ed a signi - ohsu.edu. cant number of microRNAs that were enriched in GABAergic 3Present address: Whitehead Institute for Biomedical Research, Massachusetts Institute of but not in glutamatergic neurons and vice versa, indicating that Technology, Cambridge, MA 02142. fi microRNA expression patterns are speci c for neurons that share This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. a common origin and neurotransmitter phenotype (11). Thus, 1073/pnas.1317528110/-/DCSupplemental.

17898–17903 | PNAS | October 29, 2013 | vol. 110 | no. 44 www.pnas.org/cgi/doi/10.1073/pnas.1317528110 Downloaded by guest on September 24, 2021 be the case for H-Ras, which appears to be palmitoylated pre- dominantly by DHHC9 (15). This modification is required for directing H-Ras to the neuronal membrane, where it mediates morphological changes and may participate in LTP and memory + formation (16). Our studies indicate that in SST neurons, DHHC9 is down-regulated by neuronal activity through a pathway that involves miR-134. The consequent reduction of DHHC9 expression disrupted localization of H-Ras from plasma membrane microdomains. Thus, although previous studies have high- lighted miR-134 as an activity-dependent microRNA localized in the synaptodendritic compartment, we show that functionally active miR-134 is produced in only a selected population of neurons where it influences the expression of targets, such as DHHC9, which influence the membrane targeting of critical signaling molecules such as H-Ras. Results Activity-Dependent Induction of miR-134 in SST-Expressing Interneurons. Previous studies in hippocampal and cortical cultures showed elevated levels of the miR-134 precursor transcript (pre–miR-134) following either treatment with brain-derived neurotrophin factor (BDNF) or KCl-induced membrane depolarization (1, 5). Using cultures of rat hippocampal neurons (DIV8), we compared the BDNF-stimulated induction of miR-134 with that of miR-132, another activity-dependent microRNA involved in the development of dendrites and dendritic spines (17, 18). Using Fig. 1. Activity-dependent induction of miR-134 is limited to interneur- – quantitative PCR (qPCR) and Taqman microRNA assays to ons. (A) BDNF marginally induced expression of pre and mature miR-134. qPCR (pre–miR-134 and pre–miR-132) and Taqman (mature miR-134) monitor levels of precursor and mature microRNAs, respectively, CELL BIOLOGY – assays were performed using RNA isolated from DIV7 cortical cultures we detected relatively small increases in pre miR-134 and mature treated with 40 ng/mL BDNF for the times indicated. Expression levels were miR-134 following BDNF treatment over a time course of 6 h normalized to actin mRNA, and fold induction represents the increase over (Fig. 1A). Levels of pre–miR-134 peaked 2 h after BDNF treat- the 0 time value (*P = 0.02, ***P < 0.0001). (B) Ratiometric microRNA sen- ment (1.6 ± 0.5-fold above basal levels) and returned to baseline sors. Sensor-134MRE contained three copies of miR-134 MRE downstream by 6 h. Correspondingly, mature miR-134 levels were not signif- of AcGFP. (C) Flow cytometric analysis of microRNA sensors coexpressed icantly elevated above basal level after 6 h. In contrast, pre–miR- with various amounts of miR-134 mimic in SH-SY5Y cells. The GFP/DsRed 132 levels increased by 18-fold over the same time period. Similar ratio of individual cells was plotted as a cumulative frequency distribution. results were obtained after treatment with 16 mM KCl. We hy- (D) Introduction of miR-134 decreased GFP mRNA levels of the miR-134 sensor in HEK293 cells, analyzed by qPCR and normalized to internal control, fi + pothesized that induction of miR-134 may be restricted to speci c DsRed. (E) Analysis of miR-134 expression in bicuculline-treated SST neu- cell types and therefore may not be apparent in assays using rons. Bicuculline treatment reduced GFP intensity in neurons containing the heterogeneous populations of cells. To assess the induction of sensor-134MRE, indicating miR-134 induction. (F) Quantification of the GFP/ + mature miR-134 in individual cells, we generated a ratiometric DsRed ratio in individual SST (Left) and pyramidal (SST-negative) (Right) sensor specific for miR-134, following a strategy that we had used neurons after bicuculline treatment. Intensity of green and red fluorescence previously for measurement of miR-132 (Fig. 1B). This approach from confocal images was analyzed by Fiji software. The average GFP/DsRed monitors miR-134 activity through its regulation of a GFP tran- ratio per cell is graphed. ***P < 0.0001, n.s. P = 0.385. (G) Induction of miR- 134 depends on bicuculline treatment in SST+ (Upper) and calretinin neurons script containing three reiterated miR-134 microRNA recogni- + tion elements (MREs) in its 3′UTR. Comparing GFP levels to (Lower). The GFP/DsRed ratios in SST and calretinin neurons containing the sensor-134MRE decreased after bicuculline treatment. ***P < 0.0001, that of an internally expressed DsRed control lacking these *P = 0.01, n.s. P > 0.05. Student t test; histograms show mean ± SEM. n.s., MREs provides a ratiometric measurement for microRNA ac- not significant. tivity. To test this system, we first assessed the efficacy of our miR-134 sensor in SH-SY5Y neuroblastoma cells using flow cytometry to analyze the GFP/DsRed ratio in individual cells. (Fig. 1F). We next analyzed sensor activity in neighboring large These studies revealed a dose-dependent response of the miR- excitatory pyramidal neurons. In these cells, the difference bet- 134 sensor to exogenous miR-134 (Fig. 1C). A control sensor ween the control and miR-134 sensors was not significantly dif- lacking MREs was completely unresponsive to added miR-134. ferent (P = 0.385; Fig. 1F). In contrast, miR-132 activity measured The effect of exogenous miR-134 was also apparent at the sensor using a specific sensor was readily apparent in these pyramidal D transcript level (Fig. 1 ). To monitor miR-134 induction in in- neurons (Fig. S1). These results indicated that miR-134 is induced + dividual cortical neurons, we analyzed activity of the miR-134 in SST neurons but not in pyramidal neurons, likely explaining sensor in primary cultures characterized using specific molecular the modest increase of miR-134 in preparations of heterogeneous markers. In particular, we examined cells that stained for the neu- cortical cultures where SST-expressing cells constitute a relatively ropeptide, somatostatin, which represent a well-characterized subtype of inhibitory interneuron. Cortical cultures were transfected with small minority. To determine whether miR-134 levels were, in either the miR-134 sensor or control at DIV7, stimulated at DIV8 fact, induced after stimulation, we repeated the assays in the presence or absence of bicuculline. With the control sensor, with bicuculline (a GABAA receptor antagonist that blocks GABAA bicuculline treatment did not significantly alter the GFP/DsRed receptors and thereby is predicted to increase neural excitability), and + examined at DIV9 using relative intensity measurements (Fiji soft- ratio (Fig. 1G). However, the miR-134 sensor in SST neurons fi responded robustly (Fig. 1G), demonstrating that miR-134 in- ware). The population of SST-expressing interneurons was identi ed + by immunostaining (Fig. 1E). The GFP/DsRed ratio for the control duction in SST neurons is activity-dependent. To determine sensor in these cells had a mean value of 1.09; the ratio for the miR- whether miR-134 activity was induced in other subtypes of inter- 134 sensor was 0.62, indicating a robust increase in miR-134 activity neurons, we examined calretinin-positive interneurons. SST- and

Chai et al. PNAS | October 29, 2013 | vol. 110 | no. 44 | 17899 Downloaded by guest on September 24, 2021 bicuculline treatment, and miR-134 activity was absent in untreated cells (Fig. 1G). These data suggested that miR-134 is inducedinatleasttwomajorsubtypes of early developing neocortical interneurons.

Palmitoylation Enzyme DHHC9 Is a Target of miR-134. We identified DHHC9 as a putative miR-134 target by using the RISC-trap assay that we had developed previously to capture miRNA– mRNA interactions (13). This assay, performed in HEK293 cells, uses an epitope-tagged dominant negative form of the RISC component, GW182, to stabilize microRNA–mRNA complexes. The endogenous DHHC9 transcript enriched by 4.5-fold in the presence of miR-134, compared with another neuronal micro- RNA, miR-124 (Fig. 2A). To confirm that DHHC9 was a miR- 134 target, we fused the DHHC 3′UTR to a luciferase cDNA and assayed luciferase activity in HEK293 cells and primary hippocampal cultures. Luciferase activity was significantly down- regulated by exogenous miR-134 in both types of cells (Fig. 2B). Within the DHHC9 3′UTR, we identified three putative miR- 134 MREs with perfect seed matches (Fig. 2C, designated M1, M2, and M3). Mutation of M1 and M3 individually partially stabilized a FLAG-DHHC9-3′UTR reporter; mutation of both sites rendered the reporter completely refractory to miR-134 regulation, compared with a scrambled microRNA and with the DHHC9 reporter lacking its 3′UTR (Fig. 2D). + We established that DHHC9 was expressed in SST interneurons (Fig. S2). To determine whether DHHC9 protein was regulated by endogenous miR-134, we generated a ratiometric reporter similar to the microRNA sensors described above but incorporating the DHHC9 3′UTR (Fig. 2E). This reporter was expressed in cortical cultures at DIV7, and cells were treated + with bicuculline or control for 24 h and examined in both SST interneurons and pyramidal neurons on DIV11 using ratiometric + analyses of fluorescence intensity. In SST cells, bicuculline significantly down-regulated GFP expression (Fig. 2E). In contrast, pyramidal neurons exhibited no difference in the mean GFP/DsRed ratio following bicuculline treatment (Fig. 2E). Fig. 2. miR-134 regulates DHHC9. (A) Identification of DHHC9 as a direct miR-134 target using RISC-trap. FLAG-tagged dominant-negative GW182 was H-Ras Palmitoylation Is Critical for Its Subcellular Localization. We + expressed with miR-134 or miR-124 mimic in HEK293 cells. RNA–RISC protein next asked whether the regulation of DHHC9 in SST neurons complexes were immunoprecipitated using an anti-FLAG antibody, and the by miR-134 affected membrane trafficking of the DHHC9 pal- associated RNAs were analyzed by qPCR. Relative DHHC9 mRNA enrichment mitoylation target, H-Ras. Most studies examining H-Ras lo- fl = re ected the increase compared with complexes containing miR-124 (*P calization have used reporter constructs, such as GFP fused to 0.028). (B) miR-134 decreased expression of reporters containing the DHHC9 the H-Ras coding sequence. To minimize confounding effects 3′UTR. Luciferase reporters containing the DHHC9 3′UTR or vector control from ectopic H-Ras expression (20), we generated a GFP fusion were expressed with 10 nM miR-134 mimic in HEK293 cells and hippocampal fi neurons (HN). Renilla reporter activity was measured 24 h posttransfection that contained only the carboxyl-terminal H-Ras traf cking do- C24 and normalized to control firefly luciferase activity (***P < 0.0001). (C) Pu- main. This domain (designated HRas ) has been shown to be tative MREs in the DHHC9 3′UTR (M1, M2, and M3) are indicated with sufficient to localize H-Ras to cell membranes and includes two underlined seed sequences for miR-134. Three nucleotides in each of the cysteine residues (C181 and C184) that, when palmitoylated, putative MREs were mutated (underlined in red). (D) Evaluation of putative promote H-Ras trafficking (Fig. 3A) (21–24). We first examined + MREs. FLAG-tagged reporter contained the full-length coding region and 3′ the subcellular localization of wild-type GFP-HRasC24 in SST UTR of DHHC9. FLAG-DHHC9 reporters with wild-type or mutant MREs were interneurons, compared with a mutant isoform that could not be expressed in HEK293 cells with miR-134 mimic or scrambled microRNA control. Mutations in M1 and M3 individually or in combination abolished miR- palmitoylated (C181S, C184S). Confocal microscopy revealed a distinct punctate pattern of wild-type GFP-HRasC24 throughout 134 regulation, as shown in Western blots and compared with the DHHC9 + B reporter lacking its 3′UTR. (E) DHHC9 3′UTR reporter was down-regulated the soma and processes of SST neurons (Fig. 3 ). The distinct + C24 upon bicuculline treatment in SST neurons. (Upper) The ratiometric DHHC9 microdomain localization of GFP-HRas is indistinguishable sensor contains the full-length DHHC9 3′UTR fused to AcGFP. DsRed is an from the expression pattern of the GFP-tagged full-length H-Ras internal control. (Lower) Cortical neurons transfected with DHHC9-sensor (GFP-HRas) (Fig. S3). In contrast, the palmitoylation-defective were treated or untreated with 20 μM bicuculline. ***P < 0.0001, n.s. P = mutant (C181S, C184S) exhibited a diffuse distribution of green 0.62. Student t test; histograms show mean ± SEM. n.s., not significant. fluorescence throughout the cell body and processes, consistent with the nonspecific targeting of mutated GFP-HRasC24 due to the absence of palmitoylation (24). To quantify the difference in GFP- calretinin-positive neurons have been identified as the main in- C24 – HRas localization, we used linescan measurements of GFP interneuronal population postnatally (P0 P14) in rat neocortex, tensity, captured across 30 μm of continuous dendritic length for + whereas other major interneuronal subtypes such as parvalbumin each SST neuron (Fig. 3C). The high-intensity peaks (max) rep- (PV)- and vasointestinal peptide-positive neurons emerge later resented the localization of GFP-HRasC24 in punta, and the lower- (19). The sensor assays in cortical culture showed that similar to intensity values represented diffused GFP (min). These measure- + SST neurons, calretinin neurons expressed miR-134 upon ments showed a significantly different profile of GFP-HRasC24

17900 | www.pnas.org/cgi/doi/10.1073/pnas.1317528110 Chai et al. Downloaded by guest on September 24, 2021 was comparable to that seen after expression of miR-134. To- gether, our data suggest that miR-134 regulates DHHC9- dependent palmitoylation and is important for H-Ras locali- + zation in SST interneurons.

Bicuculline Treatment Changes H-Ras Localization in an miR-134– Dependent Manner in SST+ Neurons. We next showed that bicuculline, like exogenous miR-134, altered localization of GFP- + HRasC24 and GFP-HRas full-length protein in SST neurons (Fig. 5 A and B and Fig. S3). To determine whether the bicuculline effect depended upon miR-134, we examined GFP- HRasC24 localization in cells cotransfected with a miR-134 locked nucleic acid (LNA) inhibitor. The inhibitor LNA-miR-134, but not a scrambled control, completely blocked the bicuculline effect (Fig. 5 C–F). These data suggest that miR-134 regulates DHHC9- dependent palmitoylation and is important for H-Ras localization + in SST neurons.

+ Fig. 3. Palmitoylation is critical for localizing H-Ras in SST neurons. (A) The C-terminal membrane-targeting domain of H-Ras contains two palmitoylation sites (designated Pal; C181 and C184) and a farnesylation motif (designated Far; CAAX). GFP fusions to the wild type and the palmitoylation mutant (C181S, C184S) are illustrated. (B) Subcellular localization of GFP- HRasC24 in SST+ neurons. Dendrites of SST+ neurons expressing either the wild-type or the palmitoylation defective H-Ras membrane-targeting domain are shown; localization of the wild-type H-Ras to microdomains was diminished by mutating the two palmitoylation sites. (C) Quantitative analysis using linescan measurement of GFP intensity in 30 μm of continuous CELL BIOLOGY dendritic length depicted in B. Each peak denotes microdomain localization; relative distance reflects the span of the segment analyzed in μm. (D) Quantification of linescan data was performed by measuring differences (Δ intensity) between the maximum (max) and minimum (min) intensity of each peak. The Δ intensity plotted represented the average of the five highest peaks in each dendrite. The wild type and palmitoylation mutant had a significant difference in Δ intensity. ***P < 0.0001 (n =10 neurons). Student t test, mean ± SEM. (Scale bars, 5 μm.)

(C181S, C184S) than the wild-type counterpart, with broader, rolling peaks. Differences (Δ intensity) between adjacent max and min value were averaged in each neuron (Fig. 3D). Com- parison of the wild-type and mutant GFP-HRasC24 localization patterns showed that localization of the mutant was signiﬁcantly more diffuse. This difference represents the maximal effect of palmitoylation on trafﬁcking of the reporter.

miR-134 Regulation of DHHC9 Regulates Trafficking of H-Ras in SST+ Neurons. To determine whether miR-134 affected GFP-HRasC24 localization, we examined the effects of scrambled control or miR- 134 mimics in dissociated cortical cultures stained immunohis- tochemically for SST expression. The distinct punctate pattern of GFP-HRasC24 was retained in the presence of control microRNA, whereas expression of miR-134 caused a diffused pattern of GFP + fluorescence in the SST cells (Fig. 4 A and B). This effect was somewhat less pronounced than with the C181S, C184S palmitoylation mutant, so we asked whether DHHC9 depletion resulted in a similar mislocalization. To examine the contribution of DHHC9 + fi + to GFP-HRasC24 localization in SST cells, we generated a plasmid Fig. 4. miR-134 and DHHC9 regulate H-Ras traf cking in SST neurons. (A and B) H-Ras localization changes after exogenous miR-134 expression. (A) that encodes a short-hairpin RNA to DHHC9 (shDHHC9) Linescan measurement of GFP intensity in control- or miR-134–expressing downstream of a tdTomato fluorescence marker. In HEK293 neurons. (B) Quantification of linescan data by measuring Δ intensity (***P = cells, shDHHC9 was capable of depleting coexpressed Flag-DHHC9 0.0004, n =15 neurons). (C) DHHC9 shRNA blocked expression of epitope- protein levels (Fig. 4 C and D) and endogenous levels of DHHC9 tagged DHHC9 in HEK293 cells. sh-control represents a nonspecific shRNA. fi (D) Constructs of shDHHC9 and sh-control in plasmids expressing tdTomato in Neuro2A cells (Fig. S4). We identi ed shDHHC9-expressing + fl are shown. (E and F) DHHC9 regulates H-Ras localization in SST neurons. cells by presence of the tdTomato uorescence and analyzed C24 GFP-HRasC24 localization in the population that was also posi- Linescan measurement of GFP intensity in dendrites expressing GFP-HRas along with sh-control or shDHHC9. (F) DHHC9 knockdown significantly re- tive for SST. Introduction of shDHHC9 increased levels of dif- duced Δ intensity (**P = 0.0024, n = 11 neurons). GFP intensity was mea- fused GFP fluorescence compared with control (Fig. 4 E and F). sured from 30 μm of continuous dendritic length (A, B, E, and F). Student t C24 Moreover, the effect of DHHC9 depletion on GFP-HRas test, mean ± SEM.

Chai et al. PNAS | October 29, 2013 | vol. 110 | no. 44 | 17901 Downloaded by guest on September 24, 2021 miR-134 and miR-132 were, in fact, expressed concurrently. Earlier studies indicated that miR-132 was expressed generally throughout the brain, including large pyramidal neurons (17, 18, 27, 29). Because the preponderance of neurons in the cortex are excitatory, we hypothesized that miR-134, at a minimum, would be expressed in this neuronal subtype. Using a ratiometric sensor approach, we confirmed that miR-134 was induced by bicuculline but found that the induction was restricted and + included SST interneurons. In previous studies, the participation of miR-134 in regulating dendritic spines was determined by examining a complex mixture of neurons from hippocampus and cortex (1, 8, 9). Unlike pyramidal neurons, mature interneurons often lack spines, and when present, they are few in number with an irregular distribution (30, 31). Consequently, regulation of spine morphology may be less important in interneurons than it is in excitatory, glutamatergic neurons. It remains possible, however, that miR-134 could be involved in the degeneration of dendritic spines in early stages of + interneuron development. Indeed, some of the SST interneurons did have a few dendritic filopodia, possibly reflecting their imma- turity (Fig. S5). Nonetheless, our findings do not support the idea that miR-134 contributes directly to dendritic spine morphology in pyramidal neurons because our sensor does not detect miR-134 activity in this population. Conceivably, the spine phenotype observed in excitatory neurons could result from miR-134 activity in + SST interneurons because these neurons innervate the distal dendrites of principal neurons, where they are believed to contribute to the integration of synaptic inputs and synaptic plasticity (32). In this study, we tested SST- and calretinin-positive neurons, which are found at relatively early postnatal days, but our experimental model did not allow us to test the interneuron subtypes that emerge in more mature brain. It is possible that miR-134 is expressed in those other interneuron subtypes as well. The analysis of Ago2 complexes from mouse cortex showed that microRNA expression profiles are largely maintained among PV-, SST-, and glutamate decarboxylase GAD2-expressing interneurons but are strikingly different from glutamatergic neurons (11). The idea that an activity-regulated microRNA could control expression of a protein palmitoyltransferase has two implications. Fig. 5. Bicuculline disrupts H-Ras microdomain localization via miR-134. (A First, it suggests that a microRNA could regulate a battery of and B) Bicuculline treatment significantly changed GFP-HRasC24 localization synaptic proteins concurrently by controlling their delivery to + in SST neurons (***P = 0.0002, n =17 neurons). (C and D) LNA-miR-134 synaptic membranes. This mode of regulation would not require blocked the bicuculline-induced change in H-Ras localization (P = 0.75, the individual mRNAs to share a common microRNA recog- n =15 neurons). Neurons were transfected with the plasmid expressing GFP- C24 nition site because a single protein palmitoyltransferase can HRas along with 50 nM LNA-miR-134. (E and F) Bicuculline effect is modify multiple protein targets. Second, this process, because it specific to miR-134. Scrambled control oligomer did not block bicuculline- induced H-Ras mislocalization (**P = 0.006, n =12 neurons). GFP intensity is indirect, would not require the microRNA and synaptic target was measured by linescan in 30 μm of continuous dendritic length, and to be colocalized in the same subcellular compartment. This microdomain localization of H-Ras was measured by quantification of the Δ could allow microRNAs to regulate proteins at the growth cone, intensity. Student t test, mean ± SEM. n.s., not significant. for example, a region generally not thought to be a prominent site of mRNA translation. Thus, modulation of protein palmi- toyltransferases by activity-regulated microRNAs could provide Discussion an avenue for influencing synaptic function that could be par- Several studies have suggested that microRNAs contribute to the ticularly relevant for mature neurons. This mode of regulation is establishment and maintenance of synaptic plasticity in developing not entirely unprecedented—miR-138 was shown to regulate ex- and mature neurons (1, 3, 25, 26). MicroRNAs regulated by pression of APT1, a depalmitoylation enzyme, in quiescent neu- neuronal activity are particularly suited for this role. The first ex- rons, activating Gα13 in the synapse and causing spine shrinkage (3, ample of an activity-regulated microRNA in mammalian brain 26). In contrast to miR-134, however, miR-138 expression is re- pressed by neuronal activity (3, 26). We used H-Ras localization to was miR-132, which regulates expression of p250GAP, a GTPase- + monitor DHHC9 activity in SST neurons. Both shRNA-mediated activating protein involved in remodeling the actin cytoskeleton reduction of DHHC9 and exogenous expression of miR-134 within dendritic spines (17, 18). miR-134 has also been proposed to delocalized H-Ras from microdomains. We propose that the + regulate dendritic spine morphology and synaptic plasticity (1, 8). effect of miR-134 on H-Ras localization in SST neurons occurs miR-134 provides a compelling model for these functions because through DHHC9 down-regulation. Exactly how changes in H-Ras unlike miR-132, it has been localized to the synaptodendritic localization contribute to interneuron function is unknown. In compartment. Of note, miR-132 has been shown to increase the pyramidal neurons, H-Ras activity is increased in glutamate- size and density of dendritic spines (27, 28), whereas miR-134 stimulated dendritic spines and axonal growth cones (20, 33). appears to have the opposite effect (1, 9). Because of their Additionally, H-Ras contributes to presynaptic neurotransmitter seemingly antagonistic actions, we set out to determine whether release via the ERK/Synapsin I pathway (34). Conceivably, effects

17902 | www.pnas.org/cgi/doi/10.1073/pnas.1317528110 Chai et al. Downloaded by guest on September 24, 2021 + of the miR-134/DHHC9/H-Ras pathway on axonal growth and of SST and calretinin-expressing neurons. Confocal microscopy was per- + other presynaptic mechanisms in SST interneurons could in- formed using Olympus FV1000 (Advanced Light Microscopy Core facility, fluence dendritic spine formation in principal cells. Finally, pre- Oregon Health and Science University). vious studies of miR-134 have largely addressed activities within the synaptodendritic compartment (1, 5, 8, 9). We observed miR- Image Analysis. For measurement of GFP and DsRed intensity from microRNA × 134 activity in the soma, suggesting additional functions. Con- sensors, Z-stack images of neurons were taken with 40 objectives and fi compressed into a single image. In each neuron, two to three dendrites were sistent with our ndings, miR-134 was not enriched in the syn- fl aptosome fraction of cortical neurons, compared with a total analyzed for intensity of uorescence using Fiji software. Statistical analysis was performed using paired t test with two-tailed P value by GraphPad Prism homogenate (35). Although some actions of miR-134 might software. For H-Ras localization, Z-stack images of neurons were taken with be localized to the soma, downstream effects could nonethe- 60× objectives with twofold magnification. Images were compressed into less involve synaptic regions through the effects of DHHC9 on a single image for analysis by LineScan tool using Metamorph software. In + H-Ras and other protein targets. SST neuron, primary dendrite projecting at least 30 μm from the soma was chosen to measure the intensity of GFP. Using peaks detected by linescan, Δ Materials and Methods intensity was calculated by subtracting the basal level intensity (min) from – Primary cortical and hippocampal neurons were prepared from E20 Sprague the maximum intensity (max) of each peak. Five highest peaks were chosen Dawley rats (Charles River) and maintained in Neurobasal medium supple- to calculate the average Δ intensity of a dendrite, and 10–15 neurons were mented with B-27 and GlutaMax (Life Technologies). Where indicated, chosen for analysis of one sample group. Statistical analysis was performed neurons were treated with 20 μM bicuculline (Tocris Bioscience) or 16 mM KCl. using paired t test with two-tailed P value by GraphPad Prism software. All animal experiments were approved by the Institutional Animal Care and Detailed information on cell culture and transfection, molecular method Use Committee at Oregon Health and Science University. including DNA constructs and mutagenesis, qRT-PCR, Western blot analysis, and RISC-trap assay is provided in SI Materials and Methods. Immunocytochemistry and Confocal Microscopy. Primary cortical neurons grown on poly-L-Lysine–coated coverslips in 12-well plate were fixed at DIV 8–12 in 4% (wt/vol) paraformaldehyde/PBS and permeablized and blocked in ACKNOWLEDGMENTS. We thank Drs. Gail Mandel, Gary Banker, Philip Stork, Haining Zong, Brian Jenkins, Anthony P. Barnes, and Shane Tillo for blocking buffer containing 2% (vol/vol) horse serum, 1% BSA, and 0.5% helpful discussions in this study. We also thank Dr. Stefanie Kaech Petrie and Triton X-100. Primary antibodies, goat anti-somatostatin (1:100; Santa Cruz Aurelie Snyder (Advanced Light Microscopy Core facility, Oregon Health and D-20) and mouse anti-calretinin (1:1,000; Swant), and secondary antibodies, Science University) for valuable advice in analysis of confocal images. This Alexa Fluor-647 Donkey anti-goat IgG and Alexa Fluor-647 goat anti-mouse work is supported by National Institutes of Health Grants NS097317 and

IgG (Life Technologies), were used in blocking buffer for immunodetection MH099099 (to R.H.G.), NS076094 (to X.A.C.), and P30 NS069305. CELL BIOLOGY

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