Recombinant Adeno-Associated Virus-Mediated microRNA Delivery into the Postnatal Mouse Brain Reveals a Role for miR-134 in Dendritogenesis in Vivo

Abstract

Recent studies using primary neuronal cultures have revealed important roles of the microRNA pathway in the regulation of neuronal development and morphology. For example, miR-134 is involved in dendritogenesis and spine development in hippocampal neurons by regulating local mRNA translation in dendrites. The in vivo roles of microRNAs in these processes are still uninvestigated, partly due to the lack of tools enabling stable in vivo delivery of microRNAs or microRNA inhibitors into neurons of the mammalian brain. Here we describe the construction and validation of a vector-based tool for stable delivery of microRNAs in vivo by use of recombinant adeno-associated virus (rAAV). rAAV-mediated overexpression of miR-134 in neurons of the postnatal mouse brain provided evidence for a negative role of miR-134 in dendritic arborization of cortical layer V pyramidal neurons in vivo, thereby confirming previous findings obtained with cultured neurons. Our system provides researchers with a unique tool to study the role of any candidate microRNA in vivo and can easily be adapted to microRNA loss-of-function studies. This platform should therefore greatly facilitate investigations on the role of microRNAs in synapse development, plasticity and behavior in vivo.

Keywords: chimeric hairpins; dendritogenesis; in vivo; miR-134; microRNA; nervous system; recombinant AAV.

Figure 1

Secondary structures and sequences of chimeric hairpins. (A) Top: structure and sequence of the hsa-miR-30a hairpin including flanking sequences required for Drosha processing. Light grey shaded sequence: miR-30a mature sequence. Dark grey shaded sequence: miR-30a* mature sequence. Drosha/DGCR8: flanking region of the miR-30a hairpin required for Drosha processing. Arrows: site of Drosha cleavage. Bottom: structure and sequence of chimeric hairpins, design 1 (see text for details). Shaded sequence: miRNAs or control sequences expressed from the chimeric hairpins. (B) Top: structure and sequence of the rno-miR-134 hairpin. Shaded sequence: miR-134 mature sequence. Bottom: Structure and sequence of chimeric hairpins, design 2 (see text for details). Shaded sequence: miRNAs or controls expressed from the chimeric hairpins.

Figure 2

Processing of the chimeric hairpins. (A) Expression of miR-134 and control sequences from chimeric hairpins, design 1. Left panel: 10 μg of the indicated plasmids were transfected into 4 DIV primary cortical neurons per 10 cm tissue culture dish. RNA was prepared for northern blotting at 7 DIV and assayed for the indicated miRNAs. Right panel: 1 DIV primary cortical neurons were induced with rAAV (crude lysate) carrying the indicated plasmids. RNA was extracted at 7 DIV for northern blot assaying for the indicated miRNAs. ImageJ software was used to extract expression data and the levels of miR-134 were normalized against the levels of endogenously expressed miR-138. (B,E) Expression of miR-134 from the miRNA134.1 (B) and the miRNA134.2 (E) construct. 4 DIV primary cortical neurons were transfected with 250 ng the indicated plasmids per well of a 24 well plate. At 7 DIV, RNA was harvested and qPCR analysis was performed. miR-134 levels were normalized against the levels of endogenously expressed U6 snRNA. Fold of induction is expressed relative to the miR-134 levels in cells transfected with AAV vector, which are arbitrarily set to 1. Results are shown as means + s.d. [(B): n = 2; (E): n = 2]. (C,D) Expression of miR-99a and miR-134 from the miRNA99a.1 (C) and the miRNA134.2 (D) construct, respectively. HEK293 cells were transfected with 8 μg of the indicated plasmids per 10 cm cell culture dish. RNA was harvested 2 days later and subjected to northern blot analysis. Expression data were extracted using ImageJ software. miR-99a levels (C) were normalized against the levels of endogenously expressed U6 snRNA.

Figure 3

Functionality of products expressed from miRNA134. 1, 2 and miRNA99a.1. (A,C) Products of the miRNA134.1 (A) and miRNA134.2 (C) constructs regulate expression of a luciferase reporter construct carrying a miR-134 perfect binding site. The indicated plasmids (or AAV vector) were co-transfected along with the pGL3-LimK1-3′UTR-134pbds construct into primary cortical neurons for luciferase assay analysis (see Materials and Methods for details). Values are expressed relative to the internal Renilla luciferase activity and normalized to the activity of the luciferase reporter when co-transfected with (A) AAV vector and (C) control1.1, which both are arbitrarily set to 1. Results are shown as means + s.d. [(A): n = 4; (C): n = 3]. Indicated p-values were calculated using Student′s t-test. (B) miR-134 expressed from the miRNA134.1 construct affects the Pum2 protein levels. 10–11 DIV hippocampal neurons were transduced with purified rAAV carrying the indicated plasmids. Eight days later whole cell extracts were prepared and subjected to western blot analysis probing with Pum2 antibody and a β-actin antibody as loading control. Here we show one out of three representative blots, on average displaying a significant 64% reduction in Pum2 protein levels. (D,E) miR-99a expressed from miRNA99a.1 regulates expression of luciferase reporter constructs carrying a miR-99a perfect binding site (psiCHECK-Hs3st2-3′UTR-99apbds) (D) as well as a construct carrying the Hs3t2-3′UTR containing a wild type miR-99a target site (psiCHECK-Hs3st2-3′UTR) (E). The indicated plasmids, AAV vector or synthetic pre-miRs were co-transfected along with luciferase reporter constructs into primary cortical neurons for luciferase assay (see Materials and Methods for details). Values are expressed relative to the internal Firefly luciferase activity and normalized to the activity of the luciferase reporter when co-transfected with (D) AAV vector and (E) synthetic pre-miR control, which arbitrarily are set to 1. Results are shown as means + s.d. [(D): n = 3; (E): n = 3]. Indicated p-values were calculated using Student's t-test.

Figure 4

miR-134 affects dendritogenesis in vivo. P0 mice were injected with purified rAAV carrying miRNA134.1, AAV vector or control1.1. Brains were harvested at P21, fixed ON and cut into 100 μm coronal brain sections. The sections were immunostained using an eGFP antibody and mounted for confocal microscopy. (A) Representative projection images of cortical layer V pyramidal neurons infected with AAV vector or miRNA134.1. Arrows point out the location of the primary apical dendrite for orientation. (B) miR-134 negatively affects dendritogenesis in vivo. Dendritic branching of cortical layer V pyramidal neurons was analyzed by Sholl analysis (see Materials and Methods for details), only considering the basal dendrites. The number of dendritic intersections is normalized to the number of intersections of neurons infected with AAV vector, which is arbitrarily set to 1. Results are shown as means + s.d. (n = 3 independent experiments; at least six neurons each from two to three different brains per experiment; (a) AAV vector: 9 brains, 69 neurons, (b) control1.1: 8 brains, 61 neurons, (c) miRNA134.1: 8 brains, 57 neurons.). Indicated p-value was calculated using Student's t-test. (C) Sholl profile of the basal dendritic length of cortical layer V pyramidal neurons. The number of dendritic intersections with each concentric circles is plotted against the distance of the individual circles from soma, 25 μm interval (Between-Subjects ANOVA: p = 0.002 and 0.013 comparing the miRNA134.1 condition to the control1.1 and AAV condition, respectively).