miR-34a regulates cell proliferation, morphology and function of newborn neurons resulting in improved behavioural outcomes

miR-34a is involved in the regulation of the fate of different cell types. However, the mechanism by which it controls the differentiation programme of neural cells remains largely unknown. Here, we investigated the role of miR-34a in neurogenesis and maturation of developing neurons and identified Doublecortin as a new miR-34a target. We found that the overexpression of miR-34a in vitro significantly increases precursor proliferation and influences morphology and function of developing neurons. Indeed, miR-34a overexpressing neurons showed a decreased expression of several synaptic proteins and receptor subunits, a decrement of NMDA-evoked current density and, interestingly, a more efficient response to synaptic stimulus. In vivo, miR-34a overexpression showed stage-specific effects. In neural progenitors, miR-34a overexpression promoted cell proliferation, in migratory neuroblasts reduced the migration and in differentiating newborn neurons modulated process outgrowth and complexity. Importantly, we found that rats overexpressing miR-34a in the brain have better learning abilities and reduced emotionality.


rAAV-mediated miR-34a overexpression in vivo is widespread and neuron-specific
rAAV can transduce both replicative and non-replicative cells, such as post-mitotic neurons of the central nervous system (CNS) in a highly efficient and stable manner without inducing cytotoxicity or cellular immune response. 1,2,3 We injected miR-34a and empty AAV expressing vectors into the cerebral lateral ventricles of rat pups at birth (P0). We verified that no signs of tissue damage were induced following brain injection of rAAV and no signs of inflammatory reaction were detected by immunostaining for the glial fibrillary acidic protein (GFAP) or the activated microglial marker, Using EGFP native fluorescence to directly detect transgene expression, we found a diffuse transduction of different brain regions also distant from the site of injection. Cells in the hippocampus, cortex, striatum, olfactory bulb, cerebellum, amygdala, hypothalamus were extensively transduced, confirming rAAV as a powerful tool to target brain cells for genetic manipulation (Supplementary Figure S4 b and data not shown). In the neocortex, we found EGFP-positive cells scattered in all layers, more numerous in layer II-III (Supplementary It is noteworthy that, despite the lack of specificity of CMV and PGK promoters, the rAAVmediated overexpression resulted neuron-specific and we never found infected glial cells (Supplementary Figure S4 d), as revealed by double labelling using antibodies against the neuronal marker NeuN and the glial marker GFAP. This observation is consistent with the previously reported tropism of rAAV toward neurons. 4, 5

Expression Constructs
The vectors used to construct and package rAAVs have been previously described, 6, 7 and were kindly provided by Hilmar Bading, University of Heidelberg, Germany. 8 Briefly, the pri-miR-34a was cloned by PCR (Finzyme) (gene ENSRNOG00000035623, from position 291 to 1124), using specific primers (MWG-Biotech, Germany) from rat genomic DNA and inserted into the XhoI and EcoV sites of Tween vector. 9, 10 Later, the region containing the CMV promoter driving the pri-miR-34a, the PGK promoter driving the enhanced GFP (EGFP) from the Tween vector was subclone into an AAV plasmid backbone, containing the woodchuck post-transcriptional regulatory element (WPRE) and the bovine growth hormone polyA (bGH), to yield the construct pAAV-miR-34a. The same pAAV-CMV-WPRE-bGH backbone carrying no miR (pAAV-empty vector), but expressing the only EGFP, was used as controls. It was obtained removing the pri-miR-34a by cutting with XbaI, blunting by Klenow polymerase and ligating with the EcoRV site of the AAV backbone. Cloning orientation was verified by diagnostic cuts followed by sequencing.
Three web-based miRNA target prediction methods were used: miRanda of miR-34a, were amplified by PCR from rat brain cDNA using specific primers (see Table   S1, Supplementary information), and subsequently, cloned into XhoI-NotI sites of the psiCheck2 vector (Promega). Moreover, to ablate the miR-34a binding site in the 3' UTR of DCX inserted into psiCheck2 vector, and specifically modify the nucleotides in the seed sequence, we designed specific primers (see Table S1 Stratagene) with SYBR green ready mix (Stratagene) and specific primers (see Table S1, Supplementary information). The primers to amplify the miR-34a were designed at the stem region of the precursor form as previously described. 11 Similar amplification results were obtained using primers and conditions from the miRcury LNA PCR system (Exigon). The expression of miR-34a and each other gene was defined, from the threshold cycle (C t ), and relative expression levels were calculated by using the 2 -Δ Δ C t method after normalization with reference to expression of 18S or 5S rRna and U6 small RNA (for miR-34a quantitation in exosome preparation). and counterstained with DAPI for detection of nuclei (red).

Supplementary Figures
(b) Confocal images positive for direct EGFP fluorescence immunostained for iba1 (blue) and counterstained with DAPI (red). rAAV ventricular injections did not affect normal architectural structure of cortical brain tissues and did not induce inflammatory responses as revealed by the staining with the anti-GFAP (glial cells) and anti-Iba1 (microglial cells). Scale bar: 10 µm.
Supplementary Figure S4 miR-34a is highly and widely expressed after single neonatal ventricular injection of rAAV and exclusively affects neuronal cells of rat brain.