• dendrites
• local transcriptome
• miRNA
• neurons
• virus
• virus-like particles

The processes of learning and memory are higher cognitive functions that necessarily require a nervous system possessing plastic properties. At the cellular scale, plasticity relies on the changes in the synaptic strength that affect neuronal connections and occurs in an activity-dependent fashion. From the earliest days of modern Neurosciences, the molecular events induced by synaptic plasticity have been thought to constitute the cellular basis of memory, as S. Ramon y Cajal had proposed in 1911.
The set of functional and morphological modifications -to which synapses are subjected- involve the regulation of the local proteome of the post-synaptic compartment. Indeed, neurons possess a strong cellular polarization that results from the differential localisation of mRNAs and protein between the somato-dendritic and the axonal compartment. Furthermore, protein synthesis is not restricted to the soma, but is also localised in dendrites.
Local translation regulation plays a pivotal role in the molecular control of synaptic plasticity. Remarkably, translational repression mediated by dendritic microRNAs is a regulatory network that contributes greatly to fine-tune synaptic strength. Indeed, microRNAs localise at the synapses and either their biogenesis, transport and function are under the control of neural activity. Consistently, dysregulation of several miRNAs has been linked to synaptic dysfunctions and neurological diseases. Nevertheless, only a tiny fraction of dendrite-specific miRNAs has been characterized so far. The main limitation resides in the lack of appropriate tools to selectively isolate and characterize the dendritic RNA pools.
Synaptosome preparation has represented for many years the only available technique to investigate synaptic compartments. In recent years, laser capture microdissection (LCM) of neuronal processes has been employed to characterized neuronal transcriptomes (Kye et al., 2007; Cajigas et al., 2012). However, these methods are unable to properly separate dendrites from axons and therefore cannot provide information regarding the pre- or post-synaptic origin of purified RNA species.

The final goal of the project is to develop a Virus-Like Particle (VLP)-based tool able to selectively isolate the local pool of miRNA molecules localised in the post-synaptic compartment. To do this, HIV-derived virus-like particles have been engineered to be directed specifically to the dendritic compartment and load local RNA species. Furthermore, an additional functionalisation of VLP has been designed to increase particle specificity towards microRNA molecules. The aim of my work is to test the effectiveness of the two-step of functionalisation the VLPs have been subjected to.
First, a zip-code element responsible for dendritic targeting has been added to the mRNA of Gag protein, the building block of virus-like particles. The dendritic localisation signal (DLS) derives from the 3’ untranslated region of the mRNA encoding the post-synaptic density protein PSD-95. The dendritic targeting of Gag-GFP mRNA bearing the DLS has been imaged in neuronal cells differentiated in vitro from mESC.
In order to increase the specific encapsulation of microRNAs, two RNA-binding domains from the protein TRBP have been fused to Gag protein. Enrichment in the miRNA fraction has been tested in VLP-producer cells expressing the chimeric GAG-TRBP protein. Extracellular VLPs were collected and the RNA cargo was extracted from purified particles. Analysis of RNA purified from functionalised Gag-TRBP VLPs showed a significative enrichment in the miRNA fraction in comparison to control VLPs. Similarly, Gag-TRBP VLPs showed a distribution profile of encapsulated RNAs peaking at the length of 20÷22 nt, consistent with an increased affinity towards miRNAs. Positive results were confirmed by RT-qPCR analysis of individual miRNAs.
As engineered VLPs proved to be a reliable tool producing promising results, the next steps will consist in the optimization of VLP production in neuronal culture. Ultimately, the characterization of dendrite-specific miRNome will be achieved through a sequencing approach on the RNA molecules purified from VLPs of neuronal origin.