In vivo methods for acute modulation of gene expression in the central nervous system

Accurate and timely expression of specific genes guarantees the healthy development and function of the brain. Indeed, variations in the correct amount or timing of gene expression lead to improper development and/or pathological conditions. Almost forty years after the first successful gene transfection in in vitro cell cultures, it is currently possible to regulate gene expression in an area-specific manner at any step of central nervous system development and in adulthood in experimental animals in vivo, even overcoming the very poor accessibility of the brain. Here, we will review the diverse approaches for acute gene transfer in vivo, highlighting their advantages and disadvantages with respect to the efficiency and specificity of transfection as well as to brain accessibility. In particular, we will present well-established chemical, physical and virus-based approaches suitable for different animal models, pointing out their current and future possible applications in basic and translational research as well as in gene therapy

Genetic interaction between PLK1 and downstream MCPH proteins in the control of centrosome asymmetry and cell fate during neural progenitor division

Alteration of centrosome function and dynamics results in major defects during chromosome segregation and is associated with primary autosomal microcephaly (MCPH). Despite the knowledge accumulated in the last few years, why some centrosomal defects specifically affect neural progenitors is not clear. We describe here that the centrosomal kinase PLK1 controls centrosome asymmetry and cell fate in neural progenitors during development. Gain- or loss-of-function mutations in Plk1, as well as deficiencies in the MCPH genes Cdk5rap2 (MCPH3) and Cep135 (MCPH8), lead to abnormal asymmetry in the centrosomes carrying the mother and daughter centriole in neural progenitors. However, whereas loss of MCPH proteins leads to increased centrosome asymmetry and microcephaly, deficient PLK1 activity results in reduced asymmetry and increased expansion of neural progenitors and cortical growth during mid-gestation. The combination of PLK1 and MCPH mutations results in increased microcephaly accompanied by more aggressive centrosomal and mitotic abnormalities. In addition to highlighting the delicate balance in the level and activity of centrosomal regulators, these data suggest that human PLK1, which maps to 16p12.1, may contribute to the neurodevelopmental defects associated with 16p11.2–p12.2 microdeletions and microduplications in children with developmental delay and dysmorphic features.JGM and DMA received predoctoral contracts from the Ministry of Education of Spain (FPI grant BES-2016-077901). This work was supported by Grant PID2019-104763RB-I00 and Ramón y Cajal contract (RYC-2014-15991), both from MINECO/AEI/FEDER (EU) to EP; and grants from the European Commission Seventh Framework Programme (ERA-NET NEURON8-Full-815-094), AEI-MICIU/FEDER (RTI2018-095582-B-I00 and RED2018-102723- T), and the iLUNG programme from the Comunidad de Madrid (B2017/BMD-3884) to MM. CNIO is a Severo Ochoa Center of Excellence (AEI-MICIU CEX2019-000891-S)

Targeted in vivo genetic manipulation of the mouse or rat brain by in utero electroporation with a triple-electrode probe

This article describes how to reliably electroporate with DNA plasmids rodent neuronal progenitors of the hippocampus; the motor, prefrontal and visual cortices; and the cerebellum in utero. As a Protocol Extension article, this article describes an adaptation of an existing Protocol and offers additional applications. The earlier protocol describes how to electroporate mouse embryos using two standard forceps-type electrodes. In the present protocol, additional electroporation configurations are possible because of the addition of a third electrode alongside the two standard forceps-type electrodes. By adjusting the position and polarity of the three electrodes, the electric field can be directed with great accuracy to different neurogenic areas. Bilateral transfection of brain hemispheres can be achieved after a single electroporation episode. Approximately 75% of electroporated embryos survive to postnatal ages, and depending on the target area, 50–90% express the electroporated vector. The electroporation procedure takes 1 h 35 min. The protocol is suitable for the preparation of animals for various applications, including histochemistry, behavioral studies, electrophysiology and in vivo imaging.LN

High-performance and reliable site-directed in vivo genetic manipulation of mouse and rat brain by in utero electroporation with a triple-electrode probe

One of the challenges for modern neuroscience is to understand the role of specific genes in the determination of cellular fate, and in the formation and physiology of neuronal-circuits. Techniques for genetic manipulation in vivo such as in utero electroporation are fundamental tools to address these issues. Here, we describe an established protocol for in utero electroporation in mouse and rat for reliable targeting of the hippocampus, the motor, prefrontal, and visual cortices, and the Purkinje cells of the cerebellum. The method is based on an electroporation configuration entailing commonly used forceps-type electrodes plus an additional third electrode. This configuration allows highly consistent direction of the electric field to the different neurogenic areas by simple and reliable adjustment of relative positions, polarities and/or dimensions of the electrodes. More than 70% of electroporated embryos survive to postnatal ages and around 60-90% express the electroporated vector, depending on the targeted area. By a single electroporation episode, the protocol enables for symmetric transfection in both brain hemispheres. The procedure requires 4 hours of preparation on the first day and it lasts 1 hour, including a surgery time of 30 mins, on the second day

Apaf1-deficient cortical neurons exhibit defects in axonal outgrowth

The establishment of neuronal polarity and axonal outgrowth are key processes affecting neuronal migration and synapse formation, their impairment likely leading to cognitive deficits. Here we have found that the apoptotic protease activating factor 1 (Apaf1), apart from its canonical role in apoptosis, plays an additional function in cortical neurons, where its deficiency specifically impairs axonal growth. Given the central role played by centrosomes and microtubules in the polarized extension of the axon, our data suggest that Apaf1-deletion affects axonal outgrowth through an impairment of centrosome organization. In line with this, centrosomal protein expression, as well as their centrosomal localization proved to be altered upon Apaf1-deletion. Strikingly, we also found that Apaf1-loss affects trans-Golgi components and leads to a robust activation of AMP-dependent protein kinase (AMPK), this confirming the stressful conditions induced by Apaf1-deficiency. Since AMPK hyper-phosphorylation is known to impair a proper axon elongation, our finding contributes to explain the effect of Apaf1-deficiency on axogenesis. We also discovered that the signaling pathways mediating axonal growth and involving glycogen synthase kinase-3β, liver kinase B1, and collapsing-response mediator protein-2 are altered in Apaf1-KO neurons. Overall, our results reveal a novel non-apoptotic role for Apaf1 in axonal outgrowth, suggesting that the neuronal phenotype due to Apaf1-deletion could not only be fully ascribed to apoptosis inhibition, but might also be the result of defects in axogenesis. The discovery of new molecules involved in axonal elongation has a clinical relevance since it might help to explain neurological abnormalities occurring during early brain development

Synaptogenesis Stimulates a Proteasome-Mediated Ribosome Reduction in Axons

Ribosomes and a subset of cellular mRNAs are trafficked into axons of developing neurons. The axonal localization of translational machinery allows new proteins to be rapidly and locally synthesized during axonal growth and pathfinding. However, in mature neurons, axonal ribosomes are significantly reduced or even absent. The mechanism that elicits this removal is currently unknown. Here, we demonstrate that synapse formation is the trigger for ribosome reduction in mature axons. In vivo analysis shows that axonal ribosome levels decrease in rat brain at a developmental stage coincident with synapse formation. Next, we observe in vitro that different synaptogenic inducers trigger an overall decrease of ribosomal proteins and rRNA in the axons of spinal motor neurons. We further observe that this process is dependent on the ubiquitin-proteasome system but not on autophagy. Together, these data identify synaptogenesis as the long missing biological trigger that leads to ribosome disappearance during axonal maturation