Author:

Abstract:

Although in the past decades remarkable progress has been made in understanding adult neurogenesis, a detailed comprehension of the mechanisms underlying the neurogenic process is currently still lacking. Questions remain regarding neural stem cell (NSC) identity and heterogeneity, how these NSCs give rise to new neurons and, eventually, the functional role and therapeutic potential of adult neurogenesis. In order to address these different issues it is essential to expand the available toolbox by designing new technology or further improving existing ones. In this thesis, I focused on the well-established viral vector platform, with a particular focus on lentiviral (LV) vector technology and combined it with RNA interference (RNAi) and Cre recombinase technology, to dissect the role of different proteins in mouse adult neurogenesis. First, we engineered and optimized RNAi-based viral vectors to result in efficient depletion of target proteins. The characterization of RNAi and the accompanying microRNAs (miRs), together with the exogenous expression of artificial miR-like elements, has led to the development of strategies for specific and potent gene silencing. Here, we studied the optimal strategy to achieve the most potent knockdown using miR-based viral vectors, evaluating the potential of polycistronic miRs in a viral vector context. We evaluated knockdown potency of a polycistron, either consisting of miRs targeting the same seed sequence or of miRs targeting different seed sequences using reporter and endogenous mRNAs as targets. In the first chapter of my work, we demonstrate that potent knockdown can be obtained in vitro and in vivo using viral vectors that encode a single miR-based short-hairpin RNA (shRNA) and report a generic and effective cloning platform for artificial miR30-based shRNAs to generate potent knockdown viral vectors. Using the above-mentioned miR platform, we investigated the role of DJ-1 and lens epithelium-derived growth factor (LEDGF/p75) in the adult neurogenic process by using LV vectors for local depletion of these proteins in the subventricular zone (SVZ) and in the migration to the olfactory bulb (OB) of the mouse brain. This model is widely used in the study of neurogenesis, migration, and functional integration of newborn neurons. DJ-1 and LEDGF/p75 were chosen as target proteins for specific reasons. Loss-of-function mutations in the DJ-1 gene lead to autosomal recessive early-onset Parkinson’s disease. DJ-1 has been described to play a protective role against oxidative stress and cell death. On the other hand, it has been observed that NSCs are more dependent on reactive oxygen species (ROS)-mediated cell signaling than other cells in the brain, with ROS significantly affecting NSCs proliferation, maintenance and differentiation. Employing the LV vector platform, we show that DJ-1 has a relevant role in the generation of new neurons in the OB as well as in their maturation. Moreover, this phenotype was reversed upon complementation with human DJ-1. Additionally, reduced proliferation upon DJ-1 depletion suggests a role for DJ-1 in the proliferation capacity of the SVZ. Further evaluation in an in vitro model of SVZ confirmed our initial observations by impairment of self-renewal, proliferation, and differentiation of these cells, and permitted us to reveal a role for PTEN in these phenotypes, coinciding with increased ROS levels and decreased mitochondrial membrane potential upon DJ-1 depletion. These results led us to propose DJ-1 as a regulator of the neurogenic process, being relevant in the protection of neural progenitors from high endogenous redox levels and to control intracellular signaling depending on the oxidative status of the cells through PTEN inhibition. Next to DJ-1, we also interrogated LEDGF/p75 in neural stem/progenitor cells (NSPCs). Being a transcriptional co-activator of stress-response genes, LEDGF/p75 has also been proposed to be involved in neuronal development and differentiation. Given the expertise, knowledge and interest on LEDGF/p75 in the collaborating research group for Molecular Virology and Gene Therapy, we addressed the role of this protein in adult neurogenesis. By injection of a LV vector expressing a miR against mouse LEDGF/p75 in the SVZ, we observed normal proliferation and migration after LEDGF/p75 depletion, suggesting that LEDGF/p75 is not involved in these processes, at least under the circumstances tested and for the evaluated time intervals. While investigating DJ-1 and LEDGF/p75, we corroborated that the single miR design resulting in potent knockdown allowed us to study the role of two different proteins in the SVZ. However, with this tool we could not answer whether the phenotypes observed are due to knockdown in the NSPCs, in the surrounding cells or a combination of both. Therefore, we developed a viral vector that targets a specific set of cells, building on the recently developed CreFlex (flip-excision) LV vectors. In the latter system, transgene expression is only triggered in the presence of Cre-recombinase. We expanded the CreFlex system to accommodate artificial miRs that can be specifically expressed upon Cre recombinase expression and studied the effect of DJ-1 and LEDGF/p75 depletion by specifically targeting nestin-expressing cells. In a first step, we demonstrated that the loop structure of the miR hairpin is flexible enough to accommodate a lox sequence. The development of a proof-of-principle CreEx(excision) vector based on this knowledge underscored the fact that the miR expression dependent on the Cre/lox technology does not affect its efficacy. As this allowed us to further develop the Cre-based knockdown viral vectors, we successfully developed the CreFlex knockdown system that rendered cell-specific and potent depletion of a target protein in vitro. In addition, we demonstrated efficient Cre-induced activation of the reporter cassette both under in vitro and in vivo conditions. When specifically targeting DJ-1 and LEDGF/p75 in the SVZ of Nestin-Cre mice, we studied their role in this model. Overall, the data obtained suggest that depletion of DJ-1 in NSPCs affects the survival/differentiation of the cells arriving in the OB contrary to the more pronounced overall effect in proliferation, migration and differentiation upon DJ-1 depletion in the stem cells and surrounding cells, suggesting that DJ-1 plays also a role in these supporting cells by a different mechanism. On the other hand, exclusive depletion of LEDGF/p75 in NSPCs again did not demonstrate an effect on proliferation. The question remains unanswered whether LEDGF/p75 is involved in the migration/survival or differentiation of cells arriving to the OB. The successful outcome of this project provided new and improved tools for in vivo gene silencing in the brain but also a very generic technology that can be applied to other biological systems.