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Role of Id4 in the adult subventricular zone of normal and injured brain

Periodic Reporting for period 1 - ID4 (Role of Id4 in the adult subventricular zone of normal and injured brain)

Reporting period: 2015-08-01 to 2017-07-31

The subventricular zone (SVZ) is one of the largest neurogenic niches in the adult brain of mammals. The SVZ harbours self-renewing neural stem cells (NSCs), which give rise to neural progenitor cells. These progenitor cells generate neuroblasts that migrate tangentially in chains to the olfactory bulb, where they differentiate into olfactory interneurons. Neurogenesis in the SVZ is tightly regulated by several intrinsic and extrinsic factors. Among extrinsic factors, the bone morphogenic protein (BMP) signalling pathway has been shown to positively regulate neurogenesis while inhibiting oligodendrocyte differentiation. However, the downstream transcriptional factors mediating the effects of BMPs on adult neurogenesis are not well characterized. Inhibitor of DNA binding 4 (Id4) is a helix-loop-helix (HLH) transcriptional regulator activated by BMP signalling. Id proteins participate in numerous cellular processes such as differentiation, proliferation and apoptosis, by dimerizing with bHLH transcription factors, preventing them from binding DNA. Id4 is strongly expressed in the developing central nervous system and regulates neural progenitor proliferation and differentiation. However, Id4 function in adult neurogenesis is unknown. Moreover, SVZ activation in response to stroke has been widely reported in several mouse models. Nevertheless, the molecular mechanisms driving this activation remain largely unknown. Understanding the mechanism triggering SVZ response to stroke can be essential to pave the way for the development of success regenerative strategies after this event. This is of vital importance for the society since stroke is the main cause of disability worldwide causing enormous personal and socioeconomics costs. Additionally, bringing an insight into stem cell regulation will path the way to better understand deregulation in a tumorigenic context.
In the present work, we sought to understand the function of Id4 in adult neurogenesis in normal and ischemic conditions. The main objectives of this project where (i) to determine the function of Id4 in adult NSC by gain and loss of function experiments and (ii) to understand the role of Id4 in SVZ-induced response upon ischemic stroke.
To date the expression of Id4 in the adult SVZ was fully characterized. We found that Id4 was expressed across the SVZ lineage. However, we found that stem cell were showing the highest levels of Id4 expression. Immuno-godd labelling followed by electron microscopy analysis confirms this expression. Moreover, BrdU incorporation paradigm to detect slow diving cells showed that a high percentage of these cells were Id4 positive suggesting that a big proportion of Id4 population correspond to quiescent stem cells. Additionally, we found Id4 expression in striatal astrocytes and ependymal cells, which are quiescent cells in the adult brain.
We then characterized the phenotype of the adult Id4-/- mouse. We observed a defect in SVZ morphology and a decreased in neurogenesis resulting in a decrease number of newborn neurons that reached the olfactory bulb, suggesting that Id4 may be playing an important role in the stem cell regulation. Complementary, electron microscopy analysis and wholemount stainings showed enlarged ventricles and defects in ependymal cell layer. These additional results open a new lead to investigate the potential role of Id4 in ependymal cell specification or maturation from radial glia cells.
In order to elucidate the Id4 function in the adult SVZ we acquired the conditional Id4 mouse line and cross it to a Tamoxifen inducible GlastCreERT2 mouse, which allowed us to delete specifically Id4 in stem cells. In vitro data showed an increase in proliferation of neurospheres cultured from adult SVZ, increasing the number of stem cells and the size of the spheres. Moreover, deletion of Id4 after differentiation showed an increase of division rate. Taken together this data suggest a role of Id4 in the maintenance of stem cell quiescence in the adult SVZ. To confirm these observations we analysed SVZ recombined cells in adult mice. Five days after Id4 deletion we observed an increase in the number of proliferating cells in the adult SVZ. We confirmed that this increase in the progenitors population occurred in detriment of stem cell pool observed as a result of Id4 deletion. However, we couldn’t detect an increase in the number of new born neurons but in oligodendrocytes in the whiter matter. Additionally, transcriptomic analysis showed deregulation of proliferative and cell adhesion pathways between others.
Finally, we studied the role of Id4 under pathological conditions. We developed tMCAO (transient Middle Cerebral Occlusion) model as stroke model. Immunofluorescence analysis of the SVZ in the tMCAO animal model at different time points idicated that SVZ proliferation upon stroke was preceded by Id4 downregulation and potentially it could be implicated in SVZ activation. However, further investigations need to be developed to fully understand the implication of Id4 in this process.
The results of this project have been disseminated in national meetings such as Journée de Biologie et Medicine in 2015 organized by University Pierre et Marie Curie (UPMC) and international congresses such as the ISSCR 2016 in San Francisco and 2017 in Basel. Additionally, the data was presented at the Doc and Postdoc Workshop organized by the ICM in 2015 and in the ICM Days in 2016. I also participated in the Marie Curie Alumni Association General Assembly in 2016 in Venice and the Chercheur en herbe program.
Moreover, in the team we have obtained the transcriptomic signature of Id4-overexpressing tumours. Strikingly, this signature overlaps with the signature of SVZ quiescent cells reported by other authors, suggesting that Id4 promote quiescence in gliomas. Taken together these data will be presented as a scientific article that will be submitted shortly in a peer-review journal.
Our results suggest so far that Id4 is involved in the maintenance of quiescence in adult SVZ stem cells. So far, the regulation of the activated stem cells has been widely investigated. However, here we have identified a master regulator of stem cell quiescence and we have unseated the classical view of Ids as inhibitors of differentiation proteins. The potential implications of these results can be extended to different contexts. Results obtained in this project suggest that Id4 downregulation may be necessary to drive NSC activation after stroke. Additionally, the results obtained in this project have served as a lead to investigate the role of Id4 in a tumorigenic context and the implication of Id4 in ependymal cell specification and/or maturation.
Schema representing Id4 dynamics in SVZ stem cells and outcome of Id4 deletion in quiescent NSC.