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Imaging of the neural stem cell origin, proliferation, and fate within the stem cell niches of the mammalian brain

Final Report Summary - NEURALSTEMIMAGING (Imaging of the neural stem cell origin, proliferation, and fate within the stem cell niches of the mammalian brain.)

This project was focused on adult neurogenesis in the subventricular zone (SVZ), to reveal the behaviour of adult progenitor cells in their own niches. Neural stem cells (NSCs) have been deeply studied, but many aspects of their behaviour in unperturbed conditions are still unknown. The elucidation of these features is crucial to understand NSCs in view of therapeutical application.

NSCs, a subpopulation of SVZ cells (B1 cells) with astroglial features, give rise to transit intermediate amplifying cells (C cells) which divide to generate dividing neuroblasts (A cells). While the lineage sequence has been determined, we aimed to clarify the cells cycle dynamics of each cell population.

The SVZ have been analyzed by the use of the 'whole mount' dissection technique, with adjustment of immunohistochemical techniques in confocal microscopy, to have a three-dimensional (3D) view of the whole lateral ventricle (LV) surface. This allowed us to analyze cell cycle dynamics without the effect of neuroblast migration.

Among the major tasks proposed in the project we focused on the number of times a C cell divide since an essential stage within the lineage is the transient amplification. Small changes in the cell cycle length or number of times C cells divide could result in very different lineage trees also determining the frequency with which B1 cells need to be recruited into mitosis to maintain a constant production of new neurons.

The use of whole mount technique allowed us to address also differences in lineages in different subregions of the SVZ providing an en face complete view of the surface of the LV. By this approach we labelled cells in the whole SVZ until the ones localised deep at the interface with blood vessels and above striatal parenchyma. C cells were identified by the expression of Mash1 transcription factor. Cell cycle length in the SVZ of adult rodents was analyzed by the use of different exogenous markers of S-phase (thymidine analogues such as 5'chloro-2'-deoxyuridine, and 5'ethynyl-2'-deoxyuridine) and endogenous mitotic markers (pHH3) according to different paradigms.

Previous studies indicated that the average cell cycle length in the SVZ is between 18 and 21h with an S-phase of 8.5-13h. Others suggest that it can be even shorter (cell cycle 13h, S-phase 4h). We measured a cell cycle length of C cells between 21 and 28 h. This is longer then what previously expected, especially since C cells were considered to be the fast dividing transit amplifying cells.

Moreover, only few C cells undergoes multiple mitosis. The analysis of G-TVA mice injected in the LV with RCAS-GFP virus allow us to label only GFAP expressing cells contacting the ventricle (i.e. B cells) and trace their progeny without dilution, confirming our previous findings.

Cell cycle dynamics in C cells were also analyzed in ageing animals. We analyzed 3, 6 months and 1 year old Fucci transgenic mouse that express a reporter protein in cells in S, G2 phase or mitosis. While both the number of C and of proliferating cells decrease with age, the ratio of the proliferating cells among the Mash1+ cells decrease less suggesting that they are depleted during ageing but do not slow down their cell cycle.

Further, we carried out a determination of EdU toxicity: number of EdU labeled cells one day after the injection is lower than the number of CldU labeled cells (occurrence of picnotic nuclei in animals labeled by EdU one day after the injection but not in animals labeled by CldU). Then, we provided a confirmation of the cell cycle dynamics of Mash1+ cells with percent of labeled mitosis method with CldU: 75 % of the CldU labeled cells undergo a second division one day after the injection while our previous data with EdU showed only 25 % of the EdU labelled cells undergoing a second division one day after the injection.

We pursued with the analysis of the cell cycle dynamics of B cells with cumulative labeling method, percent of labeled mitosis method and double analogue method. Then, analysis of the cell cycle dynamics of A cells with double analogue method and percent of labeled mitosis (only at short time after the injection).

We also started to analyse the cell cycle dynamics during regeneration after AraC treatment. For reasons of time and complexity, this latter study will not be included in the main publication coming from this project.

The final results consist of a better understanding of the cell cycle dynamics of neural stem cells in vivo, within their niche, in order to establish if they really act as stem cells or rather as progenitors (what is more likely, at present, and rather counterintuitive with respect to the current knowledge). This knowledge could be important in defining future strategies for the in vivo modulation of activity of such cells, as well as to adopt a more realistic approach to the therapeutic use of neural stem cells in patients with neurological diseases.