The molecular regulation of neural stem cell lineage progression in the postnatal subventricular zone by Galectin-3

Neurogenesis continues postnatally in two major neural stem cell (NSC) niches: The subventricular zone (SVZ) and dentate gurus of the hippocampus. SVZ NSCs self-renew and produce transit amplifying progenitor cells that, in turn, divide and give rise to neuroblasts. These neuroblasts migrate to the olfactory bulbs, via the rostral migratory stream (RMS), where they terminally differentiate into mature neurons. The postnatal SVZ (pSVZ) is more gliogenic than its adult counterpart (aSVZ), contributing to robust postnatal astrocytogenesis and oligodendrogenesis in the surrounding brain parenchyma. Studies examining Galectin-3 (Gal-3) in the aSVZ showed it has functions in regulating neuroblast migration, microglial activation, oligodendrocytic differentiation, and angiogenesis. However, the role of Gal-3 in pSVZ lineage progression is unknown. This thesis aims to unravel the roles of Gal-3 in regulating pSVZ lineage progression, fate choices, and NSC activation. In doing so, the thesis tackles the molecular pathways possibly involved in mediating the effects of Gal-3.
I found through co-immunoprecipitation that Gal-3 was bound to β-catenin and both proteins were co-expressed in the aSVZ. In addition, expression of Gal-3 and Wnt/β-catenin signalling were downregulated as SVZ cells progressed through the lineage and became migratory. I hypothesised that Gal-3 may regulate lineage progression through regulation of Wnt/β-catenin signalling. To explore this hypothesis, Gal-3 overexpression, knockdown or control plasmids were co-electroporated with a Wnt/β-catenin reporter into the SVZ of postnatal day two mice. I found lineage progression was not altered by Gal-3 overexpression. Surprisingly, contrary to evidence described in the cancer literature, Gal-3 overexpression reduced Wnt/β-catenin signalling. This was accompanied by an acute reduction in proliferation. Also, more cells expressed p27/Kip1 in the SVZ, and more cells migrated into the RMS, suggesting increased cell cycle exit. However, NSC proliferation and clonal neurosphere forming capacity were not altered by Gal-3 overexpression, indicating that NSC activation was not influenced by Gal-3. While olfactory neuronogenesis was not altered by Gal-3 overexpression, striatal astrocytogenesis was increased while oligodendrogenesis was dampened. Further experiments revealed phosphorylation of Smad proteins 1/5/8 was increased in vivo and in vitro after Gal-3 overexpression. These findings indicate that Gal-3 positively regulated BMP signalling in the SVZ, possibly contributing to Gal-3's pro-gliogenic effects.
Taken together, this thesis supports a model whereby a subpopulation of Gal-3-responsive pSVZ cells reacted to Gal-3 overexpression by acutely exiting the cell cycle, and possibly through the same mechanisms, switched from oligodendrocytic to astrocytic fate. These cellular responses might have been brought about, at least partially, by acute suppression of Wnt/β-catenin and activation of BMP signalling. These novel findings emphasise the regulatory actions of Gal-3 on pSVZ lineage progression through Wnt/β- catenin and BMP signalling.

Keywords: Wnt/B-catenin
Astrocytes
BMP
Oligodendrocytes
Subventricular zone
Neural stem cells
Galectin-3
cell signalling
Advisor: Szele, F