Thrombospondin 4 deficiency in mouse impairs neuronal migration in the early postnatal and adult brain

Abstract

In the post-natal rodent brain, neuronal precursors originating from the sub-ventricular zone (SVZ) migrate over a long distance along the rostral migratory stream (RMS) to eventually integrate the olfactory bulb neuronal circuitry. In order to identify new genes specifically expressed in the RMS, we have screened the Allen Brain Atlas Database. We focused our attention on Thrombospondin 4 (Thbs4), one of the 5 members of the Thrombospondin family of large, multidomain, extracellular matrix proteins. In post-natal and adult brain Thbs4 mRNA and protein are specifically expressed in the neurogenic regions, including the SVZ and along the entire RMS. RMS cells expressing Thbs4 are GFAP (Glial Fibrillary Acidic Protein) positive astrocytes. Histological analysis in both wild-type and Thbs4 knock-out mice revealed no major abnormality in the general morphology of these neurogenic regions. Nevertheless, immunostaining for doublecortin demonstrates that in Thbs4-KO, migration of newly formed neurons along the RMS is somehow impaired, with several neurons migrating out of the RMS. This is further supported by a Bromodeoxyuridine-based in vivo approach showing a decrease in the number of newly born neuronal precursors reaching the olfactory bulb, while proliferation in the SVZ is not affected compared to wild-type, both in young animals (P15) and in adults (8 to 12 weeks of age). Corroborating this observation, the number of Parvalbumin- and Calbindin-immunoreactive interneurons in the olfactory bulb is also reduced in Thbs4-KO. Together, these observations support a role for the astrocyte-secreted protein Thbs4 in the migration of newly form neurons within the RMS to the olfactory bulb.

Introduction

In the post-natal rodent brain, neurogenesis persists throughout adulthood, in mainly two neurogenic regions: the subventricular zone (SVZ) of the lateral ventricles, and the subgranular zone (SGZ) of the dentate gyrus. While in the latter, newly generated neuronal cells migrate over a short distance and differentiate into a single neuronal type (excitatory glutamatergic granule neurons), immature neurons generated within the SVZ migrate over a long distance to reach the olfactory bulb (OB) and differentiate into distinct neuronal types (the majority becoming GABA- or dopaminergic, and few being glutamatergic). In the SVZ, the neural stem cells (NSCs) are specialized astrocytes (Type B cells). These cells give rise to rapidly dividing transit-amplifying cells (Type C cells), which in turn generate neuronal precursors (Type A cells). These later migrate along the so-called rostral migratory stream (RMS) through a process called tangential migration involving homophilic interaction of neuronal precursors, surrounded by astrocytes. Once they reach the OB, immature neurons switch to radial migration, differentiate into granule and periglomerular neurons, and eventually integrate existing neuronal circuitry. Neurogenesis in the adult brain has been the subject of numerous reviews in the past decade (for recent reviews, see Basak and Taylor, 2009, Cayre et al., 2009, Faigle and Song, 2013, Ihrie and Alvarez-Buylla, 2011, Kriegstein and Alvarez-Buylla, 2009, Ming and Song, 2011, Whitman and Greer, 2009, Zhao et al., 2008).

Numerous studies have led to the identification of intrinsic and extrinsic factors, such as growth and trophic factors, transcription factors and cell adhesion molecules, involved in various aspects of adult neurogenesis, including neural stem cells, proliferation of immature neurons, their migration through the RMS and their differentiation in the OB (see references aforementioned). In particular, the importance of the extracellular environment surrounding the migrating cells has been emphasized. Indeed, several molecules of the extracellular matrix (ECM) are expressed along the RMS, and are crucial for migration. These include tenascin C, heparan- and chondroitin sulfate proteoglycans, laminins, matrix metalloproteases, thrombospondin 1, netrin, and reelin (reviewed in Dityatev et al., 2010, Kazanis and ffrench-Constant, 2011).

In this study, we have screened the Allen Brain Atlas Database to identify genes specifically expressed in the RMS. We report here the implication of the ECM protein Thrombospondin-4 (Thbs4) in early postnatal and adult mouse neurogenesis. Because of their ability to bind various ligands, proteins of the thrombospondin family have been involved in the regulation of diverse cellular processes, including cell proliferation and migration, ECM remodeling, and various pathologies (vascular and heart pathologies, cancers, skeletal pathologies) (reviewed in Adams, 2001, Mosher and Adams, 2012, Risher and Eroglu, 2012). We show here that Thbs4 is expressed in the post-natal mouse brain in RMS astrocytes. In Thbs4 knock-out mice, although proliferation of neuronal precursors in the SVZ is not affected, a decrease in the number of early born neurons reaching the olfactory bulb implicates the ECM protein Thbs4 in neuron migration along the RMS.