Deletion of lysophosphatidic acid receptor LPA₁ reduces neurogenesis in the mouse dentate gyrus

Abstract

Neurogenesis persists in certain regions of the adult brain including the subgranular zone of the hippocampal dentate gyrus wherein its regulation is essential, particularly in relation to learning, stress and modulation of mood. Lysophosphatidic acid (LPA) is an extracellular signaling phospholipid with important neural regulatory properties mediated by specific G protein-coupled receptors, LPA_1–5. LPA₁ is highly expressed in the developing neurogenic ventricular zone wherein it is required for normal embryonic neurogenesis, and, by extension may play a role in adult neurogenesis as well. By means of the analyses of a variant of the original LPA₁-null mutant mouse, termed the Malaga variant or “maLPA₁-null,” which has recently been reported to have defective neurogenesis within the embryonic cerebral cortex, we report here a role for LPA₁ in adult hippocampal neurogenesis. Proliferation, differentiation and survival of newly formed neurons are defective in the absence of LPA₁ under normal conditions and following exposure to enriched environment and voluntary exercise. Furthermore, analysis of trophic factors in maLPA₁-null mice demonstrated alterations in brain-derived neurotrophic factor and insulin growth factor 1 levels after enrichment and exercise. Morphological analyses of doublecortin positive cells revealed the anomalous prevalence of bipolar cells in the subgranular zone, supporting the operation of LPA₁ signaling pathways in normal proliferation, maturation and differentiation of neuronal precursors.

Introduction

It is well accepted that the vertebrate brain continues to produce new neurons throughout adulthood (Altman and Bayer, 1990, Eriksson et al., 1998, Alvarez-Buylla et al., 2002, Gage, 2002). Most of these cells are produced in the subgranular zone of the dentate gyrus (DG) in the hippocampus, with an estimated 9000 (rat) or 800–1600 (mouse) new cells generated per day (Cameron and McKay, 2001, Christie and Cameron, 2006). Neuronal DG progenitors proliferate, migrate, and differentiate into granular neurons and subsequently integrate into the hippocampal circuitry wherein they become functional. Though controversial (Leuner et al., 2006), numerous studies have provided evidence that newly formed neurons are significantly involved in cognition (Macklis, 2001, Shors et al., 2001, van Praag et al., 2002, Shors, 2004, Aimone et al., 2006). Accordingly, the clinical importance of this hippocampal neurogenesis becomes manifest in age-related deficiencies (West, 1993a, Persson et al., 2006), neural damage (Kuhn et al., 2001) or psychiatric disorders (Eisch and Nestler, 2002, Silva et al., 2006).

The survival of newly generated DG neurons is known to be positively modulated by a variety of external factors including learning tasks (Gould et al., 1999, Dobrossy et al., 2003), environmental enrichment (Kempermann et al., 1997, Olson et al., 2006) and exercise (Van Praag et al., 1999a, Van Praag et al., 1999b, Eadie et al., 2005). Postnatal neural progenitor cells are influenced by similar regulatory gene cascades and growth factors to those that control proliferation and differentiation during development (Vaccarino et al., 2001, Esposito et al., 2005). Identified neuronogenic molecules like IGF-I (Åberg et al., 2003, Sun et al., 2005), Sonic Hedgehog (Ahn and Joyner 2005), EGF or FGF-2 (Kuhn et al., 1997) and many environmental niche factors (Gage, 2002, Alvarez-Borda et al., 2004, Fabel et al., 2003, Battista et al., 2006) influence the overall process, while others are currently being elucidated.

Lysophospholids have recently emerged as important influences on normal nervous system development (Chun et al., 2002, Anliker and Chun, 2004, Moolenar et al., 2004, Chun, 2005, Chun, 2007). Lysophosphatidic acid (LPA) is a simple phospholipid that can act as an extracellular signal through at least 5 specific G protein-coupled receptors, LPA_1–5 (Chun et al., 2002, Anliker and Chun, 2004, Ishii et al., 2004, Lee et al., 2006). During development, LPA₁ is expressed in neural progenitor cells suggesting a regulatory function in neurogenesis (Hecht et al., 1996). In vivo LPA₁ expression has been detected in the hippocampus wherein it seems to be predominantly restricted to oligodendrocytes (Weiner et al., 1998, Handford et al., 2001) while only hardly detectable levels of LPA₁ mRNA were found in neurons (Allard et al., 1998). However, a reasonable level of neuronal hippocampal LPA₁ expression has been demonstrated under ex vivo circumstances (Tabuchi et al., 2000, Fujiwara et al., 2003, Pilpel and Segal, 2006) or in immortalized hippocampal progenitor cells (Rhee et al., 2006). Exogenous delivery of LPA has demonstrated LPA₁ receptor-mediated functions including morphophysiological changes in neural progenitors (Dubin et al., 1999, Kingsbury et al., 2003, Fukushima, 2004, Fukushima and Morita, 2006, Fukushima et al., 2000, Fukushima et al., 2007). Likewise, LPA delivery to hippocampal neurons is known to increase the tyrosine phosphorylation of FAK (Derkinderen et al., 1998), regulate cell death (Holtsberg et al., 1998), mimic neurotrophic effects (Fujiwara et al., 2003) or mediate synaptic changes associated with spatial memory (Dash et al., 2004). Effects of LPA₁ loss on interneuron-mediated rhythms in vivo (Cunningham et al., 2006) and over-expression gain on synapse formation (Pilpel and Segal, 2006) have been reported in the hippocampus. In addition, the presence of modulators of lysophosphatidic acid activity has been also demonstrated in the adult hippocampus (Brauer et al., 2003).

At present, null animals have been obtained for most of the known LPA receptors by targeted gene disruption, all of them being mice (Choi et al., 2008). Receptor loss-of-function studies using LPA₁-null or LPA₁-/LPA₂-double null mice have suggested centrally mediated behavioral defects and relatively minor morphological cerebral alterations, although the initially generated mutation was associated with ∼ 50% perinatal lethality that may have a CNS component, along with defective olfaction (Contos et al., 2000, Contos et al., 2002, Harrison et al., 2003, Roberts et al., 2005). Recently, the propagation of the original mixed background strain of LPA₁-null mice (Contos et al., 2000) in our laboratories, led to a stable variant of LPA₁-null mice called the “Malaga variant” (reported as “maLPA₁-null” mice). These mutants exhibited improved perinatal viability and showed altered cortical neurogenesis and increased cell death during brain development that caused a reduction of cortical layer cellularity in adults, indicating the action of as yet unidentified genetic modifiers of LPA₁ that influence cortical neurogenesis (Estivill-Torrús et al., 2008). Here we report that maLPA₁-null mice display reduced postnatal DG neurogenesis under both basal and environmentally enriched conditions.