Expression by midbrain dopamine neurons of Sema3A and 3F receptors is associated with chemorepulsion in vitro but a mild in vivo phenotype

Abstract

Here we explore the role of semaphorin 3A and 3F (Sema3A, Sema3F) in the formation of the mesotelencephalic pathway. We show that Sema3A and 3F are expressed in the ventral mesencephalon (VM) of E13.5 rat embryos; the receptors Neuropilin 1 and Neuropilin 2, and co-receptors L1CAM, NrCAM, and Plexins A1 and A3 but not A4 are expressed by VM dopaminergic neurons; these neurons bind Sema3A and 3F in vitro which induces collapse of their growth cones and elicits, with different potencies, a repulsive response; and this response is absent in axons from Nrp1 and Nrp2 null embryos. Despite these in vitro effects, only very mild anatomical defects were detected in the organization of the mesotelencephalic pathway in embryonic and adult Nrp1 or Nrp2 null mice. However, the dopaminergic meso-habenular pathway and catecholaminergic neurons in the parafascicular and paraventricular nuclei of the thalamus were significantly affected in Nrp2 null mice. These data are consistent with a model whereby Sema3A and 3F, in combination with other guidance molecules, contributes to the navigation of DA axons to their final synaptic targets.

Introduction

In the wiring of the central nervous system (CNS), axons frequently travel long distances following very precise pathways before synapsing with specific target cells, a process that requires a series of structured spatio-temporal signals. While progress has been made in elucidating the signals regulating the formation of several major tracts in the CNS (Garcez et al., 2007, Henion and Schwarting, 2007, Lindwall et al., 2007, Price et al., 2006, Webber and Raz, 2006) the molecular and topographical cues that make possible the formation of the meso-telencephalic dopaminergic pathway (MTp) is only beginning to be understood.

The MTp is a key component in the basal ganglia thalamo-cortical loop and its disturbance, as a consequence of neurodegenerative diseases like Parkinson's disease (Barzilai et al., 2000, Betarbet et al., 2005, Di Monte, 2003, Hague et al., 2005, Lim and Lim, 2003, Moore et al., 2005) can lead to severe motor and behavioral deficits. The interest in devising restorative therapies for patients suffering from these diseases in part motivates elucidation of the cues involved in the formation of this pathway. The MTp originates in DA neurons in the ventral mesencephalon (VM) whose axons grow rostrally to traverse the diencephalon in a large but well-defined pathway, the medial forebrain bundle (MFB), and reach the telencephalon to synapse on neurons in the nucleus accumbens (mesolimbic pathway) and the striatum (mesostriatal pathway) (Altman and Bayer, 1981, Bayer et al., 1994, Bayer et al., 1995, Voorn et al., 1988). A much smaller group of DA axons originates in rostral cells of the VM, branches off the MFB at the mesencephalon–diencephalon boundary and gives rise to the meso-habenular pathway (MHp) whose axons run along the fasciculus retroflexus and synapse on the lateral habenula (Phillipson and Griffith, 1980, Skagerberg et al., 1984). The MHp has been implicated in the integration of descending output pathways from limbic hypothalamic and striatal forebrain regions and feedback control of the MTp (Gruber et al., 2007, Skagerberg et al., 1984).

During the last decade it has become increasingly clear that bound and soluble signals acting through ligand-receptor signaling can modify growth cone behavior allowing for normal axon pathfinding. Some of these proteins are located on cell surfaces or are part of the extracellular matrix (e.g., laminin, glycans) (Benson et al., 2001, Huber et al., 2003), while others, including semaphorins (Chen et al., 1998, Chen et al., 1998, Goshima et al., 2000, Goshima et al., 2002), netrins (Winberg et al., 1998, Leonardo et al., 1997, MacLennan et al., 1997, Shirasaki et al., 1996), ephrins (Barnes et al., 2003, Knoll and Drescher, 2002), and slits (Bagri et al., 2002, Marillat et al., 2002, Niclou et al., 2000) are secreted by neighboring tissue confining the growth of distinct axonal systems to specific pathways. In vitro studies indicate that attractive and repulsive signals produced in the VM and diencephalon are indeed involved in the organization of the ventro-mesencephalic DA (vDA) axons into the MTp (Gates et al., 2004, Nakamura et al., 2000). Netrin1 (Lin et al., 2005, Osborne et al., 2005) and Sema3C (Hernandez-Montiel et al., 2008) have been reported to function as attractant signals acting on vDA axons. However, while disturbances of Netrin1 signaling give rise to structural and functional defects of the DA system (Flores et al., 2005), it is not yet clear how deletion of Sema3C impacts the formation of the MTp.

A few chemorepellent factors have also been implicated in the organization of DA axons. Slits acting through Robo receptors (Brose et al., 1999) expressed by DA neurons (Lin and Isacson, 2006, Lin et al., 2005) may prevent DA axons from crossing the midline (Kawano et al., 2003, Lin et al., 2005) while EphrinB2 and its receptor EphB1 have been implicated in the topographical guidance of the MTp (Yue et al., 1999). Class 3 semaphorins are potent axonal repellents and have been recently implicated in the organization of the MTp. Sema3F, a secreted semaphorin with potent repulsive effects on cortical and sympathetic axons has been shown to repel DA axons (Hernandez-Montiel et al., 2008, Kolk et al., 2009, Yamauchi et al., 2009) and to induce aberrant growth of DA axons in Neuropilin 2 (Nrp2) null mutant mice (Kolk et al., 2009, Yamauchi et al., 2009) while Sema3A, whose mRNA has also been shown to be expressed in the mesencephalon, has been reported to be ineffective on DA axons (Hernandez-Montiel et al., 2008). The receptors for Sema3A and 3F, Neuropilin 1 (Nrp1) and Nrp2, respectively, have been reported to be expressed in the VM and by vDA neurons during development (Hernandez-Montiel et al., 2008, Kolk et al., 2009) and in the adult brain. However, it is not clear whether the plexin coreceptors recruited to the Sema3A/Nrp1 or the Sema3F/Nrp2 complex to transduce the semaphorin signal (Nakamura et al., 2000, Rohm et al., 2000, Takahashi and Strittmatter, 2001, Takahashi et al., 1999; Suto et al., 2005; Yaron et al., 2005), or the cell adhesion molecules L1CAM and NrCAM (Castellani et al., 2000, Castellani et al., 2002, Falk et al., 2005), which are also required for the full manifestation of the repulsive effect of Sema3A and 3F, are expressed by vDA neurons during the organization of the MTp.

Here we show that Sema3A and Sema3F are strongly expressed in the mesencephalon at the time the vDA axons are growing toward the striatum, and that, as previously reported (Hernandez-Montiel et al., 2008, Kolk et al., 2009, Yamauchi et al., 2009), the receptors Nrp1 and Nrp2 are expressed by vDA neurons and their axons enabling them to bind to both Sema3A and 3F. We show that the transducers PlexinA1 and A3, but not A4, and the coreceptors L1CAM and NrCAM, are expressed by vDA neurons. In line with these observations, in in vitro experiments Sema3A and 3F induced with different potencies the collapse of DA growth cones and a significant repulsive response on DA axons. However, the potency with which these semaphorins modify the response of the DA axon in vitro contrasted with the finding of very mild defects in the gross anatomical organization of the VM DA system as well as the lack of a behavioral phenotype in Nrp1 and Nrp2 knockout mice. The results suggest that Sema3A and 3F signals play a role in the organization of the DA MTp, but that their absence is largely compensated leaving the nigrostriatal and meso-limbic synaptic organization largely unimpaired.