Ascending midbrain dopaminergic axons require descending GAD65 axon fascicles for normal pathfinding

Abstract

The Nigrostriatal pathway (NSP) is formed by dopaminergic axons that project from the ventral midbrain to the dorsolateral striatum as part of the medial forebrain bundle. Previous studies have implicated chemotropic proteins in the formation of the NSP during development but little is known of the role of substrate-anchored signals in this process. We observed in mouse and rat embryos that midbrain dopaminergic axons ascend in close apposition to descending GAD65-positive axon bundles throughout their trajectory to the striatum. To test whether such interaction is important for dopaminergic axon pathfinding, we analyzed transgenic mouse embryos in which the GAD65 axon bundle was reduced by the conditional expression of the diphtheria toxin. In these embryos we observed dopaminergic misprojection into the hypothalamic region and abnormal projection in the striatum. In addition, analysis of Robo1/2 and Slit1/2 knockout embryos revealed that the previously described dopaminergic misprojection in these embryos is accompanied by severe alterations in the GAD65 axon scaffold. Additional studies with cultured dopaminergic neurons and whole embryos suggest that NCAM and Robo proteins are involved in the interaction of GAD65 and dopaminergic axons. These results indicate that the fasciculation between descending GAD65 axon bundles and ascending dopaminergic axons is required for the stereotypical NSP formation during brain development and that known guidance cues may determine this projection indirectly by instructing the pathfinding of the axons that are part of the GAD65 axon scaffold.

The discovery of the growth cone and its influence on the study of axon guidance

“…Together, the response to these signals, mediated by membrane receptors on the growth cone, is accompanied by changes in cytoskeletal organization of the growth cone and its adhesion to the substrate as it courses through the developing brain (Figure 4 ). Current knowledge confirms that the instructive directional signals of both neurotropic proteins and contact guidance cues are integrated by growth cones, resulting in stereotypical axon route finding (Myers and Gomez, 2011 ; Bonanomi et al, 2012 ; Moore et al, 2012 ; Leung et al, 2013 ; Garcia-Peña et al, 2014 ).…”

“…For example, we have observed interaction between axons from opposite sides of the developing mouse hindbrain that is relevant for their decussation (Sandoval-Minero and Varela-Echavarría, 2008 ) and the anatomical coupling of sensory projections to motor axons influences sensory axon projection (Wang et al, 2011 ). Moreover, our group has recently shown that ascending midbrain dopaminergic axons grow in close apposition to descending GAD65-positive axons and that this interaction appears to participate in stereotypical dopaminergic projection (Garcia-Peña et al, 2014 ). We also observed that previously known misprojections of dopaminergic axons in embryos lacking the chemotropic molecules Slit1 and Slit2 or their receptors Robo1 and Robo2 (Dugan et al, 2011 ) were accompanied by severe alterations of the underlying GAD65 axon scaffold.…”

“…E13.5 rat embryo pretectum (P1) explants exert an attractive effect upon axon GABA/TH fascicles growing from ventral midbrain explants (VM) in collagen gel cultures. Cultured gels were immunostained for GABA (red) and TH (green) (Garcia-Peña et al, 2014 ).…”

Roles of Semaphorins in Neurodegenerative Diseases

“…Paradoxically, the Sema3A found to promote neurodegeneration in PD pathogenesis, at the same time may be useful for steam cell transplantation therapy in PD patients [96][97][98][99]. Given that semaphorins participate in the formation of the nigrostriatal pathway during prenatal development, they have also been proposed to guide axons to their appropriate targets after possible cell replacement therapy with dopaminergic neurons [96,[100][101][102][103][104][105]. Embryonic stem cells differentiated to tyrosine hydroxylase-expressing neurons have been shown to have similar phenotype, expression of neuropilins, and response to Class 3 semaphorins than embryonic ventral mesencephalon neurons [96,97,106].…”

Cell-Cell and Cell-Matrix Interactions during Axons Guidance

“…Npn-1 depletion also affects fasciculation and targeting of cranial nerves and Schwann cells migration [87]. Interestingly, altered projections of descending GAD65-positive fascicles from the MTG tract, present in double knockout mice for Slit chemotropic protein receptors Robo1/2, modify the nigrostriatal projection (NP) of dopaminergic neurons, impairing both tracts interactions, probably by the absence of homophilic Robo-Robo interactions and heterophilic interaction with NCAM proteins [88], explaining some of the Slit1/2 independent role of Robo-expressing axons during NP projection [89]. These results show that besides their role of mediating attraction or repulsive responses, some receptors for chemotropic proteins also mediate axons fasciculation by homo-and heterophilic interactions, and this role could be concealed or dismissed by the more characterized chemotropic response.…”