Roles of the GPI-anchored proteins PrP and Thy-1 in axon growth and regeneration
Final Report Abstract
The goal of this study was to determine if the GPI-anchored proteins Thy-1 and PrP possess axon growth promoting properties and promote axon regeneration. Moreover, the question was addressed whether the reggie proteins might participate as mediators of Thy-1 and PrP clustering and signal transduction. We also asked whether PrP might promote trafficking of functionally important cell adhesion proteins and participate in the recruitment of cargo for growth cones and spine synapses. After generation of a new monoclonal antibody against Thy-1 we were able to show that zebrafish retinal ganglion cells (RGCs) upregulate Thy-1 after optic nerve lesion. Downregulation of Thy-1 by specific morpholinos (MOs), in turn, impaired RGC differentiation in vitro and axon regeneration in two independent experiments. As this suggested that Thy-1 has axon growth supportive functions we also analysed its role in 1-2 d old mouse hippocampal neurons in vitro. Overexpression of Thy-1 led to increased neurite length whereas its downregulation had the opposite effect: it impaired cell differentiation and neurite elongation. Thy-1 was closely associated with the reggie proteins and its co-clustering enhanced reggie-associated signal transduction connected to growth whereas downregulation caused the opposite. These data suggest a growth promoting function of Thy-1 during hippocampal neuran differentiation and during axon regeneration in the fish visual pathway. With specific antibodies against zebrafish PrP-2 we demonstrate that PrP-2 is also upregulated after optic nerve lesion and during axon regeneration. Functional assays using PrP-2 specific MOs reduced the PrP-2 protein concentration significantly and blocked RGCs differentiation in vitro and axon regeneration in vivo. Moreover, mouse PrPc seemed to possess a growth-promoting influence on hippocampal neurons in vitro. By analyzing neurons from PrP knock out mice, we observed that growth cones of hippocampal neurons were smaller and had fewer N-cadherin cargo vesicles which indicates that PrP is involved in trafficking of cargo to important regions of the neuron. Accordingly, we also discovered that trafficking of the NMDA receptor is impaired in PrP knock out mice. Neurons of these mice formed fewer synapses in vitro. Ongoing studies suggest that this site specific trafficking involves the reggie proteins and their interaction with Rab11. Altogether, our data show that Thy-1 expression and re-expression is beneficial for regeneration of zebrafish RGCs after lesion as well as for the differentiation of mouse hippocampal neurons. Likewise, zebrafish PrP-2 and its homologue in mouse, PrPc, proved to possess growth promoting properties and to enhance the target directed delivery of growth associated proteins to the growth cone and postsynaptic receptors to spines.
Publications
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(2011) Prion protein promotes growth cone development through reggie/flotillin-dependent N-cadherin trafficking. J Neurosci 31:18013-18025
Bodrikov V, Solis GP, Stuermer CA
