doi:?10.1083/jcb.200210174. strong axonal sprouting distal to a spinal cord lesion. Moreover, fostered PRG3 expression promoted complex motor-behavioral recovery compared to wild type controls as revealed in the Schnell swim test (SST). Thus, PRG3 emerges as a developmental RasGRF1-dependent conductor of filopodia formation and axonal growth enhancer. PRG3-induced neurites resist brain injury-associated outgrowth inhibitors and contribute to functional recovery after spinal cord lesions. Here, we provide evidence that PRG3 operates as an essential neuronal growth promoter in the nervous system. Maintaining PRG3 expression in aging brain may turn back the developmental clock for neuronal regeneration and plasticity. and neuronal morphology shape by PRG3 We further investigated PRG3 location and found it expressed in axon tips of primary neurons (Fig. 2 A). Endogenous PRG3 was located at the tip of actin-rich growth cones of cortical neurons (Fig. 2 A; Fig. S 2). Interestingly, Kynurenic acid primary astrocytes were almost immuno-negative for PRG3 (Fig. S 2). To investigate whether PRG3 has a general impact on neuronal morphology independently of the type of neurons, we studied this gene in cerebellar neurons. PRG3 expression in rat granule neurons caused extensive formation of neurites and filopodia in comparison to GFP expressing control granule neurons (Fig. 2 B, C). Electron microscopy studies of hippocampal synapses revealed post-synaptic (Fig. 2 D-G) and occasional pre-synaptic location of PRG3 (Fig. 2 H-K). Immuno-histochemistry of brain cryo-sections identified hippocampal neurons with high PRG3 levels in the adult mouse brain (Fig. 2 N). Open in a separate window Figure 2 PRG3 is located at pre-synaptic domains and electroporation [55]. (P) Representative example of electroporated brain section showing pyramidal neurons positive for GFP expressing pyramidal neurons (left) and PRG3-positive pyramidal cells (right). Neuronal morphology was analysed at postnatal day 10 (P10). Scale bar represents 20 m. High-power magnifications of boxed areas show spines and spine-like membrane protrusions, which are indicated by arrowheads. Scale bar represents 20 m. (Q) Number of protrusions per m dendrite were quantified in 70 m confocal stacks. Neurons electoroporated with PRG3 show significantly more protrusions per m compared to GFP electroporated neurons. Values are given as mean SEM. (N=5). Statistical analysis was performed using two tailed student’s t-test. P value was set as * = p 0.05: ** = p 0.01; *** = p 0.001. For assessments we performed electroporation of mouse embryonic cortical neurons at embryonic day 13 (Fig. 2 O) with GFP control and PRG3 constructs (Fig. 2 P). Noteworthy, neonates survived the procedure without obvious constraints and were sacrificed at postnatal day 10 (P10). In depth morphometric investigations of single pyramidal neurons displayed a higher protrusion density of PRG3 positive neurons. These data demonstrate that PRG3 operates on neural shape and filopodia in vivo (Fig. 2 P). PRG3 C-terminal domain promotes neurite growth and branching PRG3 and PRG5 are both the smallest PRG family members with the shortest intracellular c-terminal (CT) domains of 46 and 47 amino acids, respectively (Fig. S 1 A). We hypothesized, that the unique CT domain of PRG3 which is absent in other PRG family members, might be causal for the enhanced differentiated neuronal phenotype. To investigate this further, we generated a PRG3 construct lacking the CT domain (PRG3CT) and another mutant construct with solely the CT domain (PRG3CT). Both constructs eliminated the effect induced by wild-type PRG3 (Fig. 3 A). We found the overexpressed CT domain primarily in the cytosol, whereas in the wild-type situation the CT domain is located at the plasma membrane. Hence, we fused the myristoylation consensus sequence of the YES-kinase ARL11 together with the PRG3CT sequence to generate a membrane-targeted PRG3CT fusion protein (PRG3CTMEM, Fig. 3 C). The PRG3 phenotype was recovered when PRG3CTMEM was Kynurenic acid expressed with respect to number of trunk branches, non-trunk branches and branch ends (Fig. 3 D, E). Neurite length measurements of GFP, PRG3CTMEM and PRG3 revealed PRG3CTMEM neurites grew significant longer compared to PRG3CT mutants and controls (Fig. 3 D, E). Thus, the subcellular localization and final position of PRG3CT is significantly linked to the functional neurite and filopodia growth promotion activity. Open in a separate window Figure 3 Plasma membrane localization of the PRG3 C-terminal domain is essential for axon outgrowth(A) PRG3 overexpression induces neurite Kynurenic acid outgrowth (PRG3, arrows) in comparison to controls (GFP). A truncated PRG3 construct of its C-terminal domain (PRG3CT) and a truncated PRG3 construct consisting of.