TY - JOUR
T1 - Role of the Wnt receptor Frizzled-1 in presynaptic differentiation and function
AU - Varela-Nallar, Lorena
AU - Grabowski, Catalina P.
AU - Alfaro, Ivn E.
AU - Alvarez, Alejandra R.
AU - Inestrosa, Nibaldo C.
N1 - Funding Information:
We would like to thank to Dr Randall Moon (University of Washington, Seattle, WA) for generously providing the Fz1-GFP and Fz1-myc constructs. This work was supported by FONDAP-Biomedicine N° 13980001, the Millennium Institute for Fundamental and Applied Biology (MIFAB), Basal Center of Excellence in Aging and Regeneration (CONICYT-PFB12/ 2007) to NCI, FONDECYT N°1080221, a FONDECYT Postdoctoral Fellowship to LV-N (N° 3070017) and a Predoctoral Fellowship form CONI-CYT to IEA.
Funding Information:
Hippocampal neurons were seeded onto poly-L-lysine-coated coverslips in 24-well culture plates at a density of 2.5 × 104 cells per well. Cells were rinsed twice in ice-cold phosphate-buffered saline (PBS) and fixed with a freshly prepared solution of 4% paraformaldehyde in PBS for 20 minutes and permeabilized for 5 minutes with 0.2% Triton X-100 in PBS. After several rinses in ice-cold PBS, cells were incubated in 0.2% gelatin in PBS (blocking solution) for 30 minutes at room temperature, followed by an overnight incubation at 4°C with primary antibodies. Cells were extensively washed with PBS and then incubated with Alexa-conjugated secondary antibodies (Molecular Probes, Carlsbad, CA, USA) for 30 minutes at 37°C. Coverslips were mounted in mounting medium and analyzed on a Zeiss LSM 5 Pascal confocal microscope. Primary antibodies used were goat anti-Fz1 (R&D Systems), rabbit anti-Synapsin I (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), rabbit anti-VAMP (Santa Cruz Biotechnology Inc.), goat anti-Synapsin I (Santa Cruz Biotechnology Inc.), goat anti-SYP (Santa Cruz Biotechnology Inc.), monoclonal anti-Bassoon antibody (Assay designs, Ann Arbor, MI, USA), and monoclonal anti-MAP1BP antibody (Sternberger Monoclonals, Baltimore, MD, USABalti-more, MDBaltimore). The monoclonal antibodies anti-PSD-95 and anti-VGlut1 were developed by and obtained from the UC Davis/NIH NeuroMab Facility, supported by NIH grant U24NS050606 and maintained by the Department of Neurobiology, Physiology and Behavior, College of Biological Sciences, University of California, Davis, CA, USA.
PY - 2009
Y1 - 2009
N2 - Background. The Wnt signaling pathway regulates several fundamental developmental processes and recently has been shown to be involved in different aspects of synaptic differentiation and plasticity. Some Wnt signaling components are localized at central synapses, and it is thus possible that this pathway could be activated at the synapse. Results. We examined the distribution of the Wnt receptor Frizzled-1 in cultured hippocampal neurons and determined that this receptor is located at synaptic contacts co-localizing with presynaptic proteins. Frizzled-1 was found in functional synapses detected with FM1-43 staining and in synaptic terminals from adult rat brain. Interestingly, overexpression of Frizzled-1 increased the number of clusters of Bassoon, a component of the active zone, while treatment with the extracellular cysteine-rich domain (CRD) of Frizzled-1 decreased Bassoon clustering, suggesting a role for this receptor in presynaptic differentiation. Consistent with this, treatment with the Frizzled-1 ligand Wnt-3a induced presynaptic protein clustering and increased functional presynaptic recycling sites, and these effects were prevented by co-treatment with the CRD of Frizzled-1. Moreover, in synaptically mature neurons Wnt-3a was able to modulate the kinetics of neurotransmitter release. Conclusion. Our results indicate that the activation of the Wnt pathway through Frizzled-1 occurs at the presynaptic level, and suggest that the synaptic effects of the Wnt signaling pathway could be modulated by local activation through synaptic Frizzled receptors.
AB - Background. The Wnt signaling pathway regulates several fundamental developmental processes and recently has been shown to be involved in different aspects of synaptic differentiation and plasticity. Some Wnt signaling components are localized at central synapses, and it is thus possible that this pathway could be activated at the synapse. Results. We examined the distribution of the Wnt receptor Frizzled-1 in cultured hippocampal neurons and determined that this receptor is located at synaptic contacts co-localizing with presynaptic proteins. Frizzled-1 was found in functional synapses detected with FM1-43 staining and in synaptic terminals from adult rat brain. Interestingly, overexpression of Frizzled-1 increased the number of clusters of Bassoon, a component of the active zone, while treatment with the extracellular cysteine-rich domain (CRD) of Frizzled-1 decreased Bassoon clustering, suggesting a role for this receptor in presynaptic differentiation. Consistent with this, treatment with the Frizzled-1 ligand Wnt-3a induced presynaptic protein clustering and increased functional presynaptic recycling sites, and these effects were prevented by co-treatment with the CRD of Frizzled-1. Moreover, in synaptically mature neurons Wnt-3a was able to modulate the kinetics of neurotransmitter release. Conclusion. Our results indicate that the activation of the Wnt pathway through Frizzled-1 occurs at the presynaptic level, and suggest that the synaptic effects of the Wnt signaling pathway could be modulated by local activation through synaptic Frizzled receptors.
UR - http://www.scopus.com/inward/record.url?scp=73249133587&partnerID=8YFLogxK
U2 - 10.1186/1749-8104-4-41
DO - 10.1186/1749-8104-4-41
M3 - Article
C2 - 19883499
AN - SCOPUS:73249133587
SN - 1749-8104
VL - 4
JO - Neural Development
JF - Neural Development
IS - 1
M1 - 41
ER -