Pαx6 Expression in Postmitotic Neurons Mediates the Growth of Axons in Response to SFRP1

During development, the mechanisms that specify neuronal subclasses are coupled to those that determine their axonal response to guidance cues. Pax6 is a homedomain transcription factor required for the specification of a variety of neural precursors. After cell cycle exit, Pax6 expression is often shut down in the precursor progeny and most postmitotic neurons no longer express detectable levels of the protein. There are however exceptions and high Pax6 protein levels are found, for example, in postmitotic retinal ganglion cells (RGCs), dopaminergic neurons of the olfactory bulb and the limbic system in the telencephalon. The function of Pax6 in these differentiating neurons remains mostly elusive. Here, we demonstrate that Pax6 mediates the response of growing axons to SFRP1, a secreted molecule expressed in several Pax6-positive forebrain territories. Forced expression of Pax6 in cultured postmitotic cortical neurons, which do not normally express Pax6, was sufficient to increment axonal length. Growth was blocked by the addition of anti-SFRP1 antibodies, whereas exogenously added SFRP1 increased axonal growth of Pax6-transfected neurons but not that of control or untransfected cortical neurons. In the reverse scenario, shRNA-mediated knock-down of Pax6 in mouse retinal explants specifically abolished RGCs axonal growth induced by SFRP1, but had no effect on RGCs differentiation and it did not modify the effect of Shh or Netrin on axon growth. Taken together these results demonstrate that expression of Pax6 is necessary and sufficient to render postmitotic neurons competent to respond to SFRP1. These results reveal a novel and unexpected function of Pax6 in postmitotic neurons and situate Pax6 and SFRP1 as pair regulators of axonal connectivity

Enrichment of conserved synaptic activity-responsive element in neuronal genes predicts a coordinated response of MEF2, CREB and SRF.

A unique synaptic activity-responsive element (SARE) sequence, composed of the consensus binding sites for SRF, MEF2 and CREB, is necessary for control of transcriptional upregulation of the Arc gene in response to synaptic activity. We hypothesize that this sequence is a broad mechanism that regulates gene expression in response to synaptic activation and during plasticity; and that analysis of SARE-containing genes could identify molecular mechanisms involved in brain disorders. To search for conserved SARE sequences in the mammalian genome, we used the SynoR in silico tool, and found the SARE cluster predominantly in the regulatory regions of genes expressed specifically in the nervous system; most were related to neural development and homeostatic maintenance. Two of these SARE sequences were tested in luciferase assays and proved to promote transcription in response to neuronal activation. Supporting the predictive capacity of our candidate list, up-regulation of several SARE containing genes in response to neuronal activity was validated using external data and also experimentally using primary cortical neurons and quantitative real time RT-PCR. The list of SARE-containing genes includes several linked to mental retardation and cognitive disorders, and is significantly enriched in genes that encode mRNA targeted by FMRP (fragile X mental retardation protein). Our study thus supports the idea that SARE sequences are relevant transcriptional regulatory elements that participate in plasticity. In addition, it offers a comprehensive view of how activity-responsive transcription factors coordinate their actions and increase the selectivity of their targets. Our data suggest that analysis of SARE-containing genes will reveal yet-undescribed pathways of synaptic plasticity and additional candidate genes disrupted in mental disease

Pαx6 Expression in Postmitotic Neurons Mediates the Growth of Axons in Response to SFRP1

During development, the mechanisms that specify neuronal subclasses are coupled to those that determine their axonal response to guidance cues. Pax6 is a homedomain transcription factor required for the specification of a variety of neural precursors. After cell cycle exit, Pax6 expression is often shut down in the precursor progeny and most postmitotic neurons no longer express detectable levels of the protein. There are however exceptions and high Pax6 protein levels are found, for example, in postmitotic retinal ganglion cells (RGCs), dopaminergic neurons of the olfactory bulb and the limbic system in the telencephalon. The function of Pax6 in these differentiating neurons remains mostly elusive. Here, we demonstrate that Pax6 mediates the response of growing axons to SFRP1, a secreted molecule expressed in several Pax6-positive forebrain territories. Forced expression of Pax6 in cultured postmitotic cortical neurons, which do not normally express Pax6, was sufficient to increment axonal length. Growth was blocked by the addition of anti-SFRP1 antibodies, whereas exogenously added SFRP1 increased axonal growth of Pax6-transfected neurons but not that of control or untransfected cortical neurons. In the reverse scenario, shRNA-mediated knock-down of Pax6 in mouse retinal explants specifically abolished RGCs axonal growth induced by SFRP1, but had no effect on RGCs differentiation and it did not modify the effect of Shh or Netrin on axon growth. Taken together these results demonstrate that expression of Pax6 is necessary and sufficient to render postmitotic neurons competent to respond to SFRP1. These results reveal a novel and unexpected function of Pax6 in postmitotic neurons and situate Pax6 and SFRP1 as pair regulators of axonal connectivity.Depto. de Bioquímica y Biología MolecularFac. de MedicinaTRUEpu

Classification of SARE-containing genes at GO indicated significant enrichment of processes related to the nervous system.

<p>The analysis yielded 112 significantly enriched GO biological processes. The table shows the 21 categories with significantly high fold enrichment that are related to nervous system development and maintenance. Rank indicates the position within the list of the total 112 when ordered from higher to lower enrichment. Neural functions are ranked in the higher positions.</p

The number of SARE containing genes is significantly enriched on genes up-regulated upon bicuculline triggering of neuronal activity.

<p>Comparative analysis of the list of SARE containing genes with three independent studies reporting mRNA changes in gene expression in the accessory olfactory bulb <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053848#pone.0053848-Upadhya1" target="_blank">[21]</a>; cortical cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053848#pone.0053848-Liu1" target="_blank">[22]</a>; and hippocampal neuronal cultures <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053848#pone.0053848-Benito2" target="_blank">[23]</a>. The table shows the lists of the SARE genes that were found to be up-regulated (upper part), or down-modulated (lower part) in each study. P-values for random coincidence are shown. Not significance coincidence was observed when compared to genes that are down-regulated.</p

Up-regulation of the mRNA of SARE containing genes and promoter activation in response to neuronal activity. A)

<p><i>Upregulation of SARE identified candidates in primary cortical neurons.</i> Primary neurons from E18 cortex were cultivated for 16 days before triggering neuronal activation with 4AP/bicuculline. RNA was obtained and transcript expression of SARE containing genes analyzed by quantitative real time RT-PCR (Q-PCR). Expression of each gene is normalized to the expression in control cells. Arc up-regulation demonstrated efficient neuronal activation. * indicates p<0,5 **<0,1, ***<0,05 (t-Student test). <b>B). </b><i>Novel SARE sequences activate transcription in response to neuronal activity.</i> Mouse genomic sequence containing SARE regulatory regions (see below) corresponding to those identified for the <i>Cux1</i> and <i>Cux2</i> genes were cloned into the pGL4.23 luciferase vector (Promega). Neurons obtained from E18,5 cortex were co-transfected with the indicated firefly luciferase reporter construct and internal control Renilla luciferase plasmid at a ratio of 4∶1. Neuronal activity was trigered with 4AP/bicuculline before measuring transcription of the reporters. Relative expression of each reporter constructs was determined by normalizing the activity of each reporter to its activity on TTX treated neurons. Data represent mean and standar deviation of results obtained in three different experiments. * indicates p<0,01, **<0,001 (t-Student test).</p

FMRP targets containing SARE sequence genes.

<p>The list of genes identified as containing SARE sequences was compared to a list of 842 reliable FMRP targets. This resulted in an overlap of 70 genes common to both lists. This represent a significant enrichment of p = 4.39096.93e-13, far from the expected random distribution of coincidences between the genome and the mouse nervous system transcriptome (see Experimental Procedures).</p

SARE regulation affects several aspects of neuron function.

<p>Examples of genes containing SARE sequences according to function, extracted from the list of 827 genes identified.</p

SARE sequences are found in intergenic and intronic genetic regions.

<p>Scheme showing consensus TFBS for SARE sequence and the SARE sequenced found in the promoter of <i>SOX14</i> (top). For the SynoR search, random relative position of the three TFBS was permitted. Table shows the number and percentages of SARE sequences classified according to their position within the genes and the diagram represents the distribution of each category.</p

Knock-down of <i>Pax6</i> blocks SFRP1 stimulated growth of retinal axons.

<p><b>a</b>) Low magnification images (a) and confocal micrographs (b) of explants from control (a) or electroporated retinal explants (b) seeded onto laminin coated coverslip and cultured in the absence or presence of recombinant SFRP1 as indicated in the panels. Explants in (a) were stained with β-tubulin III whereas those in (b) were co-electroporated with CAG-GFP and shRNA control or shRNA targeting <i>Pax6</i> and axons were visualized by GFP expression. Graph shows quantification of the proportion of total axon longer than 900 (a) or 200 (b) µm. Note that knocking-down <i>Pax6</i> inhibits the axonal response stimulated by SFRP1. Bar indicates 300 (a) and 150 µm (b). Data are expressed as the mean ± SD. (*) p<0.05 comparing stimulated populations; (**) p<0.01 and (***) p<0.001 compared with control.</p