Regulation of Cdh1-APC Function in Axon Growth by Cdh1 Phosphorylation

The ubiquitin ligase Cdh1–anaphase promoting complex (Cdh1–APC) plays a key role in the control of axonal morphogenesis in the mammalian brain, but the mechanisms that regulate neuronal Cdh1–APC function remain incompletely understood. Here, we have characterized the effect of phosphorylation of Cdh1 at cyclin-dependent kinase (Cdk) sites on Cdh1–APC function in neurons. We replaced nine conserved sites of Cdk-induced Cdh1 phosphorylation with alanine (9A) or aspartate (9D) to mimic hypo- or hyper-phosphorylation, respectively. We found that the 9A mutation triggered the proteasome-dependent degradation of Cdh1, and conversely the 9D mutation stabilized Cdh1 in neuronal cells. However, the phosphomimic 9D Cdh1 protein failed to associate with the APC core protein Cdc27. In addition, whereas wild-type and 9A Cdh1 predominantly localized to the nucleus, the 9D Cdh1 protein accumulated in the cytoplasm in neurons. Importantly, in contrast to wild-type and 9A Cdh1, the 9D Cdh1 mutant failed to inhibit axon growth in primary cerebellar granule neurons. Collectively, our results suggest that phosphorylation of neuronal Cdh1 at Cdk sites triggers the stabilization of an inactive form of Cdh1 that accumulates in the cytoplasm, leading to the inhibition of Cdh1–APC function in the control of axon growth. Thus, phosphorylation of Cdh1 may represent a critical mechanism regulating Cdh1–APC function in the nervous system

The proteoglycan NG2 is complexed with alpha-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid (AMPA) receptors by the PDZ glutamate receptor interaction protein (GRIP) in glial progenitor cells - Implications for glial-neuronal signaling

The proteoglycan NG2 is expressed by immature glial cells in the developing and adult central nervous system. Using the COOH-terminal region of NG2 as bait in a yeast two-hybrid screen, we identified the glutamate receptor interaction protein GRIP1, a multi-PDZ domain protein, as an interacting partner. NG2 exhibits a PDZ binding motif at the extreme COOH terminus which binds to the seventh PDZ domain of GRIPl. In addition to the published expression in neurons, GRIPl is expressed by immature glial cells. GRIPl is known to bind to the GluRB subunit of the AMPA glutamate receptor expressed by subpopulations of neurons and immature glial cells. In cultures of primary oligodendrocytes, cells coexpress GluRB and NG2. A complex of NG2, GRIP1, and GluRB can be precipitated from transfected mammalian cells and from cultures of primary oligodendrocytes. Furthermore, NG2 and GRIP can be coprecipitated from developing brain tissue. These data suggest that GRIPl acts as a scaffolding molecule clustering NG2 and AMPA receptors in immature glia. In view of the presence of synaptic contacts between neurons and NG2-positive glial cells in the hippocampus and the close association of NG2-expressing glial cells with axons, we suggest a role for the NG2-AMPA receptor complex in glial-neuronal recognition and signaling

The Centrosomal E3 Ubiquitin Ligase FBXO31-SCF Regulates Neuronal Morphogenesis and Migration

<div><p>Neuronal development requires proper migration, polarization and establishment of axons and dendrites. Growing evidence identifies the ubiquitin proteasome system (UPS) with its numerous components as an important regulator of various aspects of neuronal development. F-box proteins are interchangeable subunits of the Cullin-1 based E3 ubiquitin ligase, but only a few family members have been studied. Here, we report that the centrosomal E3 ligase FBXO31-SCF (Skp1/Cullin-1/F-box protein) regulates neuronal morphogenesis and axonal identity. In addition, we identified the polarity protein Par6c as a novel interaction partner and substrate targeted for proteasomal degradation in the control of axon but not dendrite growth. Finally, we ascribe a role for FBXO31 in dendrite growth and neuronal migration in the developing cerebellar cortex. Taken together, we uncovered the centrosomal E3 ligase FBXO31-SCF as a novel regulator of neuronal development.</p> </div

FBXO31 regulates dendrite growth and neuronal migration in the developing cerebellum. A.

<p>HEK 293T cell lysates transfected with mycFBXO31 along with control or bi-cistronic FBXO31 RNAi #1/CMV-GFP plasmids were probed with α-myc antibody. 14-3-3ß served as a loading control. <b>B.</b> Snapshots of 3D-reconstructed cerebellar granule neurons from rat pups electroporated with control plasmid or with FBXO31 RNAi #1/CMV-GFP bi-cistronic plasmid at P4 and analyzed at P9. Arrows indicate dendrites and arrowheads indicate axons of granule neurons. Scale bar equals 50 µm. <b>C.</b> Histogram showing dendrite length measurements for control or FBXO31 knockdown neurons. A total of 84 neurons were analyzed for dendrite length measurements (n = 3, mean±SEM, unpaired t-test, ***p<0.001). <b>D.</b> Coronal sections of rat pup cerebellum electroporated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057530#pone-0057530-g004" target="_blank">Figure 4B</a>. IGL = internal granular layer, ML = molecular layer, EGL = external granular layer. Scale bar equals 50 µm. <b>E.</b> Histogram showing percentage of migrated neurons in EGL, ML or IGL. A total of 3637 neurons were analyzed. (n = 3, mean±SEM, two-way ANOVA ***p<0.001, n.s. = not significant). <b>F.</b> Histogram showing distance of granule neuron cell bodies from the pial surface. A total of 681 neurons were analyzed. (n = 3, mean±SEM, two-way ANOVA ***p<0.001).</p

FBXO31-SCF targets Par6c to proteasomal degradation. A.

<p>Transfected HEK 293T cells lysates were subjected to immunoprecipitation with α-Flag antibody and immunoblotted with α-myc antibody. <b>B.</b> Transfected HEK 293T cells lysates were subjected to immunoprecipitation with α-myc antibody and immunoblotted with α-Flag antibody. <b>C.</b> Schematic showing various Par6c deletion mutants and their interaction with FBXO31. <b>D.</b> Granule neurons were transfected with Par6c plasmid at DIV0 and treated with DMSO or lactacystin for 10 hours prior to lysis at DIV 3. Lysates were immunoblotted with α-myc antibody. 14-3-3ß served as a loading control. <b>E.</b> and <b>F.</b> HEK 293T cells (E) and granule neurons (F) were transfected with mycPar6c plasmid together with FBXO31 RNAi plasmids or respective control vectors as indicated. Cell lysates were immunoblotted with α-myc and α-Flag antibodies. 14-3-3ß served as a loading control. <b>G.</b> Cerebellar granule neurons were transfected with mycPar6c plasmid together with FBXO31 RNAi #1 plasmid or respective control vector at DIV 2. Centrosomal purification was performed at DIV 6 using sucrose density gradient centrifugation. The fractions were probed with α-myc antibody. γ-tubulin served as a positive control for centrosomal protein. The histogram shows Par6c levels relative to γ-tubulin at the centrosome (N = 3, mean±SEM, unpaired t-test, *p<0.05). <b>H.</b> HEK 293T cells were co-transfected with mycPar6c and GFP-FBXO31 WT or ΔF plasmids together with respective control vectors. Cell lysates were subjected to immunoprecipitation with α-myc antibody and immunoblotted with α-ubiquitin antibody. <b>I.</b> HEK 293T cells were co-transfected with mycPar6c and GFP-FBXO31 WT plasmids together with respective control vectors. Cell lysates were subjected to immunoprecipitation with α-myc antibody and immunoblotted with K48-specific α-ubiquitin antibody. <b>J.</b> HEK 293T cells were co-transfected with mycPar6c and GFP-FBXO31 WT plasmids together with respective control vectors. Cell lysates were subjected to immunoprecipitation with anti-myc antibody and immunoblotted with K63-specific anti-ubiquitin antibody.</p