Regulation of Neuronal Morphogenesis and Positioning by Ubiquitin-Specific Proteases in the Cerebellum

Ubiquitin signaling mechanisms play fundamental roles in the cell-intrinsic control of neuronal morphogenesis and connectivity in the brain. However, whereas specific ubiquitin ligases have been implicated in key steps of neural circuit assembly, the roles of ubiquitin-specific proteases (USPs) in the establishment of neuronal connectivity have remained unexplored. Here, we report a comprehensive analysis of USP family members in granule neuron morphogenesis and positioning in the rodent cerebellum. We identify a set of 32 USPs that are expressed in granule neurons. We also characterize the subcellular localization of the 32 USPs in granule neurons using a library of expression plasmids encoding GFP-USPs. In RNAi screens of the 32 neuronally expressed USPs, we uncover novel functions for USP1, USP4, and USP20 in the morphogenesis of granule neuron dendrites and axons and we identify a requirement for USP30 and USP33 in granule neuron migration in the rodent cerebellar cortex in vivo. These studies reveal that specific USPs with distinct spatial localizations harbor key functions in the control of neuronal morphogenesis and positioning in the mammalian cerebellum, with important implications for our understanding of the cell-intrinsic mechanisms that govern neural circuit assembly in the brain

The ubiquitin receptor S5a/Rpn10 links centrosomal proteasomes with dendrite development in the mammalian brain

SummaryProteasomes drive the selective degradation of protein substrates with covalently linked ubiquitin chains in eukaryotes. Although proteasomes are distributed throughout the cell, specific biological functions of the proteasome in distinct subcellular locales remain largely unknown. We report that proteasomes localized at the centrosome regulate the degradation of local ubiquitin conjugates in mammalian neurons. We find that the proteasomal subunit S5a/Rpn10, a ubiquitin receptor that selects substrates for degradation, is essential for proteasomal activity at centrosomes in neurons and thereby promotes the elaboration of dendrite arbors in the rodent brain in vivo. We also find that the helix-loop-helix protein Id1 disrupts the interaction of S5a/Rpn10 with the proteasomal lid and thereby inhibits centrosomal proteasome activity and dendrite elaboration in neurons. Together, our findings define a function for a specific pool of proteasomes at the neuronal centrosome and identify a biological function for S5a/Rpn10 in the mammalian brain

Expression and subcellular locale of USPs in granule neurons.

<p><b>A</b>. Analysis of USP gene expression in primary granule neurons. Neurons were isolated from P6 rat pups. After one or five days in vitro (DIV), total neuronal mRNA was isolated, reverse transcribed to cDNA, and analyzed by quantitative RT-PCR using <i>gapdh</i> as a control gene. Asterisks indicate significant changes in expression between DIV1 and DIV5 (P<0.05, t-test). <b>B</b>. Subcellular localization of neuronally expressed USPs in primary granule neurons. Cells isolated from P6 rat pups were cultured in vitro and at DIV2 transfected with expression plasmids encoding the indicated GFP-tagged USP. To visualize the entirety of the neuron, cells were cotransfected with plasmids encoding mCherry. Two days after transfection, cells were fixed and subjected to immunocytochemical analyses using the GFP and dsRED antibodies. Staining with the DNA dye bisbenzimide (Hoechst) was used to visualize the cell nucleus. An enlarged view of the localization of each USP in neurons is shown in the indicated panel. Bar = 10μm. <b>C</b>. Subcellular localization of neuronally expressed USPs in 293T cells. <b>D</b>. Summary of the subcellular localization of USPs in neurons. Abbreviations: Cent—Centrosome; Cyt—cytoplasm; ER—endoplasmic reticulum; Mito—mitochondria; Mt—microtubules; Nuc—nucleus; Nos—nucleolus; Ves—vesicles.</p

Regulation of granule neuron dendrite morphogenesis by USP1 and USP4.

<p><b>A</b>. Cerebellar granule neurons were prepared as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117076#pone.0117076.g002" target="_blank">Fig. 2B</a>, transfected at DIV2 and analyzed at DIV5. Total dendrite length in granule neurons transfected with each RNAi plasmid was normalized to the total dendrite length of control U6 transfected neurons. Asterisks indicate statistically significant effects (P<0.005, t-test). <b>B, C</b>. USP4 knockdown stimulates dendrite growth. Granule neurons were transfected as in panel A, and analyzed for dendrite length. Arrows and arrowheads indicate dendrites and axons, respectively. USP4 knockdown significantly increased total dendrite length (<i>P</i><0.001, ANOVA, n = 3; 209 cells counted). <b>D-E</b>. USP1 knockdown simplifies granule neuron dendrite arbors. Neurons transfected with GFP and USP1 RNAi or U6 control plasmid were subjected to immunocytochemistry using the GFP and the MAP2 antibodies. Arrows and arrowheads indicate dendrites and axons, respectively. <b>F</b>. Lysates of 293T cells transfected with GFP-USP1 together with U6 or the indicated USP1 RNAi plasmid were immunoblotted with the indicated antibodies. <b>G-K</b>. Granule neurons were transfected as in A with the USP1 RNAi plasmid or control U6 plasmid. USP1 knockdown significantly reduced primary and secondary/tertiary dendrite numbers and increased length of longest dendrite (<i>P</i><0.001 for 3H; <i>P</i><0.001 for 3I; <i>P</i><0.005 for 3J, ANOVA, n = 3; 206 cells), but had little or no effect on total dendrite length. Arrows and arrowheads indicate dendrites and axons, respectively. <b>L-N</b>. USP1 and USP4 RNAi regulate dendrite development <i>in vivo</i>. P4 rat pups were injected with plasmids encoding GFP together with the control U6 RNAi plasmid or the indicated RNAi plasmid targeting USP1 or USP4. Dendrite length and primary dendrite number of transfected cells were determined following immunohistochemistry of coronal sections of GFP-positive cerebella. For panel 3M, 81 cells and for panel 3N, 131 cells were counted. USP4 knockdown significantly increased total granule neuron dendrite length (<i>P</i><0.005, <i>t</i>-test) and reduced primary granule neuron dendrite number (<i>P</i><0.001, <i>t</i>-test) <i>in vivo</i>. The size of all scale bars is 20μm. Arrows and arrowheads indicate dendrites and axons, respectively.</p

Inhibition of DNA Binding by Differential Sumoylation of Heat Shock Factors

Covalent modification of proteins by the small ubiquitin-related modifier SUMO regulates diverse biological functions. Sumoylation usually requires a consensus tetrapeptide, through which the binding of the SUMO-conjugating enzyme Ubc9 to the target protein is directed. However, additional specificity determinants are in many cases required. To gain insights into SUMO substrate selection, we have utilized the differential sumoylation of highly similar loop structures within the DNA-binding domains of heat shock transcription factor 1 (HSF1) and HSF2. Site-specific mutagenesis in combination with molecular modeling revealed that the sumoylation specificity is determined by several amino acids near the consensus site, which are likely to present the SUMO consensus motif to Ubc9. Importantly, we also demonstrate that sumoylation of the HSF2 loop impedes HSF2 DNA-binding activity, without affecting its oligomerization. Hence, SUMO modification of the HSF2 loop contributes to HSF-specific regulation of DNA binding and broadens the concept of sumoylation in the negative regulation of gene expression