The lipid kinase PIK3C3 (also called Vps34) regulates both the endosomal and autophagic pathways. However, the effect of inactivating PIK3C3 on neuronal endosomal versus autophagic processes in vivo has not been studied. We generated mice in which Pik3c3 was conditionally deleted in differentiated sensory neurons. Within a few days after Pik3c3 deletion, mutant largediameter myelinated neurons accumulated numerous enlarged vacuoles and ubiquitin-positive aggregates and underwent rapid degeneration. By contrast, Pik3c3-deficient small-diameter unmyelinated neurons accumulated excessive numbers of lysosome-like organelles and degenerated more slowly. These differential degenerative phenotypes are unlikely caused by a disruption in the autophagy pathway, because inhibiting autophagy alone by conditional deletion of Atg7 results in a completely distinct phenotype in all sensory neurons (i.e., formation of very large intracellular inclusion bodies and slow degeneration over a period of several months). More surprisingly, a noncanonical PIK3C3-independent LC3-positive autophagosome formation pathway was activated in Pik3c3-deficient small-diameter neurons. Analyses of Pik3c3/Atg7 double mutant neurons revealed that this unconventional initiation pathway still depends on ATG7. Our studies represent in vivo characterization of PIK3C3 functions in mammals and provide insights into the complexity of neuronal endo-lysosomal and autophagic pathways. P IK3C3 (also known as Vps34) is a class III phosphatidylinositol-3-kinase that specifically catalyzes the formation of phosphatidylinositol-3-phosphate (PI3P) (1). Studies in invertebrate organisms as well as in nonneuronal cells showed that PIK3C3/Vps34 regulates multiple aspects of both the endocytic/ endosomal and autophagic pathways (2-5). In yeast, there are two distinct Vps34 complexes: complex I (Vps34, Vps15, Atg6, and Atg14) is involved in autophagy, and complex II (Vps34, Vps15, Atg6, and Vps38) functions in the vacuolar proteinsorting process (6). In mammals, homologs of Vps15 and Atg6 are p150 and Beclin1, respectively (7), and evidence exists for Beclin1-independent functions of PIK3C3 in the endocytic pathways (8). A mammalian homolog of complex I (PIK3C3, p150, Beclin1, and Atg14L) activates autophagy, and a homolog of complex II (PIK3C3, p150, Beclin1, and UVRAG/Vps38) regulates trafficking at late endosomes (7). Interestingly, Bif-1 and Rubicon can interact with complex II to promote autophagy (9, 10). The chemical inhibitor of PIK3C3 (3-MA or wortmanin) has been frequently used as an inhibitor for autophagy in numerous studies, including those studying autophagy in neurons (11)(12)(13)(14). However, because of the lack of genetic studies on Pik3c3 in mammals, it is not clear how inactivating PIK3C3 in neurons in vivo differentially affects endosomal versus autophagic processes.Neurons are highly susceptible to disruptions in both endocytic and autophagic pathways. Genetic mutations in ubiquitously expressed proteins regulating the endocytic (15,16) Lowe (23,...

Small ubiquitin-like modifier (SUMO1-3) conjugation plays a critical role in embryogenesis. Embryos deficient in the SUMO-conjugating enzyme Ubc9 die at the early postimplantation stage. ;Sumo3 +/À with Sumo3 À/À mice, and these rare mice were considerably smaller than littermates of the other genotypes. Thus, our findings suggest that expression levels and not functional differences between SUMO2 and SUMO3 are critical for normal embryogenesis.

Background and Purpose SUMO conjugation is a post-translational modification associated with many human diseases. Characterization of the SUMO-modified proteome is pivotal to defining the mechanistic link between SUMO conjugation and such diseases. This is particularly evident for SUMO2/3 conjugation, which is massively activated after brain ischemia/stroke, and is believed to be a protective response. The purpose of this study was to perform a comprehensive analysis of the SUMO3-modified proteome regulated by brain ischemia using a novel SUMO transgenic mouse. Methods To enable SUMO proteomics analysis in vivo, we generated transgenic mice conditionally expressing tagged SUMO1-3 paralogues. Transgenic mice were subjected to 10 minutes forebrain ischemia and 1 hour of reperfusion. SUMO3-conjugated proteins were enriched by anti-FLAG affinity purification and analyzed by LC-MS/MS. Results Characterization of SUMO transgenic mice demonstrated that all 3 tagged SUMO paralogues were functionally active, and expression of exogenous SUMOs did not modify the endogenous SUMOylation machinery. Proteomics analysis identified 112 putative SUMO3 substrates of which 91 candidates were more abundant in the ischemia group than the sham group. Data analysis revealed processes/pathways with putative neuroprotective functions, including glucocorticoid receptor signaling, RNA processing and SUMOylation-dependent ubiquitin conjugation. Conclusions The identified proteins/pathways modulated by SUMOylation could be the key to understanding the mechanisms linking SUMOylation to neuroprotection, and thus provide new promising targets for therapeutic interventions. The new transgenic mouse will be an invaluable platform for analyzing the SUMO-modified proteome in models of human disorders, and thereby help to mechanistically link SUMOylation to the pathological processes.

Transient cerebral ischemia dramatically activates small ubiquitin-like modifier (SUMO2/3) conjugation. In cells exposed to 6 h of transient oxygen/glucose deprivation (OGD), a model of ischemia, SUMOylation increases profoundly between 0 and 30 min following re-oxygenation. To elucidate the effect of transient OGD on SUMO conjugation of target proteins, we exposed neuroblastoma B35 cells expressing HA-SUMO3 to transient OGD and used stable isotope labeling with amino acids in cell culture (SILAC) to quantify OGD-induced changes in levels of specific SUMOylated proteins. Lysates from control and OGD-treated cells were mixed equally, and HA-tagged proteins were immunoprecipitated and analyzed by 1D-SDS-PAGE-LC-MS/MS. We identified 188 putative SUMO3-conjugated proteins, including numerous transcription factors and coregulators, and PIAS2 and PIAS4 SUMO ligases, of which 22 were increased or decreased >±2-fold. In addition to SUMO3, the levels of protein-conjugated SUMO1 and SUMO2, as well as ubiquitin, were all increased. Importantly, protein ubiquitination induced by OGD was completely blocked by gene silencing of SUMO2/3. Collectively, these results suggest several mechanisms for OGD-modulated SUMOylation, point to a number of signaling pathways that may be targets of SUMO-based signaling and recovery from ischemic stress, as well as demonstrate a tightly controlled crosstalk between the SUMO and ubiquitin conjugation pathways.

Small ubiquitin-like modifier (SUMO1, 2, 3) is a group of proteins that conjugate to lysine residues of target proteins thereby modifying their activity, stability, and subcellular localization. A large number of SUMO target proteins are transcription factors and other nuclear proteins involved in gene expression. Furthermore, SUMO conjugation plays key roles in genome stability, quality control of newly synthesized proteins, proteasomal degradation of proteins and DNA damage repair. Any marked increase in levels of SUMO-conjugated proteins is therefore expected to have a major impact on the fate of cells. We show here that SUMO conjugation is activated in human astrocytic brain tumors. Levels of both SUMO1- and SUMO2/3-conjugated proteins were markedly increased in tumor samples. The effect was least pronounced in low-grade astrocytoma (WHO Grade II) and most pronounced in glioblastoma multiforme (WHO Grade IV). We also found a marked rise in levels of Ubc9, the only SUMO conjugation enzyme identified so far. Blocking SUMO1-3 conjugation in glioblastoma cells by silencing their expression blocked DNA synthesis, cell growth and clonogenic survival of cells. It also resulted in DNA-PK-dependent phosphorylation of H2AX, indicative of DNA double-strand damage, and G2/M cell cycle arrest. Collectively, these findings highlight the pivotal role of SUMO conjugation in DNA damage repair processes and imply that the SUMO conjugation pathway could be a new target of therapeutic intervention aimed at increasing the sensitivity of glioblastomas to radio- and chemotherapy.

Heat shock protein, e.g. HSP70, can be induced in human skeletal muscle undergoing exercise training, and plays important role in adaptation to stress. This study was designed to investigate the effects of high-intensity strength training and low-intensity endurance training on the HSP70 response to exercise, bearing in mind whether HSP70 is induced in the well-trained muscle during low-intensity endurance training. Six well-trained rowers (male, aged 18 years) underwent a training program which consisted of 3 weeks high-intensity training (HIT) and 3 weeks low-intensity endurance training (ET), followed by 1 week of recovery each (R1 and R2, respectively). HSP70 (2.5 microg total protein loaded) was determined by Western blot with reference to a series of known amount of standard HSP70. HSP70 mRNA was analyzed by RT-PCR, and the relative percentage change was referred to the baseline level (before training). HSP70 increased significantly at the end of HIT (from 51 to 73 ng), decreased at the end of R1(66 ng), and remained unchanged throughout ET and R2. HSP70 mRNA increased significantly after HIT (257%) and decreased gradually afterwards (194%, 166%, and 119% for R1, ET, and R2, respectively). It can be concluded that: (1) HSP70 was induced by high-intensity training, but not by endurance training at low intensity, and (2) there was a discrepancy in terms of HSP70 regulation between the protein and mRNA levels, suggesting that posttranscriptional regulation may play a role in HSP70 expression in human skeletal muscle in response to exercise.

A recent theory suggests that endocytosis is involved in uptake and intracellular transport of electrotransfected plasmid DNA (pDNA). The goal of the current study was to understand if approaches used previously to improve endocytosis of gene delivery vectors could be applied to enhancing electrotransfection efficiency (eTE). Results from the study showed that photochemically induced endosomal escape, which could increase poly-L-lysine (PLL)-mediated gene delivery, decreased eTE. The decrease could not be blocked by treatment of cells with endonuclease inhibitors (aurintricarboxylic acid and zinc ion) or antioxidants (L-glutamine and ascorbic acid). Chemical treatment of cells with an endosomal trafficking inhibitor that blocks endosome progression, bafilomycin A1, resulted in a significant decrease in eTE. However, treatment of cells with lysosomotropic agents (chloroquine and ammonium chloride) had little effects on eTE. These data suggested that endosomes played important roles in protecting and intracellular trafficking of electrotransfected pDNA.

Ubiquitylation is a posttranslational protein modification that modulates various cellular processes of key significance, including protein degradation and DNA damage repair. In animals subjected to transient cerebral ischemia, ubiquitin-conjugated proteins accumulate in Triton-insoluble aggregates. Although this process is widely considered to modulate the fate of postischemic neurons, few attempts have been made to characterize the ubiquitin-modified proteome in these aggregates. We performed proteomics analyses to identify ubiquitylated proteins in postischemic aggregates. Mice were subjected to 10 minutes of forebrain ischemia and 4 hours of reperfusion. The hippocampi were dissected, aggregates were isolated, and trypsin-digested after spiking with GG-BSA as internal standard. K-e-GG-containing peptides were immunoprecipitated and analyzed by label-free quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. We identified 1,664 peptides to 520 proteins containing at least one K-e-GG. Sixty-six proteins were highly ubiquitylated, with 10 or more K-e-GG peptides. Based on selection criteria of greater than fivefold increase and Po0.001, 763 peptides to 272 proteins were highly enriched in postischemic aggregates. These included proteins involved in important neuronal functions and signaling pathways that are impaired after ischemia. Results of this study could serve as an important platform to uncover the mechanisms linking insoluble ubiquitin aggregates to the functions of postischemic neurons.