Targeted disruption of neuronal 19S proteasome subunits induces the formation of ubiquitinated inclusions in the absence of cell death

Proteasome-mediated proteolysis is a major protein degradation mechanism in cells and its dysfunction has been implicated in the pathogenesis of several neurodegenerative diseases, each with the common features of neuronal death and formation of ubiquitinated inclusions found within neurites, the cell body, or nucleus. Previous models of proteasome dysfunction have employed pharmacological inhibition of the catalytic subunits of the 20S proteasome core, or the genetic manipulation of specific subunits resulting in altered proteasome assembly. In this study, we report the use of dominant negative subunits of the 19S regulatory proteasome complex that mediate the recognition of ubiquitinated substrates as well as the removal of the poly-ubiquitin chain. Interestingly, while each mutant subunit-induced inclusion formation, like that seen with pharmacological inhibition of the 20S proteasome, none was able to induce apoptotic death, or trigger activation of macroautophagy, in either dopaminergic cell lines or primary cortical neurons. This finding highlights the dissociation between the mechanisms of neuronal inclusion formation and the induction of cell death, and represents a novel cellular model for Lewy body-like inclusion formation in neurons.     

Host cystathionine-γ lyase derived hydrogen sulfide protects against Pseudomonas aeruginosa sepsis

Hydrogen sulfide (H₂S) has recently been recognized as a novel gaseous transmitter with several anti-inflammatory properties. The role of host- derived H₂S in infections by Pseudomonas aeruginosa was investigated in clinical and mouse models. H₂S concentrations and survival was assessed in septic patients with lung infection. Animal experiments using a model of severe systemic multidrug-resistant P. aeruginosa infection were performed using mice with a constitutive knock-out of cystathionine-γ lyase (Cse) gene (Cse^-/-) and wild-type mice with a physiological expression (Cse^+/+). Experiments were repeated in mice after a) treatment with cyclophosphamide; b) bone marrow transplantation (BMT) from a Cse^+/+ donor; c) treatment with H₂S synthesis inhibitor aminooxyacetic acid (ΑΟΑΑ) or propargylglycine (PAG) and d) H₂S donor sodium thiosulfate (STS) or GYY3147. Bacterial loads and myeloperoxidase activity were measured in tissue samples. The expression of quorum sensing genes (QS) was determined in vivo and in vitro. Cytokine concentration was measured in serum and incubated splenocytes. Patients survivors at day 28 had significantly higher serum H₂S compared to non-survivors. A cut- off point of 5.3 μΜ discriminated survivors with sensitivity 92.3%. Mortality after 28 days was 30.9% and 93.7% in patients with H₂S higher and less than 5.3 μΜ (p = 7 x 10⁻⁶). In mice expression of Cse and application of STS afforded protection against infection with multidrug-resistant P. aeruginosa. Cyclophosphamide pretreatment eliminated the survival benefit of Cse^+/+ mice, whereas BMT increased the survival of Cse^-/- mice. Cse^-/- mice had increased pathogen loads compared to Cse^+/+ mice. Phagocytic activity of leukocytes from Cse^-/- mice was reduced but was restored after H₂S supplementation. An H₂S dependent down- regulation of quorum sensing genes of P.aeruginosa could be demonstrated in vivo and in vitro. Endogenous H₂S is a potential independent parameter correlating with the outcome of P. aeruginosa. H₂S provides resistance to infection by MDR bacterial pathogens.