Microglial TNFR2 signaling regulates the inflammatory response after CNS injury in a sex-specific fashion.

Microglia, the resident immune cells of the central nervous system (CNS), play a major role in damage progression and tissue remodeling after acute CNS injury, including ischemic stroke (IS) and spinal cord injury (SCI). Understanding the molecular mechanisms regulating microglial responses to injury may thus reveal novel therapeutic targets to promote CNS repair. Here, we investigated the role of microglial tumor necrosis factor receptor 2 (TNFR2), a transmembrane receptor previously associated with pro-survival and neuroprotective responses, in shaping the neuroinflammatory environment after CNS injury. By inducing experimental IS and SCI in Cx3cr1CreER:Tnfrsf1bfl/fl mice, selectively lacking TNFR2 in microglia, and corresponding Tnfrsf1bfl/fl littermate controls, we found that ablation of microglial TNFR2 significantly reduces lesion size and pro-inflammatory cytokine levels, and favors infiltration of leukocytes after injury. Interestingly, these effects were paralleled by opposite sex-specific modifications of microglial reactivity, which was found to be limited in female TNFR2-ablated mice compared to controls, whereas it was enhanced in males. In addition, we show that TNFR2 protein levels in the cerebrospinal fluid (CSF) of human subjects affected by IS and SCI, as well as healthy donors, significantly correlate with disease stage and severity, representing a valuable tool to monitor the inflammatory response after acute CNS injury. Hence, these results advance our understanding of the mechanisms regulating microglia reactivity after acute CNS injury, aiding the development of sex- and microglia-specific, personalized neuroregenerative strategies.

Premature mortality in persons with epilepsy and schizophrenia: A population-based nationwide cohort study.

OBJECTIVE: To determine the mortality for persons with epilepsy and schizophrenia by absolute and relative measures. METHODS: This is a population-based nationwide cohort study of persons born in Denmark from 1960 to 1987 who were alive and residing in Denmark on their 25th birthday. We identified persons diagnosed with epilepsy and schizophrenia prior to their 25th birthday and followed them to death, emigration, or December 31, 2012, whichever came first. The primary outcome was overall mortality. Data were analyzed using Cox regressions. RESULTS: Persons were followed for 24 167 573 person years; the median was 15 years. The mortality rate ratio was 4.4 (95% confidence interval [CI] = 4.1-4.7) for persons with epilepsy, 6.6 (95% CI = 6.1-7.1) for persons with schizophrenia, and 12.8 (95% CI = 9.1-18.1) for persons with both disorders, compared with persons without these disorders. The estimated cumulative mortality at the age of 50 years was 3.1% (95% CI = 3.0-3.1) for persons without epilepsy and schizophrenia, 10.7% (95% CI = 9.7-11.8) for persons with epilepsy, 17.4% (95% CI = 16.0-18.8) for persons with schizophrenia, and 27.2% (95% CI = 15.7-40.1) for persons with both disorders. SIGNIFICANCE: Persons with epilepsy and schizophrenia have very high mortality; more than one in four persons with both disorders died between the age of 25 and 50 years, indicating that these patients need special clinical attention.

Txl1 and Txc1 are co-factors of the 26S proteasome in fission yeast.

The 26S proteasome is a large proteolytic particle present in the cytosol and nucleus of eukaryotic cells. Most intracellular proteins, including those affected by oxidative damage, are degraded by the proteasome. The human thioredoxin, Txnl1, is known to associate with the 26S proteasome and thereby equips proteasomes with redox capabilities. Here, we characterize the fission yeast orthologue of Txnl1, called Txl1. Txl1 associates with the 26S proteasome via its C-terminal domain. This domain is also found in the uncharacterized protein, Txc1, which was also found to interact with 26S proteasomes. A txl1 null mutant, but not a txc1 null, displayed a synthetic growth defect with cut8, encoding a protein that tethers the proteasome to the nuclear membrane. Txc1 is present throughout the cytoplasm and nucleus, whereas Txl1 co-localizes with 26S proteasomes in both wild-type cells and in cut8 mutants, indicating that Txl1 is tightly associated with 26S proteasomes, while Txc1 might be only transiently bound to the complex. Finally, we show that Txl1 is an active thioredoxin. Accordingly, Txl1 was able to reduce and mediate the degradation of an oxidized model proteasome substrate in vitro. Thus, Txl1 and Txc1 are proteasome co-factors connected with oxidative stress.

Thioredoxin Txnl1/TRP32 is a redox-active cofactor of the 26 S proteasome.

The 26 S proteasome is a large proteolytic machine, which degrades most intracellular proteins. We found that thioredoxin, Txnl1/TRP32, binds to Rpn11, a subunit of the regulatory complex of the human 26 S proteasome. Txnl1 is abundant, metabolically stable, and widely expressed and is present in the cytoplasm and nucleus. Txnl1 has thioredoxin activity with a redox potential of about -250 mV. Mutant Txnl1 with one active site cysteine replaced by serine formed disulfide bonds to eEF1A1, a substrate-recruiting factor of the 26 S proteasome. eEF1A1 is therefore a likely physiological substrate. In response to knockdown of Txnl1, ubiquitin-protein conjugates were moderately stabilized. Hence, Txnl1 is the first example of a direct connection between protein reduction and proteolysis, two major intracellular protein quality control mechanisms.

Ubxd1 is a novel co-factor of the human p97 ATPase.

The AAA ATPase complex known as p97 or VCP in mammals and Cdc48 in yeast is connected to a multitude of cellular pathways, including membrane fusion, protein folding, protein degradation and activation of membrane-bound transcription factors. The mechanism by which p97 participates in such a broad spectrum of cellular functions appears to be via recruiting certain specific co-factors. Here we isolate and characterize the human protein Ubxd1, a novel co-factor of p97. We show that Ubxd1 is a stable protein that localizes to the cytoplasm and nucleus and is highly enriched in centrosomes. In mice Ubxd1 is widely expressed, but especially abundant in brain. Curiously, Ubxd1 does not associate with p97 via its UBX domain, but via its PUB domain which binds the extreme C-terminus of p97. Phosphorylation of the penultimate tyrosine residue in p97 completely abolishes Ubxd1 interaction. Ternary complexes of Ubxd1, p47, and p97 were detected in vitro. Inhibition of Ubxd1 expression by siRNA did not affect the degradation of bulk protein or a model substrate of the ERAD pathway, indicating that Ubxd1 directs p97 activity to specialized functions in vivo.

Characterisation of the nascent polypeptide-associated complex in fission yeast.

The nascent polypeptide-associated complex (NAC) is an abundant and phylogenetically conserved protein complex. It is composed of two subunits and interacts with nascent polypeptide chains emerging from the ribosome. It has been proposed to protect the nascent chains from premature interaction with other cell proteins, but has also been found to associate with DNA junctions, and to be involved in other processes including transcription regulation and mitochondrial protein import.Here, we characterize NAC in fission yeast. We find that NAC is associated with ribosomes, while a significant fraction remains in a free form. The NAC alpha subunit contains a ubiquitin-associated (UBA) domain, which is found in several proteins involved in the ubiquitin-proteasome pathway for protein degradation. However, NAC does not associate with ubiquitin chains and mutants lacking NAC did not exhibit any obvious defects in protein degradation. Accordingly, we find that the NAC UBA domain belongs to an ancient and distinct subgroup of the UBA family. In contrast to the situation with budding yeast, fission yeast cells devoid of NAC were not temperature sensitive. However, they displayed resistance to the amino acid analogue canavanine, in accordance with the idea that NAC is involved in protein quality control.

Ubiquitin-binding proteins: similar, but different.

Covalent modification of proteins with ubiquitin is a common regulatory mechanism in eukaryotic cells. Typically, ubiquitinated proteins are targeted for degradation by the 26 S proteasome. However, more recently the ubiquitin signal has also been connected with many other cell processes, including endocytosis, vesicle fusion, DNA repair and transcriptional silencing. Hence ubiquitination may be comparable with phosphorylation in its importance as an intracellular switch, controlling various signal-transduction pathways. Similar to the regulation of the extent of phosphorylation by kinases and phosphatases, specific sets of ubiquitinating/deubiquitinating enzymes control the degree of ubiquitination. A large number of ubiquitin-binding proteins act at different steps in the downstream pathways, followed by the ubiquitinated protein. Different families of ubiquitin-binding proteins have been described. UBA (ubiquitin-associated) domain-containing proteins is the largest family and includes members involved in different cell processes. The smaller groups of UIM (ubiquitin-interacting motif), GAT [GGA (Golgi-associated gamma-adaptin homologous) and Tom1 (target of Myb 1)], CUE (coupling of ubiquitin conjugation to endoplasmic reticulum degradation), UEV [ubiquitin E2 (ubiquitin-conjugating enzyme) variant] and NZF (nuclear protein localization gene 4 zinc finger) domain-containing proteins appear to have more specialized functions. Here we discuss functional and structural properties of ubiquitin-binding proteins.