The Highwire Ubiquitin Ligase Promotes Axonal Degeneration by Tuning Levels of Nmnat Protein

Axonal degeneration is a hallmark of many neuropathies, neurodegenerative diseases, and injuries. Here, using a Drosophila injury model, we have identified a highly conserved E3 ubiquitin ligase, Highwire (Hiw), as an important regulator of axonal and synaptic degeneration. Mutations in hiw strongly inhibit Wallerian degeneration in multiple neuron types and developmental stages. This new phenotype is mediated by a new downstream target of Hiw: the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (Nmnat), which acts in parallel to a previously known target of Hiw, the Wallenda dileucine zipper kinase (Wnd/DLK) MAPKKK. Hiw promotes a rapid disappearance of Nmnat protein in the distal stump after injury. An increased level of Nmnat protein in hiw mutants is both required and sufficient to inhibit degeneration. Ectopically expressed mouse Nmnat2 is also subject to regulation by Hiw in distal axons and synapses. These findings implicate an important role for endogenous Nmnat and its regulation, via a conserved mechanism, in the initiation of axonal degeneration. Through independent regulation of Wnd/DLK, whose function is required for proximal axons to regenerate, Hiw plays a central role in coordinating both regenerative and degenerative responses to axonal injury.     

Cedar Virus: A Novel Henipavirus Isolated from Australian Bats

The genus Henipavirus in the family Paramyxoviridae contains two viruses, Hendra virus (HeV) and Nipah virus (NiV) for which pteropid bats act as the main natural reservoir. Each virus also causes serious and commonly lethal infection of people as well as various species of domestic animals, however little is known about the associated mechanisms of pathogenesis. Here, we report the isolation and characterization of a new paramyxovirus from pteropid bats, Cedar virus (CedPV), which shares significant features with the known henipaviruses. The genome size (18,162 nt) and organization of CedPV is very similar to that of HeV and NiV; its nucleocapsid protein displays antigenic cross-reactivity with henipaviruses; and it uses the same receptor molecule (ephrin- B2) for entry during infection. Preliminary challenge studies with CedPV in ferrets and guinea pigs, both susceptible to infection and disease with known henipaviruses, confirmed virus replication and production of neutralizing antibodies although clinical disease was not observed. In this context, it is interesting to note that the major genetic difference between CedPV and HeV or NiV lies within the coding strategy of the P gene, which is known to play an important role in evading the host innate immune system. Unlike HeV, NiV, and almost all known paramyxoviruses, the CedPV P gene lacks both RNA editing and also the coding capacity for the highly conserved V protein. Preliminary study indicated that CedPV infection of human cells induces a more robust IFN-β response than HeV.     

Amino acid variations resulting in functional and nonfunctional zebrafish P2X<Subscript>1</Subscript> and P2X<Subscript>5.1</Subscript> receptors

Several zebrafish P2X receptors (zP2X(1), zP2X(2), and zP2X(5.1)) have been reported to produce little or no current although their mammalian orthologs produce functional homomeric receptors. We isolated new cDNA clones for these P2X receptors that revealed sequence variations in each. The new variants of zP2X(1) and zP2X(5.1) produced substantial currents when expressed by Xenopus oocytes, however the new variant of zP2X(2) was still nonfunctional. zP2X(2) lacks two lysine residues essential for ATP responsiveness in other P2X receptors; however introduction of these two lysines was insufficient to allow this receptor to function as a homotrimer. We also tested whether P2X signaling is required for myogenesis or synaptic communication at the zebrafish neuromuscular junction. We found that embryonic skeletal muscle expressed only one P2X receptor, P2X(5.1). Antisense knockdown of P2X(5.1) eliminated skeletal muscle responsiveness to ATP but did not prevent myogenesis or behaviors that require functional transmission at the neuromuscular junction.     

Generation of diversity in the innate immune system: macrophage heterogeneity arises from gene-autonomous transcriptional probability of individual inducible genes.

Microbial products such as LPS stimulate macrophages to produce a wide diversity of inducible gene products needed for immediate host defense and priming of an appropriate acquired immune response. In this study, we have examined LPS-inducible gene expression in subclones of a mouse macrophage cell line, RAW264, using cDNA microarrays. Even archetypal target genes such as TNF-alpha were not induced in all subclones, and there was no absolute correlation between expression of pairs of genes. Nevertheless, the array analysis revealed clusters of genes that were more likely to be coexpressed. RAW264 cells stably transfected with luciferase reporter genes driven by LPS-responsive promoters revealed the same kind of clonal heterogeneity. The results indicate that each LPS-inducible gene has its own inherent probability of activation in response to LPS.     

Identification of the human hepatic microsomal glucose-6-phosphatase enzyme

The glucose-6-phosphatase enzyme protein of the human hepatic microsomal glucose-6-phosphatase system was identified as a 36.5 kDa polypeptide. The 36.5 kDa glucose-6-phosphatase enzyme protein was shown to be absent in the microsomes isolated from a patient previously diagnosed as having a type 1a glycogen storage disease.