Protein Citrullination: A Proposed Mechanism for Pathology in Traumatic Brain Injury.

Protein citrullination is a calcium-driven post-translational modification proposed to play a causative role in the neurodegenerative disorders of Alzheimer's disease, multiple sclerosis (MS), and prion disease. Citrullination can result in the formation of antigenic epitopes that underlie pathogenic autoimmune responses. This phenomenon, which is best understood in rheumatoid arthritis, may play a role in the chronic dysfunction following traumatic brain injury (TBI). Despite substantial evidence of aberrations in calcium signaling following TBI, there is little understanding of how TBI alters citrullination in the brain. The present investigation addressed this gap by examining the effects of TBI on the distribution of protein citrullination and on the specific cell types involved. Immunofluorescence revealed that controlled cortical impact in rats profoundly up--regulated protein citrullination in the cerebral cortex, external capsule, and hippocampus. This response was exclusively seen in astrocytes; no such effects were observed on the status of protein citrullination in neurons, oligodendrocytes or microglia. Further, proteomic analyses demonstrated that the effects of TBI on citrullination were confined to a relatively small subset of neural proteins. Proteins most notably affected were those also reported to be citrullinated in other disorders, including prion disease and MS. In vivo findings were extended in an in vitro model of simulated TBI employing normal human astrocytes. Pharmacologically induced calcium excitotoxicity was shown to activate the citrullination and breakdown of glial fibrillary acidic protein, producing a novel candidate TBI biomarker and potential target for autoimmune recognition. In summary, these findings demonstrate that the effects of TBI on protein citrullination are selective with respect to brain region, cell type, and proteins modified, and may contribute to a role for autoimmune dysfunction in chronic pathology following TBI.

N-acetyl-aspartyl-glutamate and inhibition of glutamate carboxypeptidases protects against soman-induced neuropathology.

N-acetyl-aspartyl-glutamate (NAAG) is the most abundant neuropeptide in the mammalian brain. In a variety of animal models of brain injury, the administration of NAAG-related compounds, or inhibitors of glutamate carboxypeptidases (GCPs; the enzymes that hydrolyze NAAG), were shown to be neuroprotective. This study determined the impact of the administration of three NAAG-related compounds, NAAG, ß-NAAG (a NAAG homologue resistant to degradation), and 2-phosphonomethyl pentanedioic acid (2-PMPA; an inhibitor of GCP enzymes), on the neuropathology that develops following exposure to the nerve agent, soman. When given 1 min after soman exposure, NAAG-related drug treatments did not alter the survival rate or body weight loss seen 24 h after rats were exposed to soman. Likewise, brain levels of both NAAG and its metabolite, N-acetyl-aspartate (NAA), were substantially decreased 24 h after soman, and in particularly vulnerable brain regions the drug treatments were unable to attenuate the reduction in NAA and NAAG levels. Histochemical study indicated there was a dramatic increase in Fluoro-Jade C (FJC) staining, indicative of neuron cell death, 24 h after soman exposure. However, in the amygdala and in the entorhinal and piriform limbic cortex, which sustained severe neuropathology following soman intoxication, single or combined injections of NAAG compounds and 2-PMPA significantly reduced the number of FJC-positive cells, and effect size estimates suggest that in some brain regions the treatments were effective. The findings suggest that NAAG neurotransmission in the central nervous system is significantly altered by soman exposure, and that the administration of NAAG-related compounds and 2-PMPA reduces neuron cell death in brain regions that sustain severe damage.

Membrane fusion tropism and heterotypic functional activities of the Nipah virus and Hendra virus envelope glycoproteins.

Nipah virus (NiV) and Hendra virus (HeV) are novel paramyxoviruses from pigs and horses, respectively, that are responsible for fatal zoonotic infections of humans. The unique genetic and biological characteristics of these emerging agents has led to their classification as the prototypic members of a new genus within the Paramyxovirinae subfamily called HENIPAVIRUS: These viruses are most closely related to members of the genus Morbillivirus and infect cells through a pH-independent membrane fusion event mediated by the actions of their attachment (G) and fusion (F) glycoproteins. Understanding their cell biological features and exploring the functional characteristics of the NiV and HeV glycoproteins will help define important properties of these emerging viruses and may provide new insights into paramyxovirus membrane fusion mechanisms. Using a recombinant vaccinia virus system and a quantitative assay for fusion, we demonstrate NiV glycoprotein function and the same pattern of cellular tropism recently reported for HeV-mediated fusion, suggesting that NiV likely uses the same cellular receptor for infection. Fusion specificity was verified by inhibition with a specific antiserum or peptides derived from the alpha-helical heptads of NiV or HeV F. Like that of HeV, NiV-mediated fusion also requires both F and G. Finally, interactions between the glycoproteins of the paramyxoviruses have not been well defined, but here we show that the NiV and HeV glycoproteins are capable of highly efficient heterotypic functional activity with each other. However, no heterotypic activity was observed with envelope glycoproteins of the morbilliviruses Measles virus and Canine distemper virus.

A variable region 3 (V3) mutation determines a global neutralization phenotype and CD4-independent infectivity of a human immunodeficiency virus type 1 envelope associated with a broadly cross-reactive, primary virus-neutralizing antibody response.

The human serum human immunodeficiency virus type 1 (HIV-1)-neutralizing serum 2 (HNS2) neutralizes many primary isolates of different clades of HIV-1, and virus expressing envelope from the same donor, clone R2, is neutralized cross-reactively by HIV-immune human sera. The basis for this cross-reactivity was investigated. It was found that a rare mutation in the proximal limb of variable region 3 (V3), 313-4 PM, caused virus pseudotyped with the R2 envelope to be highly sensitive to neutralization by monoclonal antibodies (MAbs) directed against conformation-sensitive epitopes at the tip of the V3 loop, such as 19b, and moderately sensitive to MAbs against CD4 binding site (CD4bs) and CD4-induced (CD4i) epitopes, soluble CD4 (sCD4), and HNS2. In addition, introduction of this sequence by mutagenesis caused enhanced sensitivity to neutralization by 19b, anti-CD4i MAb, and HNS2 in three other primary HIV-1 envelopes and by anti-CD4bs MAb and sCD4 in one of the three. The 313-4 PM sequence also conferred increased infectivity for CD4(+) CCR5(+) cells and the ability to infect CCR5(+) cells upon all of these four and two of these four HIV-1 envelopes, respectively. Neutralization of R2 by HNS2 was substantially inhibited by the cyclized R2 V3 35-mer synthetic peptide. Similarly, the peptide also had some lesser efficacy in blocking neutralization of R2 by other sera or of neutralization of other primary viruses by HNS2. Together, these results indicate that the unusual V3 mutation in the R2 clone accounts for its uncommon neutralization sensitivity phenotype and its capacity to mediate CD4-independent infection, both of which could relate to immunogenicity and the neutralizing activity of HNS2. This is also the first primary HIV-1 isolate envelope glycoprotein found to be competent for CD4-independent infection.