The Role of the Medial Prefrontal Cortex in Regulating Social Familiarity-Induced Anxiolysis

Overcoming specific fears and subsequent anxiety can be greatly enhanced by the presence of familiar social partners, but the neural circuitry that controls this phenomenon remains unclear. To overcome this, the social interaction (SI) habituation test was developed in this lab to systematically investigate the effects of social familiarity on anxiety-like behavior in rats. Here, we show that social familiarity selectively reduced anxiety-like behaviors induced by an ethological anxiogenic stimulus. The anxiolytic effect of social familiarity could be elicited over multiple training sessions and was specific to both the presence of the anxiogenic stimulus and the familiar social partner. In addition, socially familiar conspecifics served as a safety signal, as anxiety-like responses returned in the absence of the familiar partner. The expression of the social familiarity-induced anxiolysis (SFiA) appears dependent on the prefrontal cortex (PFC), an area associated with cortical regulation of fear and anxiety behaviors. Inhibition of the PFC, with bilateral injections of the GABAA agonist muscimol, selectively blocked the expression of SFiA while having no effect on SI with a novel partner. Finally, the effect of D-cycloserine, a cognitive enhancer that clinically enhances behavioral treatments for anxiety, was investigated with SFiA. D-cycloserine, when paired with familiarity training sessions, selectively enhanced the rate at which SFiA was acquired. Collectively, these outcomes suggest that the PFC has a pivotal role in SFiA, a complex behavior involving the integration of social cues of familiarity with contextual and emotional information to regulate anxiety-like behavior

A novel frameshift GP1BB mutation causes autosomal dominant macrothrombocytopenia with decreased vWF receptor expression but normal platelet aggregation

GP1bβ is a component of the von Willebrand factor (vWF) receptor complex that is necessary for platelet formation and activation. A novel frameshift variant in GP1BB has been identified in a family with macrothrombocytopenia. The variant leads to a protein that is 101 amino acids longer than wild type with loss of the transmembrane domain. As there is no defect in platelet aggregation, the family are classified as heterozygous carriers of a Bernard–Soulier syndrome-related mutation. The levels of the vWF receptor on platelets are reduced to 50% of the controls, with the presence of large platelets but normal platelet aggregation demonstrating that decreased vWF receptor expression impacts proplatelet formation but not platelet function

Differentiation and cell density upregulate cytochrome <i>c</i> levels in megakaryoblastic cell lines: Implications for analysis of <i>CYCS</i>-associated thrombocytopenia

<div><p>Mutations in the cytochrome <i>c</i> gene (<i>CYCS</i>) cause autosomal dominant thrombocytopenia by an unknown mechanism. While attempting to generate megakaryoblastic cell lines exogenously expressing cytochrome <i>c</i> variants, we discovered that endogenous cytochrome <i>c</i> expression increased both upon induction of differentiation with the phorbol ester phorbol 12-myristate 13-acetate (PMA), and as cell density increased. A concomitant increase in cytochrome <i>c</i> oxidase subunit II in response to PMA, but not cell higher cell density, suggests upregulation of the mitochondrial respiratory chain may be a specific feature of differentiation. These results highlight the likely importance of cytochrome <i>c</i> in both differentiating and proliferating cells, and illustrate the unsuitability of megakaryoblastic lines for modeling <i>CYCS</i>-associated thrombocytopenia.</p></div

Effect of PMA on cytochrome <i>c</i> expression in MEG-01, U937 and HeLa cells.

<p>Cells plated at 2 x 10⁵ cells/mL, incubated in the presence of 5 nM (MEG-01) or 10 nM (U937, HeLa) PMA for 72 h. Whole cell lysates (20 μg) analysed by western blot for expression of cyt <i>c</i> and actin. Fold change in expression relative to 0 h. Results presented as individual values with mean, n = 3.</p

Changes in expression of cyt <i>c</i>, MTCO2, Prdx3 and actin in untreated and PMA treated Set-2 cells.

<p>Changes in expression of cyt <i>c</i>, MTCO2, Prdx3 and actin in untreated and PMA treated Set-2 cells.</p

Cytochrome <i>c</i> expression in SET-2 cells.

<p><b>A</b>. Western blot analysis of cell lysate (20 μg) prepared from SET-2 clonal lines stably transfected with pcDNA3 (SET2-empty), pcDNA3-hWT (SET2-hWT-1 & -2) or pcDNA3-hG41S (SET2-hG41S-1 & -2) probed with anti-cytochrome <i>c</i> (upper panel) or anti-actin (lower panel). Numbers below each lane are fold change in expression compared to SET2-empty. <b>B–D</b>. SET-2 cells were plated at 2 x 10⁵ cells/mL, treated with or without 10 nM PMA for 72 h, and whole cell lysates (20 μg) analysed for the expression of cyt <i>c</i>, MTCO2, Prdx3 and actin. The western blot shown (<b>B</b>) is representative of three independent experiments. Fold change in expression of cyt <i>c</i> (i), MTCO2 (ii), Prdx3 (iii) and actin (iv) relative to 0 h in PMA treated (<b>C</b>) or untreated (<b>D</b>) cells. Results presented as individual values with mean, n = 3.</p

Interspecies variation in the functional consequences of mutation of cytochrome c

The naturally occurring human cytochrome c variant (G41S) is associated with a mild autosomal dominant thrombocytopenia (Thrombocytopenia Cargeeg) caused by dysregulation of platelet production. The molecular basis of the platelet production defect is unknown. Despite high conservation of cytochrome c between human and mouse (91.4% identity), introducing the G41S mutation into mouse cytochrome c in a knockin mouse (Cycs (G41S/G41S)) did not recapitulate the low platelet phenotype of Thrombocytopenia Cargeeg. While investigating the cause of this disparity we found a lack of conservation of the functional impact of cytochrome c mutations on caspase activation across species. Mutation of cytochrome c at residue 41 has distinct effects on the ability of cytochrome c to activate caspases depending on the species of both the cytochrome c and its binding partner Apaf-1. In contrast to our previous results showing the G41S mutation increases the ability of human cytochrome c to activate caspases, here we find this activity is decreased in mouse G41S cytochrome c. Additionally unlike wildtype human cytochrome c, G41S cytochrome c is unable to activate caspases in Xenopus embryo extracts. Taken together these results demonstrate a previously unreported species-specific component to the interaction of cytochrome c with Apaf-1. This suggests that the electrostatic interaction between cytochrome c and Apaf-1 is not the sole determinant of binding, with additional factors controlling binding specificity and affinity. These results have important implications for studies of the effects of cytochrome c mutations on the intrinsic apoptosis pathway

A Novel Bcl-2-Like Inhibitor of Apoptosis Is Encoded by the Parapoxvirus Orf Virus▿

Apoptotic cell death forms part of the host defense against virus infection. We tested orf virus, a member of the poxvirus family, for the ability to inhibit apoptosis and found that orf virus-infected cells were fully resistant to UV-induced changes in cell morphology, caspase activation, and DNA fragmentation. By using a library of vaccinia virus-orf virus recombinants, we identified an orf virus gene (ORFV125) whose presence was linked with the inhibition of apoptosis. The 173-amino-acid predicted protein had no clear homologs in public databases other than those encoded by other parapoxviruses. However, ORFV125 possessed a distinctive C-terminal domain which was necessary and sufficient to direct the protein to the mitochondria. We determined that ORFV125 alone could fully inhibit UV-induced DNA fragmentation, caspase activation, and cytochrome c release and that its mitochondrial localization was required for its antiapoptotic function. In contrast, ORFV125 did not prevent UV-induced activation of c-Jun NH2-terminal kinase, an event occurring upstream of the mitochondria. These features are comparable to the antiapoptotic properties of the mitochondrial regulator Bcl-2. Furthermore, bioinformatic analyses revealed sequence and secondary-structure similarities to Bcl-2 family members, including characteristic residues of all four Bcl-2 homology domains. Consistent with this, the viral protein inhibited the UV-induced activation of the proapoptotic Bcl-2 family members Bax and Bak. ORFV125 is the first parapoxvirus apoptosis inhibitor to be identified, and we propose that it is a new antiapoptotic member of the Bcl-2 family