Silencing Relaxin-3 in Nucleus Incertus of Adult Rodents: A Viral Vector-based Approach to Investigate Neuropeptide Function

Relaxin-3, the most recently identified member of the relaxin peptide family, is produced by GABAergic projection neurons in the nucleus incertus (NI), in the pontine periventricular gray. Previous studies suggest relaxin-3 is a modulator of stress responses, metabolism, arousal and behavioural activation. Knockout mice and peptide infusions in vivo have significantly contributed to understanding the function of this conserved neuropeptide. Yet, a definitive role remains elusive due to discrepancies between models and a propensity to investigate pharmacological effects over endogenous function. To investigate the endogenous function of relaxin-3, we generated a recombinant adeno-associated viral (rAAV) vector expressing microRNA against relaxin-3 and validated its use to knock down relaxin-3 in adult rats. Bilateral stereotaxic infusion of rAAV1/2 EmGFP miR499 into the NI resulted in significant reductions in relaxin-3 expression as demonstrated by ablation of relaxin-3-like immunoreactivity at 3, 6 and 9 weeks and by qRT-PCR at 12 weeks. Neuronal health was unaffected as transduced neurons in all groups retained expression of NeuN and stained for Nissl bodies. Importantly, qRT-PCR confirmed that relaxin-3 receptor expression levels were not altered to compensate for reduced relaxin-3. Behavioural experiments confirmed no detrimental effects on general health or well-being and therefore several behavioural modalities previously associated with relaxin-3 function were investigated. The validation of this viral vector-based model provides a valuable alternative to existing in vivo approaches and promotes a shift towards more physiologically relevant investigations of endogenous neuropeptide function.     

New ketomethylene inhibitor analogues: synthesis and assessment of structural determinants for N-domain selective inhibition of angiotensin-converting enzyme

Angiotensin-converting enzyme (ACE) is a zinc metallopeptidase containing two homologous domains. While the C-domain plays a major role in blood pressure regulation, the N-domain hydrolyzes the antifibrotic agent N-acetyl-Ser-Asp-Lys-Pro. Thus, N-domain selective (N-selective) inhibitors could be useful in the treatment of conditions relating to excessive tissue fibrosis. New keto-ACE analogues were designed that contained functionalities considered important for N-selective inhibitor RXP407 binding, namely, a P(2) Asp, N-acetyl group, and C-terminal amide. Such functionalities were incorporated to assess the structural determinants for N-selective binding in a novel inhibitor template. Inhibitors containing a C-terminal amide and modified P(2)' group were poor inhibitors of the N-domain, with several of these displaying improved inhibition of the C-domain. Molecules with both a C-terminal amide and P(2) Asp were also poor inhibitors and not N-selective. Compounds containing a free C-terminus, a P(2) Asp and protecting group displayed a change of more than 1000-fold N-selectivity compared with the parent molecule. Molecular docking models revealed interaction of these P(2) groups with S(2) residues Tyr369 and Arg381. This study emphasizes the importance of P(2) functionalities in allowing for improved N-selective binding and provides further rationale for the design of N-selective inhibitors, which could be useful in treating tissue fibrosis.     

The intracellular redox state is a core determinant of mitochondrial fusion

Mitochondrial hyperfusion has recently been shown to function as a cellular stress response, providing transient protection against apoptosis and mitophagy. However, the mechanisms that mediate this response remain poorly understood. In this study, we demonstrate that oxidized glutathione (GSSG), the core cellular stress indicator, strongly induces mitochondrial fusion. Biochemical and functional experiments show that GSSG induces the generation of disulphide‐mediated mitofusin oligomers, in a process that also requires GTP hydrolysis. Our data outline the molecular events that prime the fusion machinery, providing new insights into the coupling of mitochondrial fusion with the cellular stress response.     

Immune response to bacteria induces dissemination of Ras-activated Drosophila hindgut cells

Although pathogenic bacteria are suspected contributors to colorectal cancer progression, cancer-promoting bacteria and their mode of action remain largely unknown. Here we report that sustained infection with the human intestinal colonizer Pseudomonas aeruginosa synergizes with the Ras1V12 oncogene to induce basal invasion and dissemination of hindgut cells to distant sites. Cross-talk between infection and dissemination requires sustained activation by the bacteria of the Imd-dTab2-dTak1 innate immune pathway, which converges with Ras1V12 signalling on JNK pathway activation, culminating in extracellular matrix degradation. Hindgut, but not midgut, cells are amenable to this cooperative dissemination, which is progressive and genetically and pharmacologically inhibitable. Thus, Drosophila hindgut provides a valuable system for the study of intestinal malignancies.     

AMPK: a nutrient and energy sensor that maintains energy homeostasis

AMP-activated protein kinase (AMPK) is a crucial cellular energy sensor. Once activated by falling energy status, it promotes ATP production by increasing the activity or expression of proteins involved in catabolism while conserving ATP by switching off biosynthetic pathways. AMPK also regulates metabolic energy balance at the whole-body level. For example, it mediates the effects of agents acting on the hypothalamus that promote feeding and entrains circadian rhythms of metabolism and feeding behaviour. Finally, recent studies reveal that AMPK conserves ATP levels through the regulation of processes other than metabolism, such as the cell cycle and neuronal membrane excitability.     

Human Fidgetin is a microtubule severing the enzyme and minus-end depolymerase that regulates mitosis

Fidgetin is a member of the AAA protein superfamily with important roles in mammalian development. Here we show that human Fidgetin is a potent microtubule severing and depolymerizing the enzyme used to regulate mitotic spindle architecture, dynamics and anaphase A. In vitro, recombinant human Fidgetin severs taxol-stabilized microtubules along their length and promotes depolymerization, primarily from their minus-ends. In cells, human Fidgetin targets to centrosomes, and its depletion with siRNA significantly reduces the velocity of poleward tubulin flux and anaphase A chromatid-to-pole motion. In addition, the loss of Fidgetin induces a microtubule-dependent enlargement of mitotic centrosomes and an increase in the number and length of astral microtubules. Based on these data, we propose that human Fidgetin actively suppresses microtubule growth from and attachment to centrosomes.     

A mathematical methodology for determining the temporal order of pathway alterations arising during gliomagenesis

Human cancer is caused by the accumulation of genetic alterations in cells. Of special importance are changes that occur early during malignant transformation because they may result in oncogene addiction and thus represent promising targets for therapeutic intervention. We have previously described a computational approach, called Retracing the Evolutionary Steps in Cancer (RESIC), to determine the temporal sequence of genetic alterations during tumorigenesis from cross-sectional genomic data of tumors at their fully transformed stage. Since alterations within a set of genes belonging to a particular signaling pathway may have similar or equivalent effects, we applied a pathway-based systems biology approach to the RESIC methodology. This method was used to determine whether alterations of specific pathways develop early or late during malignant transformation. When applied to primary glioblastoma (GBM) copy number data from The Cancer Genome Atlas (TCGA) project, RESIC identified a temporal order of pathway alterations consistent with the order of events in secondary GBMs. We then further subdivided the samples into the four main GBM subtypes and determined the relative contributions of each subtype to the overall results: we found that the overall ordering applied for the proneural subtype but differed for mesenchymal samples. The temporal sequence of events could not be identified for neural and classical subtypes, possibly due to a limited number of samples. Moreover, for samples of the proneural subtype, we detected two distinct temporal sequences of events: (i) RAS pathway activation was followed by TP53 inactivation and finally PI3K2 activation, and (ii) RAS activation preceded only AKT activation. This extension of the RESIC methodology provides an evolutionary mathematical approach to identify the temporal sequence of pathway changes driving tumorigenesis and may be useful in guiding the understanding of signaling rearrangements in cancer development.     

Polymorphisms in the mitochondrial ribosome recycling factor EF-G2mt/MEF2 compromise cell respiratory function and increase atorvastatin toxicity

Mitochondrial translation, essential for synthesis of the electron transport chain complexes in the mitochondria, is governed by nuclear encoded genes. Polymorphisms within these genes are increasingly being implicated in disease and may also trigger adverse drug reactions. Statins, a class of HMG-CoA reductase inhibitors used to treat hypercholesterolemia, are among the most widely prescribed drugs in the world. However, a significant proportion of users suffer side effects of varying severity that commonly affect skeletal muscle. The mitochondria are one of the molecular targets of statins, and these drugs have been known to uncover otherwise silent mitochondrial mutations. Based on yeast genetic studies, we identify the mitochondrial translation factor MEF2 as a mediator of atorvastatin toxicity. The human ortholog of MEF2 is the Elongation Factor Gene (EF-G) 2, which has previously been shown to play a specific role in mitochondrial ribosome recycling. Using small interfering RNA (siRNA) silencing of expression in human cell lines, we demonstrate that the EF-G2mt gene is required for cell growth on galactose medium, signifying an essential role for this gene in aerobic respiration. Furthermore, EF-G2mt silenced cell lines have increased susceptibility to cell death in the presence of atorvastatin. Using yeast as a model, conserved amino acid variants, which arise from non-synonymous single nucleotide polymorphisms (SNPs) in the EF-G2mt gene, were generated in the yeast MEF2 gene. Although these mutations do not produce an obvious growth phenotype, three mutations reveal an atorvastatin-sensitive phenotype and further analysis uncovers a decreased respiratory capacity. These findings constitute the first reported phenotype associated with SNPs in the EF-G2mt gene and implicate the human EF-G2mt gene as a pharmacogenetic candidate gene for statin toxicity in humans.     

Research ethics education for community-engaged research: a review and research agenda

Community engagement is increasingly becoming an integral part of research. "Community-engaged research" (CEnR) introduces new stakeholders as well as unique challenges to the protection of participants and the integrity of the research process. We--a group of representatives of CTSA-funded institutions and others who share expertise in research ethics and CEnR--have identified gaps in the literature regarding (1) ethical issues unique to CEnR; (2) the particular instructional needs of academic investigators, community research partners, and IRB members; and (3) best practices for teaching research ethics. This paper presents what we know, as well as what we still need to learn, in order to develop quality research ethics educational materials tailored to the full range of stakeholder groups in CEnR.