Probes for INS

Delineating the basic cellular components of cortical inhibitory circuits remains a fundamental issue in order to understand their specific contributions to microcircuit function. It is still unclear how current classifications of cortical interneuron subtypes relate to biological processes such as their developmental specification. Here we identified the developmental trajectory of neurogliaform cells (NGCs), the main effectors of a powerful inhibitory motif recruited by long-range connections. Using in vivo genetic lineage-tracing in mice, we report that NGCs originate from a specific pool of 5-HT3AR-expressing Hmx3+ cells located in the preoptic area (POA). Hmx3-derived 5-HT3AR+ cortical interneurons (INs) expressed the transcription factors PROX1, NR2F2, the marker reelin but not VIP and exhibited the molecular, morphological and electrophysiological profile of NGCs. Overall, these results indicate that NGCs are a distinct class of INs with a unique developmental trajectory and open the possibility to study their specific functional contribution to cortical inhibitory microcircuit motifs.

In response to myocardial infarction (MI), cardiac macrophages regulate inflammation and scar formation. We hypothesized that macrophages undergo polarization state changes over the MI time course and assessed macrophage polarization transcriptomic signatures over the first week of MI. C57BL/6 J male mice (3-6 months old) were subjected to permanent coronary artery ligation to induce MI, and macrophages were isolated from the infarct region at days 1, 3, and 7 post-MI. Day 0, no MI resident cardiac macrophages served as the negative MI control. Whole transcriptome analysis was performed using RNA-sequencing on n = 4 pooled sets for each time. Day 1 macrophages displayed a unique pro-inflammatory, extracellular matrix (ECM)-degrading signature. By flow cytometry, day 0 macrophages were largely F4/80highLy6Clow resident macrophages, whereas day 1 macrophages were largely F4/80lowLy6Chigh infiltrating monocytes. Day 3 macrophages exhibited increased proliferation and phagocytosis, and expression of genes related to mitochondrial function and oxidative phosphorylation, indicative of metabolic reprogramming. Day 7 macrophages displayed a pro-reparative signature enriched for genes involved in ECM remodeling and scar formation. By triple in situ hybridization, day 7 infarct macrophages in vivo expressed collagen I and periostin mRNA. Our results indicate macrophages show distinct gene expression profiles over the first week of MI, with metabolic reprogramming important for polarization. In addition to serving as indirect mediators of ECM remodeling, macrophages are a direct source of ECM components. Our study is the first to report the detailed changes in the macrophage transcriptome over the first week of MI.

Abstract

BACKGROUND:

Progressive familial intrahepatic cholestasis type 3 (PFIC3) often leads to end-stage liver disease before adulthood with limited therapeutic options, due to impaired ABCB4 dependent phospholipid transport to bile. To restore ABCB4 function we propose adeno-associated virus serotype 8 (AAV8)-mediated gene therapy directed to the liver, although achieving stable transgene expression in hyperproliferative tissue is challenging. By restoring the phospholipid content in bile to levels that prevent liver damage, this study aims for stable hepatic ABCB4 expression and long-term correction of the phenotype in a murine model of PFIC3.

METHODS:

Ten weeks old Abcb4-/- mice received a single dose of AAV8-hABCB4 (n=10) or AAV8-GFP (n=7) under control of a liver specific promoter via tail vein injection. Animals were sacrificed either 10 or 26 weeks after vector administration to assess transgene persistence, after being challenged with a 0.1% cholate diet for 2 weeks. Periodic evaluation of plasma cholestatic markers was performed and bile duct cannulation enabled analysis of biliary phospholipids. Liver fibrosis and the Ki67 proliferation index were assessed by (immuno-)histochemistry.

RESULTS:

Stable transgene expression was achieved in all animals that received AAV8-hABCB4 up to 26 weeks after administration, which restored biliary phospholipid excretion to levels that ameliorate liver damage. This resulted in normalization of plasma cholestatic markers, prevented progressive liver fibrosis and reduced hepatocyte proliferation for the duration of the study.

CONCLUSION:

Liver-directed gene therapy provides stable hepatic ABCB4 expression and long-term correction of the phenotype in a murine model of PFIC3, encouraging translational studies to verify clinical feasibility.

LAY SUMMARY:

Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a severe genetic liver disease that results from impaired transport of lipids to bile, which makes the bile toxic to liver cells. Because therapeutic options are currently limited, this study aims to evaluate gene therapy to correct the underlying genetic defect in a mouse model of this disease. By introducing a functional copy of the missing gene in liver cells of mice, we were able to restore lipid transport to bile and strongly reduce damage to the liver. Also proliferation of liver cells was reduced, which contributes to long term correction of the phenotype. Limitations of the mouse model requires further studies to evaluate if this approach can be applied in PFIC3 patients.

Abstract

RATIONALE:

Given the paucity of effective treatments for Idiopathic Pulmonary Fibrosis (IPF), new insights into the deleterious mechanisms controlling lung fibroblast activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies. Transforming growth factor β (TGF-β) is the main pro-fibrotic factor, but its inhibition is associated with severe side effects due to its pleiotropic role.

OBJECTIVES:

We hypothesized that downstream non-coding effectors of TGF-β in fibroblasts may represent new effective therapeutic targets whose modulation may be well-tolerated.

METHODS:

We investigated the whole non-coding fraction of TGF-β-stimulated lung fibroblast transcriptome to identify new genomic determinants of lung fibroblast differentiation into myofibroblast. Differential expression of the long non-coding RNA DNM3OS and its associated miRNAs was validated in a murine model of pulmonary fibrosis and in IPF tissue samples. Distinct and complementary antisense oligonucleotide-based strategies aiming at interfering with DNM3OS were used to elucidate the role of DNM3OS and its associated miRNAs in IPF pathogenesis.

MEASUREMENTS AND MAIN RESULTS:

We identified DNM3OS as a fibroblast-specific critical downstream effector of TGF-β-induced lung myofibroblast activation. Mechanistically, DNM3OS regulates this process in trans by giving rise to three distinct profibrotic mature miRNAs (i.e. miR-199a-5p/3p and miR-214-3p), which influence both SMAD and non-SMAD components of TGF-β signaling in a multifaceted way. In vivo, we showed that interfering with DNM3OS function not only prevents lung fibrosis but also improves established pulmonary fibrosis.

CONCLUSION:

Pharmacological approaches aiming at interfering with DNM3OS may represent new effective therapeutic strategies in IPF.

The HSV1 virion host shutoff (vhs) protein is an endoribonuclease that binds to the cellular translation initiation machinery and degrades associated mRNAs, resulting in shut-off of host protein synthesis. Hence its unrestrained activity is considered to be lethal, and it has been proposed that vhs is regulated by two other virus proteins, VP22 and VP16. We have found that during infection, translation of vhs requires VP22 but not the VP22-VP16 complex. Moreover, in the absence of VP22, vhs is not overactive against cellular or viral transcripts. In transfected cells, vhs was also poorly translated, correlating with aberrant localization of its mRNA. Counterintuitively, vhs mRNA was predominantly nuclear in cells where vhs protein was detected. Likewise, transcripts from co-transfected plasmids were also retained in the same nuclei where vhs mRNA was located, while polyA binding protein (PABP) was relocalised to the nucleus in a vhs-dependent manner, implying a general block to mRNA export. Co-expression of VP16 and VP22 rescued cytoplasmic localization of vhs mRNA but failed to rescue vhs translation. We identified a 230-nucleotide sequence in the 5' region of vhs that blocked its translation and, when transferred to a heterologous GFP transcript, reduced translation without altering mRNA levels or localization. We propose that expression of vhs is tightly regulated by a combination of inherent untranslatability and auto-induced nuclear retention of its mRNA that results in a negative feedback loop, with nuclear retention but not translation of vhs mRNA being the target of rescue by the vhs-VP16-VP22 complex.IMPORTANCE A myriad of gene expression strategies has been discovered through studies carried out on viruses. This report concerns the regulation of the HSV1 vhs endoribonuclease, a virus factor that is important for counteracting host antiviral responses by degrading their mRNAs, but which must be regulated during infection to ensure that it does not act against and inhibit the virus itself. We show that regulation of vhs involves multifaceted post-transcriptional cellular and viral processes, including aberrant mRNA localization and a novel, autoregulated negative feedback loop to target its own and co-expressed mRNAs for nuclear retention, an activity that is relieved by co-expression of two other virus proteins, VP22 and VP16. These studies reveal the interplay of strategies by which multiple virus-encoded factors co-ordinate gene expression at the time they are needed. These findings are broadly relevant to both virus and cellular gene expression.