Measuring Nonapoptotic Caspase Activity with a Transgenic Reporter in Mice.

The protease caspase-3 is a key mediator of apoptotic programmed cell death. But weak or transient caspase activity can contribute to neuronal differentiation, axonal pathfinding, and synaptic long-term depression. Despite the importance of sublethal, or nonapoptotic, caspase activity in neurodevelopment and neural plasticity, there has been no simple method for mapping and quantifying nonapoptotic caspase activity (NACA) in rodent brains. We therefore generated a transgenic mouse expressing a highly sensitive and specific fluorescent reporter of caspase activity, with peak signal localized to the nucleus. As a proof of concept, we first obtained evidence that NACA influences neurophysiology in an amygdalar circuit. Then focusing on the amygdala, we were able to quantify a sex-specific persistent elevation in caspase activity in females after restraint stress. This simple in vivo caspase activity reporter will facilitate systems-level studies of apoptotic and nonapoptotic phenomena in behavioral and pathologic models.

HER2 Isoforms Uniquely Program Intratumor Heterogeneity and Predetermine Breast Cancer Trajectories During the Occult Tumorigenic Phase.

HER2-positive breast cancers are among the most heterogeneous breast cancer subtypes. The early amplification of HER2 and its known oncogenic isoforms provide a plausible mechanism in which distinct programs of tumor heterogeneity could be traced to the initial oncogenic event. Here a Cancer rainbow mouse simultaneously expressing fluorescently barcoded wildtype (WTHER2), exon-16 null (d16HER2), and N-terminally truncated (p95HER2) HER2 isoforms is used to trace tumorigenesis from initiation to invasion. Tumorigenesis was visualized using whole-gland fluorescent lineage tracing and single-cell molecular pathology. We demonstrate that within weeks of expression, morphologic aberrations were already present and unique to each HER2 isoform. Although WTHER2 cells were abundant throughout the mammary ducts, detectable lesions were exceptionally rare. In contrast, d16HER2 and p95HER2 induced rapid tumor development. d16HER2 incited homogenous and proliferative luminal-like lesions which infrequently progressed to invasive phenotypes whereas p95HER2 lesions were heterogenous and invasive at the smallest detectable stage. Distinct cancer trajectories were observed for d16HER2 and p95HER2 tumors as evidenced by oncogene-dependent changes in epithelial specification and the tumor microenvironment. These data provide direct experimental evidence that intratumor heterogeneity programs begin very early and well in advance of screen or clinically detectable breast cancer. IMPLICATIONS: Although all HER2 breast cancers are treated equally, we show a mechanism by which clinically undetected HER2 isoforms program heterogenous cancer phenotypes through biased epithelial specification and adaptations within the tumor microenvironment.

A cancer rainbow mouse for visualizing the functional genomics of oncogenic clonal expansion.

Field cancerization is a premalignant process marked by clones of oncogenic mutations spreading through the epithelium. The timescales of intestinal field cancerization can be variable and the mechanisms driving the rapid spread of oncogenic clones are unknown. Here we use a Cancer rainbow (Crainbow) modelling system for fluorescently barcoding somatic mutations and directly visualizing the clonal expansion and spread of oncogenes. Crainbow shows that mutations of ß-catenin (Ctnnb1) within the intestinal stem cell results in widespread expansion of oncogenes during perinatal development but not in adults. In contrast, mutations that extrinsically disrupt the stem cell microenvironment can spread in adult intestine without delay. We observe the rapid spread of premalignant clones in Crainbow mice expressing oncogenic Rspondin-3 (RSPO3), which occurs by increasing crypt fission and inhibiting crypt fixation. Crainbow modelling provides insight into how somatic mutations rapidly spread and a plausible mechanism for predetermining the intratumor heterogeneity found in colon cancers.

Slow-release delivery enhances the pharmacological properties of oral 5-hydroxytryptophan: mouse proof-of-concept.

5-hydroxytryptophan (5-HTP) has shown therapeutic promise in a range of human CNS disorders. But native 5-HTP immediate release (IR) is poorly druggable, as rapid absorption causes rapid onset of adverse events, and rapid elimination causes fluctuating exposure. Recently, we reported that 5-HTP delivered as slow-release (SR) in mice augmented the brain pro-serotonergic effect of selective serotonin reuptake inhibitors (SSRIs), without the usual adverse events associated with 5-HTP IR. However, our previous study entailed translational limitations, in terms of route, dose, and duration. Here we modeled oral 5-HTP SR in mice by administering 5-HTP via the food. We modeled oral SSRI treatment via fluoxetine in the water, in a regimen recapitulating clinical pharmacokinetics and pharmacodynamics. 5-HTP SR produced plasma 5-HTP levels well within the range enhancing brain 5-HT function in humans. 5-HTP SR robustly increased brain 5-HT synthesis and levels. When administered with an SSRI, 5-HTP SR enhanced 5-HT-sensitive behaviors and neurotrophic mRNA expression. 5-HTP SR's pro-serotonergic effects were stronger in mice with endogenous brain 5-HT deficiency. In a comprehensive screen, 5-HTP SR was devoid of overt toxicological effects. The present preclinical data, appreciated in the context of published 5-HTP clinical data, suggest that 5-HTP SR could represent a new therapeutic approach to the plethora of CNS disorders potentially treatable with a pro-serotonergic drug. 5-HTP SR might in particular be therapeutically relevant when brain 5-HT deficiency is pathogenic and as an adjunctive augmentation therapy to SSRI therapy.

Effect of mutant IkappaB on cytokine-induced activation of NF-kappaB in cultured human RPE cells.

PURPOSE: The nuclear transcription factor (NF)-kappaB is a central regulator of multiple inflammatory cytokines. The current study was conducted to determine whether infection of human retinal pigment epithelial (RPE) cells by adenovirus carrying a mutant inhibitory (I)-kappaB (IkappaB) transgene inhibits cytokine-induced activity of NF-kappaB and expression of NF-kappaB-dependent cytokines by preventing degradation of IkappaB. The persistence of recombinant protein expression and function after the viral infection was also examined. METHODS: Cultured human RPE cells were infected with adenovirus encoding either beta-galactosidase (LacZ) or mutant IkappaB and were treated with interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha. IkappaB protein expression was determined by Western blot. NF-kappaB nuclear translocation was evaluated by immunofluorescence, and functional NF-kappaB activation was determined by luciferase reporter assay. NF-kappaB-dependent cytokine gene expression was determined by reverse transcription-polymerase chain reaction. IL-1beta-induced monocyte chemoattractant protein (MCP)-1 protein secretion was measured by enzyme-linked immunosorbent assay. RESULTS: Stimulation of RPE cells with IL-1beta or TNF-alpha caused rapid degradation of the endogenous, but not mutant, IkappaB protein. Expression of the mutant IkappaB isoform inhibited cytokine-stimulated NF-kappaB nuclear translocation, NF-kappaB transcriptional activity, NF-kappaB-dependent gene expression, and secretion of MCP-1. Significant levels of mutant IkappaB protein were expressed for at least 7 weeks after infection. CONCLUSIONS: Infection of human RPE by an adenoviral vector carrying a mutant IkappaB transgene blocks NF-kappaB activation and expression of multiple NF-kappaB-dependent cytokine genes over an extended period. This technique will be useful to determine the role of NF-kappaB in experimental proliferative vitreoretinopathy (PVR), and may offer a novel approach to treatment of PVR with a gene therapy approach.