Wistar Kyoto Rats Display Anhedonia In Consumption but Retain Some Sensitivity to the Anticipation of Palatable Solutions.

The Wistar Kyoto (WKY) rat has been proposed as a model of depression-like symptoms. However, anhedonia-a reduction in the response to normatively rewarding events-as a central depression symptom has yet to be fully assessed in this model. We compared WKY rats and Wistar controls, with stress-susceptibility examined by applying mild unpredictable stress to a subset of each group. Anhedonia-like behavior was assessed using microstructural analysis of licking behavior, where mean lick cluster size reflects hedonic responses. This was combined with tests of anticipatory contrast, where the consumption of a moderately palatable solution (4% sucrose) is suppressed in anticipation of a more palatable solution (32% sucrose). WKY rats displayed greatly attenuated hedonic reactions to sucrose overall, although their reactions retained some sensitivity to differences in sucrose concentration. They displayed normal reductions in consumption in anticipatory contrast, although the effect of contrast on hedonic reactions was greatly blunted. Mild stress produced overall reductions in sucrose consumption, but this was not exacerbated in WKY rats. Moreover, mild stress did not affect hedonic reactions or the effects of contrast. These results confirm that the WKY substrain expresses a direct behavioral analog of anhedonia, which may have utility for increasing mechanistic understanding of depression symptoms.

A Negative Regulatory Mechanism Involving 14-3-3ζ Limits Signaling Downstream of ROCK to Regulate Tissue Stiffness in Epidermal Homeostasis.

ROCK signaling causes epidermal hyper-proliferation by increasing ECM production, elevating dermal stiffness, and enhancing Fak-mediated mechano-transduction signaling. Elevated dermal stiffness in turn causes ROCK activation, establishing mechano-reciprocity, a positive feedback loop that can promote tumors. We have identified a negative feedback mechanism that limits excessive ROCK signaling during wound healing and is lost in squamous cell carcinomas (SCCs). Signal flux through ROCK was selectively tuned down by increased levels of 14-3-3ζ, which interacted with Mypt1, a ROCK signaling antagonist. In 14-3-3ζ(-/-) mice, unrestrained ROCK signaling at wound margins elevated ECM production and reduced ECM remodeling, increasing dermal stiffness and causing rapid wound healing. Conversely, 14-3-3ζ deficiency enhanced cutaneous SCC size. Significantly, inhibiting 14-3-3ζ with a novel pharmacological agent accelerated wound healing 2-fold. Patient samples of chronic non-healing wounds overexpressed 14-3-3ζ, while cutaneous SCCs had reduced 14-3-3ζ. These results reveal a novel 14-3-3ζ-dependent mechanism that negatively regulates mechano-reciprocity, suggesting new therapeutic opportunities.

Estradiol induces gene proximity and MLL-MLLT3 fusion in an activation-induced cytidine deaminase-mediated pathway.

Epidemiological data have linked birth control formulations to an increased risk of infant acute leukemia involving MLL rearrangements. Reverse transcription polymerase chain reaction (RT-PCR) studies showed that 10 nM estradiol enhanced MLL transcription in addition to its common translocation partners, MLLT2 (AF4) and MLLT3 (AF9). The same concentration of estradiol triggered MLL and MLLT3 co-localization without affecting the interaction of genes located on the same chromosomes. Estradiol also stimulated the generation of MLL-MLLT3 fusion transcripts as seen by RT-PCR. RNAi knockdown of activation-induced cytidine deaminase (AICDA) suppressed the induction of MLL-MLLT3 fusion transcript formation observed with estradiol. Additionally, chromatin immunoprecipitation (ChIP) analysis showed estradiol dependent localization of AICDA in MLL intron 11, upstream of a hotspot for both DNA cleavage and rearrangement, but not downstream within intron 12. Combined, these studies show that levels of estradiol consistent with that observed during pregnancy have the potential to initiate MLL fusions through an AICDA-mediated mechanism.

A systematic description of MLL fusion gene formation.

Rearrangements of the MLL gene involve multiple partners and are implicated in both therapy related acute leukemia [tAL] and infant acute leukemia. For these diseases, recently compiled clinical data confirms an elevated frequency of such breakpoints within a 4 kb tract between exon 11 and a region of structural instability adjacent to exon 12. Linked primarily to cases of tAL, interference with topoisomerase II activity may either contribute to the initial DNA lesion directly or indirectly by, for example, providing a physical block to transcription progression. Alternatively, sites of fragmentation may be mis-repaired, guided by intergenic spliced transcripts of the participating genes. Co-transcription of MLL and potential fusion partners may provide the localization that enhances the probability of gene interaction. An indirect role for the leukemogenic activity of topoisomerase II inhibitors would imply that the negative consequences of their use may be separated from their therapeutic effects.

Microstructural analysis of negative anticipatory contrast: A reconsideration of the devaluation account.

An animal's appetitive behavior is not a fixed response to current stimulation but can be affected by the anticipation of future events. For example, rats regularly given access to a moderately valued solution followed by a higher value solution (e.g., 4 % sucrose → 32 % sucrose) consume less of the initial solution than in control conditions where the initial solution is not followed by a higher value solution (e.g., 4 % sucrose → 4 % sucrose). Previous analyses have suggested that this negative anticipatory contrast effect does not depend on the "expectation" of a valuable stimulus producing a functional devaluation of a currently available stimulus of lesser value. In a within-subjects anticipatory contrast procedure, this study revealed that both consumption and the mean size of licking clusters were smaller for a 4 % sucrose solution on days when it preceded 32 % sucrose than on days when 4 % preceded 4 %. Since lick cluster size typically bears a positive monotonic relationship with the concentration of palatable solutions, this reduction is indicative of a decrease in the palatability/hedonic value of the solution subject to contrast. As such, we provide direct evidence that negative anticipatory contrast does produce a functional devaluation of the solution, thus challenging prevailing theoretical assumptions.

Efficient conversion of astrocytes to functional midbrain dopaminergic neurons using a single polycistronic vector.

Direct cellular reprogramming is a powerful new tool for regenerative medicine. In efforts to understand and treat Parkinson's Disease (PD), which is marked by the degeneration of dopaminergic neurons in the midbrain, direct reprogramming provides a valuable new source of these cells. Astrocytes, the most plentiful cells in the central nervous system, are an ideal starting population for the direct generation of dopaminergic neurons. In addition to their potential utility in cell replacement therapies for PD or in modeling the disease in vitro, astrocyte-derived dopaminergic neurons offer the prospect of direct in vivo reprogramming within the brain. As a first step toward this goal, we report the reprogramming of astrocytes to dopaminergic neurons using three transcription factors - ASCL1, LMX1B, and NURR1 - delivered in a single polycistronic lentiviral vector. The process is efficient, with 18.2±1.5% of cells expressing markers of dopaminergic neurons after two weeks. The neurons exhibit expression profiles and electrophysiological characteristics consistent with midbrain dopaminergic neurons, notably including spontaneous pacemaking activity, stimulated release of dopamine, and calcium oscillations. The present study is the first demonstration that a single vector can mediate reprogramming to dopaminergic neurons, and indicates that astrocytes are an ideal starting population for the direct generation of dopaminergic neurons.

Characterisation of complex formation between members of the Mycobacterium tuberculosis complex CFP-10/ESAT-6 protein family: towards an understanding of the rules governing complex formation and thereby functional flexibility.

We have previously shown that the secreted M. tuberculosis complex proteins CFP-10 and ESAT-6 form a tight, 1:1 complex, which may represent their functional form. In the work reported here a combination of yeast two-hybrid and biochemical analysis has been used to characterise complex formation between two other pairs of CFP-10/ESAT-6 family proteins (Rv0287/Rv0288 and Rv3019c/Rv3020c) and to determine whether complexes can be formed between non-genome paired members of the family. The results clearly demonstrate that Rv0287/Rv0288 and Rv3019c/3020c form tight complexes, as initially observed for CFP-10/ESAT-6. The closely related Rv0287/Rv0288 and Rv3019c/Rv3020c proteins are also able to form non-genome paired complexes (Rv0287/Rv3019c and Rv0288/Rv3020c), but are not capable of binding to the more distantly related CFP-10/ESAT-6 proteins.