Dysregulation of REST-regulated coding and non-coding RNAs in a cellular model of Huntington's disease

Huntingtin (Htt) protein interacts with many transcriptional regulators, with widespread disruption to the transcriptome in Huntington's disease (HD) brought about by altered interactions with the mutant Htt (muHtt) protein. Repressor Element-1 Silencing Transcription Factor (REST) is a repressor whose association with Htt in the cytoplasm is disrupted in HD, leading to increased nuclear REST and concomitant repression of several neuronal-specific genes, including brain-derived neurotrophic factor (Bdnf). Here, we explored a wide set of HD dysregulated genes to identify direct REST targets whose expression is altered in a cellular model of HD but that can be rescued by knock-down of REST activity. We found many direct REST target genes encoding proteins important for nervous system development, including a cohort involved in synaptic transmission, at least two of which can be rescued at the protein level by REST knock-down. We also identified several microRNAs (miRNAs) whose aberrant repression is directly mediated by REST, including miR-137, which has not previously been shown to be a direct REST target in mouse. These data provide evidence of the contribution of inappropriate REST-mediated transcriptional repression to the widespread changes in coding and non-coding gene expression in a cellular model of HD that may affect normal neuronal function and survival.

Rescue of gene expression by modified REST decoy oligonucleotides in a cellular model of Huntington's disease

Transcriptional dysfunction is a prominent hallmark of Huntington's disease (HD). Several transcription factors have been implicated in the aetiology of HD progression and one of the most prominent is repressor element 1 (RE1) silencing transcription factor (REST). REST is a global repressor of neuronal gene expression and in the presence of mutant Huntingtin increased nuclear REST levels lead to elevated RE1 occupancy and a concomitant increase in target gene repression, including brain-derived neurotrophic factor. It is of great interest to devise strategies to reverse transcriptional dysregulation caused by increased nuclear REST and determine the consequences in HD. Thus far, such strategies have involved RNAi or mutant REST constructs. Decoys are double-stranded oligodeoxynucleotides corresponding to the DNA-binding element of a transcription factor and act to sequester it, thereby abrogating its transcriptional activity. Here, we report the use of a novel decoy strategy to rescue REST target gene expression in a cellular model of HD. We show that delivery of the decoy in cells expressing mutant Huntingtin leads to its specific interaction with REST, a reduction in REST occupancy of RE1s and rescue of target gene expression, including Bdnf. These data point to an alternative strategy for rebalancing the transcriptional dysregulation in HD.

Factors governing cellular convection in orographic precipitation

The development of shallow cellular convection in warm orographic clouds is investigated through idealized numerical simulations of moist flow over topography using a cloud-resolving numerical model. Buoyant instability, a necessary element for moist convection, is found to be diagnosed most accurately through analysis of the moist Brunt–Väisälä frequency (N_m) rather than the vertical profile of θ_e. In statically unstable orographic clouds (N_m^2) < 0), additional environmental and terrain-related factors are shown to have major effects on the amount of cellularity that occurs in 2D simulations. One of these factors, the basic-state wind shear, may suppress convection in 2D yet allow for longitudinal convective roll circulations in 3D. The presence of convective structures within an orographic cloud substantially enhanced the maximum rainfall rates, precipitation efficiencies, and precipitation accumulations in all simulations.

From a discrete to a continuum model of cell dynamics in one dimension

Multiscale modeling is emerging as one of the key challenges in mathematical biology. However, the recent rapid increase in the number of modeling methodologies being used to describe cell populations has raised a number of interesting questions. For example, at the cellular scale, how can the appropriate discrete cell-level model be identified in a given context? Additionally, how can the many phenomenological assumptions used in the derivation of models at the continuum scale be related to individual cell behavior? In order to begin to address such questions, we consider a discrete one-dimensional cell-based model in which cells are assumed to interact via linear springs. From the discrete equations of motion, the continuous Rouse [P. E. Rouse, J. Chem. Phys. 21, 1272 (1953)] model is obtained. This formalism readily allows the definition of a cell number density for which a nonlinear "fast" diffusion equation is derived. Excellent agreement is demonstrated between the continuum and discrete models. Subsequently, via the incorporation of cell division, we demonstrate that the derived nonlinear diffusion model is robust to the inclusion of more realistic biological detail. In the limit of stiff springs, where cells can be considered to be incompressible, we show that cell velocity can be directly related to cell production. This assumption is frequently made in the literature but our derivation places limits on its validity. Finally, the model is compared with a model of a similar form recently derived for a different discrete cell-based model and it is shown how the different diffusion coefficients can be understood in terms of the underlying assumptions about cell behavior in the respective discrete models.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine (DCVC) resulted in a >1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine ( DCVC) resulted in a > 1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Comparing a discrete and continuum model of the intestinal crypt

The integration of processes at different scales is a key problem in the modelling of cell populations. Owing to increased computational resources and the accumulation of data at the cellular and subcellular scales, the use of discrete, cell-level models, which are typically solved using numerical simulations, has become prominent. One of the merits of this approach is that important biological factors, such as cell heterogeneity and noise, can be easily incorporated. However, it can be difficult to efficiently draw generalizations from the simulation results, as, often, many simulation runs are required to investigate model behaviour in typically large parameter spaces. In some cases, discrete cell-level models can be coarse-grained, yielding continuum models whose analysis can lead to the development of insight into the underlying simulations. In this paper we apply such an approach to the case of a discrete model of cell dynamics in the intestinal crypt. An analysis of the resulting continuum model demonstrates that there is a limited region of parameter space within which steady-state (and hence biologically realistic) solutions exist. Continuum model predictions show good agreement with corresponding results from the underlying simulations and experimental data taken from murine intestinal crypts.

Improving a convection-permitting model simulation of a cold air outbreak

A convection-permitting local-area model was used to simulate a cold air outbreak crossing from the Norwegian Sea into the Atlantic Ocean near Scotland. A control model run based on an operational configuration of the Met Office UKV high-resolution (1.5 km grid spacing) NWP model was compared to satellite, aircraft and radar data. While the control model captured the large-scale features of the synoptic situation, it was not able to reproduce the shallow (<1.5 km) stratiform layer to the north of the open cellular convection. Liquid water paths were found to be too low in both the stratiform and convective cloud regions. Sensitivity analyses including a modified boundary-layer diagnosis to generate a more well-mixed boundary layer and inhibition of ice formation to lower temperatures improved cloud morphology and comparisons with observational data. Copyright © 2013 Royal Meteorological Society and British Crown Copyright, the Met Office

A mathematical model of the sterol regulatory element binding protein 2 cholesterol biosynthesis pathway

Cholesterol is one of the key constituents for maintaining the cellular membrane and thus the integrity of the cell itself. In contrast high levels of cholesterol in the blood are known to be a major risk factor in the development of cardiovascular disease. We formulate a deterministic nonlinear ordinary differential equation model of the sterol regulatory element binding protein 2 (SREBP-2) cholesterol genetic regulatory pathway in an hepatocyte. The mathematical model includes a description of genetic transcription by SREBP-2 which is subsequently translated to mRNA leading to the formation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a main precursor of cholesterol synthesis. Cholesterol synthesis subsequently leads to the regulation of SREBP-2 via a negative feedback formulation. Parameterised with data from the literature, the model is used to understand how SREBP-2 transcription and regulation affects cellular cholesterol concentration. Model stability analysis shows that the only positive steady-state of the system exhibits purely oscillatory, damped oscillatory or monotic behaviour under certain parameter conditions. In light of our findings we postulate how cholesterol homestasis is maintained within the cell and the advantages of our model formulation are discussed with respect to other models of genetic regulation within the literature.