Human disc cells in monolayer vs 3D culture: cell shape, division and matrix formation

BACKGROUND: The relationship between cell shape, proliferation, and extracellular matrix (ECM) production, important aspects of cell behavior, is examined in a little-studied cell type, the human annulus cell from the intervertebral disc, during monolayer vs three-dimensional (3D) culture. RESULTS: Three experimental studies showed that cells respond specifically to culture microenvironments by changes in cell shape, mitosis and ECM production: 1) Cell passages showed extensive immunohistochemical evidence of Type I and II collagens only in 3D culture. Chondroitin sulfate and keratan sulfate were abundant in both monolayer and 3D cultures. 2) Cells showed significantly greater proliferation in monolayer in the presence of platelet-derived growth factor compared to cells in 3D. 3) Cells on Matrigel™-coated monolayer substrates became rounded and formed nodular colonies, a finding absent during monolayer growth. CONCLUSIONS: The cell's in vivo interactions with the ECM can regulate shape, gene expression and other cell functions. The shape of the annulus cell changes markedly during life: the young, healthy disc contains spindle shaped cells and abundant collagen. With aging and degeneration, many cells assume a strikingly different appearance, become rounded and are surrounded by unusual accumulations of ECM products. In vitro manipulation of disc cells provides an experimental window for testing how disc cells from given individuals respond when they are grown in environments which direct cells to have either spindle- or rounded-shapes. In vitro assessment of the response of such cells to platelet-derived growth factor and to Matrigel™ showed a continued influence of cell shape even in the presence of a growth factor stimulus. These findings contribute new information to the important issue of the influence of cell shape on cell behavior

Structural Olfactory Nerve Changes in Patients Suffering from Idiopathic Intracranial Hypertension

BACKGROUND: Complications of idiopathic intracranial hypertension (IIH) are usually caused by elevated intracranial pressure (ICP). In a similar way as in the optic nerve, elevated ICP could also compromise the olfactory nerve system. On the other side, there is growing evidence that an extensive lymphatic network system around the olfactory nerves could be disturbed in cerebrospinal fluid disorders like IIH. The hypothesis that patients with IIH suffer from hyposmia has been suggested in the past. However, this has not been proven in clinical studies yet. This pilot study investigates whether structural changes of the olfactory nerve system can be detected in patients with IIH. METHODOLOGY/PRINCIPAL FINDINGS: Twenty-three patients with IIH and 23 matched controls were included. Olfactory bulb volume (OBV) and sulcus olfactorius (OS) depth were calculated by magnetic resonance techniques. While mean values of total OBV (128.7±38.4 vs. 130.0±32.6 mm(3), p=0.90) and mean OS depth (8.5±1.2 vs. 8.6±1.1 mm, p=0.91) were similar in both groups, Pearson correlation showed that patients with a shorter medical history IIH revealed a smaller OBV (r=0.53, p<0.01). In untreated symptomatic patients (n=7), the effect was greater (r=0.76, p<0.05). Patients who suffered from IIH for less than one year (n=8), total OBV was significantly smaller than in matched controls (116.6±24.3 vs. 149.3±22.2 mm(3), p=0.01). IIH patients with visual disturbances (n=21) revealed a lower OS depth than patients without (8.3±0.9 vs. 10.8±1.0 mm, p<0.01). CONCLUSIONS/SIGNIFICANCE: The results suggest that morphological changes of the olfactory nerve system could be present in IIH patients at an early stage of disease

Multisite Phosphorylation Provides an Effective and Flexible Mechanism for Switch-Like Protein Degradation

Phosphorylation-triggered degradation is a common strategy for elimination of regulatory proteins in many important cell signaling processes. Interesting examples include cyclin-dependent kinase inhibitors such as p27 in human and Sic1 in yeast, which play crucial roles during the G1/S transition in the cell cycle. In this work, we have modeled and analyzed the dynamics of multisite-phosphorylation-triggered protein degradation systematically. Inspired by experimental observations on the Sic1 protein and a previous intriguing theoretical conjecture, we develop a model to examine in detail the degradation dynamics of a protein featuring multiple phosphorylation sites and a threshold site number for elimination in response to a kinase signal. Our model explains the role of multiple phosphorylation sites, compared to a single site, in the regulation of protein degradation. A single-site protein cannot convert a graded input of kinase increase to much sharper output, whereas multisite phosphorylation is capable of generating a highly switch-like temporal profile of the substrate protein with two characteristics: a temporal threshold and rapid decrease beyond the threshold. We introduce a measure termed temporal response coefficient to quantify the extent to which a response in the time domain is switch-like and further investigate how this property is determined by various factors including the kinase input, the total number of sites, the threshold site number for elimination, the order of phosphorylation, the kinetic parameters, and site preference. Some interesting and experimentally verifiable predictions include that the non-degradable fraction of the substrate protein exhibits a more switch-like temporal profile; a sequential system is more switch-like, while a random system has the advantage of increased robustness; all the parameters, including the total number of sites, the threshold site number for elimination and the kinetic parameters synergistically determine the exact extent to which the degradation profile is switch-like. Our results suggest design principles for protein degradation switches which might be a widespread mechanism for precise regulation of cellular processes such as cell cycle progression

Pilocarpine-Induced Status Epilepticus in Rats Involves Ischemic and Excitotoxic Mechanisms

The neuron loss characteristic of hippocampal sclerosis in temporal lobe epilepsy patients is thought to be the result of excitotoxic, rather than ischemic, injury. In this study, we assessed changes in vascular structure, gene expression, and the time course of neuronal degeneration in the cerebral cortex during the acute period after onset of pilocarpine-induced status epilepticus (SE). Immediately after 2 hr SE, the subgranular layers of somatosensory cortex exhibited a reduced vascular perfusion indicative of ischemia, whereas the immediately adjacent supragranular layers exhibited increased perfusion. Subgranular layers exhibited necrotic pathology, whereas the supergranular layers were characterized by a delayed (24 h after SE) degeneration apparently via programmed cell death. These results indicate that both excitotoxic and ischemic injuries occur during pilocarpine-induced SE. Both of these degenerative pathways, as well as the widespread and severe brain damage observed, should be considered when animal model-based data are compared to human pathology

Parallel Odor Processing by Two Anatomically Distinct Olfactory Bulb Target Structures

The olfactory cortex encompasses several anatomically distinct regions each hypothesized to provide differential representation and processing of specific odors. Studies exploring whether or not the diversity of olfactory bulb input to olfactory cortices has functional meaning, however, are lacking. Here we tested whether two anatomically major olfactory cortical structures, the olfactory tubercle (OT) and piriform cortex (PCX), differ in their neural representation and processing dynamics of a small set of diverse odors by performing in vivo extracellular recordings from the OT and PCX of anesthetized mice. We found a wealth of similarities between structures, including odor-evoked response magnitudes, breadth of odor tuning, and odor-evoked firing latencies. In contrast, only few differences between structures were found, including spontaneous activity rates and odor signal-to-noise ratios. These results suggest that despite major anatomical differences in innervation by olfactory bulb mitral/tufted cells, the basic features of odor representation and processing, at least within this limited odor set, are similar within the OT and PCX. We predict that the olfactory code follows a distributed processing stream in transmitting behaviorally and perceptually-relevant information from low-level stations

Loss of STOP Protein Impairs Peripheral Olfactory Neurogenesis

International audienceIn conclusion, STOP protein seems to be involved in the establishment of synapses in the olfactory glomerulus. Our results indicate that the olfactory system of STOP null mice is a well-suited experimental model (1) for the study of the mechanism of action of STOP protein in synaptic function/plasticity and (2) for pathophysiological studies of the mechanisms of altered neuronal connections in schizophrenia

Galanin Receptor 1 Deletion Exacerbates Hippocampal Neuronal Loss after Systemic Kainate Administration in Mice

Galanin is a neuropeptide with a wide distribution in the central and peripheral nervous systems and whose physiological effects are mediated through three G protein-coupled receptor subtypes, GalR1, GalR2, and GalR3. Several lines of evidence indicate that galanin, as well as activation of the GalR1 receptor, is a potent and effective modulator of neuronal excitability in the hippocampus.In order to test more formally the potential influence of GalR1 on seizure-induced excitotoxic cell death, we conducted functional complementation tests in which transgenic mice that exhibit decreased expression of the GalR1 candidate mRNA underwent kainate-induced status epilepticus to determine if the quantitative trait of susceptibility to seizure-induced cell death is determined by the activity of GalR1. In the present study, we report that reduction of GalR1 mRNA via null mutation or injection of the GalR1 antagonist, galantide, prior to kainate-induced status epilepticus induces hippocampal damage in a mouse strain known to be highly resistant to kainate-induced neuronal injury. Wild-type and GalR1 knockout mice were subjected to systemic kainate administration. Seven days later, Nissl and NeuN immune- staining demonstrated that hippocampal cell death was significantly increased in GalR1 knockout strains and in animals injected with the GalR1 antagonist. Compared to GalR1-expressing mice, GalR1-deficient mice had significantly larger hippocampal lesions after status epilepticus.Our results suggest that a reduction of GalR1 expression in the C57BL/6J mouse strain renders them susceptible to excitotoxic injury following systemic kainate administration. From these results, GalR1 protein emerges as a new molecular target that may have a potential therapeutic value in modulating seizure-induced cell death

Segmental Duplications Arise from Pol32-Dependent Repair of Broken Forks through Two Alternative Replication-Based Mechanisms

The propensity of segmental duplications (SDs) to promote genomic instability is of increasing interest since their involvement in numerous human genomic diseases and cancers was revealed. However, the mechanism(s) responsible for their appearance remain mostly speculative. Here, we show that in budding yeast, replication accidents, which are most likely transformed into broken forks, play a causal role in the formation of SDs. The Pol32 subunit of the major replicative polymerase Polδ is required for all SD formation, demonstrating that SDs result from untimely DNA synthesis rather than from unequal crossing-over. Although Pol32 is known to be required for classical (Rad52-dependant) break-induced replication, only half of the SDs can be attributed to this mechanism. The remaining SDs are generated through a Rad52-independent mechanism of template switching between microsatellites or microhomologous sequences. This new mechanism, named microhomology/microsatellite-induced replication (MMIR), differs from all known DNA double-strand break repair pathways, as MMIR-mediated duplications still occur in the combined absence of homologous recombination, microhomology-mediated, and nonhomologous end joining machineries. The interplay between these two replication-based pathways explains important features of higher eukaryotic genomes, such as the strong, but not strict, association between SDs and transposable elements, as well as the frequent formation of oncogenic fusion genes generating protein innovations at SD junctions

Cerebral cortex expression of Gli3 is required for normal development of the lateral olfactory tract

<div><p>Formation of the lateral olfactory tract (LOT) and innervation of the piriform cortex represent fundamental steps to allow the transmission of olfactory information to the cerebral cortex. Several transcription factors, including the zinc finger transcription factor Gli3, influence LOT formation by controlling the development of mitral cells from which LOT axons emanate and/or by specifying the environment through which these axons navigate. <i>Gli3</i> null and hypomorphic mutants display severe defects throughout the territory covered by the developing lateral olfactory tract, making it difficult to identify specific roles for <i>Gli3</i> in its development. Here, we used <i>Emx1Cre</i>;<i>Gli3</i>^<i>fl/fl</i> conditional mutants to investigate LOT formation and colonization of the olfactory cortex in embryos in which loss of <i>Gli3</i> function is restricted to the dorsal telencephalon. These mutants form an olfactory bulb like structure which does not protrude from the telencephalic surface. Nevertheless, mitral cells are formed and their axons enter the piriform cortex though the LOT is shifted medially. Mitral axons also innervate a larger target area consistent with an enlargement of the piriform cortex and form aberrant projections into the deeper layers of the piriform cortex. No obvious differences were found in the expression patterns of key guidance cues. However, we found that an expansion of the piriform cortex temporally coincides with the arrival of LOT axons, suggesting that <i>Gli3</i> affects LOT positioning and target area innervation through controlling the development of the piriform cortex.</p></div

The location of olfactory receptors within olfactory epithelium is independent of odorant volatility and solubility

<p>Abstract</p> <p>Background</p> <p>Our objective was to study the pattern of olfactory receptor expression within the dorsal and ventral regions of the mouse olfactory epithelium. We hypothesized that olfactory receptors were distributed based on the chemical properties of their ligands: e.g. receptors for polar, hydrophilic and weakly volatile odorants would be present in the dorsal region of olfactory epithelium; while receptors for non-polar, more volatile odorants would be distributed to the ventral region. To test our hypothesis, we used micro-transplantation of cilia-enriched plasma membranes derived from dorsal or ventral regions of the olfactory epithelium into Xenopus oocytes for electrophysiological characterization against a panel of 100 odorants.</p> <p>Findings</p> <p>Odorants detected by ORs from the dorsal and ventral regions showed overlap in volatility and water solubility. We did not find evidence for a correlation between the solubility and volatility of odorants and the functional expression of olfactory receptors in the dorsal or ventral region of the olfactory epithelia.</p> <p>Conclusions</p> <p>No simple clustering or relationship between chemical properties of odorants could be associated with the different regions of the olfactory epithelium. These results suggest that the location of ORs within the epithelium is not organized based on the physico-chemical properties of their ligands.</p