Simulation of developing human neuronal cell networks

BACKGROUND: Microelectrode array (MEA) is a widely used technique to study for example the functional properties of neuronal networks derived from human embryonic stem cells (hESC-NN). With hESC-NN, we can investigate the earliest developmental stages of neuronal network formation in the human brain. METHODS: In this paper, we propose an in silico model of maturating hESC-NNs based on a phenomenological model called INEX. We focus on simulations of the development of bursts in hESC-NNs, which are the main feature of neuronal activation patterns. The model was developed with data from developing hESC-NN recordings on MEAs which showed increase in the neuronal activity during the investigated six measurement time points in the experimental and simulated data. RESULTS: Our simulations suggest that the maturation process of hESC-NN, resulting in the formation of bursts, can be explained by the development of synapses. Moreover, spike and burst rate both decreased at the last measurement time point suggesting a pruning of synapses as the weak ones are removed. CONCLUSIONS: To conclude, our model reflects the assumption that the interaction between excitatory and inhibitory neurons during the maturation of a neuronal network and the spontaneous emergence of bursts are due to increased connectivity caused by the forming of new synapses.BioMed Central open acces

Functional expression of NF1 tumor suppressor protein: association with keratin intermediate filaments during the early development of human epidermis

BACKGROUND: NF1 refers to type 1 neurofibromatosis syndrome, which has been linked with mutations of the large NF1 gene. NF1 tumor suppressor protein, neurofibromin, has been shown to regulate ras: the NF1 protein contains a GTPase activating protein (GAP) related domain which functions as p21rasGAP. Our studies have previously demonstrated that the NF1 protein forms a high affinity association with cytokeratin 14 during the formation of desmosomes and hemidesmosomes in cultured keratinocytes. METHODS: The expression of NF1 protein was studied in developing human epidermis using western transfer analysis, indirect immunofluorescence, confocal laser scanning microscopy, immunoelectron microscopy, and in situ hybridization. RESULTS: The expression of NF1 protein was noted to be highly elevated in the periderm at 8 weeks estimated gestational age (EGA) and in the basal cells at 8–14 weeks EGA. During this period, NF1 protein was associated with cytokeratin filaments terminating to desmosomes and hemidesmosomes. NF1 protein did not display colocalization with α-tubulin or actin of the cytoskeleton, or with adherens junction proteins. CONCLUSIONS: These results depict an early fetal period when the NF1 tumor suppressor is abundantly expressed in epidermis and associated with cytokeratin filaments. This period is characterized by the initiation of differentiation of the basal cells, maturation of the basement membrane zone as well as accentuated formation of selected cellular junctions. NF1 tumor suppressor may function in the regulation of epidermal histogenesis via controlling the organization of the keratin cytoskeleton during the assembly of desmosomes and hemidesmosomes

NF1 tumor suppressor in skin:expression in response to tissue trauma and in cellular differentiation

Abstract Type 1 neurofibromatosis (NF1) syndrome is caused by a mutation of the NF1 gene. NF1 protein (neurofibromin) contains a domain which is related to the GTPase activating protein (GAP) and accelerates the switch of active Ras-GTP to inactive Ras-GDP. The clinical symptoms of NF1 patients include e.g. the formation of benign neurofibroma tumors and hyperpigmented lesions of the skin. The NF1 protein has been referred to as a tumor suppressor since cells of malignant schwannomas of NF1 patients may display loss of heterozygosity of the NF1 gene. In the present study, the expression of the NF1 gene was investigated during tissue repair in human skin. Elevated NF1 protein levels were seen in a fibroblastic cell population of healing wounds. In vitro studies were designed to investigate NF1 expression in dermal fibroblasts under the influence of growth factors that are operative during wound healing. Platelet-derived growth factor (PDGF) isoforms AB and BB and transforming growth factor β1 (TGFβ1) elevated NF1 mRNA levels in cultured dermal fibroblasts. In further studies, histological examination on apparently healthy skin of NF1 patients revealed frequently small masses of neurofibromatous tissue at the vicinity of hair follicles. Thus, action of the NF1 gene appears to be an integral part of normal tissue repair. Enhanced NF1 tumor suppressor expression may serve to limit excessive fibrosis in wound healing. As Ras proteins play a role in the regulation of cell differentiation and formation of cell junctions, the functional expression of NF1 protein was elucidated using differentiating keratinocytes as an in vitro model system. The results demonstrate that an intense NF1 tumor suppressor signal on intermediate filaments was temporally limited to the period in which the formation of desmosomes takes place. In analogy to NF1 protein, a rapid elevation of NF1 mRNA level was detected following initiation of differentiation. Interestingly, NF1 mRNA hybridization signal polarized towards cell-cell contact zones. This finding recognizes a potential way for post-transcriptional modification of NF1 expression and targeting of translation to subplasmalemmal location. The results demonstrate that the function of NF1 protein is associated with the formation of cell junctions, and thus to cellular communication

Class III β-Tubulin Is a Component of the Mitotic Spindle in Multiple Cell Types

The findings of this study show that Class III β-tubulin is a component of the mitotic spindle in multiple cell types. Class III β-tubulin has been widely used as a neuron-specific marker, but it has been detected also in association with breast and pancreatic cancers. In this study, we describe a novel finding of Class III β-tubulin in a subpopulation of cells in malignant peripheral nerve sheath tumor. The findings of this study also show that Class III β-tubulin is expressed by normal mesenchymal and epithelial cells (fibroblasts and keratinocytes), two transitional cell carcinoma cell lines, and neurofibroma Schwann cells, as shown by immunolabeling and Western transfer analysis using two different Tuj-1 antibodies that are specific for Class III β-tubulin. The corresponding mRNA was detected using RT-PCR and whole human genome microarrays. Both antibodies localized Class III β-tubulin to the mitotic spindle and showed a colocalization with α-tubulin. The immunoreaction became visible in early prophase, and the most intense immunoreaction was detected during metaphase and anaphase when microtubules were connected to the kinetochores on chromosomes. Class III β-tubulin–specific immunoreaction lasted to the point when the midbody of cytokinesis became detectable. (J Histochem Cytochem 56:1113–1119, 2008

Mimicking exercise in vitro - effects of myotube contractions and mechanical stretch on omics

The number of studies using skeletal muscle (SkM) cell culture models to study exercise in vitro are rapidly expanding. Progressively, more comprehensive analysis methods, such as different omics approaches including transcriptomics, proteomics and metabolomics have been used to examine the intra- and extracellular molecular responses to exercise mimicking stimuli in cultured myotubes. Among other techniques, exercise-like electrical pulse stimulation (EL-EPS) and mechanical stretch of SkM cells are the two most commonly used methods to mimic exercise in vitro. In this mini-review we focus on these two approaches and their effects on the omics of myotubes and/or cell culture media. Furthermore, besides traditional two-dimensional (2D) methods, the use of three-dimensional (3D) SkM approaches are increasing in the field of in vitro exercise mimicry. Our aim with this mini-review is to provide the reader with an up-to-date overview of the 2D and 3D models and the use of omics approaches to study the molecular response to exercise in vitro.peerReviewe