Clonal Neuronal Cell Lines Research Articles

SerpinA3N is a novel hypothalamic gene upregulated by a high-fat diet and leptin in mice

BackgroundEnergy homeostasis is regulated by the hypothalamus but fails when animals are fed a high-fat diet (HFD), and leptin insensitivity and obesity develops. To elucidate the possible mechanisms underlying these effects, a microarray-based transcriptomics approach was used to identify novel genes regulated by HFD and leptin in the mouse hypothalamus.ResultsMouse global array data identified serpinA3N as a novel gene highly upregulated by both a HFD and leptin challenge. In situ hybridisation showed serpinA3N expression upregulation by HFD and leptin in all major hypothalamic nuclei in agreement with transcriptomic gene expression data. Immunohistochemistry and studies in the hypothalamic clonal neuronal cell line, mHypoE-N42 (N42), confirmed that alpha 1-antichymotrypsin (α1AC), the protein encoded by serpinA3, is localised to neurons and revealed that it is secreted into the media. SerpinA3N expression in N42 neurons is upregulated by palmitic acid and by leptin, together with IL-6 and TNFα, and all three genes are downregulated by the anti-inflammatory monounsaturated fat, oleic acid. Additionally, palmitate upregulation of serpinA3 in N42 neurons is blocked by the NFκB inhibitor, BAY11, and the upregulation of serpinA3N expression in the hypothalamus by HFD is blunted in IL-1 receptor 1 knockout (IL-1R1−/−) mice.ConclusionsThese data demonstrate that serpinA3 expression is implicated in nutritionally mediated hypothalamic inflammation.

Generation of a Phenotypic Array of Hypothalamic Neuronal Cell Models to Study Complex Neuroendocrine Disorders

Knowledge of how the brain achieves its diverse central control of basic physiology is severely limited by the virtual absence of appropriate cell models. Isolation of clonal populations of unique peptidergic neurons from the hypothalamus will facilitate these studies. Herein we describe the mass immortalization of mouse primary hypothalamic cells in monolayer culture, resulting in the generation of a vast representation of hypothalamic cell types. Subcloning of the heterogeneous cell populations resulted in the establishment of 38 representative clonal neuronal cell lines, of which 16 have been further characterized by analysis of 28 neuroendocrine markers. These cell lines represent the first available models to study the regulation of neuropeptides associated with the control of feeding behavior, including neuropeptide Y, ghrelin, urocortin, proopiomelanocortin, melanin-concentrating hormone, neurotensin, proglucagon, and GHRH. Importantly, a representative cell line responds appropriately to leptin stimulation and results in the repression of neuropeptide Y gene expression. These cell models can be used for detailed molecular analysis of neuropeptide gene regulation and signal transduction events involved in the direct hormonal control of unique hypothalamic neurons, not yet possible in the whole brain. Such studies may contribute information necessary for the strategic design of therapeutic interventions for complex neuroendocrine disorders, such as obesity.

Proton-gated channels in PC12 cells.

Acid-sensing ion channels (ASICs) are expressed in various sensory and central neurons. The functional role of these channels remains elusive. Complex subunit combinations and lack of specific blockers for native receptors are likely to contribute to the difficulty of resolving the function of ASICs. Finding a neuronal cell line, which expresses a single population of ASICs, should prove to be useful in delineating the function of individual ASICs. Using patch-clamp, Ca(2+)-imaging, and RT-PCR techniques, we have explored the existence of ASICs in PC12 cells, a clonal neuronal cell line. Fast drops of extracellular pH activated transient inward currents in PC12 cells with pH(0.5) at 6.0-6.2. The ASICs in PC12 cells were selective for Na(+) with significant Ca(2+) permeability. Currents in PC12 cells were blocked by the nonselective ASIC blocker amiloride. PcTX1, a specific homomeric ASIC1a blocker, also blocked the ASIC currents with an IC(50) of approximately 1.5 nM. RT-PCR demonstrated the existence of ASIC1a transcript in both undifferentiated and nerve growth factor-differentiated PC12 cells. Our data suggest that PC12 cells likely contain a single population of functional proton-gated channel-homomeric ASIC1a. It might be an ideal neuronal cell line for the study of physiological and potential pathological roles of this key subunit of ASICs.

Oxidative Mechanisms in β-Amyloid Cytotoxicity

Amyloid β-peptide has been demonstrated to be toxic for primary and clonal neuronal cell lines in vitro. Oxidative mechanisms have been implicated in this pathway at several points, including the aggregation of β-amyloid necessary for cytotoxic activity, generation of radicals by the peptide itself, and intracellularly in response to toxic β-amyloid peptides. Supporting an oxidative hypothesis are the observations that cells mount a stress response to β-amyloid similar to that seen in response to oxidative stress and that they may be rescued from cytotoxicity by antioxidants, inhibitors of oxidative enzyme metabolism, and overexpression of antioxidant enzymes. Although the source(s) of the oxygen radicals has not yet been identified, altered antioxidant enzyme levels and oxidative by-products in Alzheimer's disease brain samples relate the in vitrostudies to the human disease.

Generation of Neuronal Cell Lines from Mitotic and Postmitotic Rat Hippocampal Cultures

Clonal neuronal cell lines of rat hippocampal origin have been developed by two alternative strategies using retroviral transduction of a temperature-sensitive simian virus 40 large tumor antigen (T ts). Initially, T ts was introduced into dividing E17 rat embryonal hippocampal cells. In method 1, cell lines were generated directly from these progenitor cells by shifting the transductants to the permissive temperature under conditions promoting proliferation. Cell lines generated by this approach derived from neuronal, glial, or bipotential lineage. Alternatively, in method 2, the E17 rat hippocampal cells were infected with the T ts gene and then cultured at the nonpermissive temperature to enrich for postmitotic pyramidal neurons. The cells were shifted to the permissive temperature to allow the T ts gene product to promote cell cycle progression and were then cloned. Both approaches yielded cells that could differentiate in culture at the nonpermissive temperature and that expressed characteristics of a neuronal phenotype, ranging from expression of neuronal proteins such as neurofilament and induction of action potentials to synapse formation. These results indicate that it is possible to generate clonal neuronal cell lines from both precursor and differentiated hippocampal neurons and provide valuable model systems for investigating hippocampal neuronal survival, maturation, and function.

Clonal neuronal cell lines in neuroendocrine systems and the regulation of pulsatile secretion of neurapeptides

Gonadotropin-releasing hormone (GnRH) neurons exert the ultimate central neural control over reproduction in vertebrates. GnRH neurons were immortalized by genetically targeted tumorigenesis in transgenic mice in order to obtain GT1 cell lines. Over the past several years, GT1 cells have become a valuable model for investigating GnRH neurobiology. These cells have been used to determine what receptor populations are present on GnRH neurons, to examine the effects of various neuromediators on GnRH release, to demonstrate that pulsatile release is an intrinsic property of GnRH neurons, and (more recently) to begin to investigate how episodic GnRH release is generated and synchronized. GT1 cells are also proving to be useful in the study of central neural effects of neurotropic factors, both with regard to their effects upon GnRH neurons and as a general model of a central nervous system neuron.

The cholecystokinin gene is abundantly co-expressed with gastrin-releasing peptide, enkephalin and neuropeptide Y genes in a clonal human neuroepithelioma cell line

The cholinergic human neuroepithelioma cell line SK-N-MCIXC expressed mRNAs for the neuropeptides cholecystokinin (CCK), neuropeptide Y, gastrin-releasing peptide (GRP) and enkephalin. The CCK transcript of about 800 nt was present at very high levels and CCK-like peptides immunoreactive to a C-terminal CCK octapeptide antiserum were present in the cell line and its medium. This clonal neuronal cell line provides a unique model system to identify cis- and trans-acting factors responsible for neuron-specific expression and regulation of the CCK gene. Further-more, the pluripotent properties of the undifierentiated cell line may open studies on neuronal differentiation at the level of co-expression of neuropeptides and transmitters.