The multimodal Munich Clinical Deep Phenotyping study to bridge the translational gap in severe mental illness treatment research.

Introduction: Treatment of severe mental illness (SMI) symptoms, especially negative symptoms and cognitive dysfunction in schizophrenia, remains a major unmet need. There is good evidence that SMIs have a strong genetic background and are characterized by multiple biological alterations, including disturbed brain circuits and connectivity, dysregulated neuronal excitation-inhibition, disturbed dopaminergic and glutamatergic pathways, and partially dysregulated inflammatory processes. The ways in which the dysregulated signaling pathways are interconnected remains largely unknown, in part because well-characterized clinical studies on comprehensive biomaterial are lacking.

Expression of LGI1 Impairs Proliferation and Survival of HeLa Cells.

The LGI1 gene was suggested to function as tumor suppressor for its ability to reduce malignant features of glioblastoma cells. In support to this proposal were the findings that overexpression of LGI1 in neuroblastoma cells inhibited proliferation and induced apoptosis. In this study we performed stable LGI1 expression in HeLa cells to examine whether the noxious effect of LGI1 might be extended to cancer cells of diverse origin.

Increased expression of LGI1 gene triggers growth inhibition and apoptosis of neuroblastoma cells.

The LGI1 gene has been implicated in the malignant progression of glioblastoma and it has also been genetically linked to a form of partial epilepsy (ADLTE). In this study, we investigated the relevance of LGI1 expression for neuroblastoma cells. The analysis of two cell lines (SH-SY5Y and SK-N-BE) revealed unpredictably low levels of LGI1 and stable cell transfection with LGI1 cDNA yielded moderate increases of LGI1 expression.

Downstream regulatory element antagonist modulator regulates Ca2+ homeostasis and viability in cerebellar neurons.

The Na+/Ca2+ exchangers NCX1, NCX2, and NCX3 are vital for the control of cellular Ca2+ homeostasis. Here, we show that a doublet of downstream regulatory element sites in the promoter of the NCX3 gene mediates transcriptional repression of NCX3 by the Ca2+-modulated transcriptional repressor downstream regulatory element antagonist modulator (DREAM). Overexpression of a DREAM EF-hand mutant insensitive to Ca2+ (EFmDREAM) in hippocampus and cerebellum of transgenic mice significantly reduced NCX3 mRNA and protein levels without modifying NCX1 and NCX2 expression.

Transcriptional regulation by cAMP and Ca2+ links the Na+/Ca2+ exchanger 3 to memory and sensory pathways.

The signaling cascades triggered by neurotrophins such as BDNF and by several neurotransmitters and hormones lead to the rapid induction of gene transcription by increasing the intracellular concentration of cAMP and Ca2+. This review examines the mechanisms by which these second messengers control transcriptional initiation at CRE promoters via transcription factor CREB, as well as at DRE sites via transcriptional repressor DREAM. The regulation of the SLC8A3 gene encoding the Na+/Ca2+ exchanger 3 (NCX3) is taken as an example to illustrate both mechanisms since it includes a CRE site in the promoter and several DRE sites in the exon 1 sequence.

Photosensitization of DNA strand breaks by three phenothiazine derivatives.

The interaction and the photosensitizing activity of three phenothiazine derivatives, fluphenazine hydrochloride (FP), thioridazine hydrochloride (TR), and perphenazine (PP), toward DNA were studied. Evidences obtained from various spectroscopic studies such as fluorimetric and linear dichroism measurements indicate that these derivatives bind to the DNA at least in two ways: intercalation and external stacking on the DNA helix, depending on their relative concentrations. Irradiation of supercoiled plasmid DNA in the presence of these phenothiazines leads to single strand breaks.

Naphthoquinolizinium derivatives as a novel platform for DNA-binding and DNA-photodamaging chromophores.

The association of the naphtho[1,2-b]quinolizinium bromide (5a) and naphtho[2,1-b]quinolizinium bromide (5b) with DNA and the propensity of these cationic arenes to damage DNA after UV-A irradiation have been studied. Spectrophotometric and fluorimetric titrations show that the two isomers 5a and 5b bind to DNA (K approximately 10(5) M(-1)). The highest affinity was observed for GC base pairs.

Control of the Na+/Ca2+ exchanger 3 promoter by cyclic adenosine monophosphate and Ca2+ in differentiating neurons.

The human gene for member 3 of solute carrier family 8 (SLC8A3), encoding the Na+/Ca2+ exchanger isoform 3 (NCX3), was identified on chromosome 14q24.2. The minimal promoter region was predicted 250 bp upstream of exon 1.

The human SLC8A3 gene and the tissue-specific Na+/Ca2+ exchanger 3 isoforms.

We have identified the human gene for member 3 of Solute Carrier family 8 (SLC8A3) by bioinformatic analysis of human genomic sequences. The gene is located on chromosome 14q24.2, and spans a region of about 150 kb.

Indolo[2,3-b]-quinolizinium bromide: an efficient intercalator with DNA-photodamaging properties.

The associative interactions of indolo[2,3-b]-quinolizinium bromide with DNA and its DNA photocleavage properties were studied in detail. Absorption and emission spectroscopy, linear dichroism, and energy-transfer measurements indicate that the indoloquinolizinium binds to DNA primarily by intercalation, with a preference for GC base pairs. In agreement with this data, the results of primer extension analysis indicate that photocleavage occurrs prevalently at the GC nucleotides.

Acridizinium salts as a novel class of DNA-binding and site-selective DNA-photodamaging chromophores.

It was demonstrated that the interaction of the aminoacridizinium salts 2a-2d with DNA depends on the substitution pattern of the chromophore. Spectrophotometric and fluorometric titrations of the acridizinium salts 2a-2d with natural and synthetic polynucleotides reveal that the degree of interaction of the acridizinium salts 2a-2d with the nucleic acid differs significantly. The binding mode of the dyes with DNA was evaluated by circular dichroism and linear dichroism spectroscopy and compared with the parent system 2c.

A polymorphic GT repeat from the human cardiac Na+Ca2+ exchanger intron 2 activates splicing.

The sequence analysis of the human intron 2 from the Na+/Ca2+ exchanger 1 (NCX1) gene has revealed a GT repeat of variable length (10-16). The 5' sequence of intron 2 exhibited significant homology (65-70%) with other minisatellite sequences. DNA segments at the 5' end of intron 2 were inserted in the NCX1 cDNA (3.

Electrophysiological characterization of ionic transport by the retinal exchanger expressed in human embryonic kidney cells.

The retinal Na+:Ca2+, K+exchanger cDNA was transiently expressed in human embryonic kidney (HEK 293) cells by transfection with plasmid DNA. The correct targeting of the expressed protein to the plasma membrane was confirmed by immunocytochemistry. The reverse exchange offrent (Ca2+ imported per Na+ extruded) was measured in whole-cell voltage-clamp experiments after intracellular perfusion with Na+ (Na+i, 128 mM) and extracellular perfusion with Ca2+ (Ca2o+, 1 mM) and Ko+ (20 mM).

Expression of an active Na+/Ca2+ exchanger isoform lacking the six C-terminal transmembrane segments.

The short isoform of the Na+/Ca2+ exchanger (67 kDa) that is produced by alternative splicing during the expression of the 6 kb canine exchanger cDNA in 293 cells was separately expressed in the same system. The protein consisted of the five N-terminal transmembrane segments and of a large portion of the main hydrophilic loop, but lacked the six C-terminal hydrophobic segments of the regular protein (108 kDa). Very high RNA levels were found after transient cell transfection with plasmid DNA encoding this truncated isoform.

Expression of Na(+)-Ca2+ exchanger with modified C-terminal hydrophobic domains and enhanced activity.

A 6-Kb canine cDNA fragment complementary to the 5' region of the 7-Kb mRNA encoding the cardiac Na+-Ca2+ exchanger was expressed in human kidney 293 cells. The mRNA products were reverse transcribed and amplified by PCR. The determined DNA sequence of the amplified DNA fragments revealed the presence of an intron that was alternatively spliced.

An alternative splicing site modifies the carboxyl-terminal trans-membrane domains of the Na+/Ca2+ exchanger.

The 6-kilobase (kb) cDNA of pTB11 clone and its 5' fragment of 3.7 kb encoding the canine heart Na+/Ca2+ exchanger (Nicoll, D.A.

Nerve growth factor production by lymphocytes.

Nerve growth factor (NGF) is a neurotrophic protein essential for the maintenance and growth of peripheral sympathetic neurons and basal forebrain cholinergic neurons. Recently, NGF has also been shown to have effects on cells of the immune system. In a search for extra neural sources of NGF, we detected NGF-specific mRNA in mouse T lymphocytes of both the CD4+ and CD8+ phenotypes with the use of an RNase protection assay, PCR, and DNA sequence analysis.

Is the heterologous expression of metabotropic glutamate receptors (mGluRs) an appropriate method to study the mGluR function? Experience with human embryonic kidney 293 cells transfected with mGluR1.

The cloning of metabotropic glutamate receptors (mgluRs) has initiated a new approach to the study of their function: the introduction of mGluR cDNA into cells that do not normally express mGluRs, thus allowing the heterologous receptor expression. We have transfected human embryonic kidney (HEK) 293 cells with the full length mGluR1a cDNA and with its truncated variant which encodes the receptor termed mGluR1T (a receptor lacking the long intracellular domain and similar to the splice variant mGluR1c). Transient transfection of HEK-293 cells with mGluR1a, but not the mGluR1T cDNA, resulted in a significant increase in inositol phosphate (IP) formation in absence of any mGluR agonists.

Gene transfer in cultured ganglion neurons by DNA/calcium phosphate co-precipitation and gene activation by NGF signal.

The co-precipitation of DNA with calcium phosphate has been successfully employed to transfect cultured chicken embryonic sensory neurons (DRG). Up to 90% of the cultured DRG neurons were transfected by this method. This has allowed a study of the intracellular second messengers involved in signal transduction and gene activation by NGF in DRG neurons.

Nerve growth factor does not influence the expression of beta amyloid precursor protein mRNA in rat brain: in vivo and in vitro studies.

We investigated the effect of NGF on amyloid precursor protein (APP) mRNA levels in the rat septal/nucleus basalis system. Total APP mRNA and APP 695 mRNA were determined in basal forebrain primary cell cultures exposed acutely and chronically to NGF (150-300 ng/ml) and, in vivo, in the septal area and striatum of rat pups after multiple intracerebroventricular injections of NGF. The trophic factor was able to affect cholinergic neurons in both paradigms, as evidenced by the significant increase of choline acetyltransferase (ChAT) activity induced by NGF in cell cultures (+80%) and in the striatum (+240%) of rat pups.

Trans-azetidine-2,4-dicarboxylic acid activates neuronal metabotropic receptors.

The expression of metabotropic glutamate receptors (mGluRs) in primary cultures of cerebellar granule neurones can be: (i) modulated by the degree of depolarization during the culture period, rendering neurones differently sensitive to agonist-stimulated inositol phosphate (IP) hydrolysis; (ii) down-regulated by specific mGluR agonists. In this culture the new rigid glutamate analogue, (+/-)-trans-azetidine-2,4-dicarboxylic acid (t-ADA) and the known mGluR agonist 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) stimulated IP formation in line with the depolarization-modified expression of mGluR1. However, the two compounds caused different patterns of mGluR down-regulation.

Polyamines modulate the function of transfected glutamate receptor mGluR1a.

Metabotropic glutamate receptor mGluR1a was expressed in human embryonic kidney 293 cells. 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) stimulated dose-dependently, phosphoinositide (PI) hydrolysis in transfected, but not in non-transfected cells. The polyamine spermine did not affect PI hydrolysis in the absence of 1S,3R-ACPD even at a concentration of 1 mM, but it potentiated the stimulatory action of 1S,3R-ACPD at 10 microM.

Functional evidence for a L-AP3-sensitive metabotropic receptor different from glutamate metabotropic receptor mGluR1.

The efficacy of mGluR agonists quisqualate and 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) in stimulating the inositol phosphate (IP) formation in primary cultures of cerebellar granule neurons correlated with mGluR1 mRNA expression and was affected by the medium KCl content. L-2-Amino-3-phosphonopropionic acid (L-AP3) mimicked the stimulatory action of mGluR agonists. Maximal stimulatory doses of mGluR agonist 1S,3R-ACPD and L-AP3 were additive, suggesting the action of L-AP3 on a receptor different from mGluR1.