Polygenic risk for schizophrenia converges on alternative polyadenylation as molecular mechanism underlying synaptic impairment.

Schizophrenia (SCZ) is a genetically heterogenous psychiatric disorder of highly polygenic nature. Correlative evidence from genetic studies indicate that the aggregated effects of distinct genetic risk factor combinations found in each patient converge onto common molecular mechanisms. To prove this on a functional level, we employed a reductionistic cellular model system for polygenic risk by differentiating induced pluripotent stem cells (iPSCs) from 104 individuals with high polygenic risk load and controls into cortical glutamatergic neurons (iNs). Multi-omics profiling identified widespread differences in alternative polyadenylation (APA) in the 3' untranslated region of many synaptic transcripts between iNs from SCZ patients and healthy donors. On the cellular level, 3'APA was associated with a reduction in synaptic density of iNs. Importantly, differential APA was largely conserved between postmortem human prefrontal cortex from SCZ patients and healthy donors, and strongly enriched for transcripts related to synapse biology. 3'APA was highly correlated with SCZ polygenic risk and affected genes were significantly enriched for SCZ associated common genetic variation. Integrative functional genomic analysis identified the RNA binding protein and SCZ GWAS risk gene PTBP2 as a critical trans-acting factor mediating 3'APA of synaptic genes in SCZ subjects. Functional characterization of PTBP2 in iNs confirmed its key role in 3'APA of synaptic transcripts and regulation of synapse density. Jointly, our findings show that the aggregated effects of polygenic risk converge on 3'APA as one common molecular mechanism that underlies synaptic impairments in SCZ.

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. HeLa cell clones stably expressing LGI1 exhibited a significant impairment of proliferation and a consistent increase of cell death when compared with control cells lacking expression of LGI1. Expression of LGI1 increased the activity of apoptosis effectors caspase-3/7; furthermore it downregulated the antiapoptotic BCL2 gene and upregulated the proapoptotic BAX gene expression, suggesting that the cause of HeLa cells death might be an increased susceptibility to apoptosis induced by LGI1. The results suggested that LGI1 is capable to restrain growth and survival of adenocarcinoma cells such as HeLa.

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. Neuroblastoma cell clones exhibited impaired cell growth and survival ability in relation to LGI1 levels. The process of growth inhibition could be discerned under experimental conditions of low cell density, since conditions of elevated cell density, which enhance the requirement for survival stimuli, resulted in massive cellular death. At high cell density, spontaneous apoptosis of LGI1 cells was clearly shown by the release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria and by phosphatydil serine exposure and nuclear fragmentation. Activation of apoptotic effectors caspase-3/7 also occurred, however, the broad caspase inhibitor Z-VAD-FMK substantially failed to block cell death. Thus the possibility that LGI1-triggered apoptosis may involve initiator caspases linked to activation of death receptors, appears unlikely. The decreased ratio of Bcl-2 to Bax suggests that apoptosis is initiated by the intrinsic mitochondrial pathway through the release of caspase-dependent and -independent apoptogenic molecules. This study provides the first evidence that LGI1 controls neuronal cell survival, suggesting its role in the development of the nervous system in relation to the pathogenesis of neuroblastoma and ADLTE.

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. Cerebellar granules from EFmDREAM transgenic mice showed increased levels of cytosolic Ca2+ and were more vulnerable to increased Ca2+ influx after partial opening of voltage-gated plasma membrane Ca2+ channels induced by increasing K+ in the culture medium but survived better in the conditions of reduced Ca2+ influx prevailing in low extracellular K+. Overexpression of NCX3 in EFmDREAM transgenic granules using a lentiviral vector restored the normal survival response to high K+ observed in wild-type granules. Thus, the downregulation of the regulator of Ca2+ homeostasis NCX3 by Ca2+-regulated DREAM is a striking example of the autoregulatory property of the Ca2+ signal in neurons.

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. The upregulation of the NCX3 by Ca2+ signals may be specifically required to establish the Ca2+ balance that regulates several physiological and pathological processes in neurons. The regulatory features and the expression pattern of SLC8A3 gene suggest that NCX3 activity could be crucial in neuronal functions such as memory formation and sensory processing.

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. The DNA photocleavage appears to be due to externally bound molecules rather than to those intercalated. The highest photocleavage activity was observed with PP and TR whereas FP was less efficient. The efficiency of the photocleavage in aerated and deaerated solutions does not change thus indicating that an involvement of singlet oxygen can be excluded. Primer extension analysis of plasmid DNA irradiated in the presence of phenothiazines indicates that photocleavage of DNA occurs predominantly at Gua and Cyt residues. Laser flash experiments carried out in the presence of 2'-deoxyguanosine 5'-monophosphate reveal an efficient electron transfer between the nucleotide and the radical cations produced by photoionization of the phenothiazines. In the presence of DNA, an electron transfer process takes place within the laser pulse from the lowest singlet state of phenothiazines to the DNA bases; the time-resolved measurements showed that the back-electron transfer is a negligible decay pathway for the charged species.

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. This was confirmed by luciferase reporter assays of pGL3-promoter constructs in transfected SH-SY5Y cells. The promoter activity was monitored during the differentiation of this cell line elicited by the sequential treatment with retinoic acid and brain-derived neurotrophic factor (BDNF). The activity was induced by cyclic AMP (cAMP) via the CRE (cAMP response element) and was stimulated by retinoic acid. The increase of intracellular Ca2+ induced by the partial depolarization of the plasma membrane with KCl down-regulated both the basal and the cAMP-stimulated transcription. The down-regulation of the latter may be mediated by the phosphorylation of the CRE-binding protein by a calmodulin-dependent kinase (CaMKII). The exposure of cells to BDNF after treatment with retinoic acid rapidly induced promoter activity during the initial five hours and phosphorylation of CRE-binding protein during the first two hours. The promoter activity was further enhanced by cAMP, but became insensitive to Ca2+. In BDNF-stimulated cells cAMP elevation caused the preferential phosphorylation of ATF1 instead of that of CRE-binding protein.

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. The full-length DNA complementary to RNA encoding the Na(+)/Ca(2+) exchanger isoform 3 (NCX3), amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) from the human neuroblastoma SH-SY5Y RNA, includes seven exons and encodes a protein of about 100 kDa. RT-PCR analysis was performed in different tissues to determine the exon composition in the region encoding the large intracellular loop of the protein. The region underwent modifications by alternative tissue-specific splicing. NCX3.2, including exon 4 but not exon 5, was found in human brain and in the neuroblastoma cell line. In human skeletal muscle two additional isoforms were identified: NCX3.3, including exons 4 and 5, and a truncated isoform (NCX3.4) produced by the skipping of both exons 3 and 4. The skipping causes a frame shift downstream of the exon 2 sequence. The new coding sequence of 25 amino acids terminates with a stop codon in exon 6. The NCX3.4 isoform (68 kDa) is truncated in the C-terminal portion of the domain first found in Drosophila Na(+)/Ca(2+) exchanger domain (Calxbeta) and lacks the C-terminal hydrophobic segments.

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. Molecular modeling studies confirm that intercalative stacking between adjacent base pairs is energetically favorable. However, it is also observed that the location of the dye in the minor groove of the DNA is energetically even more favorable. Upon UVA irradiation, the indoloquinolizinium causes single-strand cleavage with an efficiency that varies with the dye-DNA ratio. This observation is rationalized in terms of more efficient photocleavage by the externally bound dye compared with the intercalated one. The kinetics of strand degradation under aerobic and anaerobic conditions suggest that a Type I reaction occurs, that is, radical-mediated DNA damage.

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. The mode of binding was investigated by CD and LD spectroscopy. Whereas quinolizinium 5a exclusively intercalates in DNA, the isomer 5b exhibits a deviation from perfect intercalation into the double helix. Moreover, efficient DNA damage was observed on UV-A irradiation in the presence of the quinolizinium salts. Primer extension analysis indicates that the photocleavage takes place preferentially at guanine-rich regions.