Disrupted Cacna1c gene expression perturbs spontaneous Ca2+ activity causing abnormal brain development and increased anxiety.

The L-type voltage-gated Ca2+ channel gene CACNA1C is a risk gene for various psychiatric conditions, including schizophrenia and bipolar disorder. However, the cellular mechanism by which CACNA1C contributes to psychiatric disorders has not been elucidated. Here, we report that the embryonic deletion of Cacna1c in neurons destined for the cerebral cortex using an Emx1-Cre strategy disturbs spontaneous Ca2+ activity and causes abnormal brain development and anxiety. By combining computational modeling with electrophysiological membrane potential manipulation, we found that neural network activity was driven by intrinsic spontaneous Ca2+ activity in distinct progenitor cells expressing marginally increased levels of voltage-gated Ca2+ channels. MRI examination of the Cacna1c knockout mouse brains revealed volumetric differences in the neocortex, hippocampus, and periaqueductal gray. These results suggest that Cacna1c acts as a molecular switch and that its disruption during embryogenesis can perturb Ca2+ handling and neural development, which may increase susceptibility to psychiatric disease.

The T-type Ca2+ Channel Cav3.2 Regulates Differentiation of Neural Progenitor Cells during Cortical Development via Caspase-3.

Here we report that the low-voltage-dependent T-type calcium (Ca2+) channel Cav3.2, encoded by the CACNA1H gene, regulates neuronal differentiation during early embryonic brain development through activating caspase-3. At the onset of neuronal differentiation, neural progenitor cells exhibited spontaneous Ca2+ activity. This activity strongly correlated with the upregulation of CACNA1H mRNA. Cells exhibiting robust spontaneous Ca2+ signaling had increased caspase-3 activity unrelated to apoptosis. Inhibition of Cav3.2 by drugs or viral CACNA1H knock down resulted in decreased caspase-3 activity followed by suppressed neurogenesis. In contrast, when CACNA1H was overexpressed, increased neurogenesis was detected. Cortical slices from Cacna1h knockout mice showed decreased spontaneous Ca2+ activity, a significantly lower protein level of cleaved caspase-3, and microanatomical abnormalities in the subventricular/ventricular and cortical plate zones when compared to their respective embryonic controls. In summary, we demonstrate a novel relationship between Cav3.2 and caspase-3 signaling that affects neurogenesis in the developing brain.

Isotopic resonance at 370 ppm deuterium negatively affects kinetics of luciferin oxidation by luciferase.

Since 1930s, it has been known that some biochemical and biological processes exhibit abnormal kinetics at a deuterium concentration in the local environment of 250-600 ppm, which is 2-4 times higher that the normal concentration of 150 ppm D. We sought to test if the kinetics of firefly luciferase oxidizing luciferin, the reaction widely used as a read-out in various biochemical assays, is also affected by an elevated deuterium content. To this end, both luciferase and luciferin substrate solutions were prepared based on water with extra deuterium added to a concentration ranging from 150 ppm and up to 10,000 ppm (1%). Upon mixing the solutions, the luminescence intensity at different times was compared with that of the corresponding control solutions with 150 ppm D. A broad negative resonance was detected (p < 10-6), with a ≈20% drop in luminescence at 370 ppm D. Given that, on average, about half of hydrogen atoms in proteins are not exchangeable in solution, this value corresponds to ≈260 ppm of deuterium in all enzyme's hydrogens, in a very good agreement with the prediction of the Isotopic resonance hypothesis.

Bioanalytical advantages of a novel recombinant apyrase enzyme in ATP-based bioluminescence methods.

Ultrasensitive measurements of intracellular ATP (intATP) based on the firefly luciferase reactions are frequently used to enumerate bacterial or mammalian cells. During clinical applications, extracellular ATP (extATP) should be depleted in biological samples since it interferes with intATP and affects the quantification of bacteria. The extATP can be eliminated by ATP-degrading enzymes but complete hydrolysis of extATP remains a challenge for today's commercial enzymes. The catalytic efficiency of ATP-degrading enzymes depends on enzyme characteristics, sample composition and the ability to deplete diphosphates, triphosphates and their complexes generated during the reaction. This phenomenon restricts the usage of bioluminescence-based ATP methods in clinical diagnostics. In light of this, we have developed a recombinant Shigella flexneri apyrase (RSFA) enzyme and analysed its ATP depletion potential with five commercial biochemical sources including potato apyrase, acid phosphatase, alkaline phosphatase, hexokinase and glycerol kinase. The RSFA revealed superior activity by completely eliminating the extracellular ATP and ATP-complexes, even in biological samples like urine and serum. Therefore, our results can potentially unwrap the chemical and bio-analytical applications of ATP-based bioluminescence tests to develop highly sensitive point-of-care diagnostics.

[6]-shogaol induces Ca²âº signals by activating the TRPV1 channels in the rat insulinoma INS-1E cells.

CONTEXT: [6]-shogaol is a vanilloid compound present in steamed ginger (Zingiber officinale), a commonly used spice. Pancreatic beta-cells respond to nutrients like glucose, amino acids and fatty acids, by an increase in the cytoplasmic free Ca²âº concentration ([Ca²âº](i)), which mediates diverse cellular processes in these cells. Some vanilloid compounds activate the transient receptor potential vanilloid receptor type 1 (TRPV1) channel. OBJECTIVE: We investigated whether [6]-shogaol could trigger Ca²âº signals in the beta-cell. METHODS: [Ca²âº](i) was measured from single INS-1E cells by microscope-based fluorometry using fura-2 as the Ca²âº indicator. RESULTS: In fura-2 loaded single rat insulinoma INS-1E cells, a widely used model of beta-cell, [6]-shogaol increased [Ca²âº](i) in a concentration-dependent manner. [Ca²âº](i) increase by [6]-shogaol was completely blocked when Ca²âº was omitted from the extracellular medium. Capsazepine, an inhibitor of the TRPV1 ion channel completely inhibited the [6]-shogaol-induced [Ca²âº](i) increase. [Ca²âº](i) increase obtained by 1 µM [6]-shogaol was greater than that obtained by 10 mM glucose. Moreover, a sub-stimulatory concentration of [6]-shogaol (300 nM), significantly enhanced the glucose-induced [Ca²âº](i) increase in these cells. CONCLUSION: We conclude that [6]-shogaol induces Ca²âº signals in the beta-cell by activating the TRPV1 channels, and it sensitizes the beta-cells to stimulation by glucose.

Small-world networks of spontaneous Ca(2+) activity.

Synchronized network activity among groups of interconnected cells is essential for diverse functions in the brain. However, most studies have been made on cellular networks in the mature brain when chemical synapses have been formed. Much less is known about the situation earlier in development. When studying neural progenitors derived from embryonic stem cells and neural progenitors from mice embryos, we found networks of gap junction coupled cells with vivid spontaneous non-random calcium (Ca(2+)) activity driven by electrical depolarization that stimulated cell growth. Network activity was revealed by single-cell live Ca(2+) imaging and further analyzed for correlations and network topology. The analysis revealed the networks to have small-world characteristics with scale-free properties. Taken together, these results demonstrate that immature cells in the developing brain organize in small-world networks that critically regulate neural progenitor proliferation.

Neural progenitors organize in small-world networks to promote cell proliferation.

Coherent network activity among assemblies of interconnected cells is essential for diverse functions in the adult brain. However, cellular networks before formations of chemical synapses are poorly understood. Here, embryonic stem cell-derived neural progenitors were found to form networks exhibiting synchronous calcium ion (Ca(2+)) activity that stimulated cell proliferation. Immature neural cells established circuits that propagated electrical signals between neighboring cells, thereby activating voltage-gated Ca(2+) channels that triggered Ca(2+) oscillations. These network circuits were dependent on gap junctions, because blocking prevented electrotonic transmission both in vitro and in vivo. Inhibiting connexin 43 gap junctions abolished network activity, suppressed proliferation, and affected embryonic cortical layer formation. Cross-correlation analysis revealed highly correlated Ca(2+) activities in small-world networks that followed a scale-free topology. Graph theory predicts that such network designs are effective for biological systems. Taken together, these results demonstrate that immature cells in the developing brain organize in small-world networks that critically regulate neural progenitor proliferation.

Perfluorooctane sulfonate induces neuronal and oligodendrocytic differentiation in neural stem cells and alters the expression of PPARγ in vitro and in vivo.

Perfluorinated compounds are ubiquitous chemicals of major concern for their potential adverse effects on the human population. We have used primary rat embryonic neural stem cells (NSCs) to study the effects of perfluorooctane sulfonate (PFOS) on the process of NSC spontaneous differentiation. Upon removal of basic fibroblast growth factor, NSCs were exposed to nanomolar concentrations of PFOS for 48 h, and then allowed to differentiate for additional 5 days. Exposure to 25 or 50 nM concentration resulted in a lower number of proliferating cells and a higher number of neurite-bearing TuJ1-positive cells, indicating an increase in neuronal differentiation. Exposure to 50 nM also significantly increased the number of CNPase-positive cells, pointing to facilitation of oligodendrocytic differentiation. PPAR genes have been shown to be involved in PFOS toxicity. By q-PCR we detected an upregulation of PPARγ with no changes in PPARα or PPARδ genes. One of the downstream targets of PPARs, the mitochondrial uncoupling protein 2 (UCP2) was also upregulated. The number of TuJ1- and CNPase-positive cells increased after exposure to PPARγ agonist rosiglitazone (RGZ, 3 µM) and decreased after pre-incubation with the PPARγ antagonist GW9662 (5 µM). RGZ also upregulated the expression of PPARγ and UCP2 genes. Meanwhile GW9662 abolished the UCP2 upregulation and decreased Ca²âº activity induced by PFOS. Interestingly, a significantly higher expression of PPARγ and UCP3 genes was also detected in mouse neonatal brain after prenatal exposure to PFOS. These data suggest that PPARγ plays a role in the alteration of spontaneous differentiation of NSCs induced by nanomolar concentrations of PFOS.

Local control of nuclear calcium signaling in cardiac myocytes by perinuclear microdomains of sarcolemmal insulin-like growth factor 1 receptors.

RATIONALE: The ability of a cell to independently regulate nuclear and cytosolic Ca(2+) signaling is currently attributed to the differential distribution of inositol 1,4,5-trisphosphate receptor channel isoforms in the nucleoplasmic versus the endoplasmic reticulum. In cardiac myocytes, T-tubules confer the necessary compartmentation of Ca(2+) signals, which allows sarcomere contraction in response to plasma membrane depolarization, but whether there is a similar structure tunneling extracellular stimulation to control nuclear Ca(2+) signals locally has not been explored. OBJECTIVE: To study the role of perinuclear sarcolemma in selective nuclear Ca(2+) signaling. METHODS AND RESULTS: We report here that insulin-like growth factor 1 triggers a fast and independent nuclear Ca(2+) signal in neonatal rat cardiac myocytes, human embryonic cardiac myocytes, and adult rat cardiac myocytes. This fast and localized response is achieved by activation of insulin-like growth factor 1 receptor signaling complexes present in perinuclear invaginations of the plasma membrane. The perinuclear insulin-like growth factor 1 receptor pool connects extracellular stimulation to local activation of nuclear Ca(2+) signaling and transcriptional upregulation through the perinuclear hydrolysis of phosphatidylinositol 4,5-biphosphate inositol 1,4,5-trisphosphate production, nuclear Ca(2+) release, and activation of the transcription factor myocyte-enhancing factor 2C. Genetically engineered Ca(2+) buffers--parvalbumin--with cytosolic or nuclear localization demonstrated that the nuclear Ca(2+) handling system is physically and functionally segregated from the cytosolic Ca(2+) signaling machinery. CONCLUSIONS: These data reveal the existence of an inositol 1,4,5-trisphosphate-dependent nuclear Ca(2+) toolkit located in direct apposition to the cell surface, which allows the local control of rapid and independent activation of nuclear Ca(2+) signaling in response to an extracellular ligand.

RET PLCγ phosphotyrosine binding domain regulates Ca2+ signaling and neocortical neuronal migration.

The receptor tyrosine kinase RET plays an essential role during embryogenesis in regulating cell proliferation, differentiation, and migration. Upon glial cell line-derived neurotrophic factor (GDNF) stimulation, RET can trigger multiple intracellular signaling pathways that in concert activate various downstream effectors. Here we report that the RET receptor induces calcium (Ca(2+)) signaling and regulates neocortical neuronal progenitor migration through the Phospholipase-C gamma (PLCγ) binding domain Tyr1015. This signaling cascade releases Ca(2+) from the endoplasmic reticulum through the inositol 1,4,5-trisphosphate receptor and stimulates phosphorylation of ERK1/2 and CaMKII. A point mutation at Tyr1015 on RET or small interfering RNA gene silencing of PLCγ block the GDNF-induced signaling cascade. Delivery of the RET mutation to neuronal progenitors in the embryonic ventricular zone using in utero electroporation reveal that Tyr1015 is necessary for GDNF-stimulated migration of neurons to the cortical plate. These findings demonstrate a novel RET mediated signaling pathway that elevates cytosolic Ca(2+) and modulates neuronal migration in the developing neocortex through the PLCγ binding domain Tyr1015.

Non-dioxin-like polychlorinated biphenyls interfere with neuronal differentiation of embryonic neural stem cells.

Developmental exposure to food contaminants, such as polychlorinated biphenyls (PCBs), has been considered as a possible cause of neurodevelopmental disorders. We have investigated the effects of noncytotoxic concentrations of PCBs 153 and 180 on spontaneous differentiation of rat embryonic neural stem cells (NSCs). Upon removal of basic fibroblast growth factor to induce spontaneous differentiation, cells were exposed to 100 nM of the selected PCBs for 48 h and analyzed after 5 days. Both PCBs 153 and 180 induced a significant increase in the number of neurite-bearing Tuj1-positive cells with a concomitant decrease in proliferating cells, as detected by FUCCI transfection and EdU staining. Measurements of spontaneous Ca²âº oscillations showed a decreased number of cells with Ca²âº activity after PCB exposure, further confirming the increase in neuronal cells. Conversely, exposure to methylmercury (MeHg), which we evaluated in parallel, led to an increased number of cells with Ca²âº activity, in agreement with the previously observed inhibition of neuronal differentiation. Analysis with quantitative PCR of the Notch pathway revealed that PCBs have a repressive action on Notch signaling, whereas MeHg activates it. Altogether, the data indicate that nanomolar concentrations of the selected non-dioxin-like PCBs and MeHg interfere in opposite directions with neuronal spontaneous differentiation of NSCs through Notch signaling. Combined exposures to PCBs and MeHg resulted in an induction of apoptosis and an antagonistic interaction on spontaneous neuronal differentiation. NSCs are further proven to be a valuable in vitro model to identify potential developmental neurotoxicants.