SynGAP regulates spine formation.

SynGAP is a brain-specific ras GTPase-activating protein that is an abundant component of the signaling complex associated with the NMDA-type glutamate receptor. We generated mutant mice lacking synGAP to study its physiological role. Homozygous mutant mice die in the first few days after birth; however, neurons from mutant embryos can be maintained in culture. Here, we report that spine and synapse formation are accelerated in cultured mutant neurons, and the spines of mature mutant neurons are significantly larger than those of wild type. Clusters of PSD-95 and subunits of AMPA-type and NMDA-type glutamate receptors accumulate in spines of mutant neurons by day 10 in vitro, whereas in wild-type neurons they are still mostly located in dendritic shafts. The frequency and amplitude of miniature EPSCs are larger in mutant neurons at day 10 in vitro, confirming that they have more functional synapses. At day 21 in vitro, the spines of mutant neurons remain significantly larger than those of wild type. The mutant phenotype at day 10 in vitro can be rescued by introduction of recombinant wild-type synGAP on day 9. In contrast, introduction of mutant synGAP with a mutated GAP domain or lacking the terminal domain that binds to PSD-95 does not rescue the mutant phenotype, indicating that both domains play a role in control of spine formation. Thus, the GAP activity of synGAP and its association with PSD-95 are important for normal regulation of spine and synapse formation in hippocampal neurons.

Human placenta-derived adherent cells improve cardiac performance in mice with chronic heart failure.

Human placenta-derived adherent cells (PDACs) are a culture-expanded, undifferentiated mesenchymal-like population derived from full-term placental tissue, with immunomodulatory, anti-inflammatory, angiogenic, and neuroprotective properties. PDA-001 (cenplacel-L), an intravenous formulation of PDAC cells, is in clinical development for the treatment of autoimmune and inflammatory diseases. We tested the therapeutic effects of PDA-001 in mice with chronic heart failure (CHF). Three weeks after transaortic constriction surgery to induce CHF, the mice underwent direct intramyocardial (IM) or i.v. injection of PDA-001 at a high (0.5 × 10(6) cells per mouse), medium (0.5 × 10(5) cells per mouse), or low (0.5 × 10(4) cells per mouse) dose. The mice were sacrificed 4 weeks after treatment. Echocardiography and ventricular catheterization showed that IM injection of PDA-001 significantly improved left ventricular systolic and diastolic function compared with injection of vehicle or i.v. injection of PDA-001. IM injection of PDA-001 also decreased cardiac fibrosis, shown by trichrome staining in the vicinity of the injection sites. Low-dose treatment showed the best improvement in cardiac performance compared with the medium- and high-dose groups. In another independent study to determine the mechanism of action with bromodeoxyuridine labeling, the proliferation rates of endothelial cells and cardiomyocytes were significantly increased by low or medium IM dose PDA-001. However, no surviving PDA-001 cells were detected in the heart 1 month after injection. In vivo real-time imaging consistently revealed that the PDA-001 cells were detectable only within 2 days after IM injection of luciferase-expressing PDA-001. Together, these results have demonstrated the cardiac therapeutic potential of PDA-001, likely through a paracrine effect.

Human placenta-derived adherent cells induce tolerogenic immune responses.

Human placenta-derived adherent cells (PDAC cells) are a culture expanded, undifferentiated mesenchymal-like population derived from full-term placental tissue, with immunomodulatory and anti-inflammatory properties. PDA-001 (cenplacel-L), an intravenous formulation of PDAC cells, is in clinical development for the treatment of autoimmune and inflammatory diseases. To elucidate the mechanisms underlying the immunoregulatory properties of PDAC cells, we investigated their effects on immune cell populations, including T cells and dendritic cells (DC) in vitro and in vivo. PDAC cells suppressed T-cell proliferation in an OT-II T-cell adoptive transfer model, reduced the severity of myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis and ameliorated inflammation in a delayed type hypersensitivity response model. In vitro, PDAC cells suppressed T-cell proliferation and inhibited Th1 and Th17 differentiation. Analysis of tissues derived from PDAC cell-treated animals revealed diminished CD86 expression on splenic DC, suggesting that they can also modulate DC populations. Furthermore, PDAC cells modulate the differentiation and maturation of mouse bone marrow-derived DC. Similarly, human DC differentiated from CD14(+) monocytes in the presence of PDAC cells acquired a tolerogenic phenotype. These tolerogenic DC failed to induce allogeneic T-cell proliferation and differentiation toward Th1, but skewed T-cell differentiation toward Th2. Inhibition of cyclo-oxygenase-2 activity resulted in a significant, but not complete, abrogation of PDAC cells' effects on DC phenotype and function, implying a role for prostaglandin E2 in PDAC-mediated immunomodulation. This study identifies modulation of DC differentiation toward immune tolerance as a key mechanism underlying the immunomodulatory activities of PDAC cells.

A role for synGAP in regulating neuronal apoptosis.

The brain-specific Ras/Rap GTPase-activating protein synGAP is a major component of the postsynaptic density at glutamatergic synapses. It is a target for phosphorylation by Ca(2+)/calmodulin-dependent protein kinase II, which up-regulates its GTPase-activating activity. Thus, SynGAP may play an important role in coupling N-methyl-D-aspartate-type glutamate receptor activation to signaling pathways downstream of Ras or Rap. Homozygous deletion of synGAP is lethal within the first few days after birth. Therefore, to study the functions of synGAP, we used the cre/loxP recombination system to produce conditional mice mutants in which gradual loss of synGAP begins at approximately 1 week, and usually becomes maximal by 3 weeks, after birth. The resulting phenotypes fall into two groups. In a small group, the level of synGAP protein is reduced to 20-25% of wild type, and they die at 2-3 weeks of age. In a larger group, the levels remain higher than approximately 40% of wild type, and they survive and remain healthy. In all mutants, however, an abnormally high number of neurons in the hippocampus and cortex undergo apoptosis, as detected by caspase-3 activation. The effect is cell autonomous, occurring only in neuronal types in which the synGAP gene is eliminated. The level of caspase-3 activation in neurons correlates inversely with the level of synGAP protein measured at 2 and 8 weeks after birth, indicating that neuronal apoptosis is enhanced by reduction of synGAP. These data show that synGAP plays a role in regulation of the onset of apoptotic neuronal death.