Activity-dependent modulation of hippocampal synaptic plasticity via PirB and endocannabinoids.

The threshold for Hebbian synaptic plasticity in the CNS is modulated by prior synaptic activity. At adult CA3-CA1 synapses, endocannabinoids play a role in this process, but how activity engages and maintains this retrograde signaling system is not well understood. Here we show that conditional deletion of Paired Immunoglobulin-like receptor B (PirB) from pyramidal neurons in adult mouse hippocampus results in deficient LTD at CA3-CA1 synapses over a range of stimulation frequencies, accompanied by an increase in LTP. This finding can be fully explained by the disengagement of retrograde endocannabinoid signaling selectively at excitatory synapses. In the absence of PirB, the NMDAR-dependent regulation of endocannabinoid signaling is lost, while CB1R-dependent and group I mGluR-dependent regulation are intact. Moreover, mEPSC frequency in mutant CA1 pyramidal cells is elevated, consistent with a higher density of excitatory synapses and altered synapse pruning. Mice lacking PirB also perform better than WT in learning and memory tasks. These observations suggest that PirB is an integral part of an NMDA receptor-mediated synaptic mechanism that maintains bidirectional Hebbian plasticity and learning via activity-dependent endocannabinoid signaling.

Developmental Sculpting of Intracortical Circuits by MHC Class I H2-Db and H2-Kb.

Synapse pruning is an activity-regulated process needed for proper circuit sculpting in the developing brain. Major histocompatibility class I (MHCI) molecules are regulated by activity, but little is known about their role in the development of connectivity in cortex. Here we show that protein for 2 MHCI molecules H2-Kb and H2-Db is associated with synapses in the visual cortex. Pyramidal neurons in mice lacking H2-Kb and H2-Db (KbDb KO) have more extensive cortical connectivity than normal. Modified rabies virus tracing was used to monitor the extent of pyramidal cell connectivity: Horizontal connectivity is greater in the visual cortex of KbDb KO mice. Basal dendrites of L2/3 pyramids, where many horizontal connections terminate, are more highly branched and have elevated spine density in the KO. Furthermore, the density of axonal boutons is elevated within L2/3 of mutant mice. These increases are accompanied by elevated miniature excitatory postsynaptic current frequency, consistent with an increase in functional synapses. This functional and anatomical increase in intracortical connectivity is also associated with enhanced ocular dominance plasticity that persists into adulthood. Thus, these MHCI proteins regulate sculpting of local cortical circuits and in their absence, the excess connectivity can function as a substrate for cortical plasticity throughout life.

The involvement of PCP proteins in radial cell intercalations during Xenopus embryonic development.

The planar cell polarity (PCP) pathway orients cells in diverse epithelial tissues in Drosophila and vertebrate embryos and has been implicated in many human congenital defects and diseases, such as ciliopathies, polycystic kidney disease and malignant cancers. During vertebrate gastrulation and neurulation, PCP signaling is required for convergent extension movements, which are primarily driven by mediolateral cell intercalations, whereas the role for PCP signaling in radial cell intercalations has been unclear. In this study, we examine the function of the core PCP proteins Vangl2, Prickle3 (Pk3) and Disheveled in the ectodermal cells, which undergo radial intercalations during Xenopus gastrulation and neurulation. In the epidermis, multiciliated cell (MCC) progenitors originate in the inner layer, but subsequently migrate to the embryo surface during neurulation. We find that the Vangl2/Pk protein complexes are enriched at the apical domain of intercalating MCCs and are essential for the MCC intercalatory behavior. Addressing the underlying mechanism, we identified KIF13B, as a motor protein that binds Disheveled. KIF13B is required for MCC intercalation and acts synergistically with Vangl2 and Disheveled, indicating that it may mediate microtubule-dependent trafficking of PCP proteins necessary for cell shape regulation. In the neural plate, the Vangl2/Pk complexes were also concentrated near the outermost surface of deep layer cells, suggesting a general role for PCP in radial intercalation. Consistent with this hypothesis, the ectodermal tissues deficient in Vangl2 or Disheveled functions contained more cell layers than normal tissues. We propose that PCP signaling is essential for both mediolateral and radial cell intercalations during vertebrate morphogenesis. These expanded roles underscore the significance of vertebrate PCP proteins as factors contributing to a number of diseases, including neural tube defects, tumor metastases, and various genetic syndromes characterized by abnormal migratory cell behaviors.

Synapse elimination and learning rules co-regulated by MHC class I H2-Db.

The formation of precise connections between retina and lateral geniculate nucleus (LGN) involves the activity-dependent elimination of some synapses, with strengthening and retention of others. Here we show that the major histocompatibility complex (MHC) class I molecule H2-D(b) is necessary and sufficient for synapse elimination in the retinogeniculate system. In mice lacking both H2-K(b) and H2-D(b) (K(b)D(b)(-/-)), despite intact retinal activity and basal synaptic transmission, the developmentally regulated decrease in functional convergence of retinal ganglion cell synaptic inputs to LGN neurons fails and eye-specific layers do not form. Neuronal expression of just H2-D(b) in K(b)D(b)(-/-) mice rescues both synapse elimination and eye-specific segregation despite a compromised immune system. When patterns of stimulation mimicking endogenous retinal waves are used to probe synaptic learning rules at retinogeniculate synapses, long-term potentiation (LTP) is intact but long-term depression (LTD) is impaired in K(b)D(b)(-/-) mice. This change is due to an increase in Ca(2+)-permeable AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. Restoring H2-D(b) to K(b)D(b)(-/-) neurons renders AMPA receptors Ca(2+) impermeable and rescues LTD. These observations reveal an MHC-class-I-mediated link between developmental synapse pruning and balanced synaptic learning rules enabling both LTD and LTP, and demonstrate a direct requirement for H2-D(b) in functional and structural synapse pruning in CNS neurons.

Neuroprotection from stroke in the absence of MHCI or PirB.

Recovery from stroke engages mechanisms of neural plasticity. Here we examine a role for MHC class I (MHCI) H2-Kb and H2-Db, as well as PirB receptor. These molecules restrict synaptic plasticity and motor learning in the healthy brain. Stroke elevates neuronal expression not only of H2-Kb and H2-Db, but also of PirB and downstream signaling. KbDb knockout (KO) or PirB KO mice have smaller infarcts and enhanced motor recovery. KO hippocampal organotypic slices, which lack an intact peripheral immune response, have less cell death after in vitro ischemia. In PirB KO mice, corticospinal projections from the motor cortex are enhanced, and the reactive astrocytic response is dampened after MCAO. Thus, molecules that function in the immune system act not only to limit synaptic plasticity in healthy neurons, but also to exacerbate brain injury after ischemia. These results suggest therapies for stroke by targeting MHCI and PirB.

Frodo proteins: modulators of Wnt signaling in vertebrate development.

The Frodo/dapper (Frd) proteins are recently discovered signaling adaptors, which functionally and physically interact with Wnt and Nodal signaling pathways during vertebrate development. The Frd1 and Frd2 genes are expressed in dynamic patterns in early embryos, frequently in cells undergoing epithelial-mesenchymal transition. The Frd proteins function in multiple developmental processes, including mesoderm and neural tissue specification, early morphogenetic cell movements, and organogenesis. Loss-of-function studies using morpholino antisense oligonucleotides demonstrate that the Frd proteins regulate Wnt signal transduction in a context-dependent manner and may be involved in Nodal signaling. The identification of Frd-associated factors and cellular targets of the Frd proteins should shed light on the molecular mechanisms underlying Frd functions in embryonic development and in cancer.

A vertebrate homolog of the cell cycle regulator Dbf4 is an inhibitor of Wnt signaling required for heart development.

Early stages of vertebrate heart development have been linked to Wnt signaling. Here we show in both gain- and loss-of-function experiments that XDbf4, a known regulator of Cdc7 kinase, is an inhibitor of the canonical Wnt signaling pathway. Depletion of endogenous XDbf4 protein did not disturb gastrulation movements or early organizer genes but resulted in embryos with morphologically defective heart and eyes and suppressed cardiac markers. These markers were restored by overexpressed XDbf4, or an XDbf4 mutant that inhibits Wnt signaling but lacks the ability to regulate Cdc7. This indicates that the function of XDbf4 in heart development is independent of its role in the cell cycle. Moreover, our data suggest that XDbf4 acts through the physical and functional interaction with Frodo, a context-dependent regulator of Wnt signaling. These findings establish an unexpected function for a vertebrate Dbf4 homolog and demonstrate the requirement for Wnt inhibition in early cardiac specification.

Nuclear localization is required for Dishevelled function in Wnt/beta-catenin signaling.

BACKGROUND: Dishevelled (Dsh) is a key component of multiple signaling pathways that are initiated by Wnt secreted ligands and Frizzled receptors during embryonic development. Although Dsh has been detected in a number of cellular compartments, the importance of its subcellular distribution for signaling remains to be determined. RESULTS: We report that Dsh protein accumulates in cell nuclei when Xenopus embryonic explants or mammalian cells are incubated with inhibitors of nuclear export or when a specific nuclear-export signal (NES) in Dsh is disrupted by mutagenesis. Dsh protein with a mutated NES, while predominantly nuclear, remains fully active in its ability to stimulate canonical Wnt signaling. Conversely, point mutations in conserved amino-acid residues that are essential for the nuclear localization of Dsh impair the ability of Dsh to activate downstream targets of Wnt signaling. When these conserved residues of Dsh are replaced with an unrelated SV40 nuclear localization signal, full Dsh activity is restored. Consistent with a signaling function for Dsh in the nucleus, treatment of cultured mammalian cells with medium containing Wnt3a results in nuclear accumulation of endogenous Dsh protein. CONCLUSIONS: These findings suggest that nuclear localization of Dsh is required for its function in the canonical Wnt/beta-catenin signaling pathway. We discuss the relevance of these findings to existing models of Wnt signal transduction to the nucleus.

Mek2 is dispensable for mouse growth and development.

MEK is a dual-specificity kinase that activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase upon agonist binding to receptors. The ERK/MAP kinase cascade is involved in cell fate determination in many organisms. In mammals, this pathway is proposed to regulate cell growth and differentiation. Genetic studies have shown that although a single Mek gene is present in Caenorhabditis elegans, Drosophila melanogaster, and Xenopus laevis, two Mek homologs, Mek1 and Mek2, are present in the mammalian cascade. The inactivation of the Mek1 gene leads to embryonic lethality and has revealed the unique role played by Mek1 during embryogenesis. To investigate the biological function of the second homolog, we have generated mice deficient in Mek2 function. Mek2 mutant mice are viable and fertile, and they do not present flagrant morphological alteration. Although several components of the ERK/MAP kinase cascade have been implicated in thymocyte development, no such involvement was observed for MEK2, which appears to be nonessential for thymocyte differentiation and T-cell-receptor-induced proliferation and apoptosis. Altogether, our findings demonstrate that MEK2 is not necessary for the normal development of the embryo and T-cell lineages, suggesting that the loss of MEK2 can be compensated for by MEK1.

Regulation of Wnt/LRP signaling by distinct domains of Dickkopf proteins.

Dickkopfs (Dkks) are secreted developmental regulators composed of two cysteine-rich domains. We report that the effects of Dkks depend on molecular context. Although Wnt8 signaling is inhibited by both Dkk1 and Dkk2 in Xenopus embryos, the same pathway is activated upon interaction of Dkk2 with the Wnt coreceptor LRP6. Analysis of individual Dkk domains and chimeric Dkks shows that the carboxy-terminal domains of both Dkks associate with LRP6 and are necessary and sufficient for Wnt8 inhibition, whereas the amino-terminal domain of Dkk1 plays an inhibitory role in Dkk-LRP interactions. Our study illustrates how an inhibitor of a pathway may be converted into an activator and is the first study to suggest a molecular mechanism for how a ligand other than Wnt can positively regulate beta-catenin signaling.