Selective synapse development determines how complex neuronal networks in the brain are formed. Complexes of postsynaptic neuroligins and LRRTMs with presynaptic neurexins contribute widely to excitatory synapse development, and mutations in these gene families increase the risk of developing psychiatric disorders. We find that LRRTM4 has distinct presynaptic binding partners, heparan sulfate proteoglycans (HSPGs). HSPGs are required to mediate the synaptogenic activity of LRRTM4. LRRTM4 shows highly selective expression in the brain. Within the hippocampus, we detected LRRTM4 specifically at excitatory postsynaptic sites on dentate gyrus granule cells. LRRTM4(-/-) dentate gyrus granule cells, but not CA1 pyramidal cells, exhibit reductions in excitatory synapse density and function. Furthermore, LRRTM4(-/-) dentate gyrus granule cells show impaired activity-regulated AMPA receptor trafficking. These results identifying cell-type-specific functions and multiple presynaptic binding partners for different LRRTM family members reveal an unexpected complexity in the design and function of synapse-organizing proteins.

SUMMARY Perturbations of cell surface synapse organizing proteins, particularly α-neurexins, contribute to neurodevelopmental and psychiatric disorders. From an unbiased screen, we identify calsyntenin-3 (alcadein-β) as a novel synapse organizing protein unique in binding and recruiting α-neurexins but not β-neurexins. Calsyntenin-3 is present in many pyramidal neurons throughout cortex and hippocampus but is most highly expressed in interneurons. The transmembrane form of calsyntenin-3 can trigger excitatory and inhibitory presynapse differentiation in contacting axons. However, calsyntenin-3 shed ectodomain, which represents about half the calsyntenin-3 pool in brain, suppresses the ability of multiple α-neurexin partners including neuroligin 2 and LRRTM2 to induce presynapse differentiation. Clstn3 −/− mice show reductions in excitatory and inhibitory synapse density by confocal and electron microscopy and corresponding deficits in synaptic transmission. These results identify calsyntenin-3 as an α-neurexin-specific binding partner required for normal functional GABAergic and glutamatergic synapse development.

SUMMARY Plasticity related gene-1 (PRG-1) is a brain-specific membrane protein related to lipid phosphate phosphatases, which acts in the hippocampus specifically at the excitatory synapse terminating on glutamatergic neurons. Deletion of prg-1 in mice leads to epileptic seizures and augmentation of EPSCs, but not IPSCs. In utero electroporation of PRG-1 into deficient animals revealed that PRG-1 modulates excitation at the synaptic junction. Mutation of the extracellular domain of PRG-1 crucial for its interaction with lysophosphatidic acid (LPA) abolished the ability to prevent hyperexcitability. As LPA application in vitro induced hyperexcitability in wild-type but not in LPA2 receptor-deficient animals, and uptake of phospholipids is reduced in PRG-1-deficient neurons, we assessed PRG-1/LPA2 receptor-deficient animals, and found that the pathophysiology observed in the PRG-1-deficient mice was fully reverted. Thus, we propose PRG-1 as an important player in the modulatory control of hippocampal excitability dependent on presynaptic LPA2 receptor signaling.

Summary Nedd4-1 is a ‘Neuronal Precursor Cell Expressed and Developmentally Downregulated Protein’ and among the most abundant E3 ubiquitin ligases in mammalian neurons. In analyses of conventional and conditional Nedd4-1 deficient mice, we found that Nedd4-1 plays a critical role in dendrite formation. Nedd4-1, the serine/threonine kinase TNIK, and Rap2A form a complex that controls Nedd4-1-mediated ubiquitination of Rap2A. Ubiquitination by Nedd4-1 inhibits Rap2A function, which reduces the activity of Rap2 effector kinases of the TNIK family and promotes dendrite growth. We conclude that a Nedd4-1/Rap2A/TNIK signaling pathway regulates neurite growth and arborization in mammalian neurons.

PTEN is a tumor suppressor frequently mutated in cancer. Recent reports implicated Nedd4-1 as the E3 ubiquitin ligase for PTEN that regulates its stability and nuclear localization. We tested the physiological role of Nedd4-1 as a PTEN regulator by using cells and tissues derived from two independently generated strains of mice with their Nedd4-1 gene disrupted. PTEN stability and ubiquitination were indistinguishable between the wild-type and Nedd4-1-deficient cells, and an interaction between the two proteins could not be detected. Moreover, PTEN subcellular distribution, showing prominent cytoplasmic and nuclear staining, was independent of Nedd4-1 presence. Finally, activation of PKB/Akt, a major downstream target of cytoplasmic PTEN activity, and the ability of PTEN to transactivate the Rad51 promoter, a measure of its nuclear function, were unaffected by the loss of Nedd4-1. Taken together, our results fail to support a role for Nedd4-1 as the E3 ligase regulating PTEN stability and subcellular localization.E3 ligase ͉ tumor suppressor ͉ P13K signal ͉ WW domain P TEN is one of the most frequently mutated genes in human cancer (1-3). In the cytoplasm, PTEN functions as a lipid phosphatase by dephosphorylating the D3 position of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ], and directly antagonizing PI3K (4-6). Consistent with a negative regulatory role for PTEN in regulation of PI3 signaling, PTEN-deficient cells and tissues exhibit defects in cell proliferation, growth, survival, death, protein translation, metabolism, migration, and structural organization (2,3,7,8).One of the most intriguing features of PTEN is its subcellular localization. Although PTEN was originally found to be a cytoplasmic protein (9), its nuclear localization in many cell types has been reported by using various independently developed monoclonal and polyclonal anti-PTEN antibodies (10)(11)(12)(13)(14). A number of attempts have been made to uncouple the cytoplasmic and nuclear roles of PTEN, yielding incongruous results. For example, differing accounts of the respective levels of cytoplasmic and nuclear PTEN throughout the stages of the cell cycle have been documented (15, 16). Functionally, nuclear PTEN has been shown to induce cell cycle arrest in certain cell types (17), whereas in others, nuclear accumulation of PTEN increased upon stimulation with proapoptotic factors and correlated with induction of apoptosis (18). Finally, nuclear-targeted PTEN was shown to impede the growth of U251MG glioblastoma cells and down-regulate p70S6K in a PKB/Akt-independent manner but was unable to inhibit cell invasion (19).Recently, published work revealed a phosphatase activityindependent nuclear role for PTEN in the regulation of chromosomal stability and repair of DNA damage (20). Wang et al. (21) and Trotman et al. (22) reported that PTEN stability and nuclear translocation were regulated by ubiquitination mediated by the ubiquitin ligase Nedd4-1. Although the Nedd4-1-mediated polyubiquitination of PTEN in the cytosol caused ...

Cystic fibrosis is caused by impaired ion transport due to mutated cystic fibrosis transmembrane conductance regulator, accompanied by elevated activity of the amiloride-sensitive epithelial Na + channel (ENaC). Here we show that knockout of the ubiquitin ligase Nedd4L (Nedd4-2) specifically in lung epithelia (surfactant protein C-expressing type II and Clara cells) causes cystic fibrosis-like lung disease, with airway mucus obstruction, goblet cell hyperplasia, massive inflammation, fibrosis, and death by three weeks of age. These effects of Nedd4L loss are likely caused by enhanced ENaC function, as reflected by increased ENaC protein levels, increased lung dryness at birth, amiloride-sensitive dehydration of lung explants, and elevated ENaC currents in primary alveolar type II cells analyzed by patch clamp recordings. Moreover, the lung defects were rescued with administration of amiloride into the lungs of young knockout pups via nasal instillation. Our results therefore suggest that the ubiquitin ligase Nedd4L can suppress the onset of cystic fibrosis symptoms by inhibiting ENaC in lung epithelia.

SummaryDendritic spines are the major transmitter reception compartments of glutamatergic synapses in most principal neurons of the mammalian brain and play a key role in the function of nerve cell circuits. The formation of functional spine synapses is thought to be critically dependent on presynaptic glutamatergic signaling. By analyzing CA1 pyramidal neurons in mutant hippocampal slice cultures that are essentially devoid of presynaptic transmitter release, we demonstrate that the formation and maintenance of dendrites and functional spines are independent of synaptic glutamate release.

Munc13 proteins are essential regulators of exocytosis. In hippocampal glutamatergic neurons, the genetic deletion of Munc13s results in the complete loss of primed synaptic vesicles (SVs) in direct contact with the presynaptic active zone membrane, and in a total block of neurotransmitter release. Similarly drastic consequences of Munc13 loss are detectable in hippocampal and striatal GABAergic neurons. We show here that, in the adult mouse retina, the two Munc13-2 splice variants bMunc13-2 and ubMunc13-2 are selectively localized to conventional and ribbon synapses, respectively, and that ubMunc13-2 is the only Munc13 isoform in mature photoreceptor ribbon synapses. Strikingly, the genetic deletion of ubMunc13-2 has little effect on synaptic signaling by photoreceptor ribbon synapses and does not prevent membrane attachment of synaptic vesicles at the photoreceptor ribbon synaptic site. Thus, photoreceptor ribbon synapses and conventional synapses differ fundamentally with regard to their dependence on SV priming proteins of the Munc13 family. Their function is only moderately affected by Munc13 loss, which leads to slight perturbations of signal integration in the retina.