Neuroligin-1 controls synaptic abundance of NMDA-type glutamate receptors through extracellular coupling.

Despite the pivotal functions of the NMDA receptor (NMDAR) for neural circuit development and synaptic plasticity, the molecular mechanisms underlying the dynamics of NMDAR trafficking are poorly understood. The cell adhesion molecule neuroligin-1 (NL1) modifies NMDAR-dependent synaptic transmission and synaptic plasticity, but it is unclear whether NL1 controls synaptic accumulation or function of the receptors. Here, we provide evidence that NL1 regulates the abundance of NMDARs at postsynaptic sites. This function relies on extracellular, NL1 isoform-specific sequences that facilitate biochemical interactions between NL1 and the NMDAR GluN1 subunit. Our work uncovers NL1 isoform-specific cis-interactions with ionotropic glutamate receptors as a key mechanism for controlling synaptic properties.

Flexible Accelerated STOP Tetracycline Operator-knockin (FAST): a versatile and efficient new gene modulating system.

We created the Flexible Accelerated STOP Tetracycline Operator (tetO)-knockin (FAST) system, an efficient method for manipulating gene expression in vivo to rapidly screen animal models of disease. A single gene targeting event yields two distinct knockin mice-STOP-tetO and tetO knockin-that permit generation of multiple strains with variable expression patterns: 1) knockout, 2) Cre-mediated rescue, 3) tetracycline-controlled transcriptional activator (tTA)-mediated misexpression, 4) tetracycline-controlled transcriptional activator (tTA)-mediated overexpression, and 5) tetracycline-controlled transcriptional silencer (tTS)-mediated conditional knockout/knockdown. Using the FAST system, multiple gain-of-function and loss-of-function strains can therefore be generated on a time scale not previously achievable. These strains can then be screened for clinically relevant abnormalities. We demonstrate the flexibility and broad applicability of the FAST system by targeting several genes encoding proteins implicated in neuropsychiatric disorders: Mlc1, neuroligin 3, the serotonin 1A receptor, and the serotonin 1B receptor.

Crystal structure of the extracellular cholinesterase-like domain from neuroligin-2.

Neuroligins (NLs) are catalytically inactive members of a family of cholinesterase-like transmembrane proteins that mediate cell adhesion at neuronal synapses. Postsynaptic neuroligins engage in Ca2+-dependent transsynaptic interactions via their extracellular cholinesterase domain with presynaptic neurexins (NRXs). These interactions may be regulated by two short splice insertions (termed A and B) in the NL cholinesterase domain. Here, we present the 3.3-A crystal structure of the ectodomain from NL2 containing splice insertion A (NL2A). The overall structure of NL2A resembles that of cholinesterases, but several structural features are unique to the NL proteins. First, structural elements surrounding the esterase active-site region differ significantly between active esterases and NL2A. On the opposite surface of the NL2A molecule, the positions of the A and B splice insertions identify a candidate NRX interaction site of the NL protein. Finally, sequence comparisons of NL isoforms allow for mapping the location of residues of previously identified mutations in NL3 and NL4 found in patients with autism spectrum disorders. Overall, the NL2 structure promises to provide a valuable model for dissecting NL isoform- and synapse-specific functions.

Neuroligin-3 is a neuronal adhesion protein at GABAergic and glutamatergic synapses.

Synaptic adhesion molecules are thought to play a critical role in the formation, function and plasticity of neuronal networks. Neuroligins (NL1-4) are a family of presumptive postsynaptic cell adhesion molecules. NL1 and NL2 isoforms are concentrated at glutamatergic and GABAergic synapses, respectively, but the cellular expression and synaptic localization of the endogenous NL3 and NL4 isoforms are unknown. We generated a panel of NL isoform-specific antibodies and examined the expression, developmental regulation and synaptic specificity of NL3. We found that NL3 was enriched in brain, where NL3 protein levels increased during postnatal development, coinciding with the peak of synaptogenesis. Subcellular fractionation revealed a concentration of NL3 in synaptic plasma membranes and postsynaptic densities. In cultured hippocampal neurons, endogenous NL3 was highly expressed and was localized at both glutamatergic and GABAergic synapses. Clustering of NL3 in hippocampal neurons by neurexin-expressing cells resulted in coaggregation of NL3 with glutamatergic and GABAergic scaffolding proteins. Finally, individual synapses contained colocalized NL2 and NL3 proteins, and coimmunoprecipitation studies revealed the presence of NL1-NL3 and NL2-NL3 complexes in brain extracts. These findings suggest that rodent NL3 is a synaptic adhesion molecule that is a shared component of glutamatergic and GABAergic synapses.

Substrate specificity of lipoprotein lipase and endothelial lipase: studies of lid chimeras.

The triglyceride (TG) lipase gene subfamily, consisting of LPL, HL, and endothelial lipase (EL), plays a central role in plasma lipoprotein metabolism. Compared with LPL and HL, EL is relatively more active as a phospholipase than as a TG lipase. The amino acid loop or "lid" covering the catalytic site has been implicated as the basis for the difference in substrate specificity between HL and LPL. To determine the role of the lid in the substrate specificity of EL, we studied EL in comparison with LPL by mutating specific residues of the EL lid and exchanging their lids. Mutation studies showed that amphipathic properties of the lid contribute to substrate specificity. Exchanging lids between LPL and EL only partially shifted the substrate specificity of the enzymes. Studies of a double chimera possessing both the lid and the C-terminal domain (C-domain) of EL in the LPL backbone showed that the role of the lid in determining substrate specificity does not depend on the nature of the C-domain of the lipase. Using a kinetic assay, we showed an additive effect of the EL lid on the apparent affinity for HDL(3) in the presence of the EL C-domain.

Complete deficiency of the low-density lipoprotein receptor is associated with increased apolipoprotein B-100 production.

OBJECTIVE: We addressed the role of the low-density lipoprotein (LDL) receptor in determining clearance rates and production rate (PR) of apolipoprotein B (apoB) in humans. METHODS AND RESULTS: Kinetic studies using endogenous labeling of apoB with deuterated leucine were performed in 7 genetically defined patients with homozygous familial hypercholesterolemia (FH) and compared with 4 controls. The fractional catabolic rates (FCR) and PRs for apoB were determined by multicompartmental modeling. The FCRs of very-low-density lipoprotein 1 (VLDL1), VLDL2, intermediate-density lipoprotein (IDL), and LDL apoB were lower in FH than in controls, with the LDL apoB FCR being significantly lower (0.148+/-0.049 versus 0.499+/-0.099 pools x d(-1); P=0.008). Whereas receptor-defective FH patients had a total apoB PR similar to controls, receptor-null FH patients had a significantly greater total apoB PR than controls (35.97+/-10.51 versus 21.32+/-4.21 mg x kg(-1) x d(-1), respectively; P=0.02). CONCLUSIONS: This first study of apoB metabolism in homozygous FH using endogenous labeling with stable isotopes demonstrates that the LDL receptor contributes significantly to the clearance of LDL from plasma but plays a lesser role in the clearance of larger apoB-containing lipoproteins. Furthermore, these data also indicate that absence of a LDL receptor in humans substantially influences the apoB PR in vivo.

Epilepsy after early-life seizures can be independent of hippocampal injury.

Prolonged early-life seizures are considered potential risk factors for later epilepsy development, but mediators of this process remain largely unknown. Seizure-induced structural damage in hippocampus, including cell loss and mossy fiber sprouting, is thought to contribute to the hyperexcitability characterizing epilepsy, but a causative role has not been established. To determine whether early-life insults that lead to epilepsy result in similar structural changes, we subjected rat pups to lithium-pilocarpine-induced status epilepticus during postnatal development (day 20) and examined them as adults for the occurrence of spontaneous seizures and alterations in hippocampal morphology. Sixty-seven percent of rats developed spontaneous seizures after status epilepticus, yet only one third of these epileptic animals exhibited visible hippocampal cell loss or mossy fiber sprouting in dentate gyrus. Most epileptic rats had no apparent structural alterations in the hippocampus detectable using standard light microscopy methods (profile counts and Timm's staining). These results suggest that hippocampal cell loss and mossy fiber sprouting can occur after early-life status epilepticus but may not be necessary prerequisites for epileptogenesis in the developing brain.