Astrid Rohlmann

α-Neurexins couple Ca2+ channels to synaptic vesicle exocytosis

Synapses are specialized intercellular junctions in which cell adhesion molecules connect the presynaptic machinery for neurotransmitter release to the postsynaptic machinery for receptor signalling. Neurotransmitter release requires the presynaptic co-assembly of Ca2+ channels with the secretory apparatus, but little is known about how synaptic components are organized. α-Neurexins, a family of >1,000 presynaptic cell-surface proteins encoded by three genes, link the pre- and postsynaptic compartments of synapses by binding extracellularly to postsynaptic cell adhesion molecules and intracellularly to presynaptic PDZ domain proteins. Using triple-knockout mice, we show that α-neurexins are not required for synapse formation, but are essential for Ca2+-triggered neurotransmitter release. Neurotransmitter release is impaired because synaptic Ca2+ channel function is markedly reduced, although the number of cell-surface Ca2+ channels appears normal. These data suggest that α-neurexins organize presynaptic terminals by functionally coupling Ca2+ channels to the presynaptic machinery.

Neuronal LRP1 Functionally Associates with Postsynaptic Proteins and Is Required for Normal Motor Function in Mice

ABSTRACT The LDL receptor-related protein 1 (LRP1) is a multifunctional cell surface receptor that is highly expressed on neurons. Neuronal LRP1 in vitro can mediate ligand endocytosis, as well as modulate signal transduction processes. However, little is known about its role in the intact nervous system. Here, we report that mice that lack LRP1 selectively in differentiated neurons develop severe behavioral and motor abnormalities, including hyperactivity, tremor, and dystonia. Since their central nervous systems appear histoanatomically normal, we suggest that this phenotype is likely attributable to abnormal neurotransmission. This conclusion is supported by studies of primary cultured neurons that show that LRP1 is present in close proximity to the N-methyl-d-aspartate (NMDA) receptor in dendritic synapses and can be coprecipitated with NMDA receptor subunits and the postsynaptic density protein PSD-95 from neuronal cell lysates. Moreover, treatment with NMDA, but not dopamine, reduces the interaction of LRP1 with PSD-95, indicating that LRP1 participates in transmitter-dependent postsynaptic responses. Together, these findings suggest that LRP1, like other ApoE receptors, can modulate synaptic transmission in the brain.

Extracellular Domains of α-Neurexins Participate in Regulating Synaptic Transmission by Selectively Affecting N- and P/Q-Type Ca2+ Channels

Neurexins constitute a large family of highly variable cell-surface molecules that may function in synaptic transmission and/or synapse formation. Each of the three known neurexin genes encodes two major neurexin variants, α- and β-neurexins, that are composed of distinct extracellular domains linked to identical intracellular sequences. Deletions of one, two, or all three α-neurexins in mice recently demonstrated their essential role at synapses. In multiple α-neurexin knock-outs, neurotransmitter release from excitatory and inhibitory synapses was severely reduced, primarily probably because voltage-dependent Ca2+ channels were impaired. It remained unclear, however, which neurexin variants actually influence exocytosis and Ca2+ channels, which domain of neurexins is required for this function, and which Ca2+-channel subtypes are regulated. Here, we show by electrophysiological recordings that transgenic neurexin 1α rescues the release and Ca2+-current phenotypes, whereas transgenic neurexin 1β has no effect, indicating the importance of the extracellular sequences for the function of neurexins. Because neurexin 1α rescued the knock-out phenotype independent of the α-neurexin gene deleted, these data are consistent with a redundant function among different α-neurexins. In both knock-out and transgenically rescued mice, α-neurexins selectively affected the component of neurotransmitter release that depended on activation of N- and P/Q-type Ca2+ channels, but left L-type Ca2+ channels unscathed. Our findings indicate that α-neurexins represent organizer molecules in neurotransmission that regulate N- and P/Q-type Ca2+ channels, constituting an essential role at synapses that critically involves the extracellular domains of neurexins.

Structure-Function Relationships in Gap Junctions

Gap junctions are metabolic and electrotonic pathways between cells and provide direct cooperation within and between cellular nets. They are among the cellular structures most frequently investigated. This chapter primarily addresses aspects of the assembly of the gap junction channel, considering the insertion of the protein into the membrane, the importance of phosphorylation of the gap junction proteins for coupling modulation, and the formation of whole channels from two hemichannels. Interactions of gap junctions with the subplasmalemmal cytoplasm on the one side and with tight junctions on the other side are closely considered. Furthermore, reviewing the significance and alterations of gap junctions during development and oncogenesis, respectively, including the role of adhesion molecules, takes up a major part of the chapter. Finally, the literature on gap junctions in the central nervous system, especially between astrocytes in the brain cortex and horizontal cells in the retina, is summarized and new aspects on their structure-function relationship included.

Deletion of α‐neurexins does not cause a major impairment of axonal pathfinding or synapse formation

α‐Neurexins are synaptic cell‐surface molecules that are required for Ca2+‐triggered exocytosis. Mice lacking all three α‐neurexins show drastically reduced neurotransmitter release at excitatory and inhibitory synapses and die early postnatally. Although previous histological analysis of newborn α‐neurexin triple mutants revealed only a moderate reduction in the density of type II synapses in the brainstem, cell culture studies proposed that neurexins are prominently involved in synapse formation. To assess the contribution of α‐neurexins to the formation and structural properties of synapses in vivo, we performed a detailed morphological analysis of the brains from surviving adult double knockout mice lacking two of the three α‐neurexins. Despite their impaired neurotransmission, we did not observe any gross anatomical defects or changes in the distribution of synaptic proteins in adult mutants. Only mild structural alterations were found: a ∼20% reduction of neuropil area in many brain regions, resulting predominantly from shortened distal dendritic branches and fewer spines, as demonstrated by Golgi impregnation of pyramidal neurons. Quantitative electron microscopy revealed ultrastructurally normal type I and II terminals and a ∼30% decrease in the density of type II synapses in the neocortex. To exclude errors in pathfinding, we investigated axonal projections in the olfactory bulb of newborn knockouts and did not observe any changes. Therefore, α‐neurexins are not essential for the formation of the vast majority of synapses in vivo but rather regulate the function of these synapses. J. Comp. Neurol. 502:261–274, 2007.

Astrocytes as rapid sensors of peripheral axotomy in the facial nucleus of rats

Facial nerve transection leads to functional and structural reactions in lesioned motor neurones and surrounding glial cells. Data from this study provide evidence that the most rapid reaction described so far consists of an increase in immunoreactivity of connexin-43 (cx-43), the predominant gap junction protein in astrocytes. The ipsilateral facial nucleus is selectively marked as early as 0.75 to 1.5 hours after axotomy, while the unlesioned side as well as the unoperated controls remain faintly stained. Thus, enhanced coupling capacity of astrocytes by gap junctions appears to be a sensitive indicator of modified neuronal-glial interaction in the CNS.

Autocellular coupling by gap junctions in cultured astrocytes: A new view on cellular autoregulation during process formation

Neocortical astrocytes make two types of gap junctions, intercellular ones create a functional syncytium, while reflexive gap junctions mediate autocellular coupling and serve unknown functions (Rohlmann and Wolff, 1996). Here, the question is addressed whether solitary astrocytes in vitro express connexin43 (Cx43) and establish gap junctions in the absence of intercellular contacts. In all media conditions tested, immunocytochemistry visualized Cx43‐expression and gap junctions irrespective of the presence or absence of intercellular contacts. Reflexive gap junctions were associated with mechanical junctions (adherent spots and fascia adherens) connecting surface membranes and cytoskelal components, respectively. Both were characteristically located along incompletely separated borders between developing processes and/or branches. In addition, Cx43‐immunoreactivity was found on some non‐junctional membranes: i) intracellular vesicle clusters sited to forming processes and at the basis of filopodia; ii) the surface membrane of filopodial subpopulations usually appearing in bunches. Results suggest changes in the resumptive role of Cx43 in cultivated astrocytes: 1) Cx43 is not confined to intercellular gap junctions, it may even selectively compose reflexive ones; 2) from intracellular stores (vesicle aggregates), Cx43 may be incorporated into the surface membrane of filopodia; 3) by contacting other parts of the same cell surface (or neighboring cells), filopodia and membrane patches carrying Cx43‐half channels may be essential in initial steps of gap junction formation; 4) the distribution of reflexive gap junctions is compatible with the hypothesis that autocellular coupling serves reorganization of cytoskeleton during the formation of cell processes and branches; 5) in general, gap junctions may be important for coordinating the cytoskeleton across intercellular contacts and within cells with complex shape. GLIA 24:121–140, 1998.

Facial nerve lesions lead to increased immunostaining of the astrocytic gap junction protein (connexin 43) in the corresponding facial nucleus of rats

After peripheral transection of the facial nerve, immunostaining of astrocytic gap junction protein changed in the corresponding brainstem nucleus of the rat. Enhanced connexin-43 immunoreactivity was restricted to the ipsilateral facial nucleus and to astrocytes surrounding lesioned motoneurons. This reaction is focally distinct, and marks only a part of the astrocytic network indicating a local plasticity of intercellular coupling. These results suggest that astrocytes work as sensors of signals which either depend on the integrity of neighboring neurons or inform about neuronal disorders.

Polarized Targeting of Neurexins to Synapses Is Regulated by their C-Terminal Sequences

Two families of cell-adhesion molecules, predominantly presynaptic neurexins and postsynaptic neuroligins, are important for the formation and functioning of synapses in the brain, and mutations in several genes encoding these transmembrane proteins have been found in autism patients. However, very little is known about how neurexins are targeted to synapses and which mechanisms regulate this process. Using various epitope-tagged neurexins in primary hippocampal neurons of wild-type and knock-out mice in vitro and in transgenic animals in vivo, we show that neurexins are trafficked throughout neurons via transport vesicles and the plasma membrane insertion of neurexins occurs preferentially in the axonal/synaptic compartment. We also observed that exit of neurexins from the ER/Golgi and correct targeting require their PDZ-binding motif at the C terminus, whereas two presumptive ER retention signals are inactive. The ubiquitous presence of neurexin-positive transport vesicles and absence of bassoon colabeling demonstrate that these carriers are not active zone precursor vesicles, but colocalization with CASK, RIM1α, and calcium channels suggests that they may carry additional components of the exocytotic machinery. Our data indicate that neurexins are delivered to synapses by a polarized and regulated targeting process that involves PDZ-domain mediated interactions, suggesting a novel pathway for the distribution of neurexins and other synaptic proteins.

Dendritic spine formation and synaptic function require neurobeachin

A challenge in neuroscience is to understand the mechanisms underlying synapse formation. Most excitatory synapses in the brain are built on spines, which are actin-rich protrusions from dendrites. Spines are a major substrate of brain plasticity, and spine pathologies are observed in various mental illnesses. Here we investigate the role of neurobeachin (Nbea), a multidomain protein previously linked to cases of autism, in synaptogenesis. We show that deletion of Nbea leads to reduced numbers of spinous synapses in cultured neurons from complete knockouts and in cortical tissue from heterozygous mice, accompanied by altered miniature postsynaptic currents. In addition, excitatory synapses terminate mostly at dendritic shafts instead of spine heads in Nbea mutants, and actin becomes less enriched synaptically. As actin and synaptopodin, a spine-associated protein with actin-bundling activity, accumulate ectopically near the Golgi apparatus of mutant neurons, a role emerges for Nbea in trafficking important cargo to pre- and postsynaptic compartments.