Mechanism-based rescue of Munc18-1 dysfunction in varied encephalopathies by chemical chaperones.

Heterozygous de novo mutations in the neuronal protein Munc18-1 are linked to epilepsies, intellectual disability, movement disorders, and neurodegeneration. These devastating diseases have a poor prognosis and no known cure, due to lack of understanding of the underlying disease mechanism. To determine how mutations in Munc18-1 cause disease, we use newly generated S. cerevisiae strains, C. elegans models, and conditional Munc18-1 knockout mouse neurons expressing wild-type or mutant Munc18-1, as well as in vitro studies. We find that at least five disease-linked missense mutations of Munc18-1 result in destabilization and aggregation of the mutant protein. Aggregates of mutant Munc18-1 incorporate wild-type Munc18-1, depleting functional Munc18-1 levels beyond hemizygous levels. We demonstrate that the three chemical chaperones 4-phenylbutyrate, sorbitol, and trehalose reverse the deficits caused by mutations in Munc18-1 in vitro and in vivo in multiple models, offering a novel strategy for the treatment of varied encephalopathies.

Unique Structural Features of Membrane-Bound C-Terminal Domain Motifs Modulate Complexin Inhibitory Function.

Complexin is a small soluble presynaptic protein that interacts with neuronal SNARE proteins in order to regulate synaptic vesicle exocytosis. While the SNARE-binding central helix of complexin is required for both the inhibition of spontaneous fusion and the facilitation of synchronous fusion, the disordered C-terminal domain (CTD) of complexin is specifically required for its inhibitory function. The CTD of worm complexin binds to membranes via two distinct motifs, one of which undergoes a membrane curvature dependent structural transition that is required for efficient inhibition of neurotransmitter release, but the conformations of the membrane-bound motifs remain poorly characterized. Visualizing these conformations is required to clarify the mechanisms by which complexin membrane interactions regulate its function. Here, we employ optical and magnetic resonance spectroscopy to precisely define the boundaries of the two CTD membrane-binding motifs and to characterize their conformations. We show that the curvature dependent amphipathic helical motif features an irregular element of helical structure, likely a pi-bulge, and that this feature is important for complexin inhibitory function in vivo.

Evolutionary Divergence of the C-terminal Domain of Complexin Accounts for Functional Disparities between Vertebrate and Invertebrate Complexins.

Complexin is a critical presynaptic protein that regulates both spontaneous and calcium-triggered neurotransmitter release in all synapses. Although the SNARE-binding central helix of complexin is highly conserved and required for all known complexin functions, the remainder of the protein has profoundly diverged across the animal kingdom. Striking disparities in complexin inhibitory activity are observed between vertebrate and invertebrate complexins but little is known about the source of these differences or their relevance to the underlying mechanism of complexin regulation. We found that mouse complexin 1 (mCpx1) failed to inhibit neurotransmitter secretion in Caenorhabditis elegans neuromuscular junctions lacking the worm complexin 1 (CPX-1). This lack of inhibition stemmed from differences in the C-terminal domain (CTD) of mCpx1. Previous studies revealed that the CTD selectively binds to highly curved membranes and directs complexin to synaptic vesicles. Although mouse and worm complexin have similar lipid binding affinity, their last few amino acids differ in both hydrophobicity and in lipid binding conformation, and these differences strongly impacted CPX-1 inhibitory function. Moreover, function was not maintained if a critical amphipathic helix in the worm CPX-1 CTD was replaced with the corresponding mCpx1 amphipathic helix. Invertebrate complexins generally shared more C-terminal similarity with vertebrate complexin 3 and 4 isoforms, and the amphipathic region of mouse complexin 3 significantly restored inhibitory function to worm CPX-1. We hypothesize that the CTD of complexin is essential in conferring an inhibitory function to complexin, and that this inhibitory activity has been attenuated in the vertebrate complexin 1 and 2 isoforms. Thus, evolutionary changes in the complexin CTD differentially shape its synaptic role across phylogeny.

Synaptic activity regulates the abundance and binding of complexin.

Nervous system function relies on precise chemical communication between neurons at specialized junctions known as synapses. Complexin (CPX) is one of a small number of cytoplasmic proteins that are indispensable in controlling neurotransmitter release through SNARE and synaptic vesicle interactions. However, the mechanisms that recruit and stabilize CPX are poorly understood. The mobility of CPX tagged with photoactivatable green fluorescent protein (pGFP) was quantified in vivo using Caenorhabditis elegans. Although pGFP escaped the synapse within seconds, CPX-pGFP displayed both fast and slow decay components, requiring minutes for complete exchange of the synaptic pool. The longer synaptic residence time of CPX arose from both synaptic vesicle and SNARE interactions, and surprisingly, CPX mobility depended on synaptic activity. Moreover, mouse CPX-GFP reversibly dispersed out of hippocampal presynaptic terminals during stimulation, and blockade of vesicle fusion prevented CPX dispersion. Hence, synaptic CPX can rapidly redistribute and this exchange is influenced by neuronal activity, potentially contributing to use-dependent plasticity.

Membrane curvature sensing by the C-terminal domain of complexin.

Complexin functions at presynaptic nerve terminals to inhibit spontaneous SNARE-mediated synaptic vesicle (SV) exocytosis, while enhancing stimulated neurotransmitter release. The C-terminal domain (CTD) of complexin is essential for its inhibitory function and has been implicated in localizing complexin to SVs via direct membrane interactions. Here we show that complexin's CTD is highly sensitive to membrane curvature, which it senses via tandem motifs, a C-terminal motif containing a mix of bulky hydrophobic and positively charged residues, and an adjacent amphipathic region that can bind membranes in either a disordered or a helical conformation. Helix formation requires membrane packing defects found on highly curved membrane surfaces. Mutations that disrupt helix formation without disrupting membrane binding compromise complexin's inhibitory function in vivo. Thus, this membrane curvature-dependent conformational transition, combined with curvature-sensitive binding by the adjacent C-terminal motif, constitute a novel mechanism for activating complexin's inhibitory function on the surface of SVs.

Synaptic vesicles position complexin to block spontaneous fusion.

Synapses continually replenish their synaptic vesicle (SV) pools while suppressing spontaneous fusion events, thus maintaining a high dynamic range in response to physiological stimuli. The presynaptic protein complexin can both promote and inhibit fusion through interactions between its α-helical domain and the SNARE complex. In addition, complexin's C-terminal half is required for the inhibition of spontaneous fusion in worm, fly, and mouse, although the molecular mechanism remains unexplained. We show here that complexin's C-terminal domain binds lipids through a novel protein motif, permitting complexin to inhibit spontaneous exocytosis in vivo by targeting complexin to SVs. We propose that the SV pool serves as a platform to sequester and position complexin where it can intercept the rapidly assembling SNAREs and control the rate of spontaneous fusion.

Three distinct amine receptors operating at different levels within the locomotory circuit are each essential for the serotonergic modulation of chemosensation in Caenorhabditis elegans.

Serotonin modulates behavioral plasticity in both vertebrates and invertebrates and in Caenorhabditis elegans regulates key behaviors, including locomotion, aversive learning and olfaction through at least four different 5-HT receptors. In the present study, we examined the serotonergic stimulation of aversive responses to dilute octanol in animals containing null alleles of these 5-HT receptors. Both ser-1 and mod-1 null animals failed to increase sensitivity to dilute octanol on food/5-HT, in contrast to wild-type, ser-4 or ser-7 null animals. 5-HT sensitivity was restored by the expression of MOD-1 and SER-1 in the AIB or potentially the AIY, and RIA interneurons of mod-1 and ser-1 null animals, respectively. Because none of these 5-HT receptors appear to be expressed in the ASH sensory neurons mediating octanol sensitivity, we identified a 5-HT(6)-like receptor, F16D3.7(SER-5), that was required for food/5-HT-dependent increases in octanol sensitivity. ser-5 null animals failed to increase octanol sensitivity in the presence of food/5-HT and sensitivity could be restored by expression of SER-5 in the ASHs. Similarly, the RNAi knockdown of ser-5 expression in the ASHs of wild-type animals also abolished 5-HT-dependent increases in octanol sensitivity, suggesting that SER-5 modulates the octanol responsiveness of the ASHs directly. Together, these results suggest that multiple amine receptors, functioning at different levels within the locomotory circuit, are each essential for the serotonergic modulation of ASH-mediated aversive responses.

Tyramine and octopamine independently inhibit serotonin-stimulated aversive behaviors in Caenorhabditis elegans through two novel amine receptors.

Biogenic amines modulate key behaviors in both vertebrates and invertebrates. In Caenorhabditis elegans, tyramine (TA) and octopamine (OA) inhibit aversive responses to 100%, but not dilute (30%) octanol. TA and OA also abolish food- and serotonin-dependent increases in responses to dilute octanol in wild-type but not tyra-3(ok325) and f14d12.6(ok371) null animals, respectively, suggesting that TA and OA modulated responses to dilute octanol are mediated by separate, previously uncharacterized, G-protein-coupled receptors. TA and OA are high-affinity ligands for TYRA-3 and F14D12.6, respectively, based on their pharmacological characterization after heterologous expression. f14d12.6::gfp is expressed in the ASHs, the neurons responsible for sensitivity to dilute octanol, and the sra-6-dependent expression of F14D12.6 in the ASHs is sufficient to rescue OA sensitivity in f14d12.6(ok371) null animals. In contrast, tyra-3::gfp appears not to be expressed in the ASHs, but instead in other neurons, including the dopaminergic CEP/ADEs. However, although dopamine (DA) also inhibits 5-HT-dependent responses to dilute octanol, TA still inhibits in dop-2; dop-1; dop-3 animals that do not respond to DA and cat-2(tm346) and Pdat-1::ICE animals that lack significant dopaminergic signaling, suggesting that DA is not an intermediate in TA inhibition. Finally, responses to TA and OA selectively desensitize after preexposure to the amines. Our data suggest that although tyraminergic and octopaminergic signaling yield identical phenotypes in these olfactory assays, they act independently through distinct receptors to modulate the ASH-mediated locomotory circuit and that C. elegans is a useful model to study the aminergic modulation of sensory-mediated locomotory behaviors.