Presynaptic neurons self-tune by inversely coupling neurotransmitter release with the abundance of CaV2 voltage-gated Ca2+ channels.

The abundance of CaV2 voltage-gated calcium channels is linked to presynaptic homeostatic plasticity (PHP), a process that recalibrates synaptic strength to maintain the stability of neural circuits. However, the molecular and cellular mechanisms governing PHP and CaV2 channels are not completely understood. Here, we uncover a previously not described form of PHP in Caenorhabditis elegans, revealing an inverse regulatory relationship between the efficiency of neurotransmitter release and the abundance of UNC-2/CaV2 channels. Gain-of-function unc-2SL(S240L) mutants, which carry a mutation analogous to the one causing familial hemiplegic migraine type 1 in humans, showed markedly reduced channel abundance despite increased channel functionality. Reducing synaptic release in these unc-2SL(S240L) mutants restored channel levels to those observed in wild-type animals. Conversely, loss-of-function unc-2DA(D726A) mutants, which harbor the D726A mutation in the channel pore, exhibited a marked increase in channel abundance. Enhancing synaptic release in unc-2DA mutants reversed this increase in channel levels. Importantly, this homeostatic regulation of UNC-2 channel levels is accompanied by the structural remodeling of the active zone (AZ); specifically, unc-2DA mutants, which exhibit increased channel abundance, showed parallel increases in select AZ proteins. Finally, our forward genetic screen revealed that WWP-1, a HECT family E3 ubiquitin ligase, is a key homeostatic mediator that removes UNC-2 from synapses. These findings highlight a self-tuning PHP regulating UNC-2/CaV2 channel abundance along with AZ reorganization, ensuring synaptic strength and stability.

C. elegans Clarinet/CLA-1 recruits RIMB-1/RIM-binding protein and UNC-13 to orchestrate presynaptic neurotransmitter release.

Synaptic transmission requires the coordinated activity of multiple synaptic proteins that are localized at the active zone (AZ). We previously identified a Caenorhabditis elegans protein named Clarinet (CLA-1) based on homology to the AZ proteins Piccolo, Rab3-interactingmolecule (RIM)/UNC-10 and Fife. At the neuromuscular junction (NMJ), cla-1 null mutants exhibit release defects that are greatly exacerbated in cla-1;unc-10 double mutants. To gain insights into the coordinated roles of CLA-1 and UNC-10, we examined the relative contributions of each to the function and organization of the AZ. Using a combination of electrophysiology, electron microscopy, and quantitative fluorescence imaging we explored the functional relationship of CLA-1 to other key AZ proteins including: RIM1, Cav2.1 channels, RIM1-binding protein, and Munc13 (C. elegans UNC-10, UNC-2, RIMB-1 and UNC-13, respectively). Our analyses show that CLA-1 acts in concert with UNC-10 to regulate UNC-2 calcium channel levels at the synapse via recruitment of RIMB-1. In addition, CLA-1 exerts a RIMB-1-independent role in the localization of the priming factor UNC-13. Thus C. elegans CLA-1/UNC-10 exhibit combinatorial effects that have overlapping design principles with other model organisms: RIM/RBP and RIM/ELKS in mouse and Fife/RIM and BRP/RBP in Drosophila. These data support a semiconserved arrangement of AZ scaffolding proteins that are necessary for the localization and activation of the fusion machinery within nanodomains for precise coupling to Ca2+ channels.

Active Zone Trafficking of CaV2/UNC-2 Channels Is Independent of ß/CCB-1 and α2δ/UNC-36 Subunits.

The CaV2 voltage-gated calcium channel is the major conduit of calcium ions necessary for neurotransmitter release at presynaptic active zones (AZs). The CaV2 channel is a multimeric complex that consists of a pore-forming α1 subunit and two auxiliary ß and α2δ subunits. Although auxiliary subunits are critical for channel function, whether they are required for α1 trafficking is unresolved. Using endogenously fluorescent protein-tagged CaV2 channel subunits in Caenorhabditis elegans, we show that UNC-2/α1 localizes to AZs even in the absence of CCB-1/ß or UNC-36/α2δ, albeit at low levels. When UNC-2 is manipulated to be trapped in the endoplasmic reticulum (ER), CCB-1 and UNC-36 fail to colocalize with UNC-2 in the ER, indicating that they do not coassemble with UNC-2 in the ER. Moreover, blocking ER-associated degradation does not further increase presynaptic UNC-2 channels in ccb-1 or unc-36 mutants, indicating that UNC-2 levels are not regulated in the ER. An unc-2 mutant lacking C-terminal AZ protein interaction sites with intact auxiliary subunit binding sites displays persistent presynaptic UNC-2 localization and a prominent increase of UNC-2 channels in nonsynaptic axonal regions, underscoring a protective role of auxiliary subunits against UNC-2 degradation. In the absence of UNC-2, presynaptic CCB-1 and UNC-36 are profoundly diminished to barely detectable levels, indicating that UNC-2 is required for the presynaptic localization of CCB-1 and UNC-36. Together, our findings demonstrate that although the pore-forming subunit does not require auxiliary subunits for its trafficking and transport to AZs, it recruits auxiliary subunits to stabilize and expand calcium channel signalosomes.SIGNIFICANCE STATEMENT Synaptic transmission in the neuron hinges on the coupling of synaptic vesicle exocytosis with calcium influx. This calcium influx is mediated by CaV2 voltage-gated calcium channels. These channels consist of one pore-forming α1 subunit and two auxiliary ß and α2δ subunits. The auxiliary subunits enhance channel function and regulate the overall level of channels at presynaptic terminals. However, it is not settled how these auxiliary subunits regulate the overall channel level. Our study in C. elegans finds that although the auxiliary subunits do not coassemble with α1 and aid trafficking, they are recruited to α1 and stabilize the channel complex at presynaptic terminals. Our study suggests that drugs that target the auxiliary subunits can directly destabilize and have an impact on CaV2 channels.

Interference with glutamate antiporter system xc - enables post-hypoxic long-term potentiation in hippocampus.

Our group previously showed that genetic or pharmacological inhibition of the cystine/glutamate antiporter, system xc -, mitigates excitotoxicity after anoxia by increasing latency to anoxic depolarization, thus attenuating the ischaemic core. Hypoxia, however, which prevails in the ischaemic penumbra, is a condition where neurotransmission is altered, but excitotoxicity is not triggered. The present study employed mild hypoxia to further probe ischaemia-induced changes in neuronal responsiveness from wild-type and xCT KO (xCT-/-) mice. Synaptic transmission was monitored in hippocampal slices from both genotypes before, during and after a hypoxic episode. Although wild-type and xCT-/- slices showed equal suppression of synaptic transmission during hypoxia, mutant slices exhibited a persistent potentiation upon re-oxygenation, an effect we termed 'post-hypoxic long-term potentiation (LTP)'. Blocking synaptic suppression during hypoxia by antagonizing adenosine A1 receptors did not preclude post-hypoxic LTP. Further examination of the induction and expression mechanisms of this plasticity revealed that post-hypoxic LTP was driven by NMDA receptor activation, as well as increased calcium influx, with no change in paired-pulse facilitation. Hence, the observed phenomenon engaged similar mechanisms as classical LTP. This was a remarkable finding as theta-burst stimulation-induced LTP was equivalent between genotypes. Importantly, post-hypoxic LTP was generated in wild-type slices pretreated with system xc - inhibitor, S-4-carboxyphenylglycine, thereby confirming the antiporter's role in this phenomenon. Collectively, these data indicate that system xc - interference enables neuroplasticity in response to mild hypoxia, and, together with its regulation of cellular damage in the ischaemic core, suggest a role for the antiporter in post-ischaemic recovery of the penumbra.

Fiduciary assignment as a potential intervention for substance use disorder: a retrospective chart review.

Background: Assignment of fiduciaries to veterans with disability payments is an intervention thought to improve quality of life; however, in veterans who use substances, a proportion of these payments may be misspent on drugs and/or alcohol. While fiduciary assignment may reduce funds available to purchase substances, clinical efficacy of this intervention in the management of substance use disorders has not been rigorously demonstrated.Objectives: The purpose of this study is to evaluate changes in clinical status before and after fiduciary assignment.Methods: This was a retrospective chart review of 50 (44 male, 6 female) veterans who were assigned a fiduciary and determined to have a substance use disorder (SUD). SUD-related data including outpatient and inpatient treatment, toxicology testing, and measures of psychosocial functioning for the three years before and after fiduciary assignment were extracted and compared.Results: Veterans were found to have higher rates of any form of employment after fiduciary assignment (Wilcoxon, Signed Ranked S-statistic = 0.22, pr = 0.02). Two changes in measures of substance use were found after fiduciary assignment. There was a reduction in positive screens for heroin (tstatistic = -2.7, p = .01), but an increase in positive screens for fentanyl (t statistic = 2.53, p = .02). There were some potentially clinically but not statistically significant trends in increased adherence with mental health appointments, number of medical hospitalizations, and rates of employment post-fiduciary assignment.Conclusions: Understanding the clinical impact of fiduciary assignment for veteran's benefits is desirable but still pending at this time.

Participation-related constructs and participation of children with additional support needs in schools.

AIM: To investigate associations between participation-related constructs and participation frequency and involvement in inclusive schools. METHOD: In this cross-sectional study, teachers of children with additional support needs, including intellectual disability, autism, and learning difficulties, completed measures. Participation-related constructs were measured using the School Participation Questionnaire; participation frequency and involvement were measured using the Participation and Environment Measure for Children and Youth. A series of multilevel linear mixed-effects regression models with maximum likelihood estimates and bootstrap confidence intervals with p-values were obtained. Final models included participation-related constructs and participation, controlling for demographic and diagnostic confounders (including age, sex, language, level of school support, and autism). RESULTS: Six hundred and eighty-eight children (448 [65.1%] males; mean age 8 years 7 months [range 4 years 10 months-12 years 13 months, standard deviation 2 years 1 months]) were assessed by 252 teachers. Across a series of models, participation-related constructs were consistently associated with more intensive participation (competence, environment, identity p < 0.001; symptoms p = 0.007), independent of confounders. More frequent participation remained associated with three of four participation-related constructs (competence, identity p < 0.001; environment p = 0.021). Age (p = 0.046), language (p = 0.002), and level of school support (p = 0.039) also remained significantly associated with frequency of participation. INTERPRETATION: Children with additional support needs in inclusive schools may have several participation barriers. Policies and interventions to improve participation are needed. WHAT THIS PAPER ADDS: Across a series of models, participation-related constructs were associated with frequency and intensity of participation. Only participation-related constructs were associated with participation intensity. Demographic and diagnostic variables were associated with frequency, not intensity, of participation. Teacher assessment is valid for assessment of participation and participation-related constructs.

BK channel density is regulated by endoplasmic reticulum associated degradation and influenced by the SKN-1A/NRF1 transcription factor.

Ion channels are present at specific levels within subcellular compartments of excitable cells. The regulation of ion channel trafficking and targeting is an effective way to control cell excitability. The BK channel is a calcium-activated potassium channel that serves as a negative feedback mechanism at presynaptic axon terminals and sites of muscle excitation. The C. elegans BK channel ortholog, SLO-1, requires an endoplasmic reticulum (ER) membrane protein for efficient anterograde transport to these locations. Here, we found that, in the absence of this ER membrane protein, SLO-1 channels that are seemingly normally folded and expressed at physiological levels undergo SEL-11/HRD1-mediated ER-associated degradation (ERAD). This SLO-1 degradation is also indirectly regulated by a SKN-1A/NRF1-mediated transcriptional mechanism that controls proteasome levels. Therefore, our data indicate that SLO-1 channel density is regulated by the competitive balance between the efficiency of ER trafficking machinery and the capacity of ERAD.

UNC-2 CaV2 Channel Localization at Presynaptic Active Zones Depends on UNC-10/RIM and SYD-2/Liprin-α in Caenorhabditis elegans.

Presynaptic active zone proteins couple calcium influx with synaptic vesicle exocytosis. However, the control of presynaptic calcium channel localization by active zone proteins is not completely understood. In a Caenorhabditis elegans (C. elegans) forward genetic screen, we find that UNC-10/RIM (Rab3-interacting molecule) and SYD-2/Liprin-α regulate presynaptic localization of UNC-2, the CaV2 channel ortholog. We further quantitatively analyzed live animals using endogenously GFP-tagged UNC-2 and active zone components. Consistent with the interaction between RIM and CaV2 in mammals, the intensity and number of UNC-2 channel puncta at presynaptic terminals were greatly reduced in unc-10 mutant animals. To understand how SYD-2 regulates presynaptic UNC-2 channel localization, we analyzed presynaptic localization of endogenous SYD-2, UNC-10, RIMB-1/RIM-BP (RIM binding protein), and ELKS-1. Our analysis revealed that although SYD-2 is the most critical for active zone assembly, loss of SYD-2 function does not completely abolish presynaptic localization of UNC-10, RIMB-1, and ELKS-1, suggesting an existence of SYD-2-independent active zone assembly. UNC-2 localization analysis in double and triple mutants of active zone components show that SYD-2 promotes UNC-2 localization by partially controlling UNC-10 localization, and ELKS-1 and RIMB-1 also contribute to UNC-2 channel localization. In addition, we find that core active zone proteins are unequal in their abundance. Although the abundance of UNC-10 at the active zone is comparable to UNC-2, SYD-2 and ELKS-1 are twice more and RIMB-1 four times more abundant than UNC-2. Together our data show that UNC-10, SYD-2, RIMB-1, and ELKS-1 control presynaptic UNC-2 channel localization in redundant yet distinct manners.SIGNIFICANCE STATEMENT Precise control of neurotransmission is dependent on the tight coupling of the calcium influx through voltage-gated calcium channels (VGCCs) to the exocytosis machinery at the presynaptic active zones. However, how these VGCCs are tethered to the active zone is incompletely understood. To understand the mechanism of presynaptic VGCC localization, we performed a C. elegans forward genetic screen and quantitatively analyzed endogenous active zones and presynaptic VGCCs. In addition to RIM, our study finds that SYD-2/Liprin-α is critical for presynaptic localization of VGCCs. Yet, the loss of SYD-2, a core active zone scaffolding protein, does not completely abolish the presynaptic localization of the VGCC, showing that the active zone is a resilient structure assembled by redundant mechanisms.

Transcriptional control of parallel-acting pathways that remove specific presynaptic proteins in remodeling neurons.

Synapses are actively dismantled to mediate circuit refinement, but the developmental pathways that regulate synaptic disassembly are largely unknown. We have previously shown that the epithelial sodium channel ENaC/UNC-8 triggers an activity-dependent mechanism that drives the removal of presynaptic proteins liprin-α/SYD-2, Synaptobrevin/SNB-1, RAB-3 and Endophilin/UNC-57 in remodeling GABAergic neurons in C. elegans (Miller-Fleming et al., 2016). Here, we report that the conserved transcription factor Iroquois/IRX-1 regulates UNC-8 expression as well as an additional pathway, independent of UNC-8, that functions in parallel to dismantle functional presynaptic terminals. We show that the additional IRX-1-regulated pathway is selectively required for the removal of the presynaptic proteins, Munc13/UNC-13 and ELKS, which normally mediate synaptic vesicle fusion and neurotransmitter release. Our findings are notable because they highlight the key role of transcriptional regulation in synapse elimination during development and reveal parallel-acting pathways that coordinate synaptic disassembly by removing specific active zone proteins.SIGNIFICANT STATEMENT:Synaptic pruning is a conserved feature of developing neural circuits but the mechanisms that dismantle the presynaptic apparatus are largely unknown. We have determined that synaptic disassembly is orchestrated by parallel-acting mechanisms that target distinct components of the active zone. Thus, our finding suggests that synaptic disassembly is not accomplished by en masse destruction but depends on mechanisms that dismantle the structure in an organized process.

Psychometric properties of the School Participation Questionnaire: Testing a measure of participation-related constructs.

AIM: To explore concurrent validity, convergent validity, interrater reliability, test-retest reliability, and Rasch model analysis of the School Participation Questionnaire (SPQ), a tool for teachers to assess personal and environmental determinants of school participation. METHOD: Teachers of children with additional support needs, including intellectual disability, autism, and learning difficulties completed measures. Data were collected using the SPQ and the Participation and Environment Measure for Children and Youth (PEM-CY). Test-retest and interrater reliability were assessed using intraclass correlation coefficients (ICCs). Internal consistency was assessed with Cronbach's alpha. Concurrent and convergent validity were explored via correlations with the PEM-CY. Further psychometrics were examined using a Rasch model. RESULTS: One hundred and eighty-seven children (136 [72.7%] male; mean age 9y [range 5y 6mo-12y 10mo, SD 2y]) were assessed by 67 teachers. Cronbach's alpha, test-retest, and interrater reliability were acceptable-excellent across each SPQ scale (alphas=0.89, 0.9, 0.94, 0.79; test-retest ICCs=0.64, 0.61, 0.78, 0.62; interrater ICCs=0.85, 0.71, 0.90, 0.81). Concurrent and convergent validity were confirmed with significant positive correlations between SPQ and PEM-CY. After Mokken and Rasch model analysis, person and item reliability were good, and unidimensionality was confirmed. Mean administration time was 8.2 minutes. INTERPRETATION: The results suggest that the SPQ is a rapid, reliable, and valid tool for assessment of participation-related indicators in schools.

Alcohol induces mitochondrial fragmentation and stress responses to maintain normal muscle function in Caenorhabditis elegans.

Chronic excessive ethanol consumption has distinct toxic and adverse effects on a variety of tissues. In skeletal muscle, ethanol causes alcoholic myopathy, which is characterized by myofiber atrophy and the loss of muscle strength. Alcoholic myopathy is more prevalent than all inherited muscle diseases combined. Current evidence indicates that ethanol directly impairs muscle organization and function. However, the underlying mechanism by which ethanol causes toxicity in muscle is poorly understood. Here, we show that the nematode Caenorhabditis elegans exhibits the key features of alcoholic myopathy when exposed to ethanol. As in mammals, ethanol exposure impairs muscle strength and induces the expression of protective genes, including oxidative stress response genes. In addition, ethanol exposure causes the fragmentation of mitochondrial networks aligned with myofibril lattices. This ethanol-induced mitochondrial fragmentation is dependent on the mitochondrial fission factor DRP-1 (dynamin-related protein 1) and its receptor proteins on the outer mitochondrial membrane. Our data indicate that this fragmentation contributes to the activation of the mitochondrial unfolded protein response (UPR). We also found that robust, perpetual mitochondrial UPR activation effectively reduces muscle weakness caused by ethanol exposure. Our results strongly suggest that the modulation of mitochondrial stress responses may provide a method to ameliorate alcohol toxicity and damage to muscle.

Prescription Drug Monitoring Program: Access in the First Year.

BACKGROUND AND OBJECTIVES: Prescription Drug Monitoring Programs (PDMP) detect high-risk prescribing and patient behaviors. This study describes the characteristics associated with documented PDMP access when prescribing opioids. METHODS: Retrospective chart review of 695 opioid prescriptions written from inpatient and outpatient medical and psychiatric settings. Data were abstracted and analyzed to identify characteristics associated with documented PDMP access. RESULTS: One-third of the charts had PDMP access documented within the week of opioid prescription; 12% showed PDMP consultation on the same day. Services varied greatly from 10.5% (inpatient medicine) to 57% (inpatient psychiatry) with regard to same-day PDMP access (P < .0001). Patient characteristics associated with PDMP access include having acute pain, current mental health treatment, and current and past substance use disorders (all P < .05). Logistic regression modeling identified three variables associated with the odds of PDMP access (c-statistic = 0.66): if the prescription originated from the inpatient medicine unit (odds ratio [OR] = 0.47, 95% confidence interval [CI] = 0.32, 0.68), or if the patient received a prescription for an opioid in the past 30 days (OR = 0.30, 95% CI = 0.10, 0.90) or had a urine toxicology screen in the past year (OR = 2.00, 95% CI = 1.40, 2.90). DISCUSSION AND CONCLUSIONS: Utilization of the PDMP varied by specialty and setting. SCIENTIFIC SIGNIFICANCE: This study is among the first to compare rates of PDMP access in a large sample by specialty and practice setting in a healthcare system with a policy requiring its access and appropriate documentation. With less than one-third adherence to the policy, additional steps to increase consistent PDMP access are warranted. (Am J Addict 2021;00:00-00).

C. elegans Punctin specifies cholinergic versus GABAergic identity of postsynaptic domains.

Because most neurons receive thousands of synaptic inputs, the neuronal membrane is a mosaic of specialized microdomains where neurotransmitter receptors cluster in register with the corresponding presynaptic neurotransmitter release sites. In many cases the coordinated differentiation of presynaptic and postsynaptic domains implicates trans-synaptic interactions between membrane-associated proteins such as neurexins and neuroligins. The Caenorhabditis elegans neuromuscular junction (NMJ) provides a genetically tractable system in which to analyse the segregation of neurotransmitter receptors, because muscle cells receive excitatory innervation from cholinergic neurons and inhibitory innervation from GABAergic neurons. Here we show that Ce-Punctin/madd-4 (ref. 5), the C. elegans orthologue of mammalian punctin-1 and punctin-2, encodes neurally secreted isoforms that specify the excitatory or inhibitory identity of postsynaptic NMJ domains. These proteins belong to the ADAMTS (a disintegrin and metalloprotease with thrombospondin repeats)-like family, a class of extracellular matrix proteins related to the ADAM proteases but devoid of proteolytic activity. Ce-Punctin deletion causes the redistribution of synaptic acetylcholine and GABAA (γ-aminobutyric acid type A) receptors into extrasynaptic clusters, whereas neuronal presynaptic boutons remain unaltered. Alternative promoters generate different Ce-Punctin isoforms with distinct functions. A short isoform is expressed by cholinergic and GABAergic motoneurons and localizes to excitatory and inhibitory NMJs, whereas long isoforms are expressed exclusively by cholinergic motoneurons and are confined to cholinergic NMJs. The differential expression of these isoforms controls the congruence between presynaptic and postsynaptic domains: specific disruption of the short isoform relocalizes GABAA receptors from GABAergic to cholinergic synapses, whereas expression of a long isoform in GABAergic neurons recruits acetylcholine receptors to GABAergic NMJs. These results identify Ce-Punctin as a previously unknown synaptic organizer and show that presynaptic and postsynaptic domain identities can be genetically uncoupled in vivo. Because human punctin-2 was identified as a candidate gene for schizophrenia, ADAMTS-like proteins may also control synapse organization in the mammalian central nervous system.

Biosynthesis of ionotropic acetylcholine receptors requires the evolutionarily conserved ER membrane complex.

The number of nicotinic acetylcholine receptors (AChRs) present in the plasma membrane of muscle and neuronal cells is limited by the assembly of individual subunits into mature pentameric receptors. This process is usually inefficient, and a large number of the synthesized subunits are degraded by endoplasmic reticulum (ER)-associated degradation. To identify cellular factors required for the synthesis of AChRs, we performed a genetic screen in the nematode Caenorhabditis elegans for mutants with decreased sensitivity to the cholinergic agonist levamisole. We isolated a partial loss-of-function allele of ER membrane protein complex-6 (emc-6), a previously uncharacterized gene in C. elegans. emc-6 encodes an evolutionarily conserved 111-aa protein with two predicted transmembrane domains. EMC-6 is ubiquitously expressed and localizes to the ER. Partial inhibition of EMC-6 caused decreased expression of heteromeric levamisole-sensitive AChRs by destabilizing unassembled subunits in the ER. Inhibition of emc-6 also reduced the expression of homomeric nicotine-sensitive AChRs and GABAA receptors in C. elegans muscle cells. emc-6 is orthologous to the yeast and human EMC6 genes that code for a component of the recently identified ER membrane complex (EMC). Our data suggest this complex is required for protein folding and is connected to ER-associated degradation. We demonstrated that inactivation of additional EMC members in C. elegans also impaired AChR synthesis and induced the unfolded protein response. These results suggest that the EMC is a component of the ER folding machinery. AChRs might provide a valuable proxy to decipher the function of the EMC further.

A single immunoglobulin-domain protein required for clustering acetylcholine receptors in C. elegans.

At Caenorhabditis elegans neuromuscular junctions (NMJs), synaptic clustering of the levamisole-sensitive acetylcholine receptors (L-AChRs) relies on an extracellular scaffold assembled in the synaptic cleft. It involves the secreted protein LEV-9 and the ectodomain of the transmembrane protein LEV-10, which are both expressed by muscle cells. L-AChRs, LEV-9 and LEV-10 are part of a physical complex, which localizes at NMJs, yet none of its components localizes independently at synapses. In a screen for mutants partially resistant to the cholinergic agonist levamisole, we identified oig-4, which encodes a small protein containing a single immunoglobulin domain. The OIG-4 protein is secreted by muscle cells and physically interacts with the L-AChR/LEV-9/LEV-10 complex. Removal of OIG-4 destabilizes the complex and causes a loss of L-AChR clusters at the synapse. Interestingly, OIG-4 partially localizes at NMJs independently of LEV-9 and LEV-10, thus providing a potential link between the L-AChR-associated scaffold and local synaptic cues. These results add a novel paradigm for the immunoglobulin super-family as OIG-4 is a secreted protein required for clustering ionotropic receptors independently of synapse formation.

A secreted complement-control-related protein ensures acetylcholine receptor clustering.

Efficient neurotransmission at chemical synapses relies on spatial congruence between the presynaptic active zone, where synaptic vesicles fuse, and the postsynaptic differentiation, where neurotransmitter receptors concentrate. Diverse molecular systems have evolved to localize receptors at synapses, but in most cases, they rely on scaffolding proteins localized below the plasma membrane. A few systems have been suggested to control the synaptic localization of neurotransmitter receptors through extracellular interactions, such as the pentraxins that bind AMPA receptors and trigger their aggregation. However, it is not yet clear whether these systems have a central role in the organization of postsynaptic domains in vivo or rather provide modulatory functions. Here we describe an extracellular scaffold that is necessary to cluster acetylcholine receptors at neuromuscular junctions in the nematode Caenorhabditis elegans. It involves the ectodomain of the previously identified transmembrane protein LEV-10 (ref. 6) and a novel extracellular protein, LEV-9. LEV-9 is secreted by the muscle cells and localizes at cholinergic neuromuscular junctions. Acetylcholine receptors, LEV-9 and LEV-10 are interdependent for proper synaptic localization and physically interact based on biochemical evidence. Notably, the function of LEV-9 relies on eight complement control protein (CCP) domains. These domains, also called 'sushi domains', are usually found in proteins regulating complement activity in the vertebrate immune system. Because the complement system does not exist in protostomes, our results suggest that some of the numerous uncharacterized CCP proteins expressed in the mammalian brain might be directly involved in the organization of the synapse, independently from immune functions.

Visually discriminating upper case letters, lower case letters and numbers.

English and number literacy are important for successful learning and testing student literacy and numeracy standards enables early identification and remediation of children who have difficulty. Rasch measures were created with the RUMM2020 computer program for the perceptual constructs of visual discrimination upper case letters, lower case letters and numbers. Thirty items for Visual Discrimination of Upper Case Letters (VDUCL), 36 for Lower Case Letters (VDLCL) and 20 for Visual Discrimination of Numbers (VDN) were presented to 324 Pre-Primary through Year 4 children, aged 4-9 years old. All students attended school in Perth, Western Australia. Eighteen of the initial 30 items for VDUCL, thirty-one of the original 36 items for VDLCL and thirteen of the original 20 items for VDN were used to create linear scales (the others were deleted due to misfit) and these clearly showed which letters and numbers children said were easy and which were hard.

Tomosyn-dependent regulation of synaptic transmission is required for a late phase of associative odor memory.

Synaptic vesicle secretion requires the assembly of fusogenic SNARE complexes. Consequently proteins that regulate SNARE complex formation can significantly impact synaptic strength. The SNARE binding protein tomosyn has been shown to potently inhibit exocytosis by sequestering SNARE proteins in nonfusogenic complexes. The tomosyn-SNARE interaction is regulated by protein kinase A (PKA), an enzyme implicated in learning and memory, suggesting tomosyn could be an important effector in PKA-dependent synaptic plasticity. We tested this hypothesis in Drosophila, in which the role of the PKA pathway in associative learning has been well established. We first determined that panneuronal tomosyn knockdown by RNAi enhanced synaptic strength at the Drosophila larval neuromuscular junction, by increasing the evoked response duration. We next assayed memory performance 3 min (early memory) and 3 h (late memory) after aversive olfactory learning. Whereas early memory was unaffected by tomosyn knockdown, late memory was reduced by 50%. Late memory is a composite of stable and labile components. Further analysis determined that tomosyn was specifically required for the anesthesia-sensitive, labile component, previously shown to require cAMP signaling via PKA in mushroom bodies. Together these data indicate that tomosyn has a conserved role in the regulation of synaptic transmission and provide behavioral evidence that tomosyn is involved in a specific component of late associative memory.

Rebalancing the motor circuit restores movement in a Caenorhabditis elegans model for TDP-43 toxicity.

Amyotrophic lateral sclerosis can be caused by abnormal accumulation of TAR DNA-binding protein 43 (TDP-43) in the cytoplasm of neurons. Here, we use a C. elegans model for TDP-43-induced toxicity to identify the biological mechanisms that lead to disease-related phenotypes. By applying deep behavioral phenotyping and subsequent dissection of the neuromuscular circuit, we show that TDP-43 worms have profound defects in GABA neurons. Moreover, acetylcholine neurons appear functionally silenced. Enhancing functional output of repressed acetylcholine neurons at the level of, among others, G-protein-coupled receptors restores neurotransmission, but inefficiently rescues locomotion. Rebalancing the excitatory-to-inhibitory ratio in the neuromuscular system by simultaneous stimulation of the affected GABA- and acetylcholine neurons, however, not only synergizes the effects of boosting individual neurotransmitter systems, but instantaneously improves movement. Our results suggest that interventions accounting for the altered connectome may be more efficient in restoring motor function than those solely focusing on diseased neuron populations.

An alpha-catulin homologue controls neuromuscular function through localization of the dystrophin complex and BK channels in Caenorhabditis elegans.

The large conductance, voltage- and calcium-dependent potassium (BK) channel serves as a major negative feedback regulator of calcium-mediated physiological processes and has been implicated in muscle dysfunction and neurological disorders. In addition to membrane depolarization, activation of the BK channel requires a rise in cytosolic calcium. Localization of the BK channel near calcium channels is therefore critical for its function. In a genetic screen designed to isolate novel regulators of the Caenorhabditis elegans BK channel, SLO-1, we identified ctn-1, which encodes an α-catulin homologue with homology to the cytoskeletal proteins α-catenin and vinculin. ctn-1 Mutants resemble slo-1 loss-of-function mutants, as well as mutants with a compromised dystrophin complex. We determined that CTN-1 uses two distinct mechanisms to localize SLO-1 in muscles and neurons. In muscles, CTN-1 utilizes the dystrophin complex to localize SLO-1 channels near L-type calcium channels. In neurons, CTN-1 is involved in localizing SLO-1 to a specific domain independent of the dystrophin complex. Our results demonstrate that CTN-1 ensures the localization of SLO-1 within calcium nanodomains, thereby playing a crucial role in muscles and neurons.