Jin, Eugene et al. (2021) International Worm Meeting "Coordination of neuronal activity and transcriptional programs in motor circuit remodeling"

Jin, Yishi, Jin, Eugene

[International Worm Meeting, 2021]

Neuronal plasticity and circuit stability are fundamental properties of brain development and function. Activity-dependent changes to neuronal connectivity often occur within a defined time window, also known as a developmental plasticity window. How neuronal activity contributes to such precise timing of neural circuit rewiring is a central question in neuroscience. Ultrastructural connectomic studies that began nearly 50 years ago revealed that during the first larval stage, the C. elegans locomotor circuit undergoes dramatic synaptic rewiring known as 'DD synapse remodeling' as postembryonic motor neurons are born to establish the mature motor circuit (White et al., 1978). Live imaging studies subsequently showed that presynaptic terminals in DD motor neurons are progressively removed from the ventral side and new synapses are formed at dorsal locations from mid to late L1 stages (Hallam and Jin, 1998). The precise timing of DD synapse remodeling has been shown to depend on several transcriptional programs and can be modulated by neuronal activity. However, it remains unclear which form of neuronal activity affects the time window of this developmental plasticity, and how neuronal activity is molecularly coupled to transcription regulation. To address this, we are using fluorescently tagged reporters for in vivo detection of the key transcription factors, such as LIN-14 and UNC-30. To precisely determine L1 developmental stages, we use P cell nuclear migration and divisions using Nomarski optics (Sulston and Horvitz, 1977). We have also generated a nuclear calcium sensor to measure activity in DD neurons during synapse remodeling. We will present our detailed findings on the changes in nuclear calcium dynamics before, during and after DD synapse remodeling. References: Hallam, S.J., and Y. Jin. 1998. Nature. 395(6697):78-82. Sulston, J.E., and H.R. Horvitz. 1977. Dev. Biol. 56:110-156. White, J.G., D.G. Albertson, and M.A. Anness. 1978. Nature. 271:764-766.

Qi, Yingchuan et al. (2009) International Worm Meeting "Identifying genes regulating postembryonic development of DD neurons by microarray."

Qi, Yingchuan, Jin, Yishi

[International Worm Meeting, 2009]

Six "dorsal D", or DD, GABAergic ventral cord motor neurons are born in the late stage of the embryonic development. Upon hatching the axonal patterns of DDs are identical to those in older larvae and adults, and the presynaptic terminals are formed along the ventral process of the DDs. While maintaining their axonal pattern, DDs undergo synaptic remodeling by switching their presynaptic terminals from innervating the ventral muscle to innervating the dorsal muscles towards the end of the L1 stage. The timing of this DD remodeling is under the control of the heterochronic gene lin-141. In loss-of-function mutants of lin-14, the synaptic remodeling of DDs takes place precociously at early L1. To understand the mechanisms underlying DD synaptic remodeling, we carried out a microarray analysis on isolated embryonic DDs from wild type and lin-14(lf). About 3,900 genes showed differential expression between wild type and lin-14(lf) mutants. The genes defining GABA properties were not altered in lin-14(lf), indicating that lin-14 does not control DDs'' GABAergic fate specification. The number of genes up-regulated in lin-14(lf) was about the same as the number of genes down-regulated in lin-14(lf). In particular, we found that a set of acetylcholine receptor genes were up-regulated in lin-14(lf) mutant. We have focused further analysis on two genes, acr-12 and acr-14, both of which are expressed in L1 DDs2. Loss of function mutations of acr-12 or acr-14 cause no obvious DD developmental defects. However, when we expressed the hyperactive form of each acetylcholine receptor gene in DDs, we observed impaired neurite outgrowth of DDs. We found similar neurite outgrowth defects in lin-14 null animals. We are testing a hypothesis that the activity of DDs is maintained at a low level to allow initial development of DDs during embryogenesis and early L1 stage and that altering the activity level impairs the DD postembryonic development. 1.Hallam, S. J. & Jin, Y. Nature 395, 78-82 (1998). 2.Cinar, H., Keles, S. & Jin, Y. Curr Biol 15, 340-6 (2005).

Cherra, Salvatore J. et al. (2017) International Worm Meeting "Deciphering the mechanisms of the ZIG-10 immunoglobulin-domain superfamily protein in maintaining synapse density."

Cherra, Salvatore J., Jin, Yishi

[International Worm Meeting, 2017]

Synapse maintenance refers to the process of preserving the number and pattern of synaptic connections, which is balanced by synapse formation and synapse elimination. Too few or too many synapses have been associated with various neurological disorders, such as autism spectrum disorders or schizophrenia. While the mechanisms of synapse formation during development have been widely investigated, how established synaptic connections are maintained for the lifetime of an organism is still under debate. Using the C. elegans locomotor circuit as a model system, we have previously shown that ZIG-10, an immunoglobulin-domain superfamily member, is required to constrain the density of excitatory synapses, but not inhibitory synapses (1). Here, we report two new signaling molecules differentially required for ZIG-10-mediated maintenance of excitatory synapses. We show that a novel guanylate kinase, MAGU-2, is required for ZIG-10 to restrict synapse density. Null mutants in magu-2 display an increase in excitatory synapses, similar to zig-10 null mutants, but double mutants of magu-2 and zig-10 display a similar number of synapses as compared to either single mutant. This suggests that MAGU-2 and ZIG-10 function in the same pathway. Our previous data showed that ZIG-10 localizes to the cell surface of excitatory neurons and epidermal cells. In the epidermis ZIG-10 activates the phagocytosis pathway to eliminate excitatory synapses. MAGU-2 displays a similar expression pattern to ZIG-10, but is only required in either neurons or epidermis. KCA-1 is a MAGU-2 interacting protein (2); however, in contrast to MAGU-2, a deletion mutation in kca-1 reduces the excitatory synapse density in zig-10 mutants. This suggests that in the absence of ZIG-10, KCA-1 drives excessive synapse formation. Therefore, we hypothesize that the ZIG-10 normally opposes KCA-1 to maintain proper synapse density. Overall, our data uncover how optimal neuronal connectivity is maintained by coordinating the formation and elimination of synapses. Further understanding of the ZIG-10/MAGU-2 pathway will provide new insights into how different cell types within the nervous system interact in order to maintain synaptic connections. 1. S. J. Cherra, 3rd, Y. Jin, A Two-Immunoglobulin-Domain Transmembrane Protein Mediates an Epidermal-Neuronal Interaction to Maintain Synapse Density. Neuron 89, 325-336 (2016). 2. T. Koorman et al., A combined binary interaction and phenotypic map of C. elegans cell polarity proteins. Nat Cell Biol 18, 337-346 (2016).

Byrd, Dana et al. (2021) International Worm Meeting "A C. elegans model for human PACS1 syndrome"

Jin, Yishi, Byrd, Dana

[International Worm Meeting, 2021]

PACS1 syndrome (also known as Schuurs-Hoeijmakers syndrome) is a human neurogenetic disorder characterized by distinctive dysmorphic facial features, intellectual disability, and developmental delays. All known PACS1 patients have de novo mutations causing an R203W amino acid substitution in a highly conserved position of the PACS1 protein. While PACS1 (Phosphofurin Acidic Cluster Sorting Protein 1) was first identified by its role in sorting the proprotein convertase furin to the trans-Golgi network, most understanding of PACS1 function has been attained through studies of cultured cells. To better understand how the R203W disease variant changes PACS1 function in vivo, we have modeled PACS1 syndrome in C. elegans. The single C. elegans pacs-1 gene encodes a protein that shares all conserved domains, and is 27% identical/46% similar to human PACS1. The furin-binding region is especially well conserved between C. elegans and humans (45% identical/70% similar) with the disease variant site, R203, absolutely conserved and denoted as R116 in C. elegans. We edited the endogenous C. elegans pacs-1 gene to generate a PACS1 syndrome model expressing PACS-1(R116W). Using drugs to probe neuronal function, we found that pacs-1(R116W) animals are resistant to the paralyzing effect of the acetylcholine esterase inhibitor aldicarb. Consistent with its neuronal function, we also found that expression of PACS1 is enriched in the nervous system. Our C. elegans pacs-1(R116W) provides the first germline-expressed model of PACS1 syndrome that can be screened for potential chemical therapeutics and further characterized to understand molecular mechanisms of the PACS1 syndrome variant.

Cherra, Salvatore et al. (2015) International Worm Meeting "A two-immunoglobulin-domain transmembrane protein mediates an epidermal-neuronal interaction to maintain synapse density."

Jin, Yishi, Cherra, Salvatore

[International Worm Meeting, 2015]

Maintenance of synaptic connections throughout the lifetime of an organism is important for learning, memory, decision-making, and adaptive behavioral outputs. Synaptic connectivity is regulated not only by neurons, but also by adjacent non-neuronal cells. In the C. elegans locomotor circuit both excitatory and inhibitory motor neurons can form direct contacts with the epidermis. Previous studies have shown that epidermis can regulate synaptic transmission at neuromuscular junctions (1). Here, we have taken advantage of a gain of function mutation in the acetylcholine receptor 2, acr-2(gf), to further investigate the role of epidermal cell surface proteins in regulating synapse maintenance and circuit function. The acr-2(gf) mutation causes an imbalance between excitatory and inhibitory neurotransmission that results in an uncoordinated locomotion and spontaneous convulsions (2). We performed an epidermal-specific RNAi screen in acr-2(gf) animals targeting cell surface proteins, and found that knockdown of zig-10 (zwei-immunoglobulin domain protein 10) significantly increased convulsion frequency. ZIG-10 was selectively expressed in non-synaptic regions of cholinergic motor neurons and on the epidermal cell surface. zig-10 null mutants formed more cholinergic excitatory synapses and exacerbated the convulsion behavior in acr-2(gf) animals. Mis-expression of zig-10 in GABAergic inhibitory motor neurons reduced GABAergic synapse number, dependent on the presence of ZIG-10 in the epidermis. Together these data suggested that ZIG-10 mediates an epidermal-neuronal interaction that is necessary and sufficient to modulate synapse number. Furthermore, we found that ZIG-10 interacted with the tyrosine kinase SRC-2 and that the ZIG-10 pathway regulates phagocytic activity of the epidermis to restrict cholinergic synapse density. Our studies demonstrate the highly specific roles of non-neuronal cells in modulating neural circuit function, through neuron-type specific maintenance of synapse density.1. T. M. Stawicki et al., Curr Biol. 21, 883-888 (2011).2. M. Jospin et al., PLoS Biol. 7, e1000265 (2009).

Noma, Kentaro et al. (2015) International Worm Meeting "Optogenetic mutagenesis using a reactive-oxygen-species generator, miniSOG."

Jin, Yishi, Noma, Kentaro

[International Worm Meeting, 2015]

Mini singlet oxygen generator (miniSOG) is a genetically encoded 106 amino-acid protein producing reactive oxygen species upon illumination of blue light [1]. MiniSOG has been used to ablate cells and inactivate protein functions [2, 3]. Reactive oxygen species are known to cause DNA damage, and such damages may cause heritable changes. To test if miniSOG can mutagenize genomic DNA in vivo, we fused miniSOG to HIS-72, the C. elegans orthologue of histone 3 (His-mSOG), and expressed it in the germline. These animals behaved and reproduced normally under ambient light. Following blue light treatment, we observed heritable mutations in the F2 progeny, such as dumpy and uncoordinated. We optimized His-mSOG-induced mutagenesis using a suppressor screen of paralyzed rpm-1(lf); syd-2(lf) mutants [4]. By quantitating the mutation frequency, we found that the His-mSOG-induced mutation rate was highest when gravid young adults were treated. Analyses of whole-genome and Sanger sequencing showed that His-mSOG induced variable mutations including single-nucleotide substitutions, deletions and a transposon insertion with a strong bias towards G:C to T:A substitutions, which are reported to be a major type of mutations in bacteriophage induced by singlet oxygen [5]. His-mSOG mutagenesis could also cause chromosomal integrations of extrachromosomal arrays at the rate comparable to Psoralen-UV mutagenesis. We envisage that His-mSOG may be combined with other techniques for genome manipulation. [1] Shu, X., et al., PLoS Biol, 2011 [2] Lin, J.Y., et al., Neuron, 2013 [3] Qi, Y.B., et al., PNAS, 2012 [4] Noma, K., et al., Genetics, 2014 [5] Decuyper-Debergh, D., et al., EMBO J, 1987.

Stawicki, Tamara et al. (2009) International Worm Meeting "Regulation of Motor Neuron Activity by Neuropeptide Signaling."

Stawicki, Tamara, Jin, Yishi

[International Worm Meeting, 2009]

The anatomical basis of the C. elegans locomotor circuit is well known. Movement is initiated by command interneurons in the head and tail of the worm. These neurons activate the cholinergic motor neurons, which generate excitatory inputs to the muscle while simultaneously activating GABA motor neurons to inhibit the contralateral muscle. However, several open questions remain, such as which kind of neurotransmission occurs between the interneurons and the cholinergic motor neurons? How do the motor neurons of the locomotor circuit communicate with one another to coordinate the propagation of the sinusoidal wave? We have recently reported the identification of a neuronal acetylcholine receptor that is expressed and functions in the cholinergic motor neurons to maintain their excitability (Stawicki et al. & Qi et al. abstracts in C. elegans Neural Topic Meeting 2008, and Jospin et al. unpublished). This AChR is composed of three a-subunits, UNC-38, UNC-63, ACR-12, and one non-a subunit, ACR-2. A gain of function mutant, acr-2(n2420), exhibits spontaneous contraction, partially resembling the classical "Shrinker" behavior caused by the loss of GABA motor neuron function. Because the command interneurons of the locomotor circuit do not release acetylcholine, we propose that the ACR-2 receptor may act to coordinate the communication between the motor neurons themselves. To test this idea and define the signaling pathway of the ACR-2 receptor in the locomotor circuit, we have examined the genetic interactions between genes regulating synaptic transmission and acr-2(n2420). We find that blocking neuropeptide release by a loss of function mutation in unc-31 significantly suppresses the behavior of acr-2(n2420) worms, whereas a mutation leading to a reduction in classical fast neurotransmission, or in gap junction function, was not able to suppress acr-2(n2420). Using cell-type specific expression studies, we find that this effect of neuropeptide signaling involves the action of unc-31 in neurons, but not in the cholinergic motor neurons. Restoring unc-31 function in motor neurons only can further suppress acr-2(n2420) behavior. Furthermore, we observed opposing effects on acr-2(n2420) behavior by the peptide processing pathway proteins egl-3 and egl-21. These observations suggest the modulation of ACR-2 receptor activity likely involves both excitatory and inhibitory peptide action. We are in the process of testing candidate peptides and receptors. Lastly, we have identified a novel suppressor of acr-2(n2420) that appears to define a potentially new calcium pathway in the motor neurons.

Sun, Yue et al. (2021) International Worm Meeting "A negative feedback mechanism regulates DLK-1 signaling in ciliated sensory neurons"

Sun, Yue, Jin, Yishi

[International Worm Meeting, 2021]

The conserved Dual-Leucine zipper Kinases (DLKs) are neuronal sensors to many forms of stress and traumatic injury. When activated under stress conditions, DLKs act as MAP3Ks upstream of JNK or p38 MAPK signaling to trigger neuroprotective responses. Constitutive activation of DLK, such as via overexpression, can lead to neurodegeneration in different disease models. Previous studies have revealed that under normal conditions, DLK protein activity is controlled by E3 ubiquitin ligase PHR(PAM/Highwire/RPM-1) mediated degradation and chaperones. However, it remains to be discovered whether there are other mechanisms regulating endogenous DLKs. To search for new regulators of C. elegans DLK-1, we performed a forward genetic screen using a GFP knock-in that tags endogenous dlk-1. We isolated a class of mutants showing ectopic accumulation of DLK-1 at dendritic endings of ciliated sensory neurons. We determined that multiple mutations cause loss-of-function in IntraFlagellar Transport (IFT) genes and impair cilia architecture and function in sensory neurons. The ectopic accumulation of DLK-1 at defective cilia activates signal transduction cascade, as we observed elevated expression of the transcription factor CEBP-1, a key downstream target of DLK-1 pathway, in many sensory neurons. However, the increased CEBP-1 expression shows selective dependency on dlk-1. To probe into the neuron-type specificity, we carried out quantitative analyses of CEBP-1 levels in several types of sensory neurons. We found that IFT defects in AWB and AWC cause increased CEBP-1 expression dependent on dlk-1, whereas similar IFT defects in ASI and ASH induce CEBP-1 elevation independent of dlk-1. Moreover, in AWC cebp-1 further represses expression of wild type DLK-1 but not a kinase-dead DLK-1, suggesting a negative feedback control of DLK-1 signaling via CEBP-1. Additionally, we found that in AWC neurons overexpression of DLK-1 causes dendrite overgrowth by activating CEBP-1. Together, we propose that in ciliated sensory neurons, DLK-1 signaling is regulated under a cell-specific feedback mechanism, which is coupled with IFT and may contribute to the maintenance of neuronal morphology.

McCulloch, Katherine et al. (2015) International Worm Meeting "Understanding motor circuit regulation through genetic suppressor analyses of acr-2(gf) ."

McCulloch, Katherine, Jin, Yishi

[International Worm Meeting, 2015]

Sinusoidal movement in C. elegans requires the coordinated opposing functions of excitatory cholinergic and inhibitory GABAergic motor neurons. A genetic screen for mutants defective in GABA signaling identified a gain-of-function mutation in acr-2 [acr-2(gf)], which causes a Valine-to-Methionine missense mutation in the highly conserved TM2 domain of a non-alpha nicotinic acetylcholine receptor subunit. acr-2 is expressed and functions specifically in cholinergic motor neurons to modulate motor circuit activity. C. elegans mutants defective in GABAergic transmission shrink in response to touch due to reductions in inhibitory signaling; however, acr-2(gf) animals shrink spontaneously. This novel convulsion behavior is the result of both increased cholinergic excitation and decreased GABAergic inhibition in the motor circuit, causing simultaneous contraction of the body wall muscle. Interestingly, mutations similar to acr-2(gf) in a human acetylcholine receptor subunit CHNRB2 have been associated with human familial epilepsies. A genetic suppressor screen for the acr-2(gf) convulsion phenotype led to the identification of its co-subunits. Analyses of other suppressors have also recovered roles for the GTL-2 TRPM channel protein, as well as novel genes, that regulate neuron activity. Further characterization of acr-2(gf) suppressors will continue to expand our understanding of the genes and pathways that regulate motor circuit function. In addition, we are complementing these classical genetic approaches with transcriptome analyses to identify genes that are differentially expressed in response to activity imbalances caused by acr-2(gf) relative to wild-type animals. These studies will advance our understanding of the molecular pathways that regulate and respond to chronic hyperactivity in motor circuits.

Zhou, Keming et al. (2013) International Worm Meeting "Acute inhibition of synaptic transmission using Mini-Singlet Oxygen-Generator (miniSOG)-mediated protein ablation."

Zhou, Keming, Jin, Yishi

[International Worm Meeting, 2013]

Synaptic transmission occurs in milliseconds and involves hundreds of proteins that reside in microdomains of the synapse. Genetic mutations that eliminate or reduce protein function have been the traditional tools in investigating the roles of synaptic proteins in C. elegans. However, chronic compensation and homeostatic responses may arise from loss of specific proteins. It is thus of high interest to develop strategies to inducibly inhibit the function of synaptic proteins. MiniSOG (mini singlet oxygen generator) is a newly engineered green fluorescent flavoprotein1. Upon blue-light illumination, miniSOG efficiently generates singlet oxygen, a very short-lived reactive oxygen species. We have previously shown that miniSOG targeted to mitochondria can inducibly kill cells2. Here, using miniSOG-tagging of synaptic proteins, we have developed an optogenetic strategy for acute inhibition of synaptic transmission. The presynaptic protein UNC-13 is essential for synaptic transmission. The long UNC-13 isoform (UNC-13L) is localized to the presynaptic active zone via its N-terminal region. UNC-13L lacking the N-terminal region is diffuse in axons. Transgenic animals expressing miniSOG-tagged UNC-13L exhibit no discernable defects when grown in the dark. Upon brief exposure to blue light the animals rapidly display behavioral impairment, resembling unc-13 loss of function, and recover after returning to normal culture condition. By electrophysiological recordings, we show that acute inactivation of miniSOG tagged full-length UNC-13L or UNC-13L lacking the N-terminal region preferentially inhibits the fast phase and slow phase of evoked release at the neuromuscular junctions, respectively. We also identify a specific role for the N-terminal region of UNC-13L in tonic release. These observations demonstrate temporal and spatial specificity of miniSOG-mediated protein ablation. We envision that miniSOG-mediated photo-ablation will be a useful tool to transiently inhibit protein function in broad cellular contexts. 1. Shu, X. et al. PLoS Biol, 2011. 9(4): p. e1001041. 2. Qi, Y. B. et al. PNAS, 2012. 109: p7499.