A MIG-15/JNK-1 MAP kinase cascade opposes RPM-1 signaling in synapse formation and learning

The Pam/Highwire/RPM-1 (PHR) proteins are conserved intracellular signaling hubs that regulate synapse formation and axon termination. The C. elegans PHR protein, called RPM-1, acts as a ubiquitin ligase to inhibit the DLK-1 and MLK-1 MAP kinase pathways. We have identified several kinases that are likely to form a new MAP kinase pathway that suppresses synapse formation defects, but not axon termination defects, in the mechanosensory neurons of rpm-1 mutants. This pathway includes: MIG-15 (MAP4K), NSY-1 (MAP3K), JKK-1 (MAP2K) and JNK-1 (MAPK). Transgenic overexpression of kinases in the MIG-15/JNK-1 pathway is sufficient to impair synapse formation in wild-type animals. The MIG-15/JNK-1 pathway functions cell autonomously in the mechanosensory neurons, and these kinases localize to presynaptic terminals providing further evidence of a role in synapse development. Loss of MIG-15/JNK-1 signaling also suppresses defects in habituation to repeated mechanical stimuli in rpm-1 mutants, a behavioral deficit that is likely to arise from impaired glutamatergic synapse formation. Interestingly, habituation results are consistent with the MIG-15/JNK-1 pathway functioning as a parallel opposing pathway to RPM-1. These findings indicate the MIG-15/JNK-1 pathway can restrict both glutamatergic synapse formation and short-term learning.


Introduction
Information is relayed throughout the nervous system via chemical synapses, and the process of synapse formation is essential for construction of neural circuitry [1,2]. In the central nervous system, most synaptic connections are glutamatergic and often referred to as central synapses.
C. elegans has two PLM mechanosensory neurons, which sense posterior gentle touch [10]. The PLM neurons form electrical synapses and glutamatergic chemical synapses with postsynaptic interneurons [10][11][12]. Initial touch sensation is thought to rely upon both electrical synapses and glutamatergic transmission. Glutamatergic transmission is also required for more complex touch-response behaviors, such as habituation to repeated tap and arousal from a sleep-like state called lethargus [13,14].
RPM-1 is the C. elegans ortholog of mouse Phr1 and Drosophila Highwire, which are collectively referred to as Pam/Highwire/RPM-1 (PHR) proteins. RPM-1 is a relatively broad regulator of synapse formation and axon termination affecting these processes in mechanosensory neurons and motor neurons [5,[15][16][17]. Studies in flies and mice have shown these are conserved RPM-1 functions [18][19][20][21][22]. Previous work showed RPM-1 functions in the mechanosensory neurons during development to regulate habituation to repeated tap stimulus, a behavioral deficit that likely results from abnormal glutamatergic synapse formation in rpm-1 mutants [23].
JNK MAP kinase pathways play a conserved role in synapse formation in the developing nervous system [37]. Studies in worms [29,30], flies [32,38] and vertebrates [39,40] have shown JNK signaling needs to be restricted for proper synapse formation. The role of JNK signaling in synapse formation has been primarily explored using the neuromuscular junction (NMJ) in different organisms. Much less is known about how JNK signaling impacts the formation of neuron-neuron, central synapses in vivo. It also remains unclear if JNK isoforms have differential roles in mediating synapse formation in different types of neurons. The importance of addressing these gaps in our knowledge is highlighted by emerging links between altered JNK signaling and synaptic dysfunction in Alzheimer's disease [41][42][43][44].
JNK activation is regulated by upstream kinase cascades that include MAP2Ks, MAP3Ks, and in some cases MAP4Ks. In vitro biochemistry and genetic studies on fly embryogenesis have shown Misshapen (Msn), called MIG-15 in worms and NIK (HGK/MAP4K4) in mammals, can function as a MAP4K that activates JNK signaling [45][46][47]. While MIG-15 and Msn regulate axon guidance in the nervous system, they do so through the cytoskeletal regulators ERM-1 and Bifocal [48,49]. To date, functional links in the nervous system between JNK signaling and MIG-15 or Msn have remained stubbornly elusive.
Using suppressor genetics, we identified several kinases that are likely to be part of a new JNK pathway that regulates glutamatergic, neuron-neuron synapse formation in the mechanosensory neurons of C. elegans. This JNK pathway is composed of MIG-15 (MAP4K), NSY-1 (MAP3K), JKK-1 (MAP2K), and JNK-1. Loss of function mutations in kinases of the MIG-15/ JNK-1 pathway specifically suppress defects in synapse formation in the mechanosensory neurons of rpm-1 mutants. Consistent with suppression results, transgenic overexpression of kinases in the MIG-15/JNK-1 pathway in wild-type animals results in impaired synapse formation. Further biochemical support for the MIG-15/JNK-1 pathway comes from our observation that NSY-1 can bind both MIG-15 and JKK-1 when expressed in HEK 293 cells.
Based on prior work, the most likely signaling model that might explain our results is RPM-1 acting as a ubiquitin ligase to inhibit an upstream kinase in the MIG-15/JNK-1 pathway, similar to RPM-1 effects on the DLK-1 and MLK-1 pathways. However, our behavioral analysis of habituation indicated the MIG-15/JNK-1 pathway is more likely to function as a parallel opposing pathway to RPM-1. These results provide the first potential link in the nervous system between the MIG-15 MAP4K and a JNK isoform, and indicate MIG-15/JNK-1 activity can affect synapse formation and short-term learning.

Results
Synapse formation defects in rpm-1 mutants, but not axon termination defects, are suppressed by jkk-1 and jnk-1 Previous studies showed that loss-of-function mutations in kinases of the DLK-1/PMK-3 p38 MAPK pathway and the MLK-1/KGB-1 JNK pathway suppress synapse formation defects in rpm-1 mutants [29,30]. A different pathway consisting of JNK-1 and an MKK7 ortholog JKK-1 functions in the nervous system to regulate locomotion, and synaptic vesicle trafficking in GABAergic motor neurons [50,51]. jkk-1 and jnk-1 also suppress synaptic position defects in cholinergic NMJs of arl-8 mutants [52]. These observations prompted us to test whether jkk-1 and jnk-1 could suppress defects in synapse formation and axon termination caused by rpm-1 loss of function (lf) in the mechanosensory neurons.
C. elegans has two PLM mechanosensory neurons, one on each side of its body. Each PLM neuron extends a single axon that terminates growth prior to the cell body of the ALM mechanosensory neuron (Fig 1A and 1B). Glutamatergic chemical synapses are formed en passant with interneurons by a collateral branch that extends from the primary axon (Fig 1A and  1D). The transgene muIs32, which expresses GFP in the mechanosensory neurons, can be used to assess PLM neurons for axon termination, as well as formation of the synaptic branch and synaptic boutons [24]. Anatomical separation of axon termination and chemical synapse formation makes the PLM neurons an ideal system to assess these two developmental processes within the same neuron.
Consistent with prior studies [5,53], we observed that rpm-1 mutants display two phenotypes in the PLM neurons. The first is failed axon termination, which results in the PLM axon extending beyond the ALM cell body and hooking towards the ventral side of the animal, which we refer to as a hook defect (Fig 1B). The second phenotype is retraction of the synaptic branch, which results from impaired synapse formation (Fig 1D) [5]. Quantitation of axon elegans mechanosensory neurons. Left blue box highlights PLM axon termination site. Right blue box highlights collateral synaptic branch and presynaptic boutons. (B) Transgene muIs32, which expresses GFP in mechanosensory neurons, was used to visualize axon termination in a wild-type animal. Axon termination defects (hook defect) in an rpm-1 single mutant, and rpm-1; jnk-1 and rpm-1; jkk-1 double mutants (magenta arrows). (C) Quantitation of axon termination defects (hook defect) for indicated genotypes. (D) PLM synaptic branch in wildtype. Synaptic branch is absent in rpm-1 mutant (orange arrow), a defect that results from failed synapse formation. In rpm-1; jnk-1 and rpm-1; jkk-1 double mutants synaptic branch is present indicating jnk-1 and jkk-1 suppress this defect. Note presynaptic boutons are below synaptic branch, and out of focal plane. (E) Quantitation of synaptic branch defects for indicated genotypes. Averages are shown from 6-10 counts (25-30 neurons/count) of young adult animals for each genotype. Error bars represent standard error of mean, and significance was determined using unpaired Student's t test with Bonferroni correction. *** p< 0.001, ns = not significant. Scale bars 10 μm.
Given that both jkk-1 and jnk-1 suppress synaptic branch defects, but not axon termination defects, we tested whether they function in the same genetic pathway. To do so, we generated rpm-1; jkk-1; jnk-1 triple mutants. We observed no further suppression of synaptic branch defects in triple mutants (Fig 1E), and again saw no suppression of axon termination defects ( Fig 1C). These results are consistent with jkk-1 and jnk-1 functioning in the same genetic pathway to specifically suppress synapse formation defects in the PLM neurons of rpm-1 mutants.
MIG-15 and NSY-1 function in the JKK-1/JNK-1 pathway to regulate synapse formation Next, we explored MAP3Ks and MAP4Ks that might exist in the JKK-1/JNK-1 pathway. We began by testing the MAP3K NSY-1 because it regulates olfactory neuron fate during development [55], and its mammalian ortholog ASK activates JNK signaling in vitro [56]. To analyze genetic interactions between rpm-1 and nsy-1, we used a previously described nsy-1 allele, ok593, that deletes the kinase domain and is likely to be a null. Similar to outcomes with jkk-1 and jnk-1, we observed suppression of synaptic branch defects in rpm-1; nsy-1 double mutants, but did not see changes in axon termination defects (Fig 2A and 2D). Quantitation showed that suppression of synaptic branch defects in rpm-1; nsy-1 double mutants was significant compared to rpm-1 single mutants ( Fig 2E). No significant decrease in axon termination defects occurred in rpm-1; nsy-1 double mutants ( Fig 2B). Importantly, no increased suppression occurred in rpm-1; jnk-1; nsy-1 triple mutants compared to either double mutant ( Fig 2E). These results are consistent with nsy-1 functioning in the same pathway as jnk-1 to suppress synapse formation defects.
Previous studies on MAP kinases regulating neuronal development in C. elegans have often identified three component pathways (MAP3K, MAP2K and MAPK) [29][30][31]. At present, potential MAP4Ks that regulate JNK signaling in neuronal development remain unknown. MIG-15 is orthologous to mammalian NIK and fly Msn, which can act as MAP4Ks in cultured cells and during embryogenesis [46,47]. In the developing C. elegans nervous system, MIG-15 regulates cell migration and axon guidance, but functional genetic links to JNK signaling in neurons remain absent [57,58]. Therefore, we tested if loss of MIG-15 function specifically suppresses synapse formation defects in rpm-1 mutants similar to nsy-1, jkk-1 and jnk-1. We used a strong loss of function allele, mu342, that is a point mutation in the MIG-15 kinase domain and likely to disrupt kinase activity [58]. Axon termination defects were not suppressed in rpm-1; mig-15 double mutants (Fig 2A and 2C). However, synaptic branch defects were significantly suppressed in rpm-1; mig-15 double mutants (Fig 2D and 2F). It should be noted that in some mig-15 mutants, the PVM cell body did not migrate normally, which is consistent with previously described cell migration defects (Fig 2D). Similar to results with nsy-1, further suppression of branch defects was not observed in rpm-1; jnk-1; mig-15 triple mutants compared to rpm-1; mig-15 double mutants. This result is consistent with mig-15 functioning in the same genetic pathway as jnk-1.
The logic of how MAPK modules operate suggests the most likely order for this putative kinase pathway from most upstream to downstream kinase would be: MIG-15/NSY-1/JKK-1/ JNK-1. However, our genetic results do not rule out the alternative possibility that these kinases are part of multiple MAPK pathways that function in parallel to suppress synapse formation defects, but not axon termination defects, caused by rpm-1 (lf). In addition, we observed a low but significant level of synaptic branch defects in mig-15 single mutants (Fig 2F). This observation, and suppression of synaptic branch defects caused by rpm-1 (lf) suggest that a balance of MIG-15 activity is required for proper synapse formation in the PLM neurons.
Loss of mig-15 or jkk-1 results in improved bouton morphology and accumulation of presynaptic components in rpm-1 mutants Previous studies suggested impaired synapse formation in rpm-1 mutants leads to retraction of the PLM synaptic branch [5]. Therefore, the absence of the synaptic branch served as a proxy for assessing synapse formation in different double mutants of rpm-1 and kinases of interest. However, there are two possible cellular explanations for why synaptic branch defects are suppressed. The first is that synapse formation is improved. Alternatively, MIG-15, NSY-1, JKK-1 and JNK-1 kinases might be required for synaptic branch retraction.
To differentiate between these two possibilities, we simultaneously labeled PLM neurons with two transgenes: 1) jsIs1114 which expresses the synaptic vesicle marker GFP::RAB-3, and 2) jsIs973 which expresses mRFP and allows the morphology of the axon, synaptic branch and synaptic boutons to be visualized. RAB-3 is a small G-protein that associates with synaptic vesicles (SV), and has been used to assess synaptogenesis in PLM neurons [8]. Consistent with prior work, we observed GFP::RAB-3 at the presynaptic boutons of wild-type animals ( Fig  3A). In confocal projections, it is possible to see synaptic boutons from both the left and right PLM neurons (PLML and PLMR) as they reside in the same z-plane in the ventral nerve cord. However, the morphology of only a single PLM axon can be visualized ( Fig 3A). We observed two phenotypic groups of rpm-1 mutants. The first, and most common, lacked one of the two PLM synaptic branches. The example shown in Fig 3A (rpm-1 left absent) is missing the left PLM synaptic branch and accompanying GFP::RAB-3 accumulations, but GFP::RAB-3 from PLMR is still present. Less frequently, we observed rpm-1 mutants in which both PLM neurons lack a synaptic branch and show no presynaptic GFP::RAB-3 accumulation (Fig 3A, rpm-1 both absent). Quantitation showed accumulation of RAB-3 always corresponded with the presence of a synaptic branch in wild-type animals ( Fig 3C). The frequency of complete synaptic branches, defined as branches with presynaptic boutons and GFP::RAB-3 accumulation, were reduced in rpm-1 mutants ( Fig 3C). In rpm-1; mig-15 and rpm-1; jkk-1 double mutants, we observed an increase in the number of complete synaptic branches and corresponding accumulation of GFP::RAB-3 at presynaptic terminals ( Fig 3B and 3C). Thus, mig-15 and jkk-1 suppression of rpm-1 results in improved synapse formation.

jnk-1, jkk-1 and nsy-1 enhances defects in synapse formation caused by colchicine
Loss of function in NSY-1, JKK-1 and JNK-1 had minimal effects on PLM synapse formation, as assessed by the presence of the synaptic branch and presynaptic boutons (Figs 1E and 2E). Nonetheless, it was possible more subtle changes might exist in morphology or number of presynaptic terminals when these kinases are perturbed.
To test this, we utilized transgenic worms that express either the synaptic vesicle marker GFP::RAB-3, or the active zone marker UNC-10::tdTOMATO in the mechanosensory neurons (Fig 4A and 4B). Transgenes with an appropriate cell fill marker (GFP or mRFP) were evaluated simultaneously to anatomically identify PLM presynaptic terminals. Both presynaptic RAB-3 and UNC-10 puncta size and number showed no significant differences when wildtype animals were compared to jnk-1, jkk-1 or nsy-1 mutants (Fig 4C and 4D). Data for nsy-1 and GFP::RAB-3 is not shown because recombinants between nsy-1 and the RAB-3 transgene could not be obtained.
To further evaluate whether these kinases might affect synapse formation outside of rpm-1 suppression, we turned to the microtubule destabilizing drug colchicine. It was previously shown that treating wild-type worms with colchicine causes synapse formation defects in PLM neurons [59]. This provided us with a pharmacological manipulation to further examine the role of the MIG-15/JNK-1 pathway in synapse formation. While no effect was observed with the vehicle DMSO, we found that low frequency synaptic branch defects caused by colchicine were enhanced in jnk-1, jkk-1 or nsy-1 mutants (Fig 4E and 4F). These observations indicate altering microtubule stability can also unveil a role for NSY-1, JKK-1 and JNK-1 in glutamatergic synapse formation.
The MIG-15/JNK-1 pathway functions cell autonomously in mechanosensory neurons to regulate synapse formation Our genetic analysis showed mig-15, nsy-1, jkk-1 and jnk-1 are likely to function in a linear genetic pathway to suppress synapse formation defects in rpm-1 mutants. Next, we wanted to determine if these kinases regulate synapse formation by functioning cell autonomously within PLM neurons, or non-cell autonomously in surrounding tissue. We addressed this with transgenic rescue experiments using double mutants of rpm-1 and different kinases. Transgenic expression of kinases was driven by the mec-7 promoter, which is expressed in mechanosensory neurons including the PLM neurons, and is not expressed in the postsynaptic interneurons or tissues that surround the PLM synapse, such as muscles or hypodermis. NSY-1, JKK-1 and JNK-1 were expressed using transgenic extrachromasomal arrays. MIG-15 was expressed using a MosSCI single-copy integrated transgene because mig-15 mutants have small body and brood size, which made deriving extrachromosomal arrays extremely difficult. Expression of each kinase in PLM neurons gave significant, robust rescue of suppression in kinase double mutants with rpm-1 (Fig 5A and 5B). For example, transgenic expression of JNK-1 (but not the negative control protein mCherry) in rpm-1; jnk-1 double mutants significantly rescued suppression of synaptic branch defects (Fig 5A and 5B). Our findings support the conclusion that MIG-15, NSY-1, JKK-1 and JNK-1 function cell autonomously in PLM mechanosensory neurons, and are consistent with these kinases functioning in a linear cascade to regulate synapse formation.

Overexpression of MIG-15, NSY-1 and JKK-1 is sufficient to impair synapse formation
Our observation that loss of MIG-15, NSY-1, JKK-1 or JNK-1 improves synapse formation defects in rpm-1 mutants suggests these kinases might inhibit synapse formation. To test this hypothesis, we transgenically overexpressed individual kinases in wild-type animals using the pan-neuronal rgef-1 promoter, which has been used previously for transgenic overexpression experiments with PLM neurons [30,53]. PLM synaptic branches were not altered in wild-type animals carrying transgenic extrachromosomal arrays that overexpress mCherry, which was used as a negative control (Fig 6A and 6B). In contrast, transgenic overexpression of MIG-15, NSY-1 or JKK-1 in wild-type worms resulted in synaptic branch defects (Fig 6A and 6B). Transgenic overexpression of JNK-1 did not lead to defects (Fig 6B). This result with JNK-1, which is likely at the bottom of a possible MIG-15/JNK-1 pathway, is not surprising given the prior finding that kinases such as the p38 MAPK PMK-3 and the JNK isoform KGB-1 generate relatively low frequency defects when transgenically overexpressed [30]. Nonetheless, our results show that overexpressing MIG-15, NSY-1 or JKK-1 is sufficient to impair synapse formation. This indicates that these kinases need to be opposed functionally or restricted for proper PLM synapse formation to occur. Furthermore, synapse formation defects caused by overexpression of MIG-15, NSY-1 and JKK-1 occurred at similar levels, which provides further evidence these kinases could function in the same MAPK pathway.

NSY-1 MAP3K binds to both MIG-15 MAP4K and JKK-1 MAP2K
Our genetic experiments suggested that MIG-15, NSY-1, JKK-1 and JNK-1 potentially function in a novel MAPK pathway that regulates synapse formation in PLM neurons. We wanted to further evaluate the biochemistry that underpins these findings.
Previous in vitro experiments showed MAP3Ks often bind to both MAP4Ks and MAP2Ks in the same pathway [60]. For example, MEKK1 (MAP3K1) binds to both the MAP4K NIK and the MAP2K MKK4 [46,61], while NSY-1 binds to the MAP2K SEK-1 [62]. To test whether similar interactions occur between the NSY-1 MAP3K and the MIG-15 MAP4K or JKK-1 MAP2K, HEK 293 cells were transfected with FLAG or HA tagged kinases and binding was assessed by coimmunoprecipitation. When NSY-1 was immunoprecipitated from transfected cell lysates, we detected coprecipitating MIG-15 ( Fig 7A) and JKK-1 (Fig 7B). NSY-1 binding to both MIG-15 and JKK-1 provides biochemical evidence that these kinases could function in a linear MAPK pathway.

NSY-1 and JNK-1 localize to presynaptic terminals
Because MIG-15, NSY-1, JKK-1 and JNK-1 affect synapse formation, it is plausible these kinases might localize to presynaptic terminals. To explore this, we generated transgenic extrachromosomal arrays that simultaneously expressed mCherry and GFP::JNK-1. As shown in Fig 8A, GFP::JNK-1 localized to the presynaptic boutons of PLM neurons. JNK-1 was also observed in the axon and synaptic branch. GFP::NSY-1 similarly localized to presynaptic boutons ( Fig 8B). We tried to determine if MIG-15 and JKK-1 localize to presynaptic terminals, but these constructs were not readily detected in PLM neurons.
The GABAergic DD motor neurons of C. elegans form synapses with dorsal body wall muscles (Fig 9A). To visualize GABAergic NMJs we used juIs1, a transgene that expresses the synaptic vesicle marker Synaptobrevin-1 fused to GFP (SNB-1::GFP). We observed regularly spaced presynaptic puncta in wild-type animals labeled with SNB-1 (Fig 9A). Consistent with previous studies, rpm-1 mutants had synapse formation defects in which large sections of the dorsal cord lacked SNB-1 puncta, and SNB-1 puncta aggregated (Fig 9A). Interestingly, MIG-15/JNK pathway regulates synapse formation synapse formation defects were unchanged in rpm-1; jkk-1 and rpm-1; nsy-1 double mutants (Fig 9A). Quantitation of SNB-1 puncta confirmed synapse formation defects in GABAergic motor neurons of rpm-1; jkk-1 or rpm-1; nsy-1 double mutants were not suppressed compared to rpm-1 single mutants (Fig 9B). We observed a small, significant defect in NMJ formation in jkk-1 single mutants (Fig 9B). This suggests JKK-1 might regulate GABAergic synapse formation. Overall, these results demonstrate that NSY-1 and JKK-1 do not affect RPM-1 regulation of synapse formation in GABAergic motor neurons the way they do in PLM mechanosensory neurons. This indicates that loss of NSY-1 and JKK-1 specifically suppresses synapse formation defects at glutamatergic, neuron-neuron connections.

nsy-1, jkk-1 and jnk-1 suppress tap habituation defects in rpm-1 mutants
The mechanosensory neurons mediate gentle touch responses, including the response to nonlocalized mechanical stimuli such as vibrations caused by tapping the plate on which worms grow. The mechanosensory neurons, including the PLM neurons, propagate sensory activity MIG-15/JNK pathway regulates synapse formation primarily via electrical synapses at sites along the primary axon ( Fig 10A) [10,63]. Glutamatergic chemical synapses are formed in an anatomically distinct location on the collateral synaptic branch, and contribute to a lesser extent to touch sensation (Fig 10A) [10,11]. Glutamatergic transmission has been implicated in short-term learning, specifically habituation to repeated tap stimuli [13,23].
Previous work showed rpm-1 mutants respond normally to touch [5,23], which is consistent with these animals having normal electrical synapses in their mechanosensory neurons [64] (Borgen and Grill, in press). In contrast, rpm-1 mutants have dramatic defects in habitation to repeated tap, which result from loss of RPM-1 function in the mechanosensory neurons [23]. This suggests that habituation defects in rpm-1 mutants are likely to result from defective chemical synapse formation. Thus, tap habituation is a reasonable behavioral readout to further test the genetic relationship between rpm-1 and kinases in the MIG-15/JNK-1 pathway. Tap habituation also provided us with a whole-animal, physiological assay that is sensitive to impacts on chemical synapse formation in the mechanosensory neurons.
As shown in Fig 10B, wild-type animals displayed habituation to repeated tap, in which responses diminish with increasing tap stimuli over time. Consistent with prior findings [23], rpm-1 mutants were defective in habituation, and maintained a high probability of response over repeated tap stimuli (Fig 10B). Tap habituation defects were suppressed in double mutants of rpm-1 with jnk-1, jkk-1 or nsy-1 (Fig 10B, 10C and 10D). Defects in body morphology and locomotion prevented us from testing mig-15 mutants. These behavioral findings show that loss of NSY-1, JKK-1 and JNK-1 suppress rpm-1 in the context of tap habituation. This finding correlates nicely with our findings on chemical synapse formation in PLM neurons. It also provides further behavioral evidence that is consistent with NSY-1, JKK-1 and JNK-1 functioning in the same pathway.

Discussion
Previous studies have shown the C. elegans PHR protein RPM-1 functions through multiple mechanisms to regulate synapse formation and axon termination [15]. One mechanism is RPM-1 acting as a ubiquitin ligase to inhibit p38 and JNK signaling pathways. It remains unclear how broad a regulator of p38 and JNK signaling RPM-1 is. Furthermore, it is unknown if there are pathways that interact with RPM-1 to specifically affect synapse formation.
Here we identify several kinases, that are likely to form a MAP kinase pathway, that specifically suppresses synapse formation defects caused by rpm-1 (lf) in the mechanosensory neurons. This pathway is composed of the MIG-15 MAP4K, the NSY-1 MAP3K, the JKK-1 MAP2K and the JNK isoform JNK-1 (Fig 10E). Mutations in kinases of the MIG-15/JNK-1 pathway suppress synapse formation defects in the mechanosensory neurons of rpm-1 mutants, and suppress accompanying deficits in habituation. Furthermore, behavioral analysis is consistent with the MIG-15/JNK-1 pathway most likely functioning as a parallel opposing pathway to RPM-1. Thus, our results suggest the MIG-15/JNK-1 pathway needs to be opposed by RPM-1 signaling for proper glutamatergic synapse formation and short-term learning ( Fig  10E, Model 2). Our results also address an important missing functional link between the MIG-15 MAP4K and JNK in the nervous system.

MIG-15/JNK-1 pathway and synapse formation
Biochemical studies and genetics on fly embryogenesis showed the orthologs of MIG-15 (Msn and NIK) activate JNK signaling [46,47]. In worms and flies, MIG-15 and Msn regulate axon guidance by functioning through the cytoskeletal regulators, ERM-1 and Bifocal, rather than acting on JNK signaling [48,49]. At present, it is unknown if MIG-15, Msn or NIK function through JNK signaling in the nervous system.
We now provide evidence that MIG-15 is likely to function in a JNK pathway to regulate synapse formation in mechanosensory neurons. If this is the case, in the developing worm nervous system MIG-15 regulates axon guidance through the actin regulator ERM-1, and regulates synapse formation through a kinase pathway that includes the JNK isoform JNK-1. Our results show mig-15 mutants have mild defects in synapse formation, and suppress the strong synapse formation defects caused by rpm-1 (lf) (Figs 2 and 3). This suggests a balance of MIG-15 signaling is important for proper glutamatergic synapse formation. The importance of our finding is accentuated by recent work showing NIK potentially mediates synapse loss during neurodegeneration in ALS [65].
Numerous observations collectively support the conclusion that mig-15, nsy-1, jkk-1 and jnk-1 function in the same pathway. 1) Suppression of synapse formation defects caused by rpm-1 is not increased when multiple kinases are eliminated compared to elimination of a single kinase (Figs 1 and 2). 2) The MIG-15/JNK-1 pathway has a specific suppressor phenotype: These kinases suppress synapse formation defects, but not axon termination defects, in the mechanosensory neurons of rpm-1 mutants (Figs 1 and 2). 3) Kinases in the MIG-15/JNK-1 pathway function cell autonomously in the mechanosensory neurons (Fig 5). 4) Transgenic overexpression of MIG-15, NSY-1 or JKK-1 causes synapse formation defects at a similar frequency (Fig 6). 5) nsy-1, jkk-1 and jnk-1 suppress defects in tap habituation in rpm-1 mutants (Fig 10). 6) Finally, our biochemical results show that NSY-1 binds to MIG-15 and JKK-1 (Fig  7). Our findings on the MIG-15/JNK-1 pathway and the order we have assigned to kinases in the pathway agrees with prior in vitro biochemistry, genetic results on fly embryogenesis, and is consistent with how MAPK cascades generally operate [45]. However, our results do not definitively rule out the alternative possibility that theses kinases could function in multiple, parallel MAPK pathways. This is important to note, as prior work has shown that NSY-1 can stimulate activation of p38 MAPK [55].
Prior work showed JKK-1 and JNK-1 function in the same pathway to regulate locomotion, synaptic vesicle trafficking and ARL-8 regulation of synaptic position [51,52]. Our results now identify a new function for JKK-1 and JNK-1 in glutamatergic synapse formation, and suggest MIG-15 and NSY-1 are likely to be further kinases in this pathway. It is unlikely impaired synaptic vesicle trafficking in jnk-1 and jkk-1 mutants would explain our findings, as we observe the opposite phenotype, an improvement in synapse formation defects in rpm-1; jkk-1 and rpm-1; jnk-1 double mutants. However, if synaptic vesicle trafficking to glutamatergic presynaptic terminals is increased in jnk-1 and jkk-1 mutants, which remains unknown, it might explain our results. Whether MIG-15 and NSY-1 are involved in ARL-8 regulation of synaptic position like JKK-1 and JNK-1 remains an open question.

MIG-15/JNK-1 pathway molecularly distinguishes axon termination from synapse formation
We have found that the MIG-15/JNK-1 pathway specifically suppresses synapse formation defects in the mechanosensory neurons of rpm-1 mutants. This differs from prior work showing the DLK-1/PMK-3 and MLK-1/KGB-1 pathways suppress both axon termination and synapse formation defects in mechanosensory neurons, as well as synapse formation defects in motor neurons of rpm-1 mutants [29,30]. Because mechanosensory neurons form glutamatergic, neuron-neuron synapses that are reminiscent of mammalian central synapses, it is reasonable to speculate that the MIG-15/JNK-1 pathway might regulate central synapse formation in other organisms.
Recent studies suggested axon termination defects are a remodeling phenotype that occurs due to retraction of the PLM synaptic branch [59,66]. This is one possible explanation for why mutants, such as rpm-1, affect both axon termination and synapse formation. However, if this were true, we would expect genetic changes to affect both axon termination and synapse formation in all cases. Our discovery of the MIG-15/JNK-1 pathway demonstrates distinct signals exist within the mechanosensory neurons that differentially influence chemical synapse formation compared to axon termination.
MIG-15/JNK-1 pathway likely functions as a parallel opposing pathway to RPM-1 Accompanying differing effects of the MIG-15/JNK-1 pathway and the DLK-1/PMK-3 pathway on synapse development are differing results on habituation to tap, a form of short-term learning, that is dependent upon chemical synapse function in the mechanosensory neurons. Prior work showed rpm-1 mutants have dramatic defects in habituation to repeated tap stimulus, which is suppressed by dlk-1 [23]. Defects in habituation stem from RPM-1 function in the mechanosensory neurons. We have found nsy-1, jkk-1 and jnk-1 also suppress habituation defects in rpm-1 mutants. However, there were important differences between these kinases and DLK-1. First, jkk-1 and jnk-1 single mutants have increased habituation (Fig 10B and  10C), which was not observed in dlk-1 mutants [23]. Second, our results show that double mutants of rpm-1 with jnk-1, jkk-1 or nsy-1 are suppressed to an intermediate habituation phenotype with neither phenotype from single mutants dominating. The most likely explanation for these results is that the MIG-15/JNK-1 pathway functions as a parallel opposing pathway to RPM-1 (Fig 10E, Model 2). This differs from the DLK-1/PMK-3 pathway, which is inhibited by RPM-1 [29]. While we favor the parallel opposing pathway interpretation of our data, it is important to note our genetic results do not rule out that RPM-1 might ubiquitinate and inhibit a kinase in the MIG-15/JNK-1 pathway, in which case the MIG-15/JNK-1 pathway would function downstream of RPM-1 (Fig 10E, Model 1).
Our finding that the MIG-15/JNK-1 pathway is likely to function as a parallel opposing pathway to RPM-1 indicates that RPM-1 could be a relatively specific inhibitor of certain p38 and JNK pathways. This is consistent with prior work showing RPM-1 binds to the PP2C phosphatase PPM-2 to specifically inhibit DLK-1 [27]. Thus, our findings continue to support the concept that RPM-1 is a relatively sophisticated regulator of intracellular signaling, and not simply a general silencer of MAPK signaling [15,27].
Our discoveries that the MIG-15/JNK-1 pathway inhibits glutamatergic synapse formation, and that impairing certain kinases in the MIG-15/JNK-1 pathway increases short-term learning have potentially important implications for neurodegenerative disease. This is particularly noteworthy, as a JNK inhibitor was shown to reduce synaptic dysfunction and improve cognitive outcomes in mouse models of Alzheimer's disease [41][42][43], and recent work showed genetically impairing JNK activation improves outcomes in ALS and Alzheimer's models [44]. The specificity of the MIG-15/JNK-1 pathway compared to the DLK-1/PMK-3 and MLK-1/ KGB-1 pathways in worms suggests inhibitors of specific JNK isoforms could have differing efficacy in neurodegenerative disease models.

Genetics & strains
The N2 isolate of C. elegans was used for all experiments and worms were maintained using standard procedures. The following mutant alleles were used in this study: rpm-1 (ju44), jnk-1 (gk7), jkk-1 (km2), nsy-1 (ok593), and mig-15 (mu342). Genotyping was done by PCR and restriction digestion. nsy-1 (ok593)  Cloning cDNA encoding jnk-1, jkk-1, nsy-1 or mig-15 were cloned with iProof High-Fidelity DNA polymerase (BIO-RAD) PCR from an N2 cDNA pool using standard conditions. cDNAs were Taq polished and TOPO cloned into pCR8 Gateway entry vector (Invitrogen). cDNA entry vectors were validated by sequencing and recombined into the necessary Gateway destination vectors to create final plasmids for microinjection or transfection into HEK 293 cells.
For construction of Pmec-7::UNC-10::tdTOMATO (pBG-GY757), tdTOMATO cDNA was initially subcloned by PCR, adding PpuMI and NheI sites to 5' and 3' ends, respectively. These enzyme sites were used to insert tdTOMATO into a Pmec-7 Gateway destination vector to create the C-terminal tagging vector pBG-GY747. unc-10 genomic DNA was TOPO cloned using pJH430 (kind gift of Dr. Mei Zhen, University of Toronto) as a PCR template, and recombined into pBG-GY747.
To build a targeting vector for mig-15 MosSCI insertion, the pCR8 mig-15 Gateway entry vector (pBG-GY596) was recombined with a Pmec-7 destination vector (pBG-GY119). The mec-7 promoter, mig-15 cDNA, and unc-54 3'UTR was amplified by PCR as a single sequence using iProof DNA polymerase. SpeI sites were included in the primers and added to the 5' and 3' ends of this construct. PCR product was TOPO cloned into pCR8 to create pBG-GY616 and verified by sequencing. pBG-GY616 plasmid was cut with SpeI and subcloned into the pCFJ350 MosSCI targeting vector.

Transgenics
Transgenic strains were derived using standard procedures. For mechanosensory neuron rescue experiments, plasmid DNA mixtures were injected in double mutants of interest with muIs32 to derive transgenic extrachromosomal arrays that used the mec-7 promoter to drive expression from jnk-1, jkk-1 or nsy-1 cDNA. mig-15 rescue was done using a MosSCI single copy integrated transgene, bggSi1, that was crossed onto rpm-1 (ju44); mig-15 (mu342) double mutants. MosSCI was necessary due to technical limitations of injecting into mig-15 mutants, which have small body and brood size. MosSCI insertion was carried out with peel-1 negative selection [67].
For overexpression experiments, kinase cDNAs were expressed using the pan-neuronal rgef-1 promoter and extrachromasomal arrays were derived using wild-type animals. PCR amplification was performed using the Roche Expand Long Template PCR system.
For localization experiments, transgenic extrachromosomal arrays were generated in wildtype animals by injecting plasmids that used the mec-3 or mec-7 promoter to express nsy-1 or jnk-1 cDNA with N-terminal GFP fusions. For colocalization, GFP::JNK-1 was injected into bggIs28. The bggIs28 transgene was generated by TMP/UV genomic integration of a transgenic array containing Pmec-7::UNC-10::tdTOMATO (injected at 25 ng/μL). We generated N and C-terminal GFP and tdTOMATO fusions with MIG-15 and JKK-1, but these constructs were not readily detected in PLM neurons across different injection concentrations using either the mec-3 or mec-7 promoters.
All injection mixes used to make extrachromasomal arrays included the co-injection marker Pmyo-2::mCherry at 1 ng/μL, and were made up to total DNA concentration of 100 ng/μL using pBluescript. S1 Table contains  Analysis of axon and synapse development PLM axon termination and synaptic branch morphology was analyzed and quantified using the transgene muIs32. L4 parent worms were grown at 23˚C and F1 progeny were picked to separate plates as L4 animals and maintained at 23˚C. Animals were scored or imaged 16-24 hours later as young adults. This procedure was used to ensure consistent developmental staging across genotypes. Animals were anesthetized in 5 μM levamisole or 1% (v/v) 1-phenoxy-2-propanol in M9 buffer on a 2% agarose pad, and mounted and visualized with a Leica DM5000 B (CTR5000) epifluorescence microscope using 40x or 63x oil immersion objectives. Images were acquired with a CCD Leica DFC345 FX camera.
The synaptic branch was scored as a proxy for synapse formation in the PLM neurons. Defects were scored by following PLM synaptic branches down to the ventral nerve cord (VNC). Branches with terminal GFP varicosities, representing presynaptic boutons, were scored as wildtype. The absence of terminal varicosities and incomplete branches, or total absence of branches were scored as mutant. A subset of mig-15 mutants had multiple collateral PLM branches, a phenotype previously documented in the DD motor neurons for mig-15 mutants [48]. If at least one complete branch reached the VNC to form a varicosity the neuron was not counted as mutant for synaptic branch. The position of the vulva was also used to assess which branch was the normal, wild-type synaptic branch in these animals. A small proportion of mig-15 mutants displayed severe migration and guidance problems; the PLM neurons from these neurons were not included in our analysis.
Axon termination was scored as mutant if the axon overextended beyond the normal termination point (prior to the ALM cell body) and curved towards the VNC. These defects were referred to as "hook" defects.
To evaluate synapse formation in the GABAergic motor neurons, we used the transgene Punc-25::SNB-1::GFP (juIs1). Analysis of SNB-1::GFP puncta was done on young adult worms grown at 25˚C. Worms were prepared for analysis as described for the PLM neurons, and anesthetized with 1% (v/v) 1-phoxy-2-propanol. Images of the dorsal cord were collected using a 40x oil immersion objective. Stretches of dorsal cord were measured using Leica Application Suite AF software, and the number of SNB-1::GFP puncta were manually counted.

Confocal microscopy
L4 worms were picked to a separate plate and young adults were imaged 18-24 hours later. Slides were prepared with the same procedure described for PLM analysis with cytosolic GFP using 5 μM levamisole. Coverslips were sealed with BIOTIUM CoverGrip Coverslip Sealant. Confocal images were collected with Leica SP8 confocal microscope under 25x or 40x objectives. The following acquisition conditions were used: Bidirectional, 1.00 AU pinhole, 2.5-3.0x scan zoom factor, 400-600Hz, HyD detectors, 150-200 gain, 512x512 format, between lines sequential acquisition. Analysis conditions were kept identical across genotypes. Leica Application Suite (LAS) software was used to define Z-stacks that were collected at 1.0 to 1.5μm intervals. Maximum intensity projections of Z-stacks were made using LAS software.

Presynaptic morphology analysis
Exported TIFF files of confocal stacks were analyzed in FIJI (ImageJ). Regions of interest (ROI) were defined manually for GFP::RAB-3 and UNC-10::tdTOMATO puncta within PLM presynaptic boutons. Mean area and number of puncta were calculated based on ROIs, and data was pooled from three separate experiments for each genotype.

Colchicine treatment
Colchicine (0.25 mM from a 0.5 M stock solution dissolved in DMSO) was added while pouring NGM plates. Control NGM plates were made by adding an equal volume of DMSO. OP50 bacteria lawns were seeded onto colchicine or DMSO plates and used within 10 days of pouring. P0 young adult worms were placed on colchicine or DMSO plates, and F1 young adults were analyzed. Biochemistry 6-cm dishes of confluent HEK 293-T cells were transfected with 10 μL of Lipofectamine using the designated amount of plasmid: pBG-GY711 (FLAG-NSY-1, 2 μg), pBG-GY738 (MIG-15-HA, 4 μg) and pBG-GY784 (HA-JKK-1, 4 μg). pBluescript was added to reach 8 μg of total DNA transfected per plate. 22-26 hrs after transfection, cells were lysed with 0.1% NP-40 lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM DTT, and 1x Pierce HALT protease inhibitor cocktail). 0.25 to 0.5 mg of total protein from transfected cells was used for coIPs. Lysates were incubated with primary antibody for 30 min and precipitated for 4 h with 10 μL protein G agarose (Roche Applied Science) at 4˚C. Precipitates were boiled in Laemmli sample buffer (Bio-Rad) with ß-mercaptoethanol (Sigma) and run on a 4-12% Bis-Tris gel (Invitrogen). Gels were transferred to PVDF membranes using Tris acetate transfer buffer and immunoblotted. Blots were visualized with HRP conjugated secondary antibodies, ECL (1:5 dilution of Supersignal West Femto (Thermo Scientific) in TBS) and x-ray film.
FLAG tagged proteins were immunoprecipitated with a mouse monoclonal anti-FLAG antibody (M2, Sigma), and immunoblotted with rabbit polyclonal anti-FLAG antibodies (Cell Signaling). HA tagged proteins were immunoprecipitated with rabbit polyclonal anti-HA antibodies (Invitrogen) and immunoblotted with a rabbit monoclonal anti-HA antibody (C29F4, Cell Signaling). For anti-HA blots, anti-rabbit light chain specific secondary antibodies were used.

Statistical analysis
PLM synapse formation and axon termination analysis. For analysis with muIs32, statistical comparisons were done with an unpaired, 2-tailed Student's t-test and Bonferroni correction. Significance was defined as p<0.05. Bar graphs represent averages from 4-10 counts (25-35 neurons/count) for each genotype from three or more independent experiments. For analysis of the synaptic branch and RAB-3::GFP accumulation, bar graphs represent averages from 6-8 counts (25-35 neurons/count) for each genotype from three or more independent experiments. For transgenic experiments, bar graphs represent averages for data from 4-6 independent transgenic lines for each genotype. Error bars represent the standard error of the mean.
GABAergic synapse formation. For juIs1 analysis, statistical comparisons were done with an unpaired, 2-tailed Student's t-test and Bonferroni correction. Significance was defined as p<0.05. Bar graphs represent averages from 27-36 total animals collected from 3 or more independent experiments for each genotype. Error bars represent the standard error of the mean.
Habituation. One plate of animals was considered n = 1, and four plates were tested in three independent experiments for each genotype (n = 12). Graphs show the mean ± SEM of responses to all stimuli, an exponential fit of mean responses, and mean ± SEM for habituation level. Student's t-tests were performed to assess statistical significance. Note there is no suppression of axon termination defects in rpm-1; jkk-1 or rpm-1; mig-15 double mutants. Shown are averages from 6-10 counts (25-30 neurons/count) of young adult animals for each genotype. Error bars represent standard error of mean, and significance was determined using unpaired Student's t test with Bonferroni correction. ns = not significant. (TIF) S1 Table. Transgenic DNA injection details. Worms of the indicated genotypes were injected with constructs at specified concentrations. Note that PCR prod. is linear DNA PCR product from Roche Expand Long Template PCR System (see Methods). Unless stated, all constructs contained unc-54 3'UTR sequence to aid neuronal expression. All transgenic constructs were injected with Pmyo-2::mCherry co-injection marker at 1 ng/μl (see Methods). (DOCX)