The Ubiquitin Ligase RPM-1 and the p38 MAPK PMK-3 Regulate AMPA Receptor Trafficking

Ubiquitination occurs at synapses, yet its role remains unclear. Previous studies demonstrated that the RPM-1 ubiquitin ligase organizes presynaptic boutons at neuromuscular junctions in C. elegans motorneurons. Here we find that RPM-1 has a novel postsynaptic role in interneurons, where it regulates the trafficking of the AMPA-type glutamate receptor GLR-1 from synapses into endosomes. Mutations in rpm-1 cause the aberrant accumulation of GLR-1 in neurites. Moreover, rpm-1 mutations enhance the endosomal accumulation of GLR-1 observed in mutants for lin-10, a Mint2 ortholog that promotes GLR-1 recycling from Syntaxin-13 containing endosomes. As in motorneurons, RPM-1 negatively regulates the pmk-3/p38 MAPK pathway in interneurons by repressing the protein levels of the MAPKKK DLK-1. This regulation of PMK-3 signaling is critical for RPM-1 function with respect to GLR-1 trafficking, as pmk-3 mutations suppress both lin-10 and rpm-1 mutations. Positive or negative changes in endocytosis mimic the effects of rpm-1 or pmk-3 mutations, respectively, on GLR-1 trafficking. Specifically, RAB-5(GDP), an inactive mutant of RAB-5 that reduces endocytosis, mimics the effect of pmk-3 mutations when introduced into wild-type animals, and occludes the effect of pmk-3 mutations when introduced into pmk-3 mutants. By contrast, RAB-5(GTP), which increases endocytosis, suppresses the effect of pmk-3 mutations, mimics the effect of rpm-1 mutations, and occludes the effect of rpm-1 mutations. Our findings indicate a novel specialized role for RPM-1 and PMK-3/p38 MAPK in regulating the endosomal trafficking of AMPARs at central synapses

The PMK-3/p38 MAPK pathway modulates GLR-1 trafficking.

<p>GLR-1::GFP fluorescence was observed along ventral cord dendrites of (A) <i>pmk-3</i> mutants, (B) <i>pmk-3 lin-10</i> double mutants, (C) <i>pmk-3 </i><i>rpm-1</i> double mutants, (D) <i>pmk-3 </i><i>rpm-1 lin-10</i> triple mutants, (E) <i>lin-10</i> mutants that express a wild-type <i>pmk-3</i> cDNA using the <i>glr-1</i> promoter, or (F) <i>lin-10</i> mutants that express a wild-type <i>dlk-1</i> cDNA using the <i>glr-1</i> promoter. Arrowheads indicate the accumulation of GLR-1::GFP in large (1–5 micron long) accretions. Arrows indicate accumulation in very large (>5 micron long) accretions (similar to those found in <i>lin-10 rpm-1</i> double mutants). (G,H,I,J) The mean size of fluorescent structures (puncta and accretions) is plotted for adult nematodes of the given genotype. (K,L) The mean number of spontaneous reversals of locomotion for young adult animals is plotted for the given genotype. Bar, 5 µm. Error bars are SEM. N = 15–25 animals for each genotype. **P<0.01 by ANOVA followed by Dunnett's Multiple Comparison to wild type. ***P<0.001 by ANOVA followed by a Bonferroni Multiple Comparison test (indicated by lines).</p

Differential requirements for RPM-1 at NMJs versus central synapses.

<p>SNB-1::GFP fluorescence was observed along the ventral cord of animals either (A,C,E,G) expressing the reporter at motorneuron NMJs via the <i>unc-25</i> promoter, or (B,D,F,H) expressing the reporter at interneuron central synapses via the <i>glr-1</i> promoter. SNB-1::GFP localization was observed in (A,B) wild-type animals, (C,D) <i>rpm-1</i> mutants, (E,F) <i>lin-10</i> mutants, or (G,H) <i>rpm-1 lin-10</i> double mutants. (I) The number of fluorescent structures (puncta) is plotted for motorneuron NMJs. (J) The mean number of puncta is plotted for interneuron central synapses. The fluorescence of (K,L) RFP::SNN-1 and (M,N) LIN-10::GFP, expressed from the <i>glr-1</i> promoter, were also observed along the ventral cord of (K,M) wild-type animals and (L,N) <i>rpm-1</i> mutants. Bar, 5 µm. Error bars are SEM. N = 15–25 animals for each genotype. ***P<0.001 by ANOVA followed by Dunnett's Multiple Comparison to wild type.</p

GLR-1 colocalization with endosome marker Synxtaxin-13.

<p>(A,E,I,M) GLR-1::GFP and (B,F,J,N) RFP::Syntaxin-13 fluorescence was observed in single confocal images of neuron cell bodies from (A–D) wild-type animals, (E–H) <i>lin-10</i> mutants, (I–L) <i>pmk-3</i> mutants, and (M–P) <i>pmk-3 lin-10</i> double mutants. (C,G,K,O) Merged images. (D,H,L,P) Binary masks (white) were created to highlight pixels with matching intensity values for both GLR-1::GFP and RFP::Syntaxin-13, indicating colocalization. (Q) The mean percent of GLR-1::GFP colocalized with Syntaxin-13 (endosomal), normalized to total cell body GLR-1::GFP. Error bars are SEM. *P<0.05, **P<0.01 by ANOVA followed by Dunnett's Multiple Comparison to wild type.</p

A model for the regulation of GLR-1 AMPAR trafficking by PMK-3 and RPM-1.

<p>GLR-1 (gray channels) endocytosis can occur via clathrin (pit indicated to left of the synapse). Gray arrows indicate major trafficking steps, positively regulated by the factor(s) indicated next to the arrows. Black arrows indicate positive (stimulatory) genetic regulatory interactions between two factors. GLR-1 endocytosis is mediated by UNC-11/AP180 and RAB-5. Once endocytosed, receptors can either be recycled to the synapse in a step requiring LIN-10, or degraded. PMK-3/p38 MAPK stimulates GLR-1 endocytosis via RAB-5 activation. PMK-3 is activated by a MAP kinase cascade, which includes MKK-4 and DLK-1. RPM-1 and FSN-1, working as an E3 ligase, negatively regulate DLK-1 (and hence p38 MAPK signaling) by ubiquitin-mediated turnover.</p

RPM-1 and PMK-3 regulate GLR-1 endocytosis.

<p>(A,B,C,D,F) The mean size of fluorescent structures (puncta and accretions) and (E,G) the mean reversal frequency is plotted for adult nematodes of the given genotype. (A) Decreased or (B) increased endocytosis suppresses or enhances the accumulation of GLR-1 in <i>lin-10</i> mutants, respectively, as well as (C) <i>rpm-1</i> mutants. Decreased endocytosis by (D,E) expression of <i>rab-5(GDP),</i> can mimic and occlude the effect of <i>pmk-3</i> mutations on GLR-1 trafficking. Increased endocytosis by (F,G) expression of <i>rab-5(GTP)</i> can block the effect of <i>pmk-3</i> mutations, thus bypassing the requirement for PMK-3. Error bars are SEM. N = 15–25 animals for each genotype. Solid lines with ** (P<0.01) or *** (P<0.001) indicate specific comparisons by ANOVA followed by a Bonferroni Multiple Comparison test. Comparisons to the wild-type control are indicated by # (P<0.05) or ## (P<0.01) using ANOVA followed by Dunnett's test.</p

RPM-1 is required for the ubiquitin-mediated turnover of GLR-1.

<p>GLR-1::GFP fluorescence was observed along ventral cord dendrites of (A) wild-type animals, (B) <i>lin-10</i> mutants, (C) <i>rpm-1</i> mutants, or (D) <i>rpm-1 lin-10</i> double mutants, all of which also express ubiquitin from the <i>nuIs89</i> transgene. The mean size (E) and the mean number (F) of fluorescent structures (puncta) are plotted for adult nematodes of the given genotype. Bar, 5 µm. Error bars are SEM. N = 15–25 animals for each genotype. *P<0.05, ***P<0.001 by ANOVA followed by a Bonferroni Multiple Comparison test (indicated by line). ##P<0.001 by ANOVA followed by Dunnett's Multiple Comparison to wild type.</p

Mutations in RPM-1.

<p>(A) The intron/exon gene structure of <i>rpm-1</i> (gray boxes) is shown at top. At bottom is the predicted protein domain structure, including the RCC1 repeats, the dual PHR domains, the Myc-bind region, the B-box, and the RING domain. The molecular nature of several known alleles is indicated. (B) Amino acid alignment of mouse Phr1, human Pam, Zebrafish Esrom, Drosophila <i>highwire</i>, and <i>C. elegans</i> RPM-1. Black highlighting indicates common identities, and gray highlighting indicates similarities. In the <i>od14</i> mutation (indicated by the arrow), glycine is replaced with glutamate at a conserved PHR domain residue.</p

RPM-1 regulates DLK-1 levels in interneurons.

<p>(A,B) GLR-1::GFP or (C,D) RFP::PMK-3 fluorescence was observed in (A,C) the neuron cell bodies and (B,D) the ventral cord neurites of wild-type animals. (E,F) Merged images. GFP::DLK-1 fluorescence from the same transgenic line was observed in (G,I) the neuron cell bodies and (H,J) the ventral cord neurites of (G,H) wild-type animals and (I,J) <i>rpm-1</i> mutants. Arrows indicate puncta of GFP::DLK-1. (K,L) The mean GFP::DLK-1 fluorescence intensity for (K) neuron cell bodies and (L) ventral cord neurites is plotted for adult nematodes of the indicated genotype. (M,N) The mean size of fluorescent structures (puncta and accretions) is plotted for adult nematodes of the given genotype. Error bars are SEM. N = 20–25 animals for each genotype. *P<0.05, ***P<0.001 by ANOVA, followed by a Bonferroni Multiple Comparison test (indicated by lines).</p

Distinct LIN-10 Domains Are Required for Its Neuronal Function, Its Epithelial Function, and Its Synaptic Localization

α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors (AMPARs) mediate excitatory neurotransmission at neuronal synapses, and their regulated localization plays a role in synaptic plasticity. In Caenorhabditis elegans, the PDZ and PTB domain-containing protein LIN-10 is required both for the synaptic localization of the AMPAR subunit GLR-1 and for vulval fate induction in epithelia. Here, we examine the role that different LIN-10 domains play in GLR-1 localization. We find that an amino-terminal region of LIN-10 directs LIN-10 protein localization to the Golgi and to synaptic clusters. In addition, mutations in the carboxyl-terminal PDZ domains prevent LIN-10 from regulating GLR-1 localization in neurons but do not prevent LIN-10 from functioning in the vulval epithelia. A mutation in the amino terminus prevents the protein from functioning in the vulval epithelia but does not prevent it from functioning to regulate GLR-1 localization in neurons. Finally, we show that human Mint2 can substitute for LIN-10 to facilitate GLR-1 localization in neurons and that the Mint2 amino terminus is critical for this function. Together, our data suggest that LIN-10 uses distinct modular domains for its functions in neurons and epithelial cells and that during evolution its vertebrate ortholog Mint2 has retained the ability to direct AMPAR localization in neurons