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Quantitation of axon termination defects in ALM neurons for indicated genotypes. Reduced frequency of axon termination defects occurs in cdk-5; rpm-1 double mutants, but not pct-1; rpm-1 double mutants. Suppression of termination defects is not increased in cdk-5; pct-1; rpm-1 triple mutants compared to cdk-5; rpm-1 double mutants. Results suggest that CDK-5 and PCT-1 do not function redundantly during axon termination. Means (bars) are shown for 6 or more counts (black dots, 20 or more worms/count) for each genotype. Error bars indicate SEM. Significance assessed using Student’s t-test. * p<0.05, ** p<0.01.

The Cdk5 kinase plays prominent roles in nervous system development, plasticity, behavior and disease. It also has important, non-neuronal functions in cancer, the immune system and insulin secretion. At present, we do not fully understand negative regulatory mechanisms that restrict Cdk5. Here, we use Caenorhabditis elegans to show that CDK-5 is inhibited by the RPM-1/FSN-1 ubiquitin ligase complex. This atypical RING ubiquitin ligase is conserved from C. elegans through mammals. Our finding originated from unbiased, in vivo affinity purification proteomics, which identified CDK-5 as a putative RPM-1 substrate. CRISPR-based, native biochemistry showed that CDK-5 interacts with the RPM-1/FSN-1 ubiquitin ligase complex. A CRISPR engineered RPM-1 substrate ‘trap’ enriched CDK-5 binding, which was mediated by the FSN-1 substrate recognition module. To test the functional genetic relationship between the RPM-1/FSN-1 ubiquitin ligase complex and CDK-5, we evaluated axon termination in mechanosensory neurons and motor neurons. Our results indicate that RPM-1/FSN-1 ubiquitin ligase activity restricts CDK-5 to control axon termination. Collectively, these proteomic, biochemical and genetic results increase our understanding of mechanisms that restrain Cdk5 in the nervous system.

Non-conventional N-methyl-d-aspartate receptors (NMDARs) containing GluN3A subunits have unique biophysical, signalling and localization properties within the NMDAR family, and are typically thought to counterbalance functions of classical NMDARs made up of GluN1/2 subunits. Beyond their recognized roles in synapse refinement during postnatal development, recent evidence is building a wider perspective for GluN3A functions. Here we draw particular attention to the latest developments for this multifaceted and unusual subunit: from finely timed expression patterns that correlate with plasticity windows in developing brains or functional hierarchies in the mature brain to new insight onto presynaptic GluN3A-NMDARs, excitatory glycine receptors and behavioural impacts, alongside further connections to a range of brain disorders.

[EN] NMDA receptors (NMDARs) are glutamate-gated excitatory ion channels with central roles in synapse plasticity, learning and memory. NMDAR complexes containing the GluN3A subunit (GluN3A-NMDARs) possess distinct biological properties. GluN3A expression is largely restricted to short windows of postnatal development, having established roles in sensorydependent postsynaptic refinement. Ongoing work in the host lab has begun to demonstrate likely involvement of GluN3A in the refinement of axonal structures. Building from these findings, this project tested the influence of GluN3A on callosal axon development, setting up CTB retrograde tracing in two different injection, studying different aspects of callosal development. Both types of CTB injections were successfully established and optimized in the host lab. Cortical injections in young (P13) mice revealed a decrease in axon targeting to S1/S2 border region of the somatosensory cortex (SSCx) in Grin3a KOs. This indicates increased promiscuity for these SSCx CPNs, consistent with prior findings from In Utero Electroporation (IUE) labelling. Other injections into the Corpus Callosum (CC) of mature mice did not identify permanent differences in CPN distribution patterns. This may be due to a transient nature of phenotypes, as indicated in prior work. Additionally, RT-qPCRs on P11 SSCx confirmed a significant upregulation of Kirrel2 mRNA in Grin3a KO mice. Kirrel2 is an adhesion molecule known to control axon guidance, being an interesting candidate. Future experiments with a larger sample numbers, as well as injections into other regions of the SSCx and at different ages, will allow a clearer understanding of the role of GluN3A in circuit development., [ES] Los receptores NMDA (NMDAR) son canales iónicos excitatorios activados por glutamato que desempeñan un papel fundamental en la plasticidad de sinapsis aprendizaje y memoria. Los complejos NMDAR que contienen la subunidad GluN3A (GluN3A-NMDARs) poseen propiedades biológicas distintas. Su se restringe temporalmente durante el desarrollo postnatal, con funciones de refinamiento postsináptico dependiente de experiencia. Trabajos realizados en este laboratorio han comenzado a demostrar la probable participación de GluN3A en el refinamiento de estructuras axonales. Así, este proyecto probó la influencia de GluN3A en el desarrollo de los axones callosos, a través del marcaje retrógrado de CTB en dos inyecciones diferentes, estudiando distintos aspectos del desarrollo calloso. Ambos tipos de inyecciones de CTB se establecieron con éxito y se optimizaron en el laboratorio. Inyecciones corticales en ratones jóvenes (P13) revelaron una disminución de la orientación de los axones hacia la región del borde S1/S2 de la corteza somatosensorial (SSCx) en los Grin3a KO. Esto indica un aumento de la promiscuidad de estos CPNs de la SSCx, lo que concuerda con los hallazgos obtenidos por IUEs. Otras inyecciones en el cuerpo calloso (CC) de ratones adultos no identificaron diferencias permanentes en los patrones de distribución de los CPNs. Esto puede deberse a la naturaleza transitoria de los fenotipos. Además, RT-qPCRs en P11 SSCx confirmaron un aumento significativo del mRNA de Kirrel2 en los ratones Grin3a KO. Kirrel2 es una molécula de adhesión que controla la guía axonal, siendo un candidato interesante. Futuros experimentos con más muestras, así como inyecciones en otras regiones de la SSCx y a diferentes edades, permitirán una comprensión más clara del papel de GluN3A en el desarrollo del circuito.

The study of autophagy in the nervous system has predominantly centered on degeneration. Evidence is now cementing crucial roles for autophagy in neuronal development and growth, especially in axonal and presynaptic compartments. A picture is emerging that autophagy typically promotes the growth of axons and reduces presynaptic stability. Nonetheless, these are not rigid principles, and it remains unclear why autophagy does not always display these relationships during axonal and presynaptic development. Recent progress has identified mechanisms underlying spatiotemporal control of autophagy in neurons and begun to unravel how autophagy is integrated with other cellular processes, such as proteasomal degradation and axon guidance. Ultimately, understanding how autophagy is regulated and its role in the developing nervous system is key to comprehending how the nervous system assembles its stereotyped yet plastic configuration. It is also likely to inform how we think about neurodevelopmental disorders and neurodegenerative diseases.

Asymmetric disassembly of the synaptonemal complex (SC) is crucial for proper meiotic chromosome segregation. However, the signaling mechanisms that directly regulate this process are poorly understood. Here we show that the mammalian Rho GEF homolog, ECT-2, functions through the conserved RAS/ERK MAP kinase signaling pathway in the C. elegans germline to regulate the disassembly of SC proteins. We find that SYP-2, a SC central region component, is a potential target for MPK-1-mediated phosphorylation and that constitutively phosphorylated SYP-2 impairs the disassembly of SC proteins from chromosomal domains referred to as the long arms of the bivalents. Inactivation of MAP kinase at late pachytene is critical for timely disassembly of the SC proteins from the long arms, and is dependent on the crossover (CO) promoting factors ZHP-3/RNF212/Zip3 and COSA-1/CNTD1. We propose that the conserved MAP kinase pathway coordinates CO designation with the disassembly of SC proteins to ensure accurate chromosome segregation.

Wapl induces cohesin dissociation from DNA throughout the mitotic cell cycle, modulating sister chromatid cohesion and higher-order chromatin structure. Cohesin complexes containing meiosis-specific kleisin subunits govern most aspects of meiotic chromosome function, but whether Wapl regulates these complexes remains unknown. We show that during C. elegans oogenesis WAPL-1 antagonizes binding of cohesin containing COH-3/4 kleisins, but not REC-8, demonstrating that sensitivity to WAPL-1 is dictated by kleisin identity. By restricting the amount of chromosome-associated COH-3/4 cohesin, WAPL-1 controls chromosome structure throughout meiotic prophase. In the absence of REC-8, WAPL-1 inhibits COH-3/4-mediated cohesion, which requires crossover-fated events formed during meiotic recombination. Thus, WAPL-1 promotes functional specialization of meiotic cohesin: WAPL-1-sensitive COH-3/4 complexes modulate higher-order chromosome structure, while WAPL-1-refractory REC-8 complexes provide stable cohesion. Surprisingly, a WAPL-1-independent mechanism removes cohesin before metaphase I. Our studies provide insight into how meiosis-specific cohesin complexes are regulated to ensure formation of euploid gametes.

Prior to the meiotic divisions, dynamic chromosome reorganizations including pairing, synapsis, and recombination of maternal and paternal chromosome pairs must occur in a highly regulated fashion during meiotic prophase. How chromosomes identify each other's homology and exclusively pair and synapse with their homologous partners, while rejecting illegitimate synapsis with non-homologous chromosomes, remains obscure. In addition, how the levels of recombination initiation and crossover formation are regulated so that sufficient, but not deleterious, levels of DNA breaks are made and processed into crossovers is not understood well. We show that in Caenorhabditis elegans, the highly conserved Serine/Threonine protein phosphatase PP4 homolog, PPH-4.1, is required independently to carry out four separate functions involving meiotic chromosome dynamics: (1) synapsis-independent chromosome pairing, (2) restriction of synapsis to homologous chromosomes, (3) programmed DNA double-strand break initiation, and (4) crossover formation. Using quantitative imaging of mutant strains, including super-resolution (3D-SIM) microscopy of chromosomes and the synaptonemal complex, we show that independently-arising defects in each of these processes in the absence of PPH-4.1 activity ultimately lead to meiotic nondisjunction and embryonic lethality. Interestingly, we find that defects in double-strand break initiation and crossover formation, but not pairing or synapsis, become even more severe in the germlines of older mutant animals, indicating an increased dependence on PPH-4.1 with increasing maternal age. Our results demonstrate that PPH-4.1 plays multiple, independent roles in meiotic prophase chromosome dynamics and maintaining meiotic competence in aging germlines. PP4's high degree of conservation suggests it may be a universal regulator of meiotic prophase chromosome dynamics.