Neurons born from progenitor cells located in discrete niches move out and migrate, sometimes long distances, to reach their final position. A variety of guidance cues pilot these neurons from their birthplace to their final location, where they stop, mature and integrate into the existing cellular network. Signaling pathways are activated in response to guidance cues or chemotactic signals and once the information is transduced, the original signaling pathway must be downregulated. Inappropriate regulation of signaling pathways causes neurons to mismigrate, lose responsiveness to new signals and/or sustain the original signaling response, causing harm or death to the cell. The ubiquitin-proteasome system is one of the most efficient methods to tightly control signaling pathways, by targeting key signaling components for degradation. This system relies on the coordinated action of 3 enzymes, named E1, E2 and E3 ligases to conjugate the small protein ubiquitin to specific signaling effectors that will be targeted for degradation. In the Simo lab, we focus on the role of the E3 ubiquitin ligase Cullin-5 RING ligase (CRL5) during neuronal migration in several areas of the nervous system, including the neocortex, hippocampus, and retina.
The multi-protein complex CRL5 is a member of the superfamily of CRL ubiquitin ligases, that contains the core proteins Cullin 5 (Cul5), RING-box protein 2 (Rbx2), and Elongins B and C (Figure 1). The CRL5 complex recruits target substrates through 38 confirmed and putative CRL5-specific substrate adaptors. We have shown that CRL5 is a novel regulator of neuronal migration and layering of several areas of the nervous system (Figure 2)(Han et al., 2020; Fairchild et al., 2018; Simó and Cooper, 2013; and Simó et al., 2010). Disruption of CRL5, by conditional depletion of Rbx2 in the nervous system using Nestin-Cre (Rbx2cKO-Nes) or the or telencephalon using Emx1-Cre (Rbx2cKO-Emx1) mouse drivers, resulted in the identical phenotype of ectopically localized PNs and disrupted cortical layering (Figure 2, left pannel). Additionally, Rbx2 mutant PNs sustained migration for longer-than-normal periods of time leading to the progressive worsening of cortical layering. Similar results were obtained using short hairpin RNA against Cul5 (shCul5) and in utero electroporation (IUE) to knock down Cul5 from migrating cortical and hippocampal neurons, observing a sustained neuron migration in a cell-autonomous fashion.
Using quantitative mass spectrometry, we identified signaling effectors differentially expressed upon Rbx2 loss. From these experiments, we uncovered several members of the ARL4C signaling pathway deregulated in these animals, including ARL4C, Cytohesin-1, Cytohesin-3, and ARF6, to be regulated by CRL5. We used in utero electroporations in the hippocampus to assess their role on neuron over-migration in the hippocampus (Figure 3).
We are currently investigating 1) the effects of ARL4C signaling in neuron migration, both in the cortex and hippocampus, 2) the CRL5-dependent regulation of ARL4C and AR4C signaling effectors, and 3) the role of ARL4C signaling in dendritogenesis.