A kinship between cranial and pelvic visceral nerves of vertebrates has been accepted for a century. Accordingly, sacral preganglionic neurons are considered parasympathetic, as are their targets in the pelvic ganglia that prominently control rectal, bladder, and genital functions. Here, we uncover 15 phenotypic and ontogenetic features that distinguish pre- and postganglionic neurons of the cranial parasympathetic outflow from those of the thoracolumbar sympathetic outflow in mice. By every single one, the sacral outflow is indistinguishable from the thoracolumbar outflow. Thus, the parasympathetic nervous system receives input from cranial nerves exclusively and the sympathetic nervous system from spinal nerves, thoracic to sacral inclusively. This simplified, bipartite architecture offers a new framework to understand pelvic neurophysiology as well as development and evolution of the autonomic nervous system.

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BackgroundLNX (Ligand of Numb Protein-X) proteins typically contain an amino-terminal RING domain adjacent to either two or four PDZ domains - a domain architecture that is unique to the LNX family. LNX proteins function as E3 ubiquitin ligases and their domain organisation suggests that their ubiquitin ligase activity may be targeted to specific substrates or subcellular locations by PDZ domain-mediated interactions. Indeed, numerous interaction partners for LNX proteins have been identified, but the in vivo functions of most family members remain largely unclear.ResultsTo gain insights into their function we examined the phylogenetic origins and evolution of the LNX gene family. We find that a LNX1/LNX2-like gene arose in an early metazoan lineage by gene duplication and fusion events that combined a RING domain with four PDZ domains. These PDZ domains are closely related to the four carboxy-terminal domains from multiple PDZ domain containing protein-1 (MUPP1). Duplication of the LNX1/LNX2-like gene and subsequent loss of PDZ domains appears to have generated a gene encoding a LNX3/LNX4-like protein, with just two PDZ domains. This protein has novel carboxy-terminal sequences that include a potential modular LNX3 homology domain. The two ancestral LNX genes are present in some, but not all, invertebrate lineages. They were, however, maintained in the vertebrate lineage, with further duplication events giving rise to five LNX family members in most mammals. In addition, we identify novel interactions of LNX1 and LNX2 with three known MUPP1 ligands using yeast two-hybrid asssays. This demonstrates conservation of binding specificity between LNX and MUPP1 PDZ domains.ConclusionsThe LNX gene family has an early metazoan origin with a LNX1/LNX2-like protein likely giving rise to a LNX3/LNX4-like protein through the loss of PDZ domains. The absence of LNX orthologs in some lineages indicates that LNX proteins are not essential in invertebrates. In contrast, the maintenance of both ancestral LNX genes in the vertebrate lineage suggests the acquisition of essential vertebrate specific functions. The revelation that the LNX PDZ domains are phylogenetically related to domains in MUPP1, and have common binding specificities, suggests that LNX and MUPP1 may have similarities in their cellular functions.

The Bridging Integrator 1 (BIN1) gene is a major susceptibility gene for Alzheimer’s disease (AD). Deciphering its pathophysiological role is challenging due to its numerous isoforms. Here we observed in Drosophila that human BIN1 isoform1 (BIN1iso1) overexpression, contrary to human BIN1 isoform8 (BIN1iso8) and human BIN1 isoform9 (BIN1iso9), induced an accumulation of endosomal vesicles and neurodegeneration. Systematic search for endosome regulators able to prevent BIN1iso1-induced neurodegeneration indicated that a defect at the early endosome level is responsible for the neurodegeneration. In human induced neurons (hiNs) and cerebral organoids, BIN1 knock-out resulted in the narrowing of early endosomes. This phenotype was rescued by BIN1iso1 but not BIN1iso9 expression. Finally, BIN1iso1 overexpression also led to an increase in the size of early endosomes and neurodegeneration in hiNs. Altogether, our data demonstrate that the AD susceptibility gene BIN1, and especially BIN1iso1, contributes to early-endosome size deregulation, which is an early pathophysiological hallmark of AD pathology.

We recently defined genetic traits that distinguish sympathetic from parasympathetic neurons, both preganglionic and ganglionic (Espinosa-Medina et al., Science 354:893–897, 2016). By this set of criteria, we found that the sacral autonomic outflow is sympathetic, not parasympathetic as has been thought for more than a century. Proposing such a belated shift in perspective begs the question why the new criterion (cell types defined by their genetic make-up and dependencies) should be favored over the anatomical, physiological and pharmacological considerations of long ago that inspired the “parasympathetic” classification. After a brief reminder of the former, we expound the weaknesses of the latter and argue that the novel genetic definition helps integrating neglected anatomical and physiological observations and clearing the path for future research.

Type of publicationArticle (peer-reviewed) AbstractNUMB is a key regulator of neurogenesis and neuronal differentiation that can be ubiquitinated and targeted for proteasomal degradation by ligand of numb proteinX (LNX) family E3 ubiquitin ligases. However, our understanding of LNX protein function in vivo is very limited. To examine the role of LNX proteins in regulating NUMB function in vivo, we generated mice lacking both LNX1 and LNX2 expression in the brain. Surprisingly, these mice are healthy, exhibit unaltered levels of NUMB protein and do not display any neuroanatomical defects indicative of aberrant NUMB function. Behavioural analysis of LNX1/LNX2 double knockout mice revealed decreased anxiety related behaviour, as assessed in the open field and elevated plus maze paradigms. By contrast, no major defects in learning, motor or sensory function were observed. Given the apparent absence of major NUMB dysfunction in LNX null animals, we performed a proteomic analysis to identify neuronal LNXinteracting proteins other than NUMB that might contribute to the anxiolytic phenotype observed. We identified and/or confirmed interactions of LNX1 and LNX2 with proteins known to have presynaptic and neuronal signalling functions, including the presynaptic active zone constituents ERC1, ERC2 and LIPRINαs (PPFIA1, PPFIA3), as well as the FBAR domain proteins FCHSD2 (nervous wreck homologue) and SRGAP2. These and other novel LNXinteracting proteins identified are promising candidates to mediate LNX functions in the central nervous system, including their role in modulating anxietyrelated behaviour.

Ligand of Numb protein X1 (LNX1) is an E3 ubiquitin ligase that contains a catalytic RING (Really Interesting New Gene) domain and four PDZ (PSD-95, DlgA, ZO-1) domains. LNX1 can ubiquitinate Numb, as well as a number of other ligands. However, the physiological relevance of these interactions in vivo remain unclear. To gain functional insights into the LNX family, we have characterised the LNX1 interactome using affinity purification and mass spectrometry. This approach identified a large number of novel LNX1-interacting proteins, as well as confirming known interactions with NUMB and ERC2. Many of the novel interactions mapped to the LNX PDZ domains, particularly PDZ2, and many showed specificity for LNX1 over the closely related LNX2. We show that PPFIA1 (liprin-α1), KLHL11, KIF7 and ERC2 are substrates for ubiquitination by LNX1. LNX1 ubiquitination of liprin-α1 is dependent on a PDZ binding motif containing a carboxyl terminal cysteine that binds LNX1 PDZ2. Surprisingly, the neuronally-expressed LNX1p70 isoform, that lacks the RING domain, was found to promote ubiquitination of PPFIA1 and KLHL11, albeit to a lesser extent than the longer RING-containing LNX1p80 isoform. Of several E3-ligases identified in the LNX1 interactome we confirm interactions of LNX1 with MID2/TRIM1 and TRIM27. On this basis we propose a model whereby LNX1p70, despite lacking a catalytic RING domain, may function as a scaffold to promote ubiquitination of its ligands through recruitment of other E3-ligases. These findings provide functional insights into the LNX protein family, particularly the neuronal LNX1p70 isoform.

The Bridging Integrator 1 (BIN1) gene is a major genetic risk factor for Alzheimer's disease (AD) but little is known about its physiological functions. In addition, deciphering its potential pathophysiological role is difficult due to its numerous isoforms expressed in different cerebral cell types. Here we took advantage of a drosophila model to assess in vivo the impact of different BIN1 isoforms on neuronal toxicity: the neuronal isoform 1 (BIN1iso1), the muscular isoform 8 (BIN1iso8) and the ubiquituous isoform 9 (BIN1iso9). We showed that contrary to BIN1iso8 and BIN1iso9, BIN1iso1 overexpression induced neurodegeneration and an accumulation of vesicles mainly labeled by endosome markers. Systematic search for endosome trafficking regulators that are able to rescue BIN1iso1-induced neurodegeneration indicated a defect in the early endosome trafficking machinery. In human induced neurons and cerebral organoids, BIN1 knock-out resulted in narrowing of the early endosomes. This phenotype was rescued by BIN1iso1 expression but not that of BIN1iso9. Finally, in accordance with our previous observation in flies, we also observed that BIN1iso1 overexpression led to an increase in size of the early endosomes in human induced neurons. Altogether, our data demonstrate that the AD genetic risk factor BIN1, and especially BIN1iso1, contributes to early-endosome size deregulation which is a very early pathophysiological feature observed in AD pathogenesis.