Disturbance of endoplasmic reticulum (ER) proteostasis is a common feature of amyotrophic lateral sclerosis (ALS). Protein disulfide isomerases (PDIs) are ER foldases identified as possible ALS biomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process. Here, we functionally characterized four ALS-linked mutations recently identified in two major PDI genes, PDIA1 and PDIA3/ERp57. Phenotypic screening in zebrafish revealed that the expression of these PDI variants induce motor defects associated with a disruption of motoneuron connectivity. Similarly, the expression of mutant PDIs impaired dendritic outgrowth in motoneuron cell culture models. Cellular and biochemical studies identified distinct molecular defects underlying the pathogenicity of these PDI mutants. Finally, targeting ERp57 in the nervous system led to severe motor dysfunction in mice associated with a loss of neuromuscular synapses. This study identifies ER proteostasis imbalance as a risk factor for ALS, driving initial stages of the disease.

The vertebrate habenulae (Hb) is an evolutionary conserved dorsal diencephalic nuclear complex that relays information from limbic and striatal forebrain regions to the ventral midbrain. One key feature of this bilateral nucleus is the presence of left-right differences in size, cytoarchitecture, connectivity, neurochemistry and/or gene expression. In teleosts, habenular asymmetry has been associated with preferential innervation of left-right habenular efferents into dorso-ventral domains of the midbrain interpeduncular nucleus (IPN). However, the degree of conservation of this trait and its relation to the structural asymmetries of the Hb are currently unknown. To address these questions, we performed the first systematic comparative analysis of structural and connectional asymmetries of the Hb in teleosts. We found striking inter-species variability in the overall shape and cytoarchitecture of the Hb, and in the frequency, strength and to a lesser degree, laterality of habenular volume at the population level. Directional asymmetry of the Hb was either to the left in D. rerio, E. bicolor, O. latipes, P. reticulata, B. splendens, or to the right in F. gardneri females. In contrast, asymmetry was absent in P. scalare and F. gardneri males at the population level, although in these species the Hb displayed volumetric asymmetries at the individual level. Inter-species variability was more pronounced across orders than within a single order, and coexisted with an overall conserved laterotopic representation of left-right habenular efferents into dorso-ventral domains of the IPN. These results suggest that the circuit design involving the Hb of teleosts promotes structural flexibility depending on developmental, cognitive and/or behavioural pressures, without affecting the main midbrain connectivity output, thus unveiling a key conserved role of this connectivity trait in the function of the circuit. We propose that ontogenic plasticity in habenular morphogenesis underlies the observed inter-species variations in habenular asymmetric morphology.

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Although progress has been made in resolving the genetic pathways that specify neuronal asymmetries in the brain, little is known about genes that mediate the development of structural asymmetries between neurons on left and right. In this study, we identify daam1a as an asymmetric component of the signalling pathways leading to asymmetric morphogenesis of the habenulae in zebrafish. Daam1a is a member of the Formin family of actin-binding proteins and the extent of Daam1a expression in habenular neuron dendrites mirrors the asymmetric growth of habenular neuropil between left and right. Local loss and gain of Daam1a function affects neither cell number nor subtype organisation but leads to a decrease or increase of neuropil, respectively. Daam1a therefore plays a key role in the asymmetric growth of habenular neuropil downstream of the pathways that specify asymmetric cellular domains in the habenulae. In addition, Daam1a mediates the development of habenular efferent connectivity as local loss and gain of Daam1a function impairs or enhances, respectively, the growth of habenular neuron terminals in the interpeduncular nucleus. Abrogation of Daam1a disrupts the growth of both dendritic and axonal processes and results in disorganised filamentous actin and α-tubulin. Our results indicate that Daam1a plays a key role in asymmetric habenular morphogenesis mediating the growth of dendritic and axonal processes in dorsal habenular neurons.