Enrico Moro

Most of the patients affected by neuronopathic forms of Mucopolysaccharidosis type II (MPS II), a rare lysosomal storage disorder caused by defects in iduronate-2-sulfatase (IDS) activity, exhibit early neurological defects associated with white matter lesions and progressive behavioural abnormalities. While neuronal degeneration has been largely described in experimental models and human patients, more subtle neuronal pathogenic defects remain still underexplored. In this work, we discovered that the axon guidance receptor Deleted in Colorectal Cancer (Dcc) is significantly dysregulated in the brain of ids mutant zebrafish since embryonic stages. In addition, thanks to the establishment of neuronal-enriched primary cell cultures, we identified defective proteasomal degradation as one of the main pathways underlying Dcc upregulation in ids mutant conditions. Furthermore, ids mutant fish-derived primary neurons displayed higher levels of polyubiquitinated proteins and P62, suggesting a wider defect in protein degradation. Finally, we show that ids mutant larvae display an atypical response to anxiety-inducing stimuli, hence mimicking one of the characteristic features of MPS II patients. Our study provides an additional relevant frame to MPS II pathogenesis, supporting the concept that multiple developmental defects concur with early childhood behavioural abnormalities.

A considerable effort has been spent in the past decades to develop targeted therapies for the treatment of demyelinating diseases, such as multiple sclerosis (MS). Among drugs with free radical scavenging activity and oligodendrocyte protecting effects, Edaravone (Radicava) has recently received increasing attention because of being able to enhance remyelination in experimental in vitro and in vivo disease models. While its beneficial effects are greatly supported by experimental evidence, there is a current paucity of information regarding its mechanism of action and main molecular targets. By using high-throughput RNA-seq and biochemical experiments in murine oligodendrocyte progenitors and SH-SY5Y neuroblastoma cells combined with molecular docking and molecular dynamics simulation, we here provide evidence that Edaravone triggers the activation of aryl hydrocarbon receptor (AHR) signaling by eliciting AHR nuclear translocation and the transcriptional-mediated induction of key cytoprotective gene expression. We also show that an Edaravone-dependent AHR signaling transduction occurs in the zebrafish experimental model, associated with a downstream upregulation of the NRF2 signaling pathway. We finally demonstrate that its rapid cytoprotective and antioxidant actions boost increased expression of the promyelinating Olig2 protein as well as of an Olig2:GFP transgene in vivo. We therefore shed light on a still undescribed potential mechanism of action for this drug, providing further support to its therapeutic potential in the context of debilitating demyelinating conditions.

Most patients affected by neuronopathic forms of Mucopolysaccharidosis type II (MPS II), a rare lysosomal storage disorder, exhibit early neurological defects associated with white matter lesions and progressive behavioural abnormalities. While neuronal degeneration has been largely described in experimental models and human patients, more subtle neuronal pathogenic defects remains still underexplored. In this work we discovered that the axon guidance receptor Deleted in Colorectal Cancer (Dcc) is significantly dysregulated in the brain ofidsmutant zebrafish since embryonic stages. In addition, thanks to the establishment of neuronal-enriched primary cell cultures from embryonic dissected brains, we identified defective proteasomal degradation as one of the main pathways underlying Dcc upregulation inidsmutant conditions. Furthermore,idsmutant fish-derived primary neurons displayed higher levels of polyubiquitinated proteins and P62, suggesting a wider defect in protein degradation. Finally, we show thatidslarvae display atypical response to anxiety-like stimuli, hence mimicking one of the characteristic features of MPS II patients. Our study provides an additional relevant frame to MPS II pathogenesis, supporting the concept that multiple developmental defects concur to early childhood behavioural abnormalities.

Huntington’s disease (HD) is a neurodegenerative disorder caused by CAG-repeat expansions in the huntingtin (HTT) gene. The resulting mutant HTT (mHTT) protein induces toxicity and cell death via multiple mechanisms and no effective therapy is available. Here, we employ a genome-wide screening in pluripotent mouse embryonic stem cells (ESCs) to identify suppressors of mHTT toxicity. Among the identified suppressors, linked to HD-associated processes, we focus on Metal response element binding transcription factor 1 (Mtf1). Forced expression of Mtf1 counteracts cell death and oxidative stress caused by mHTT in mouse ESCs and in human neuronal precursor cells. In zebrafish, Mtf1 reduces malformations and apoptosis induced by mHTT. In R6/2 mice, Mtf1 ablates motor defects and reduces mHTT aggregates and oxidative stress. Our screening strategy enables a quick in vitro identification of promising suppressor genes and their validation in vivo, and it can be applied to other monogenic diseases.

Multiple complex intracellular cascades contributing to Hunter syndrome (mucopolysaccharidosis type II) pathogenesis have been recognized and documented in the past years. However, the hierarchy of early cellular abnormalities leading to irreversible neuronal damage is far from being completely understood. To tackle this issue, we have generated two novel iduronate-2-sulfatase (IDS) loss of function human neuronal cell lines by means of genome editing. We show that both neuronal cell lines exhibit no enzymatic activity and increased GAG storage despite a completely different genotype. At a cellular level, they display reduced differentiation, significantly decreased LAMP1 and RAB7 protein levels, impaired lysosomal acidification and increased lipid storage. Moreover, one of the two clones is characterized by a marked decrease of the autophagic marker p62, while none of the two mutants exhibit marked oxidative stress and mitochondrial morphological changes. Based on our preliminary findings, we hypothesize that neuronal differentiation might be significantly affected by IDS functional impairment.

Among multiple cytoprotective mechanisms, eukaryotic cells exhibit a complex transcriptional program relying on the Nrf2 transcription factor, which is generally recruited upon biological stressors including oxidative-stress-based cellular insults. The relevance of this master regulator has remarkably emerged in recent years in several research fields such as cancer, inflammatory disorders and age-related neurological diseases. Here, we document the generation and characterization of a novel Nrf2/ARE pathway biosensor fish which exhibits a dynamic spatiotemporal expression profile during the early developmental stages. The transgenic line is responsive to known Nrf2 pathway modulators but also to Edaravone, which direct activity on the Nrf2 pathway has never been documented in a live transgenic fish model. We also show that the reporter is faithfully activated during fin regeneration, and its degree of expression is slightly affected in a glucocerebrosidase (Gba1) morphant zebrafish model. Therefore, this novel transgenic fish may represent a valuable tool to be exploited for the characterization of zebrafish models of human diseases, as well as for primary high-throughput drug screening.

Lysosomal storage disorders (LSDs) are a group of 60 rare inherited diseases characterized by a heterogeneous spectrum of clinical symptoms, ranging from severe intellectual disabilities, cardiac abnormalities, visceromegaly, and bone deformities to slowly progressive muscle weakness, respiratory insufficiency, eye defects (corneal clouding and retinal degeneration), and skin alterations

Axonal trajectories and neural circuit activities strongly rely on a complex system of molecular cues that finely orchestrate the patterning of neural commissures. Several of these axon guidance molecules undergo continuous recycling during brain development, according to incompletely understood intracellular mechanisms, that in part rely on endocytic and autophagic cascades. Based on their pivotal role in both pathways, lysosomes are emerging as a key hub in the sophisticated regulation of axonal guidance cue delivery, localization, and function. In this review, we will attempt to collect some of the most relevant research on the tight connection between lysosomal function and axon guidance regulation, providing some proof of concepts that may be helpful to understanding the relation between lysosomal storage disorders and neurodegenerative diseases.

Precise temporal coordination of signaling processes is pivotal for cellular differentiation during embryonic development. A vast number of secreted molecules are produced and released by cells and tissues, and travel in the extracellular space. Whether they induce a signaling pathway and instruct cell fate, however, depends on a complex network of regulatory mechanisms, which are often not well understood. The conserved bilateral left-right asymmetrically formed habenulae of the zebrafish are an excellent model for investigating how signaling control facilitates the generation of defined neuronal populations. Wnt signaling is required for habenular neuron type specification, asymmetry and axonal connectivity. The temporal regulation of this pathway and the players involved have, however, remained unclear. We find that tightly regulated temporal restriction of Wnt signaling activity in habenular precursor cells is crucial for the diversity and asymmetry of habenular neuron populations. We suggest a feedback mechanism whereby the tumor suppressor Wnt inhibitory factor Wif1 controls the Wnt dynamics in the environment of habenular precursor cells. This mechanism might be common to other cell types, including tumor cells.