Endoplasmic Reticulum Protein Wolfram Syndrome 1b Deficiency Suppresses Mauthner-cell Axon Regeneration

Wolfram syndrome is a rare autosomal recessive disease characterized by childhood-onset diabetes mellitus and optic nerve atrophy,as well as a neurodegenerative disease.Its neurological symptoms include but are not limited to visual field defects,cerebellar ataxia,epilepsy,anxiety,and cognitive impairment.Neuroimaging results showed that a proportion of patients developed whole-brain atrophy,especially in the cerebellum,medulla,and pons.These patients died in middle age due to respiratory failure caused by whole-brain atrophy,which brought tremendous physical and psychological harm.Currently,Wolfram syndrome is considered to be caused mainly by mutations in the gene WFS1,which encodes the endoplasmic reticulum(ER)transmembrane protein Wolframin(WSF1).The WFS1 protein is abundant in the pancreas,and it has been shown that the WFS1 protein plays a crucial role in glucose metabolism.WFS1 protein is also abundant in the brain,especially in the hippocampus,hypothalamus,cerebellum,and brainstem.It is worth mentioning that the WFS1 protein might be a novel regulator of Alzheimer’s disease.As a neurodegenerative disease,Wolfram syndrome might lead to axonal degeneration,which is a common feature between neurodegenerative disease and spinal cord injury(SCI).Axonal degeneration is not only a target for treatment of neurodegenerative diseases but also a breakthrough for repair of SCI.Therefore,it is crucial to investigate how to alleviate axonal degeneration and promote axonal regeneration.However,the role of the WFS1 protein in axonal regeneration in the central nervous system(CNS)is unknown.Zebrafish is an ideal in vivo model for studying the CNS diseases and shares nearly 80%homology with human genes.Axons in the adult mammalian CNS show an inability to regenerate,whereas Mauthner-cells are a pair of motor neurons in the zebrafish brainstem whose axons show strong regenerative capacity,making zebrafish an ideal model for studying SCI.There are two isoforms,wfs1a and wfs1b,within the zebrafish genome that are homologous to the human gene WFS1,the only one copy in human genome.Therefore,we established a model of spinal cord injury in zebrafish to investigate the impact of the gene wfs1b,which has higher conservation to gene WFS1,on Mauthner cell axon regeneration,allowing us to better understand the pathological mechanisms of Wolfram syndrome and providing potential therapeutic targets for patients.In our study,in situ hybridization results showed that both wfs1a and wfs1b are expressed in the CNS of zebrafish at the early stage.Subsequently,the phylogenetic tree results showed that wfs1b has higher evolutionary conservation with the human gene WFS1.Therefore,we established a model of the wfs1b globally deficient zebrafish line using gene editing to explore the effect of wfslb knockout on Mauthner cell axon regeneration in the zebrafish.It was found that wfs1b knockout not only reduced the axonal regeneration ability of Mauthner cells,but also inhibited the functional recovery of Mauthner cells after axonal regeneration,and one of the reasons for this phenomenon was that wfs1b knockout caused an ER stress response.The wfs1b knockout resulted in an increase in the mRNA expression of the unfolded protein response(UPR)signaling pathway activated by the ER stress response at the molecular level,as well as a rupture of the ER ultrastructure at the morphological level.By using pharmacological methods,the application of 4-Phenylbutyric acid(4-PBA),an ER stress inhibitor,promoted the recovery of the axonal regeneration capacity of Mauthner cell in the wfs1b-/-zebrafish larvae.Administration of Tunicamycin(TM),an activator of ER stress,inhibited the axonal regeneration capacity of Mauthner cells in normal zebrafish larvae.Furthermore,retro-complementation of wfs1b at the single-cell level promoted the Mauthner cell axon regeneration in wfs1b-/-zebrafish larvae.In summary,our studies found that wfs1b knockout inhibited Mauthner cell axon regeneration through the ER stress signaling pathway by using gene editing,in situ hybridization,single-cell electroporation,in vivo imaging,two-photon axotomy,transmission electron microscopy,behavioral assays,and so on.Our findings provided new evidence for therapeutic targets in Wolfram syndrome and SCI.