Deciphering the role of BMP signalling pathway in Hereditary Spastic Paraplegia: identification of a novel therapeutic approach in zebrafish knockout models for atlastin-1 (spg3a) and spastin (spg4)

Hereditary Spastic Paraplegias (HSPs) are a miscellaneous group of uncommon and not curable neurologic disorders characterised by length-dependent axonopathy of motor neurons and associated with a spastic gait. Thanks to next-generation sequencing (NGS) and whole genome sequencing (WGS) technologies, more than eighty spastic paraplegia genes (SPGs) have been identified so far, and their protein products seem to lead to the disruption of many cellular activities such as axonal transport and development, membrane trafficking, lipid metabolism, and oxidative stress. Moreover, the loss of many SPGs have been associated with the upregulation of the Bone Morphogenetic Protein (BMP) signalling which might be considered e pluribus unum in the development of many forms of Spastic Paraplegias as well as a novel therapeutic strategy. Unfortunately, the well-known BMP inhibitors (Dorsomorphin and LDN-193189) resulted toxic and failed clinical trials. Nowadays, zebrafish represents an attractive model for studying human genetic diseases and a very powerful model for high-throughput drug screening (HTS) to assess both efficacy and toxicity. Drug development for neurological diseases is very challenging and to overcome the high failure rate of novel candidates, drug repurposing is a strategic choice. We generated two zebrafish knockout (KO) models for atlastin-1 (atl-1/spg3a) and spastin (spast/spg4) by CRISPR/Cas9 technology to dissect the molecular mechanisms of HSPs. Atl-1 KO zebrafish grow at the same rate as wild-type (WT) animals, but KO embryos have hyperbranched and thicker motor neurons, impairment of locomotion at larval stage, and show loss of skin pigmentation during adulthood. Unlike literature data, our atl-1 KO animal did not show an alteration of the BMP activity. Spast KO zebrafish, instead, showed a reduced body length and hatching rate, together with a defective tubulin acetilation in distal axons, but no changes were recorded in larval locomotor activity. Moreover, spast KO also showed a major sensitivity to ER stress and an hyperactivation of the of the BMP pathway. Surprisingly, these two events result co-dependent, and the BMP seemed to trigger the ER stress. The HTS performed on the Prestwick chemical library led to the identification of the nalidixic acid as an efficient inhibitor of the BMP activity in Tg(BMPRE:EGFP)ia18 BMP zebrafish reporter line. Interestingly, this novel molecule was also able to rescue the ER stress phenotype of spast KO zebrafish as well as LDN-193189 BMP inhibitor. These results indicate that both atl-1 and spast KO zebrafish lines are two valid tools for translational medicine, the BMP pathway has a driving role toward ER stress event, and that the nalidixic acid might represent a safe novel modulator of the BMP signalling able to ameliorate the ER stress phenotype observed in spg4 HSP.