Hereditary spastic paraplegia (HSP) is a group of currently incurable disorders caused by the degeneration of corticospinal upper motor neuron axons, which are among the longest in the body. Recent research suggests that a few unifying pathways could link many genetically distinct HSP subtypes. Investigating these pathways and the pathological mechanisms affecting them is crucial to advance the development of effective treatments for HSP and related neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease, multiple sclerosis, peripheral neuropathies and various motor neuron diseases.

This project focuses on endoplasmic reticulum (ER) proteins atlastins (ATLs), particularly ATL1, as ATL1 gene mutations cause the most frequent childhood-onset form of HSP. Using CRISPRi, I engineered ATL1 knock-down (KD) into the i³ human induced pluripotent stem cells (iPSCs) and utilised iPSC-derived neurons to investigate the roles of ATL1 in the ER and other cellular compartments and processes commonly affected in HSP. Preliminary findings showed fewer ER junctions in ATL1 KD neurons, consistent with known atlastin’s role in ER tubule fusion. Additionally, ER proteins were highly enriched in a protein list with altered abundance in ATL1 KD neurons determined through proteomics. Moreover, abnormalities in the endo-lysosomal system were identified in ATL1-depleted neurons. They had slightly fewer and larger lysosomes with reduced degradative capacity. Additionally, neuronal endosomal tubules were elongated, pointing towards a potential tubule fission defect which could lead to missorting of diverse cargo, such as lysosomal hydrolases. Lipidomics and neutral lipid staining revealed higher triglyceride content in neurons lacking ATL1. Moreover, blocking lipolysis degradation led to lipid droplet (LD) accumulation, with significantly more LDs in ATL1 KD neurons. The cellular content of other lipid classes was also altered, indicating potential lipid imbalance resulting from ATL1 loss. Finally, developing ATL1-depleted neurons exhibited altered morphology, with a significantly longer dominant neurite. Despite the detected phenotypes, the lack of ATL1 had no effect on neuronal viability.

These observations demonstrate that atlastin deficiency impacts cellular processes beyond its conventional role in ER shaping. In addition, the findings and tools developed in this study provide a foundation to investigate underlying mechanisms and pathways connecting the phenotypes resulting from ATL1 deficiency.