The hereditary spastic paraplegias (HSP) are a group of over 80 neurodegenerative conditions and the most common cause of inherited spasticity and associated disability. This K08 proposal focuses on prototypical forms of HSP in children caused by biallelic loss-of-function variants in four genes that encode subunits of the adaptor protein complex 4 (AP-4): AP4B1, AP4M1, AP4E1, and AP4S1. Progressive degeneration of the cortico-spinal tracts renders most children with AP-4-associated HSP wheelchair-dependent by the age of 10 years. Currently, there are no therapies that halt disease progression, and few patients are known to have survived into adulthood, highlighting the urgency for research into the fundamental biology of HSP. The AP-4 complex is crucial for the intracellular trafficking of transmembrane proteins, including the autophagy-related protein ATG9A. How altered trafficking of ATG9A leads to impaired neurodevelopment and axonal degeneration and how ATG9A distribution can be restored is currently unknown. In this proposal, I will address this unmet question by developing neuronal models of AP-4 deficiency and testing novel modulators of AP-4-dependent protein trafficking. In preliminary experiments, we have systematically screened small molecule modulators of ATG9A trafficking using a cell-based phenotypic assay that measures ATG9A distribution as a surrogate of AP-4 function. We identified several modulators of ATG9A distribution. I will test the hypothesis that these restore trafficking and function of ATG9A in vitro in neurons derived from AP-4-HSP patients and in vivo in an ap4b1-/- zebrafish model. This proposal presents a five-year research career development program focused on the study of AP-4 in HSP to expand the breadth and depth of understanding the role of protein trafficking and autophagy in this group of diseases. The goal is the establishment of a cross-organismal screening platform to identify and develop novel modulators of protein trafficking for the treatment of HSP. The candidate is currently a resident in Child Neurology at the Department of Neurology at Boston Children's Hospital and Harvard Medical School. The outlined proposal builds on the candidate's previous research on protein trafficking, autophagy and neurodegeneration and integrates new domains of expertise in cell biology, advanced microscopy, and iPSC-derived neurons and genetically-engineered zebrafish to model human diseases. These skills are reflected in his mentoring team consisting of primary mentor, Dr. Mustafa Sahin, and a scientific advisory committee consisting of Dr. Craig Blackstone, Dr. Thomas Schwarz, Dr. Annapurna Poduri and Dr. Leonard Zon. The proposed experiments and didactic work will position the candidate with a unique set of cross-disciplinary skills that will enable his transition to independence as a physician-scientist in the field of translational neuroscience in childhood-onset neurological diseases.