Abstract The disruption of the homeostasis in neurons often results in degeneration, an irreversible mechanism that still is not well understood. Neurodegeneration is often accompanied by lysosomal dysfunction, which is characterized by increased luminal pH levels, alterations to the proteolytic capacity, and disruption of the activity of lysosomal transmembrane proteins. Fibroblasts and induced neurons from the most severe variant (p.R126X) of a rare pediatric-onset neurodevelopmental disease called TBCK syndrome show a strong phenotype associated with lysosomal dysfunction; however, the mechanism by which the deficiency of the TBC1-containing kinase (TBCK) protein alters the effective clearance of senescent or damage proteins and organelles remains unclear. The p.R126X, also known as the “Boricua” mutation because it affects children from Puerto Rican ancestry, is a frameshift variant that causes the truncation of the TBCK protein. Proteomic studies performed in our lab showed that TBCK interacts with the c-Jun N-terminal kinase-interacting protein 4 (JIP4), and the deficiency of TBCK disrupts the protein levels of JIP4, which also correlate with more stationary lysosomes in axons from TBCK-deficient induced neurons (TBCK-deficient iNeu). Therefore, I hypothesize that TBCK deficiency impairs JIP4 stability leading to the disruption of the positioning, motility, and maturation of lysosomes. The first aim of this project will test the maturation and motility of lysosomes and the role effect of JIP4 disruption in lysosomal function. To test this, I will define the maturation state of lysosomes of TBCK-deficient iNeu using a pH-sensitive-LAMP1 reporter and confocal microscopy. Also, I will perform time-lapse live imaging analysis to assess the motility of lysosome in axons from TBCK-deficient iNeu, and I will disrupt the levels of JIP4 in control iNeu to observe whether lysosomes reflect the same phenotype as TBCK-deficient iNeu. The second aim will determine whether introducing a plasmid containing the TBCK sequence into TBCK-deficient iNeu restitutes JIP4 protein levels and restores the functionality of lysosomes. Also, I will use HEK cells to evaluate which domain of the TBCK protein, pseudo-kinase, TBC, and rhodanese, interacts with JIP4. This aim will reveal the role of TBCK in the modulation of JIP4 protein levels and the domain involved on this novel interaction. Overall, this project will define the relevance of the novel interaction between TBCK and JIP4 and how the disruption of the levels of JIP4 is implicated in the maturation and motility of lysosomes.