FIGURE 6:

AP-4 regulates neuronal lysosome composition, function, and transport. Schematic representation of the effect of AP-4 loss on neuronal lysosome trafficking and function. In AP-4 i³Neurons there are lysosomes in the soma that contain reduced levels of certain lysosomal enzymes, such as PPT-1 and Cathepsin L. Magnified insets of the TGN area in the soma of the neurons show molecular detail of cargo potentially regulated by AP-4 action. Upon loss of AP-4 complex function, concentration and transport of Sortilin out of the TGN is affected when compared with healthy Control i³Neurons. Thus strong Sortilin accumulation in the TGN area is observed in the AP-4 i³Neurons. ATG9A, previously well-established cargo whose sorting out of the TGN is dependent on AP-4 complex function, shows a similar strong accumulation in TGN area, validating these CRISPRi-based AP-4 i³Neurons as a model for studying neuronal membrane trafficking events regulated by AP-4 complex. Also, unlike healthy Control i³Neurons, where few LAMP1-positive vesicles are observed in axons, AP-4 loss causes axonal lysosomes (LAMP1-positive vesicles) to build up in dystrophies/swellings. Additionally, there is increased accumulation of JIP3/MAPK8IP3, a regulator of retrograde axonal lysosome transport, in these swellings. We propose that the accumulation of cytosolic JIP3 in the axonal swellings in AP-4 i³Neurons could also be due to absence/reduced levels of these interacting partners on lysosomes, whose sorting from TGN to the organelle is AP-4 dependent, as with ATG9A and potentially Sortilin.