The daunting complexity of the brain emerges from the large number of neurons it contains and their compartmentalized synaptic interactions at axon terminals and dendrites. Generation of functional neuronal networks requires robust, unambiguous developmental processes to ensure synapse-specific neuronal partner choice and subsequent maintenance mechanisms to keep neurons and particularly synapses healthy and functional over a long time. Defects in wiring and maintenance mechanisms are associated with neuropsychiatric and neurodegenerative disorders. Having regard to the importance of protein quality control mechanism both during development and function of the nervous system, in this doctoral work, I investigated possible local roles of lysosomal degradation pathways including ubiquitous and neuron-specific endolysosomal degradation and autophagy at axon terminals. Using live imaging in intact Drosophila brains and novel acidification-sensing degradation probes, first, we reported a direct live observation of local protein degradation at axon terminals in large, acidified compartments. These acidic, degradative endocytic compartments undergo continuous flux of fusion and fission of smaller compartments that is reflected by their molecular composition at a given time. Therefore, we named these compartments ‘local hubs’ as they behave as sort-and-degrade stations for local protein turnover at axon terminals. Secondly, we reported differential, cargo-specific sorting of plasma and synaptic vesicle membrane proteins into distinct hubs via two molecularly distinct pathways. Although plasma membrane protein sorting and degradation depends on ubiquitous Rab GTPase, Rab7, synaptic vesicle membrane protein sorting and degradation is Rab7-independent and operated by previously characterized synaptic vesicle proteins V100 and n-Syb. V100, as a subunit of a proton pump, particularly affects acidification of synaptic vesicles hubs, whereas n-Syb is required for the delivery of golgi-derived microvesicles containing acidic hydrolases into synaptic vesicle hubs. Interestingly, autophagy does not overlap with any of these local degradation pathways. Following their formation at axon terminals, they enter in axons without engaging in any fusion/fission events, hence morphologically and dynamically distinct from local hub compartments. Despite several reports on formation of autophagosomes at axon terminals, potential physiological roles it may exert still remain largely unknown, especially during neural circuit assembly. Live imaging of developing Drosophila photoreceptor axon terminals with autophagosome markers revealed their formation at the tip of synaptogenic filopodia followed by destabilization of these structures. Consistent with this observation, loss of function analyses of autophagy in developing Drosophila photoreceptors revealed increased stability of synaptogenic filopodia and subsequent increase in synapse numbers. More importantly, autophagy-deficient neurons connect to several aberrant synaptic partners causing neuronal miswiring. Finally, adult flies with miswired brains due to loss of autophagy show distinct and predictable behavioral phenotypes such as prolonged, repetitive visual attention to objects. Interestingly, development at colder temperatures exerts similar effect on filopodial stability as in loss of autophagy where axonal filopodia slow down and stabilize more synaptogenic filopodia. This effect on filopodia stability further leads to increased synapse formation and recruitment of aberrant synaptic partners changing brain wiring pattern. Collectively, these results demonstrate that filopodia kinetics play an important role to restrict or facilitate synaptic partnerships between neurons in close proximity during brain wiring. In conclusion, my doctoral work contributed to better understanding of local functions of protein degradation machineries and developmental temperature during brain wiring and maintenance. Unexpected roles of such cellular mechanisms and external factors in establishing proper neuronal circuits point to the fact that combinatorial action of several factors in time and space during brain development contribute to the final outcome, a functional brain.
