Understanding tubulin availability and regulation during neuronal development

Axons are the up-to-meter long cellular processes of neurons that form the biological cables wiring nervous systems. Axons must be sustained for an organism's lifetime which makes them key lesion sites during healthy ageing, in injury and neurodegenerative diseases. Axon degeneration is considered as the cause rather than consequence for neuron decay in the context of various neurodegenerative diseases. Parallel bundles of microtubules (MTs) are the structural backbones of axons which provide the highways for life-sustaining long-distance transport between cell bodies and the distal growth cones or synaptic endings. MTs are polymers made of alpha/beta-tubulin heterodimers. Little is known about how tubulin heterodimers are made available in axons, i.e., large distances away from the transcriptional machinery in the cell body. The aim of my Ph.D. project was to explore the possibility that tubulin can be provided in axons through local biogenesis, i.e., local translation and chaperone-mediated folding. Using single molecule in situ hybridisation approaches in Drosophila primary neurons, I found alpha/beta-tubulin mRNA is present in axons, which were depleted upon deficiency of tubulin genes and significantly reduced upon loss of kinesin-1 and/or -3 function, suggesting involvement of active mRNA transport. I found axonal localisation of ribosomal proteins, P-body components and detected active axonal translation using puromycin assays. Furthermore, I could show that at least two of the necessary chaperone machineries (prefoldin, CCT, TBC) are present in axons. Of these, TBC is the most specific for tubulin processing, presenting the last step in the folding process. Using systematic knockdown of TBCA-E, I tested how each TBC subunit impacts axon length, MT polymerisation (assessed via Eb1), the presence of mRNA in axons and protein levels of alpha/beta-tubulin (via Western blots). The results revealed differential effects when knocking down individual subunits. This suggests that the complex does not act in an all-or-nothing fashion, but that there are either different maturation stages of tubulins that cause different phenotypic outcomes, or that individual subunits have additional roles outside the complex.