Vesicle trafficking characteristics of Axon-carrying Dendrite neurons and axon initial segment plasticity

Abstract

Generally, mammalian neurons have a single axon and multiple branched dendrites emerging from the cell body. Notably, in Axon-carrying Dendrite (AcD) neurons the axon emerges from a dendrite instead of the soma. Electrophysiological studies have shown that AcD neurons can generate action potentials (APs) with lower activation thresholds when receiving their signal in their AcD, and that they fire more often during ripples: oscillatory patterns involved in memory consolidation. However, more research on the cellular biology of these neurons needs to be done. In this research I investigated the cellular biology of AcD neurons and the trafficking characteristics of vesicles passing through their axon initial segment (AIS) region. I showed that AcD neurons can develop in dissociated primary rat hippocampal cultures independent of the in vivo environment. Moreover, I found that spatial and temporal localisation of the AIS-specific proteins Ankyrin-G (AnkG) and Tripartite motif-containing protein 46 (TRIM46) to the AIS of AcD neurons during neuronal development is similar to nonAcD neurons. Earlier research found that pre-synaptic vesicles associated with Ras associated binding protein 3A (Rab3A) travel slightly faster over the AIS into the axon of AcD neurons then in nonAcD neurons. I investigated whether this faster transport is specific for Rab3A-associated pre-synaptic vesicles, by using two additional vesicles markers: Neuropeptide Y (NPY) and Lysosome-associated membrane protein 1 (LAMP1). I found no difference for the trafficking speed over the AIS between AcD neurons and nonAcD neurons, suggesting that the faster trafficking of Rab3A-associated vesicles in AcD neurons might indeed be specific for pre-synaptic vesicles. Lastly, I investigated the effect of AIS plasticity, the structurally remodelling of the AIS as a response to changes in neuronal activity, on the AIS-specific scaffolding protein AnkG and microtubule bundler TRIM46. Earlier studies have reported contradicting results on the effect of AIS plasticity on the distribution of AnkG. Moreover, studies investigating the effect on TRIM46, a critical microtubule organiser in the AIS, are lacking. In this study I used treatment with N-methyl-D-aspartate (NMDA) to investigate the effect of short-term AIS plasticity on these two important AIS components. I found that NMDA treatment did not result in changes of AnkG or TRIM46 distribution in the AIS, suggesting that neurons do not undergo rapid activity-depended remodelling of the AnkG-associated neuronal membrane or the TRIM46-associated microtubule bundles in the AIS. Moreover, I looked at the TRIM46 turnover rate in the AIS in neurons at basal neuronal activity and after treatment with NMDA. Preliminary data suggest that short-term AIS plasticity results in a higher TRIM46 turnover rate in the AIS. With this research I contributed to the unravelling of the cellular biology of AcD neurons and the vesicle trafficking characteristics in their AIS. Moreover, this research provided insight into the effect of short-term AIS plasticity on the molecular organisation of the AIS.