Spatio-temporal control of neurotrophin trafficking and signalling in primary neurons cultured inside microfluidic chambers
Creators: name: Menendez, Guillermo
Publisher: name: Imperial College London
Year: 2010

description: My PhD project was aimed at applying microfluidics technology to the study of longrange neurotrophin signalling. Neurotrophins are target-derived growth and survival factors that, among other functions, prevent innervating neurons from undergoing apoptosis. Neurotrophins and their receptors have been shown to be transported retrogradely in axons of spinal cord neurons, following a pathway they share with the tetanus neurotoxin binding fragment (TeNT Hc), and which is controlled by the small GTPase Rab7. The Rab7-dependent pathway is thought to trigger downstream signalling events such as the phosphorylation of the transcription factor CREB, important in promoting neuronal survival and differentiation, but direct evidence for this had not yet been provided. To provide direct evidence of this functional relationship between Rab7 activity and CREB phosphorylation, I established microfluidic cultures of spinal cord motor and sensory neurons, in which axonal networks can be treated independently of cell bodies. I used a microfabrication technique known as soft lithography to produce microfluidic chambers. They consist of two parallel compartments interconnected by an array of microgrooves. In this culture system, dorsal root ganglia (DRG) neurons cultured in one of the compartments (somato-dendritic side) can be chemoattracted by gradients of nerve growth factor (NGF) to grow their axons preferentially into the other compartment (axonal side). Control studies by immunofluorescence confocal microscopy of CREB phosphorylation following direct stimulation of DRG cell bodies with NGF in mass cultures and in microfluidic chambers showed no significant differences between these two systems, confirming that the signalling cascade remains unmodified in microfluidic cell cultures. Time-course analysis of CREB phosphorylation in DRG neurons prepared from E18.5 embryos surprisingly revealed a lack of response following NGF stimulation of axon terminals in microfluidic cultures. I tried DRG cultures from E14.5 embryos because a fraction of the total population of DRG neurons during development undergo apoptosis at around E15-E16 if they fail to reach their target tissues. In these cultures, CREB phosphorylation could be observed when stimulating axons with NGF in microfluidic chambers. These results suggest that this long-range signalling pathway is active during a period of development when DRG neurons depend critically on their supply of targetderived neurotrophins, but it is down-regulated at later developmental stages. To gain some further insight into the mechanisms controlling this long range signalling response, and specifically to study the role of Rab7 in this context, I infected E14.5 DRG neurons with lentivirus carrying wild type or a dominant negative mutant of Rab7 (Rab7T22N) coupled to a fluorescent tag. Overexpression of mCherry Rab7T22N affected CREB phosphorylation, significantly reducing the signal generated in distal axons. To confirm this result, I prepared lentivirus carrying shRNA sequences targeting Rab7, and analysed the response to axonal NGF after knocking down the endogenous protein. This different approach also abolished CREB phosphorylation after NGF stimulation of the axonal network in microfluidic chambers. My results provide a direct link between Rab7 activity and downstream effects of the signalling cascade initiated by neurotrophins at axonal networks in compartmentalised microfluidic chambers.