Assay of Rab17 and its guanine nucleotide exchange factor Rabex-5 in the dendrites of hippocampal neurons

Neurons are functionally and morphologically compartmentalized into axons and dendrites, and the localization of specifi c proteins within these compartments is critical to the proper formation of neuronal networks, which includes neurite morphogenesis and synapse formation. The small GTPase Rab17 is specifi cally localized in dendrites and is not found in axons, and it regulates the dendrite morphogenesis and postsynaptic development of mouse hippocampal neurons. However, the spatiotemporal regulation of Rab17 is poorly understood. We recently identifi ed Rabex-5, originally described as a Rab5-guanine nucleotide exchange factor (GEF), as a physiological Rab17-GEF that promotes translocation of Rab17 from the cell body to the dendrites of developing hippocampal neurons. Knockdown of Rab17 in mouse hippocampal neurons resulted in reductions in dendrite growth, branch numbers, fi lopodium density, and active synapse numbers. Knockdown of Rab17-GEF Rabex-5 in hippocampal neurons resulted in decreased targeting of Rab17 to the dendrites, which led to a reduction in dendrite growth. In this chapter we describe the assay procedures for analyzing Rab17 and Rabex-5 in cultured mouse hippocampal neurons, and we particularly focus on the measurement of total dendrite (or axon) length and total dendrite (or axon) branch numbers, fi lopodium density, number of active synapses, and dendritic Rab17 signals.