Regulation of basal and activity-mediated AMPAR endocytosis by Protein Kinase D (PKD)

Abstract

AMPARs are one of the four different types of glutamate-gated ion channels in the mammalian brain, allowing influx of Na+ and efflux of K+ and thus depolarisation of the neuronal membrane. They are critical for correct brain functioning, as they represent the main mediators of excitatory synaptic transmission and as such are key for intercellular communication, brain development and, importantly, learning and memory. AMPARs are highly motile, undergoing constitutive and activity-mediated endocytosis, recycling and surface lateral diffusion among their different cellular pools. Notably, regulation of this trafficking is instrumental to mediate long-term potentiation (LTP) and long-term depression (LTD), cellular mechanisms of synaptic plasticity, which promote a long-lasting enhancement or decrease in synaptic strength, respectively, and are widely believed to be the main contributors to learning and memory. However, the mechanisms behind that regulation are not fully understood yet. The protein kinase D family of serine/threonine kinases consists of three isoforms in mammalian cells, and is activated downstream of DAG and PKC to participate in the regulation of processes such as vesicle fission from the trans-Golgi network and actin cytoskeleton remodelling. All three isoforms are expressed in neurons from an early embryonic stage, where they modulate tissue-specific processes such as the establishment and maintenance of neuronal polarity, neuroprotection against early oxidative stress and, importantly, synaptic plasticity through stabilisation of filamentous actin via cofilin inactivation during the expression of LTP and the phosphorylation of remaining surface NMDARs during the expression of LTD. Despite these observations, PKD has not been linked yet to AMPAR trafficking regulation. Therefore, in this thesis I aimed to elucidate whether PKD controls AMPAR trafficking in primary neuronal cells in basal and/or in activity-mediated conditions, and to shed light on the molecular mechanism that underlies this regulation. This work presents compelling evidence that PKD acts as a promoter of AMPAR endocytosis in primary hippocampal neurons in both basal and activity-mediated conditions. Short-term pharmacological inhibition of PKD via treatment with the small-molecule inhibitor CRT0066101 led to an increase in surface and synaptic AMPAR levels and slowed down AMPAR surface trafficking dynamics. Conversely, expression of a constitutively active PKD mutant promoted a decrease in AMPAR synaptic levels while increasing its localisation at early endosomes. Moreover, it was found that PKD activity is necessary for the decrease in surface AMPAR levels in response to both agonist- and NMDA treatments. Finally, I provide evidence that phosphorylation of the PKD substrate and Rab5 effector Rabaptin-5 at S407, the PKD phosphorylation site, is necessary for the correct regulation of basal AMPAR synaptic levels and for the endocytosis of AMPAR in response to NMDA treatment. Together, these findings identify PKD as a novel regulator of AMPAR endocytosis, presumably through the phosphorylation of Rabapatin-5 and subsequent Rab5 activation.