Investigations into novel functions of JNK in neurons: Regulation of post-synaptic scaffold proteins by JNK3

• Applicant: Professorin Dr. Sarah A. Shoichet
• Subject Area: Developmental Neurobiology ; Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2014 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 261102178

In our previously published work, we have detected de novo truncations of the MAPK10 gene, encoding the kinase JNK3, in patients with cognitive disorders. We demonstrated that the truncated proteins lack normal catalytic activity, which supports our hypothesis that loss of normal JNK3 contributes to the disease phenotype in the patients, and provides the basis for our studies on novel functions of JNK3 in neurons, which we describe in detail in this proposal. In summary, we have shown that several disease-associated synaptic scaffold proteins, including PSD-95 and SAP102, as well as Shank- and CRMP-family proteins, are able to interact with JNK3. Moreover, we have identified a novel JNK phosphorylation site in SAP102 and generated an antibody that specifically recognises the phosphorylated form. We will use this antibody, together with phospho-specific antibodies for PSD-95, to assess the status of endogenous phosphorylated SAP102 and PSD-95 in neurons under different conditions. We will also further delineate the nature of the JNK-PSD-95/SAP102 interaction, and investigate how JNK influences the scaffolding properties of these molecules. We additionally hypothesise that phosphorylation of both of these synapse-associated proteins by JNK is critical for their subcellular localisation, potentially in response to synaptic activity. Given the location of JNK binding and phosphorylation of PSD-95 and SAP102, specific protein-protein interactions and subsequent signalling may also be affected by these events. We will investigate how JNK phosphorylation of SAP102 influences its binding to selected neuronal proteins, including e.g. Nedd4, which has been implicated in SAP102 mono-ubiquitination. Using photo-activation and photo-bleaching strategies, we will also assess the impact of JNK regulation on the mobility of this scaffold molecule into and out of dendritic spines. We will further assess the effects of JNK regulation on AMPA receptor surface expression and on AMPAR- and NMDAR-mediated currents. For all of these experiments, we will take advantage of virus-mediated gene delivery for expression of proteins in primary neurons, and we will make use of commercially available small molecule and peptide inhibitors. We will also make use of our tagged JIP1-JBD small protein inhibitor, which likewise inhibits total JNK in cell culture experiments. In the long-term, this tagged protein also can be used to generate a mouse model in which total JNK activity can be inhibited in an inducible manner, thereby establishing a system that would enable investigations into JNK regulation of synaptic scaffold molecules in vivo.

• DFG Programme: Research Grants
• Participating Institution: Charité - Universitätsmedizin Berlin; Campus Charité Mitte; Neurowissenschaftliches Forschungszentrum