The role of protein phosphatase-1 inhibitor-2 in synaptic scaling

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Brain neurons can sense the activity level of neuron networks. If the activity levels are too low or too high, the neurons can adjust the communication level between the neurons. The way that this is accomplished is not completely understood. In previous work, the researcher found one interesting way that involves a protein called protein phosphatase-1 (PP1) that is regulated by another protein called inhibitor-2 (I-2). In this project, the researcher will investigate the role of these two proteins in regulating neuron communications. This work will lay the foundation for future studies in animals to understand how the brain adapts to visual experience or deprivation. Adaptation to environmental light is important for daily life, so these studies will have broad impact on understanding vision in other animals, including humans. The project will provide opportunities for a postdoctoral fellow, undergraduate, and high school students, including underrepresented minorities and women, to be trained in research in this EPSCoR state. Studies will focus on the function of an abundant enzyme, protein phosphatase-1 (PP1), and its regulator, inhibitor-2 (I-2), in bi-directional synaptic scaling. The study will also determine how I-2 regulates PP1 function in the dephosphorylation of serine 295 (Ser295) on postsynaptic density protein 95 kilo Dalton (PSD95), a molecule critical for synapse architecture and neuronal communication. The impact of I-2 regulation by myosin light chain kinases (MLCK) via I-2 phosphorylation at serine 43 (Ser43) will be determined in the context of both PP1 function on PSD95 dephosphorylation at Ser295 and synaptic scaling. Using primary rat cortical neurons as a model system, the PI will apply bicuculline to enhance neuronal activity or tetrodotoxin (TTX) to decrease neuronal activity. I-2 will be knocked-down (KD) by expression of I-2 short hairpin RNA fragment RNA (ShRNA), and its effect on PSD95 phosphorylation at Ser295 will be examined by western blotting. The effect of I-2 KD on synaptic transmission will be examined by electrophysiological recording. The identity of the MLCK isoform responsible for I-2 phosphorylation at Ser43 will be determined by examining the effect of ShRNAs of various MLCK isoforms on I-2pS43 by western blotting. MLCK isoform effects on bi-directional synaptic scaling will be determined using electrophysiological recordings of neurons expressing the corresponding ShRNA.