Regulation of Synaptic Structure and Function by Drugs of Abuse

Nairn laboratory projects carried out with support of the Yale/NIDA Neuroproteomics Center are briefly described below. Some of the recent work supported by the Discovery Proteomics, Targeted Proteomics, and Bioinformatics and Biostatistics Cores is briefly described.

1) ARPP-16 is a striatal-enriched inhibitor of protein phosphatase 2A regulated by MAST3 kinase (Musante et al., 2017). We continue to work on the function of ARPP-16, a small acid-soluble protein highly expressed in medium spiny neurons (MSNs) of striatum. This work continues to be carried out in collaboration with Center Investigator Jane Taylor. Ongoing work, supported by the Discovery and Targeted Proteomics Cores, is aimed at using the BioID proximity biotinylation approach to assess the PP2A interactome and the signaling processes it regulates in striatal MSNs.

2) Striatin-1 is a B subunit of protein phosphatase PP2A that we have found regulates dendritic arborization and spine development in striatal neurons (see Li et al., 2018). As a member of the striatin family of B subunits, striatin-1 is a core component together with PP2A of a multi-protein complex called STRIPAK, the striatin-interacting phosphatase and kinase complex. With the support of the Discovery Core, we used LC-MS/MS to identify proteins from striatum and striatal neurons in culture that include PP2A and the STRIPAK complex. We followed up from these studies by investigating the role of striatin-1 in neuronal maturation. Reduced expression of striatin-1 resulted in increased dendritic complexity and an increased number of dendritic spines, classified as stubby spines. Reduction of striatin-1 did not result in deficits in neuronal connectivity, as we observed no abnormalities in synapse formation or in spontaneous excitatory postsynaptic currents, with the latter work being done in collaboration with Center Investigator, Dr. Marina Picciotto. Together these results suggest that striatin-1 is a regulator of medium spiny neuron development in the striatum.

3) Cell-type specific psychostimulant effects on the neuronal translatome (R21 DA040454; Carlyle et al., 2018). We were funded by NIDA for an R21 grant that proposed to develop biochemical methods for the purification and quantitative profiling of ribosome-affiliated RNA footprints and nascent polypeptides from specific neuronal cell types, combined with integrated methods of data analysis, to characterize all stages of control over the translatome. By integrating mRNA-seq and its ability to reliably quantify isoforms, with ribosome profiling and LC-MS/MS, a more complete understanding of gene regulation at the isoform level can be obtained. As part of the data analysis related to this study, we have been supported by the Bioinformatics Core who have developed an approach termed EMPire to integrate mRNA, ribosomal footprint and proteomic data. Specifically, an expectation maximization algorithm was designed to relate mRNA transcript abundance to protein isoforms from LC-MS/MS, and this was extended to allow analysis of ribosomal footprinting results (see Carlyle et al., 2018). In this approach, we leveraged the principle that most cell types, and even tissues, predominantly express a single principal isoform to set isoform-level mRNA-seq quantifications as priors to guide and improve allocation of footprints or peptides to isoforms. Through tightly integrated mRNAseq, ribosome footprinting and/or LC-MS/MS proteomics we have found that a principal isoform can be identified in over 80% of gene products in homogenous HEK293 cell culture and over 70% of proteins detected in complex human brain tissue. Defining isoforms in experiments with matched RNA-seq and translatomic/proteomic data increases the functional relevance of such datasets and will further broaden our understanding of multi-level control of gene expression.

4) A targeted mass spectrometry-based approach for quantitation of proteins enriched in the postsynaptic density (PSD) (Wilson et al., 2019). The PSD is a structural, electron-dense region of excitatory glutamatergic synapses, which is involved in a variety of cellular and signaling processes in neurons. The PSD is comprised of a large network of proteins, many of which have been implicated in a wide variety of neuropsychiatric disorders. Biochemical fractionation combined with mass spectrometry analyses have enabled an in-depth understanding of the protein composition of the PSD. However, the PSD composition may change rapidly in response to stimuli; and robust and reproducible methods to thoroughly quantify changes in protein abundance are warranted. With the support of the Discovery and Targeted Proteomics Cores, we developed two types of targeted mass spectrometry-based assays for quantitation of PSD-enriched proteins. In total, we quantified 50 PSD proteins in a targeted, parallel reaction monitoring (PRM) assay using heavy-labeled, synthetic internal peptide standards; and were able to identify and quantify over 2,100 proteins through a pre-determined spectral library using a data independent acquisition (DIA) approach in PSD fractions isolated from mouse cortical brain tissue. This work was recently published (Wilson et al., 2019).

5) Collaboration with other Center Investigators (Bertholomey et al., 2018; Miller et al., 2018; Torregrossa et al., 2019). We continue to collaborate on a number of projects with the Taylor, Picciotto and Torregrossa labs.

Literature Cited

Bertholomey, M.L., Stone, K.L., Lam, T.T., Bang, S., Wu, W., Nairn, A.C., Taylor, J.R., Torregrossa, M.M. (2018) Phosphoproteomic analysis of the amygdala response to adolescent glucocorticoid exposure reveals G-protein coupled receptor kinase 2 (GRK2) as a target for reducing motivation for alcohol. Proteomes Special Issue on Neuroproteomics 6(4), 41 (PMCID: PMC6313880, PMID:30322021)

Carlyle, B.C., Kitchen, R.R., Zhang, J., Wilson, R., Lam, T.T., Rozowsky, J.S., Williams, K., Sestan, N., Gerstein, M.B., Nairn, A.C. (2018) Isoform level interpretation of high-throughput proteomic data enabled by deep integration with RNA-seq. J. Proteome Research 17(10), 3431-3444 (PMCID:PMC6392456, PMID: 30125121).

Li, D., Musante, V., Zhou, W., Picciotto, M.R., Nairn, A.C. (2018) Striatin-1 is a B subunit of protein phosphatase PP2A that regulates dendritic arborization and spine development in striatal neurons. J.Biol.Chem. 293(28):11179-11194 (PMCID: PMC6052221, PMID: 29802198).

Miller, M.B., Wilson, R.S., Lam, T.T., Nairn, A.C., Picciotto, M.R., (2018) Evaluation of the phosphoproteome of mouse alpha 4/beta 2-containing nicotinic acetylcholine receptors in vitro and in vivo. Proteomes Special Issue on Neuroproteomics 6(4), 42 (PMCID: PMC6313896, PMID: 30326594).

Musante, V., Li L., Kanyo, J., Lam, T.T., Colangelo, C.M., Cheng, S.K., Brody, H., Greengard, P., Le Novère N., Nairn, A.C. (2017) Reciprocal regulation of ARPP-16 by PKA and MAST3 kinases provides a cAMP-regulated switch in protein phosphatase 2A inhibition. eLife 2017 June 14;6. pii: e24998 (PMCID:PMC5515580, PMID:28613156).

Torregrossa, M., MacDonald, M., Stone, K.L., Lam, T.T., Nairn, A.C., Taylor, J.R. (2019) Phosphoproteomic analysis of cocaine memory extinction and reconsolidation in the nucleus accumbens, Psychopharmacology Epub ahead of print on Nov. 8, 2018 (PMCID:PMC6374162, PMID: 30411139).

Wilson, R. Rauniyar, N., Sakaue, F., Lam, T., Williams, K., Nairn, A. (2019) Development of targeted mass spectrometry-based approaches for quantitation of proteins enriched in the postsynaptic density (PSD), Proteomes Special Issue on Neuroproteomics, 7(2) (PMCID:PMC6630806, PMID:30986977).