crossmark Research © 2016 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC)*□S Farr Niere‡§¶ʈ§§, Sanjeev Namjoshi‡§ §§, Ehwang Song**, Geoffrey A. Dilly‡§¶, Grant Schoenhard‡‡, Boris V. Zemelman‡§¶, Yehia Mechref**, and Kimberly F. Raab-Graham‡§¶ʈ‡‡¶¶ Many biological processes involve the mechanistic/mam- ally alters the expression of proteins associated with malian target of rapamycin complex 1 (mTORC1). Thus, epilepsy, Alzheimer’s disease, and autism spectrum the challenge of deciphering mTORC1-mediated func- disorder—neurological disorders that exhibit elevated tions during normal and pathological states in the central mTORC1 activity. Through a protein–protein interaction nervous system is challenging. Because mTORC1 is at the network analysis, we have identified common proteins core of translation, we have investigated mTORC1 func- shared among these mTORC1-related diseases. One such tion in global and regional protein expression. Activation protein is Parkinson protein 7, which has been implicated of mTORC1 has been generally regarded to promote in Parkinson’s disease, yet not associated with epilepsy, translation. Few but recent works have shown that sup- Alzheimers disease, or autism spectrum disorder. To ver- pression of mTORC1 can also promote local protein syn- ify our finding, we provide evidence that the protein ex- thesis. Moreover, excessive mTORC1 activation during pression of Parkinson protein 7, including new protein diseased states represses basal and activity-induced pro- synthesis, is sensitive to mTORC1 inhibition. Using a tein synthesis. To determine the role of mTORC1 activa- mouse model of tuberous sclerosis complex, a disease tion in protein expression, we have used an unbiased, that displays both epilepsy and autism spectrum disorder large-scale proteomic approach. We provide evidence phenotypes and has overactive mTORC1 signaling, that a brief repression of mTORC1 activity in vivo by ra- we show that Parkinson protein 7 protein is elevated in pamycin has little effect globally, yet leads to a significant the dendrites and colocalizes with the postsynaptic remodeling of synaptic proteins, in particular those pro- marker postsynaptic density-95. Our work offers a com- teins that reside in the postsynaptic density. We have also prehensive view of mTORC1 and its role in regulating re- found that curtailing the activity of mTORC1 bidirection- gional protein expression in normal and diseased states. Molecular & Cellular Proteomics 15: 10.1074/ From the ‡Center for Learning and Memory, University of Texas, mcp.M115.055079, 426–444, 2016. Austin, 1 University Station C7000, Texas 78712; §Institute for Cell and Molecular Biology, University of Texas, Austin, Texas; ¶Institute for Neuroscience, University of Texas, Austin, Texas; ʈWaggoner The mechanistic/mammalian target of rapamycin complex Center for Alcohol and Addiction Research, University of Texas, Aus- 1 (mTORC1)1 is a serine/threonine protein kinase that is highly tin, Texas; **Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409; ‡‡Pain Therapeutics, Inc., 7801 N Capital of Texas Hwy, #260, Austin, Texas 78731 1 The abbreviations used are: mTORC1, mechanistic/mammalian Received August 28, 2015, and in revised form, September 15, target of rapamycin complex 1; AD, Alzheimer’s disease; AHA, azi- 2015 dohomoalanine; APP, amyloid precursor protein; ASD, autism spec- Published, MCP Papers in Press, September 29, 2015, DOI trum disorder; BONCAT, bioorthogonal noncanonical amino acid 10.1074/mcp.M115.055079 tagging; CI, confidence interval; cKO, conditional knockout; Cre, Cre- Author contributions: FN and KRG designed research. FN and SN recombinase; DAVID, database for annotation, visualization and inte- conducted experiments and analyzed data. SN performed bioinfor- grated discovery; DIV, day in vitro; Dlg4, discs large homolog 4; matics analyses. ES and YM conducted mass spectrometry analyses. DMSO, dimethyl sulfoxide; EASE, expression analysis systematic GS facilitated mass spectrometry experiments and provided technical explorer; FDA, Food and Drug Administration; GAP-43, growth-asso- advice. GAD and BVZ provided the virus and performed stereotaxic ciated protein-43; GO, gene ontology; GRIN, glutamate receptor, injections. FN, SN, and KRG wrote the manuscript. ionotropic NMDA subtype; ICC, immunocytochemistry; GluN, gluta- 426 Molecular & Cellular Proteomics 15.2 mTORC1 Rapidly Shifts Expression of Disease Proteomes expressed in many cell types (1). In the brain, mTORC1 tightly in protein expression. Herein, we provide evidence that coordinates different synaptic plasticities — long-term poten- mTORC1 activation bidirectionally regulates protein expres- tiation (LTP) and long-term depression (LTD) — the molecular sion, especially in the PSD where roughly an equal distribution correlates of learning and memory (2–5). Because mTORC1 is of proteins dynamically appear and disappear. Remarkably, at the core of many synaptic signaling pathways downstream using protein–protein interaction networks facilitated the of glutamate and neurotrophin receptors, many hypothesize novel discovery that PARK7, a protein thus far only implicated that dysregulated mTORC1 signaling underlies cognitive def- in Parkinson’s disease, (1) is up-regulated by increased icits observed in several neurodegenerative diseases (3, mTORC1 activity, (2) resides in the PSD only when mTORC1 6–17). For example, mTORC1 and its downstream targets are is active, and (3) is aberrantly expressed in a rodent model of hyperactive in human brains diagnosed with Alzheimer’s dis- TSC, an mTORC1-related disease that has symptoms of ep- ease (AD) (18–20). Additionally in animal models of autism ilepsy and autism. Collectively, these data provide the first spectrum disorder (ASD), altered mTORC1 signaling contrib- comprehensive list of proteins whose abundance or subcel- utes to the observed synaptic dysfunction and aberrant net- lular distributions are altered with acute changes in mTORC1 work connectivity (13, 15, 21–27). Furthermore, epilepsy, activity in vivo. which is common in AD and ASD, has enhanced mTORC1 activity (28–32). EXPERIMENTAL PROCEDURES Phosphorylation of mTORC1, considered the active form, is Sample Preparation—We used three sets of paired sibling male generally regarded to promote protein synthesis (33). Thus, Sprague-Dawley rats (seven to 9-weeks old) that were housed to- many theorize that diseases with overactive mTORC1 arise gether. Within each pair, one received rapamycin (10 mg/kg) and the from excessive protein synthesis (14). Emerging data, how- other received an equal volume of DMSO (carrier, control) via intra- ever, show that suppressing mTORC1 activation can trigger peritoneal (intraperitoneal) injection. After one hour, the animals were sacrificed. Cortices were homogenized, nuclei were pelleted by low local translation in neurons (34, 35). Pharmacological antag- speed centrifugation (100 ϫ g), and the resulting supernatant was onism of N-methyl-D-aspartate (NMDA) receptors, a subtype analyzed as cell lysates (L). To obtain synaptoneurosomes, homoge- of glutamate receptors that lies upstream of mTOR activation, nized cortices were processed as described (39). An aliquot of the promotes the synthesis of the voltage-gated potassium chan- synaptoneurosome fraction from each animal was solubilized in 1% ϫ nel, Kv1.1, in dendrites (34, 35). Consistent with these results, triton X-100 (10min) and centrifuged (12,000 g) to yield a triton X-100-soluble fraction (soluble, S) and a triton X-100-insoluble frac- in models of temporal lobe epilepsy there is a reduction in the tion (pellet, PSD). To prepare protein for LC-MS/MS, lysates, soluble, expression of voltage-gated ion channels including Kv1.1 (30, and PSD fractions were further solubilized in SDS-sample buffer. 31, 36). Interestingly in a model of focal neocortical epilepsy, SDS-solubilized fractions were run on a 10% SDS-polyacrylamide gel overexpression of Kv1.1 blocked seizure activity (37). Be- (4min). The migration was stopped as the ladder began to separate. cause both active and inactive mTORC1 permit protein syn- Gel plugs containing the sample were sectioned and sent for mass spectrometry analysis. Animal experiments were performed accord- thesis, we sought to determine the proteins whose expression ing to the National Institutes of Health’s Guide for the Care and Use is altered when mTORC1 phosphorylation is reduced in vivo. of Laboratory Animals and approved by the UT-Austin Institutional Rapamycin is an FDA-approved, immunosuppressive drug Care and Usage Committee. that inhibits mTORC1 activity (38). We capitalized on the Western Blot Analysis—Proteins were separated by SDS-poly- ability of rapamycin to reduce mTORC1 activity in vivo and the acrylamide gel electrophoresis (PAGE). To visualize the proteins, we used the following antibodies: rabbit anti-phospho-mTOR Ser2448 unbiased approach of mass spectrometry to identify changes (1:2000; Cell Signaling, Danvers, MA), mouse anti-mTOR (1:5000; Life Technologies, Grand Island, NY), mouse anti-Park7 (1:2000; Novus Biologicals, Littleton, CO) mouse anti-PSD-95 (1:10,000; NeuroMab, mate receptor, NMDA subtype; KAR, kainic