Neuroscience 343 (2017) 190–212 NEURON-SPECIFIC SUMO KNOCKDOWN SUPPRESSES GLOBAL GENE EXPRESSION RESPONSE AND WORSENS FUNCTIONAL OUTCOME AFTER TRANSIENT FOREBRAIN ISCHEMIA IN MICE LIN ZHANG, a,b XIAOZHI LIU, a,b HUAXIN SHENG, a suppression of global gene expression response in post- SHUAI LIU, a YING LI, a,c JULIA Q. ZHAO, a ischemic brain due to SUMO knockdown has a negative a a DAVID S. WARNER, WULF PASCHEN * AND effect on post-ischemic neurologic function. Together, our a WEI YANG * data provide a basis for future studies to mechanistically a Multidisciplinary Neuroprotection Laboratories, Department of link SUMOylation to neurologic function in health and dis- Anesthesiology, Duke University Medical Center, Durham, NC, USA ease. Ó 2016 IBRO. Published by Elsevier Ltd. All rights b Department of Neurosurgery, The Fifth Central Hospital of reserved. Tianjin, Tianjin, China c Department of Cardiology, The Fifth Central Hospital of Tianjin, Tianjin, China Key words: brain ischemia, SUMO, microarray, knockdown, transgenic mice. Abstract—Small ubiquitin-like modifier (SUMO) conjugation (SUMOylation) plays key roles in neurologic function in health and disease. Neuronal SUMOylation is essential for emotionality and cognition, and this pathway is dramatically INTRODUCTION activated in post-ischemic neurons, a neuroprotective response to ischemia. It is also known from cell culture Small ubiquitin-like modifier (SUMO) conjugation studies that SUMOylation modulates gene expression. How- (SUMOylation) is a post-translational protein ever, it remains unknown how SUMOylation regulates neu- modification whereby SUMOs are covalently conjugated ronal gene expression in vivo, in the physiologic state and to lysine residues in target proteins (Flotho and after ischemia, and modulates post-ischemic recovery of Melchior, 2013). This is an energy-dependent process neurologic function. To address these important questions, that is regulated via the action of activating (E1, ATP- we used a SUMO1-3 knockdown (SUMO-KD) mouse in which dependent), conjugating (E2), and ligating (E3) enzymes. a Thy-1 promoter drives expression of 3 distinct microRNAs In mammalian cells, three SUMO isoforms have been against SUMO1-3 to silence SUMO expression specifically in identified: SUMO1, SUMO2, and SUMO3. SUMO2 and neurons. Wild-type and SUMO-KD mice were subjected to SUMO3 are highly homologous, and are usually referred transient forebrain ischemia. Microarray analysis was per- formed in hippocampal CA1 samples, and neurologic func- to as SUMO2/3. SUMO1, however, shares only about tion was evaluated. SUMOylation had opposite effects on 50% homology with SUMO2/3. Notably, while SUMO1 neuronal gene expression before and after ischemia. In the knockout mice and SUMO3 knockout mice do not show physiological state, most genes regulated by SUMOylation any obvious abnormality, SUMO2 knockout is lethal in were up-regulated in SUMO-KD compared to wild-type mice. the embryonic stage (Evdokimov et al., 2008; Wang Brain ischemia/reperfusion significantly modulated the et al., 2014b). expression levels of more than 400 genes in wild-type mice, SUMOylation can modify the activity, stability, and with a majority of those genes upregulated. The extent of function of target proteins, and thereby modulate almost this post-ischemic transcriptome change was suppressed all major cellular pathways (Flotho and Melchior, 2013). in SUMO-KD mice. Moreover, SUMO-KD mice exhibited sig- Our knowledge about the significance of SUMO conjuga- nificantly worse functional outcome. This suggests that tion in cellular homeostasis is primarily based on results from cell culture experiments. In the physiologic state, *Corresponding authors. Address: Department of Anesthesiology, most SUMO1 is conjugated to target proteins, predomi- Duke University Medical Center, Box 3094, 152 Sands Building, nately the Ran GTPase-activating protein-1 (RanGAP1). Research Drive, Durham, NC 27710, USA. E-mail addresses: [email protected] (W. Paschen), wei. Thus, under stress conditions, there is only small change [email protected] (W. Yang). in SUMO1 modification profiles. In contrast, most Abbreviations: ANOVA, analysis of variance; Arc, activity-regulated SUMO2/3 is present as free SUMO in the physiologic cytoskeleton-associated protein; GFP, green fluorescent protein; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; state. However, under a variety of stress conditions, MAPK, mitogen-activated protein kinase; miRNA, microRNA; OGD, including heat, metabolic, and oxidative stress, SUMO2/3 oxygen/glucose deprivation; PCA, principal component analysis; conjugation is dramatically activated (Saitoh and Hinchey, qPCR, quantitative real-time PCR; RanGAP1, Ran GTPase- 2000; Yang et al., 2012). activating protein-1; rCBF, regional cerebral blood flow; SUMO, small ubiquitin-like modifier; SUMO-KD, SUMO1-3 knockdown; WI, wild-type Many of the SUMO targets are nuclear proteins ischemia; WS, wild-type sham. involved in gene expression (Golebiowski et al., 2009; http://dx.doi.org/10.1016/j.neuroscience.2016.11.036 0306-4522/Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved. 190 L. Zhang et al. / Neuroscience 343 (2017) 190–212 191 Yang et al., 2014). It has been reported that SUMOylation mice were previously generated in our laboratory (Wang can both activate and suppress transcription. In most et al., 2014a). In this transgenic mouse model, the trans- cases, however, gene expression is suppressed when gene contains 3 distinct miRNAs that target SUMO1, 2, the respective transcription factor is SUMO-conjugated and 3, and are expressed under the control of the (Gill, 2005; Chymkowitch et al., 2015). The mechanisms neuron-specific Thy1 promoter. Green fluorescent protein that link SUMO conjugation to gene expression are still (GFP) is co-expressed as indicator of transgene expres- not fully understood. Generally, experimental studies sion. SUMO-KD mice have been backcrossed with have focused on individual transcription factors to clarify C57Bl/6 mice for more than 10 generations. Male the role of SUMOylation in expression of target genes. SUMO-KD and wild-type littermates (2–3 months old) Recently, chromatin immunoprecipitation coupled with were used in this study. next-generation sequencing (ChIP-seq) was used to more completely define these mechanisms (Liu et al., 2012; Transient forebrain ischemia Neyret-Kahn et al., 2013; Seifert et al., 2015). These stud- ies were carried out in cell cultures. A genome-wide anal- Transient forebrain ischemia was performed as described ysis of the effects of SUMOylation on gene expression previously (Yang et al., 2008c). Briefly, anesthesia was in vivo has not yet been performed. induced with 5% isoflurane and maintained with 1.5– SUMO conjugation plays a pivotal role in neurologic 1.8% isoflurane during surgery. The rectal temperature function in the physiologic and pathologic state. For was maintained at 37.0 °C ± 0.2 °C by surface heating example, SUMOylation is essential for emotionality and or cooling. PE-10 tubes were inserted into the right cognition, and silencing SUMO1-3 expression femoral artery and the right internal jugular vein to contin- specifically in neurons impairs episodic and fear uously monitor arterial blood pressure and to withdraw memories (Wang et al., 2014a). However, the pathways blood, respectively. Forebrain ischemia was induced by that link SUMOylation to memory processes have not a combination of 10-min bilateral common carotid artery been identified. Furthermore, SUMOylation is associated occlusion, and blood withdrawal-induced hypotension with brain ischemia/stroke and degenerative diseases (mean arterial blood pressure = 30 mmHg). To end the (Yang et al., 2008b,c; Flotho and Melchior, 2013; ischemic episode, the carotid arteries were de-occluded Krumova and Weishaupt, 2013). Transient brain ischemia and withdrawn blood was re-infused. Sham-operated is a severe form of metabolic stress that triggers dramatic mice underwent the same procedures except carotid activation of SUMO2/3 conjugation, and to a lesser artery occlusion and blood withdrawal. To determine extent, SUMO1 conjugation (Yang et al., 2014). It has whether SUMO knockdown had any effect on blood flow been proposed that this is a protective response that reduction in our transient forebrain ischemia model, a shields neurons from damage induced by ischemia cohort of mice was subjected to blood flow measure- (Yang et al., 2008a, 2016; Lee and Hallenbeck, 2013). ments. Before inducing ischemia, a microprobe (Moor, Indeed, results from in vitro and in vivo studies support Delaware, DE, USA) was affixed to the surface of the right this notion. For example, neurons in which SUMO2/3 parietal skull to monitor regional cerebral blood flow expression is silenced by lentiviral delivery of SUMO2 (rCBF) in the middle cerebral artery territory by Laser and SUMO3 microRNAs (miRNAs), are highly sensitive Doppler flowmetry (Moor). to transient oxygen/glucose deprivation (OGD, an experi- mental model that simulates ischemia in cells), whereas Tissue sample preparation transgenic mice overexpressing SUMO conjugating enzyme Ubc9, have higher levels of SUMO1- and At the indicated times of reperfusion, mice were SUMO2/3-conjugated proteins and smaller infarcts after sacrificed, and brains were quickly removed. CA1 and stroke (Datwyler et al., 2011; Lee et al., 2011). However, cortex samples were excised in a cryostat set at we still do not know the role of SUMO conjugation in post- À20 °C. Tissue samples were stored at