LSD1 modulates stress-evoked transcription of immediate early genes and emotional behavior Francesco Rusconia,1, Barbara Grilloa,2, Luisa Ponzonib,2, Silvia Bassanic, Emanuela Toffoloa, Leda Paganinia, Alessandra Malleid, Daniela Braidab, Maria Passafaroc, Maurizio Popolid, Mariaelvina Salab,c, and Elena Battagliolia,c,1 aLaboratory of Neuroepigenetics, Dipartimento di Biotecnologie Mediche e Medicina Traslazionale and Centro di Eccellenza per le Malattie Neurodegenerative (CEND), Università degli Studi di Milano, 20133 Milan, Italy; bDipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, 20129 Milan, Italy; cNational Research Council Institute of Neuroscience, 20129 Milan, Italy; and dLaboratory of Neuropsychopharmacology and Functional Neurogenomics, Dipartimento di Scienze Farmacologiche e Biomolecolari and CEND, Università degli Studi di Milano, 20133 Milan, Italy Edited by Huda Akil, University of Michigan, Ann Arbor, MI, and approved February 19, 2016 (received for review June 18, 2015) Behavioral changes in response to stressful stimuli can be con- of target genes (12–14) suggests that modulation of exon E8a trolled via adaptive epigenetic changes in neuronal gene expres- splicing may represent a mechanism for fine-tuning the transcrip- sion. Here we indicate a role for the transcriptional corepressor tion of selected targets. The generation of a mouse model lacking Lysine-Specific Demethylase 1 (LSD1) and its dominant-negative microexon E8a (neuroLSD1KO mice) allowed us to characterize splicing isoform neuroLSD1, in the modulation of emotional be- neuroLSD1 as an in vivo modulator of hippocampal excitability. havior. In mouse hippocampus, we show that LSD1 and neuroLSD1 We reported that shifts in the LSD1/neuroLSD1 ratio in response can interact with transcription factor serum response factor (SRF) to activity contribute dynamically to the control of neuronal ex- and set the chromatin state of SRF-targeted genes early growth citability by participating in the transcriptional mechanism of ho- response 1 (egr1) and c-fos. Deletion or reduction of neuroLSD1 in meostatic adaptation (15). In this work we implicate LSD1 and mutant mice translates into decreased levels of activating histone neuroLSD1 in shaping emotional behavior as fine tuners of psy- marks at egr1 and c-fos promoters, dampening their psychosocial chosocial stress-evoked transcription of IEGs. We found that the stress-induced transcription and resulting in low anxiety-like behav- complete lack (neuroLSD1KO) or reduction (neuroLSD1 hetero- KO ior. Administration of suberoylanilide hydroxamine to neuroLSD1 zygosity, hereafter neuroLSD1HET) of neuroLSD1 in the mouse mice reactivates egr1 and c-fos transcription and restores the be- brain causes low anxiety-like behavior. At the molecular level, NEUROSCIENCE havioral phenotype. These findings indicate that LSD1 is a molecular knocking out neuroLSD1 results in H3K4 hypomethylation and H3 transducer of stressful stimuli as well as a stress-response modifier. hypoacetylation of early growth response 1 (egr1)andc-fos pro- Indeed, LSD1 expression itself is increased acutely at both the moters, decreasing their stress-induced transcription. This impair- transcriptional and splicing levels by psychosocial stress, suggest- ment hinders the acquisition of stress-related plasticity relevant to ing that LSD1 is involved in the adaptive response to stress. anxiety behavior. Moreover, in wild-type animals, LSD1 tran- scription and alternative splicing are directly modulated by psy- epigenetics | stress | immediate early genes | LSD1 | SRF chosocial stress, indicating that LSD1 and neuroLSD1 not only participate in the transduction of stressful stimuli but also rep- ynamic changes in neuronal chromatin through histone resent stress-response modifiers. Dposttranslational modifications affect complex functions such as learning, memory, and emotional behavior (1). Seminal studies have Significance shown that mice experiencing different forms of stress, including psychosocial stress, promote stress-related plasticity through epige- In mammals, different forms of stress, including psychosocial netic changes at specific genes, including brain-derived neurotrophic stress, can affect several aspects of health, fostering mood and factor (BDNF) and immediate early genes (IEGs) (2–4). These anxiety disorders in humans. However, a lack of knowledge modifications induce contrasting structural and functional changes in about the mechanisms underlying the brain physiology of the the hippocampus and the amygdala (5), brain areas responsible for stress response hinders the development of new therapeutic the expression of anxiety-like behavior (5–8). A decrease in neural strategies. We describe the role of the epigenetic enzyme activity in the hippocampus caused by the loss of dendritic arbors Lysine-Specific Demethylase 1 (LSD1) in the transduction path- and spines is associated with posttraumatic stress disorder and re- way that translates social stress into an altered transcriptional current depressive illness (5). Therefore, an important challenge for physiology of plasticity genes in hippocampus. In particular, we molecular psychiatry is a better understanding of the epigenetic show that in the brain LSD1 is finely tuned by a mammal- regulation of plasticity gene transcription in response to stress (9). restricted splicing-based mechanism, and its demethylase activ- Lysine-Specific Demethylase 1(LSD1)alsoknownaslysinede- ity consistently affects anxiety-like behavior in mice. This work methylase 1A (KDM1A) is an epigenetic transcriptional corepressor, addresses a fundamental mechanism, explaining a brain-related tightly associated to Corepressor of REST (CoREST) and histone phenotype as a result of genome–environment interactions deacetylase 2 (HDAC2). It removes methyl groups from mono- and mediated by the epigenome. di-methylated lysine 4 of histone H3 (H3K4), erasing a histone mark of active transcription (10). In mammals, neurospecific splicing of Author contributions: F.R., M. Popoli, M.S., and E.B. designed research; F.R., B.G., L. Ponzoni, microexon E8a generates the dominant-negative splicing isoform of S.B., E.T., L. Paganini, and A.M. performed research; M. Passafaro contributed new reagents/ LSD1 (neuroLSD1), which is required for the acquisition of proper analytic tools; F.R., B.G., L. Ponzoni, S.B., E.T., L. Paganini, A.M., D.B., M. Passafaro, M.S., and neurite morphology inherent in neuronal maturation (11). Although E.B. analyzed data; and F.R. and E.B. wrote the paper. conventional LSD1 acts as a constitutive repressor through its H3K4 The authors declare no conflict of interest. demethylase activity, neuroLSD1 is unable to repress transcription This article is a PNAS Direct Submission. (11, 12). It has been shown recently that neuroLSD1 lacks dimeth- Freely available online through the PNAS open access option. ylated H3K4 (H3K4me2) demethylase activity, confirming its role 1To whom correspondence may be addressed. Email: [email protected] or elena. as a dominant-negative LSD1 isoform (13). The neuroLSD1-con- [email protected]. taining complex also can act as transcriptional activator by ac- 2B.G. and L. Ponzoni contributed equally this work. quiring H3K9me2 and H4K20 demethylase activity (13, 14). In This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. this context, the finding that LSD1 and neuroLSD1 share a subset 1073/pnas.1511974113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1511974113 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 the elevated plus maze (EPM), we observed that neuroLSD1KO mice spent more time in and made more entries into the open arms than their wild-type littermates (Fig. 1A). A trend for increased time and number of entries in the open arms also was observed in neuroLSD1HET mice. The total number of entries into each arm of the maze was comparable in all genotypes, and no differences in motor function were observed (Fig. S2). In the marble-burying test (MBT), post hoc analysis indicated a significant decrease in the number of buried marbles in both neuroLSD1KO and heterozygous mice compared with wild-type littermates (Fig. 1B). Both geno- types showed a trend to increased latency to the first burial (Fig. 1B). In the novelty-suppressed feeding (NSF) test, both neuroLSD1KO and neuroLSD1HET mice exhibited significantly decreased latency to feed in the center of the arena compared with wild-type littermates. No difference was found in the food intake among genotypes (Fig. 1C). Taken together, the results of be- havioral tests consistently show that complete lack (neuroLSD1KO mice) or reduction (neuroLSD1HET mice) of neuroLSD1 expres- Fig. 1. NeuroLSD1-mutant mice feature a low anxiety-like phenotype. sion affects emotional behavior, resulting in a low-anxiety phenotype. Anxiety-like behavior is evaluated by comparing neuroLSD1KO and neuroLSD1HET mice with wild-type littermates. The anxiety trend is shown on the y axis. LSD1 and NeuroLSD1 Are Modulated by Stress and Regulate Stress- = = (A) Time spent in the open arm time (F2,27 4.65, P 0.018), entries into the Evoked Transcription of IEGs. To shed light on the molecular = = open arm (F2,27 6.16, P 0.0063), and total entries (which did not show any mechanism whereby reduction of neuroLSD1 gives rise to the difference among genotypes) evaluated in the EPM. (B) Number of marbles = = = = low anxiety-like phenotype, we investigated