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Active, phosphorylated fingolimod inhibits histone deacetylases and facilitates fear extinction memory

Nature Neuroscience volume 17, pages 971–980 (2014)Cite this article

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Abstract

FTY720 (fingolimod), an FDA-approved drug for treatment of multiple sclerosis, has beneficial effects in the CNS that are not yet well understood, independent of its effects on immune cell trafficking. We show that FTY720 enters the nucleus, where it is phosphorylated by sphingosine kinase 2 (SphK2), and that nuclear FTY720-P binds and inhibits class I histone deacetylases (HDACs), enhancing specific histone acetylations. FTY720 is also phosphorylated in mice and accumulates in the brain, including the hippocampus, inhibits HDACs and enhances histone acetylation and gene expression programs associated with memory and learning, and rescues memory deficits independently of its immunosuppressive actions. Sphk2−/− mice have lower levels of hippocampal sphingosine-1-phosphate, an endogenous HDAC inhibitor, and reduced histone acetylation, and display deficits in spatial memory and impaired contextual fear extinction. Thus, sphingosine-1-phosphate and SphK2 play specific roles in memory functions and FTY720 may be a useful adjuvant therapy to facilitate extinction of aversive memories.

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Figure 1: FTY720-P is produced in the nucleus by SphK2.
Figure 2: FTY720-P enhances specific histone acetylations.
Figure 3: FTY720-P inhibits class I HDACs.
Figure 4: FTY720-P competes with S1P for binding to HDAC1.
Figure 5: FTY720 administration-facilitated contextual fear extinction in SCID mice correlates with formation of hippocampal FTY720-P, inhibition of HDACs and enhanced histone acetylation and memory-related gene expression.
Figure 6: Treatment with FTY720 enhances LTP, but does not alter basal synaptic transmission of Schaffer-collateral synapses.
Figure 7: SphK2 knockout mice have reduced hippocampal histone acetylations and learning deficits.
Figure 8: SAHA administration to SphK2 knockout mice reversed the increased HDAC activity, reinstated hippocampal histone acetylations and rescued their extinction deficit in contextual fear conditioning.

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References

  1. Brinkmann, V. et al. Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis. Nat. Rev. Drug Discov. 9, 883–897 (2010).

    Article  CAS  PubMed  Google Scholar 

  2. Cyster, J.G. & Schwab, S.R. Sphingosine-1-phosphate and lymphocyte egress from lymphoid organs. Annu. Rev. Immunol. 30, 69–94 (2012).

    Article  CAS  PubMed  Google Scholar 

  3. Foster, C.A. et al. Brain penetration of the oral immunomodulatory drug FTY720 and its phosphorylation in the central nervous system during experimental autoimmune encephalomyelitis: consequences for mode of action in multiple sclerosis. J. Pharmacol. Exp. Ther. 323, 469–475 (2007).

    Article  CAS  PubMed  Google Scholar 

  4. Hla, T. & Brinkmann, V. Sphingosine 1-phosphate (S1P): physiology and the effects of S1P receptor modulation. Neurology 76, S3–S8 (2011).

    Article  CAS  PubMed  Google Scholar 

  5. Hait, N.C. et al. Regulation of histone acetylation in the nucleus by sphingosine-1-phosphate. Science 325, 1254–1257 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Alvarez, S.E. et al. Sphingosine-1-phosphate is a missing cofactor for the E3 ubiquitin ligase TRAF2. Nature 465, 1084–1088 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Igarashi, N. et al. Sphingosine kinase 2 is a nuclear protein and inhibits DNA synthesis. J. Biol. Chem. 278, 46832–46839 (2003).

    Article  CAS  PubMed  Google Scholar 

  8. Kazantsev, A.G. & Thompson, L.M. Therapeutic application of histone deacetylase inhibitors for central nervous system disorders. Nat. Rev. Drug Discov. 7, 854–868 (2008).

    Article  CAS  PubMed  Google Scholar 

  9. Fischer, A., Sananbenesi, F., Mungenast, A. & Tsai, L.H. Targeting the correct HDAC(s) to treat cognitive disorders. Trends Pharmacol. Sci. 31, 605–617 (2010).

    Article  CAS  PubMed  Google Scholar 

  10. Bressi, J.C. et al. Exploration of the HDAC2 foot pocket: Synthesis and SAR of substituted N-(2-aminophenyl)benzamides. Bioorg. Med. Chem. Lett. 20, 3142–3145 (2010).

    Article  CAS  PubMed  Google Scholar 

  11. Finnin, M.S. et al. Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors. Nature 401, 188–193 (1999).

    Article  CAS  PubMed  Google Scholar 

  12. Choi, J.W. et al. FTY720 (fingolimod) efficacy in an animal model of multiple sclerosis requires astrocyte sphingosine 1-phosphate receptor 1 (S1P1) modulation. Proc. Natl. Acad. Sci. USA 108, 751–756 (2011).

    Article  CAS  PubMed  Google Scholar 

  13. Mandala, S. et al. Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists. Science 296, 346–349 (2002).

    Article  CAS  PubMed  Google Scholar 

  14. Allende, M.L. et al. Mice deficient in sphingosine kinase 1 are rendered lymphopenic by FTY720. J. Biol. Chem. 279, 52487–52492 (2004).

    Article  CAS  PubMed  Google Scholar 

  15. Briard, E. et al. BZM055, an iodinated radiotracer candidate for PET and SPECT imaging of myelin and FTY720 brain distribution. ChemMedChem 6, 667–677 (2011).

    Article  CAS  PubMed  Google Scholar 

  16. Bredy, T.W. et al. Histone modifications around individual BDNF gene promoters in prefrontal cortex are associated with extinction of conditioned fear. Learn. Mem. 14, 268–276 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Lattal, K.M., Barrett, R.M. & Wood, M.A. Systemic or intrahippocampal delivery of histone deacetylase inhibitors facilitates fear extinction. Behav. Neurosci. 121, 1125–1131 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Bredy, T.W. & Barad, M. The histone deacetylase inhibitor valproic acid enhances acquisition, extinction, and reconsolidation of conditioned fear. Learn. Mem. 15, 39–45 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Vecsey, C.G. et al. Histone deacetylase inhibitors enhance memory and synaptic plasticity via CREB:CBP-dependent transcriptional activation. J. Neurosci. 27, 6128–6140 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Stefanko, D.P., Barrett, R.M., Ly, A.R., Reolon, G.K. & Wood, M.A. Modulation of long-term memory for object recognition via HDAC inhibition. Proc. Natl. Acad. Sci. USA 106, 9447–9452 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kipnis, J., Cohen, H., Cardon, M., Ziv, Y. & Schwartz, M. T cell deficiency leads to cognitive dysfunction: implications for therapeutic vaccination for schizophrenia and other psychiatric conditions. Proc. Natl. Acad. Sci. USA 101, 8180–8185 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Brynskikh, A., Warren, T., Zhu, J. & Kipnis, J. Adaptive immunity affects learning behavior in mice. Brain Behav. Immun. 22, 861–869 (2008).

    Article  CAS  PubMed  Google Scholar 

  23. Gräff, J. et al. Epigenetic priming of memory updating during reconsolidation to attenuate remote fear memories. Cell 156, 261–276 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Kappos, L. et al. A placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis. N. Engl. J. Med. 362, 387–401 (2010).

    Article  CAS  PubMed  Google Scholar 

  25. Yirmiya, R. & Goshen, I. Immune modulation of learning, memory, neural plasticity and neurogenesis. Brain Behav. Immun. 25, 181–213 (2011).

    Article  CAS  PubMed  Google Scholar 

  26. Guan, J.S. et al. HDAC2 negatively regulates memory formation and synaptic plasticity. Nature 459, 55–60 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Peleg, S. et al. Altered histone acetylation is associated with age-dependent memory impairment in mice. Science 328, 753–756 (2010).

    Article  CAS  PubMed  Google Scholar 

  28. Derecki, N.C. et al. Regulation of learning and memory by meningeal immunity: a key role for IL-4. J. Exp. Med. 207, 1067–1080 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Mauceri, D., Freitag, H.E., Oliveira, A.M., Bengtson, C.P. & Bading, H. Nuclear calcium-VEGFD signaling controls maintenance of dendrite arborization necessary for memory formation. Neuron 71, 117–130 (2011).

    Article  CAS  PubMed  Google Scholar 

  30. Stefansson, H. et al. Common variants conferring risk of schizophrenia. Nature 460, 744–747 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Brzózka, M.M., Radyushkin, K., Wichert, S.P., Ehrenreich, H. & Rossner, M.J. Cognitive and sensorimotor gating impairments in transgenic mice overexpressing the schizophrenia susceptibility gene Tcf4 in the brain. Biol. Psychiatry 68, 33–40 (2010).

    Article  PubMed  CAS  Google Scholar 

  32. McQuown, S.C. et al. HDAC3 is a critical negative regulator of long-term memory formation. J. Neurosci. 31, 764–774 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Orlandini, M., Marconcini, L., Ferruzzi, R. & Oliviero, S. Identification of a c-fos-induced gene that is related to the platelet-derived growth factor/vascular endothelial growth factor family. Proc. Natl. Acad. Sci. USA 93, 11675–11680 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Patterson, S.L. et al. Some forms of cAMP-mediated long-lasting potentiation are associated with release of BDNF and nuclear translocation of phospho-MAP kinase. Neuron 32, 123–140 (2001).

    Article  CAS  PubMed  Google Scholar 

  35. Alarcón, J.M. et al. Chromatin acetylation, memory, and LTP are impaired in CBP+/− mice: a model for the cognitive deficit in Rubinstein-Taybi syndrome and its amelioration. Neuron 42, 947–959 (2004).

    Article  PubMed  Google Scholar 

  36. Levenson, J.M. et al. Regulation of histone acetylation during memory formation in the hippocampus. J. Biol. Chem. 279, 40545–40559 (2004).

    Article  CAS  PubMed  Google Scholar 

  37. Liu, H. et al. Molecular cloning and functional characterization of a novel mammalian sphingosine kinase type 2 isoform. J. Biol. Chem. 275, 19513–19520 (2000).

    Article  CAS  PubMed  Google Scholar 

  38. Blondeau, N. et al. Distribution of sphingosine kinase activity and mRNA in rodent brain. J. Neurochem. 103, 509–517 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Liang, J. et al. Sphingosine-1-phosphate links persistent STAT3 activation, chronic intestinal inflammation, and development of colitis-associated cancer. Cancer Cell 23, 107–120 (2013).

    Article  CAS  PubMed  Google Scholar 

  40. Fujita, Y. et al. Vorinostat, a histone deacetylase inhibitor, facilitates fear extinction and enhances expression of the hippocampal NR2B-containing NMDA receptor gene. J. Psychiatr. Res. 46, 635–643 (2012).

    Article  PubMed  Google Scholar 

  41. Day, J.J. & Sweatt, J.D. Epigenetic mechanisms in cognition. Neuron 70, 813–829 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Zovkic, I.B. & Sweatt, J.D. Epigenetic mechanisms in learned fear: implications for PTSD. Neuropsychopharmacology 38, 77–93 (2013).

    Article  CAS  PubMed  Google Scholar 

  43. Deogracias, R. et al. Fingolimod, a sphingosine-1 phosphate receptor modulator, increases BDNF levels and improves symptoms of a mouse model of Rett syndrome. Proc. Natl. Acad. Sci. USA 109, 14230–14235 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Peters, J., Dieppa-Perea, L.M., Melendez, L.M. & Quirk, G.J. Induction of fear extinction with hippocampal-infralimbic BDNF. Science 328, 1288–1290 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Soliman, F. et al. A genetic variant BDNF polymorphism alters extinction learning in both mouse and human. Science 327, 863–866 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Hawk, J.D. et al. NR4A nuclear receptors support memory enhancement by histone deacetylase inhibitors. J. Clin. Invest. 122, 3593–3602 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Lee, Y.S. & Silva, A.J. The molecular and cellular biology of enhanced cognition. Nat. Rev. Neurosci. 10, 126–140 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Maren, S. Seeking a spotless mind: extinction, deconsolidation, and erasure of fear memory. Neuron 70, 830–845 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Tronson, N.C., Corcoran, K.A., Jovasevic, V. & Radulovic, J. Fear conditioning and extinction: emotional states encoded by distinct signaling pathways. Trends Neurosci. 35, 145–155 (2012).

    Article  CAS  PubMed  Google Scholar 

  50. Beaudoin, G.M. III et al. Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex. Nat. Protoc. 7, 1741–1754 (2012).

    Article  CAS  PubMed  Google Scholar 

  51. Fitting, S. et al. Synaptic dysfunction in the hippocampus accompanies learning and memory deficits in human immunodeficiency virus type-1 Tat transgenic mice. Biol. Psychiatry 73, 443–453 (2013).

    Article  CAS  PubMed  Google Scholar 

  52. Plendl, W. & Wotjak, C.T. Dissociation of within- and between-session extinction of conditioned fear. J. Neurosci. 30, 4990–4998 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Varvel, S.A., Hamm, R.J., Martin, B.R. & Lichtman, A.H. Differential effects of delta 9-THC on spatial reference and working memory in mice. Psychopharmacology (Berl.) 157, 142–150 (2001).

    Article  CAS  Google Scholar 

  54. Lazenka, M.F., Selley, D.E. & Sim-Selley, L.J. DeltaFosB induction correlates inversely with CB receptor desensitization in a brain region-dependent manner following repeated delta-THC administration. Neuropharmacology 77, 224–233 (2014).

    Article  CAS  PubMed  Google Scholar 

  55. Kerns, R.T. et al. Ethanol-responsive brain region expression networks: implications for behavioral responses to acute ethanol in DBA/2J versus C57BL/6J mice. J. Neurosci. 25, 2255–2266 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Zhang, L., Wang, L., Ravindranathan, A. & Miles, M.F. A new algorithm for analysis of oligonucleotide arrays: application to expression profiling in mouse brain regions. J. Mol. Biol. 317, 225–235 (2002).

    Article  CAS  PubMed  Google Scholar 

  57. Chen, J., Bardes, E.E., Aronow, B.J. & Jegga, A.G. ToppGene Suite for gene list enrichment analysis and candidate gene prioritization. Nucleic Acids Res. 37, W305–W311 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Baker, E.J., Jay, J.J., Bubier, J.A., Langston, M.A. & Chesler, E.J. GeneWeaver: a web-based system for integrative functional genomics. Nucleic Acids Res. 40, D1067–D1076 (2012).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by US National Institutes of Health (NIH) grant R37GM043880 to S.S. Behavioral studies were supported by 5P01DA009789 to A.H.L. and R21AG042745 to L.E.W. LTP studies were supported by R01NS057758 to T.M.R. The Lipidomics core was supported in part by NIH grant P30CA16059 to the Massey Cancer Center. Modeling studies were supported by National Natural Science Foundation of China grant 91029704 to C.L. We thank R. Proia (US National Institutes of Health) for providing the Sphk2−/− mice, B.L. Mason for technical assistance and S. Lima for discussions.

Author information

Author notes

  1. Nitai C Hait and Laura E Wise: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, Virginia, USA

    Nitai C Hait, Jeremy C Allegood, Dorit Avni, Sheldon Milstien & Sarah Spiegel

  2. Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, Virginia, USA

    Nitai C Hait, Jeremy C Allegood, Dorit Avni, Sheldon Milstien & Sarah Spiegel

  3. Department of Pharmacology and Toxicology, Virginia Commonwealth University School of Medicine, Richmond, Virginia, USA

    Laura E Wise, Megan O'Brien, Michael F Miles & Aron H Lichtman

  4. Department of Anatomy and Neurobiology, Virginia Commonwealth University School of Medicine, Richmond, Virginia, USA

    Thomas M Reeves & Pamela E Knapp

  5. State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    Junyan Lu & Cheng Luo

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Contributions

N.C.H. designed, performed research and analyzed; L.E.W. performed behavior studies and analyzed data; S.M. and S.S. developed the concept, created figures and wrote the manuscript. J.C.A., D.A. and M.O. performed research; J.L. and C.L. performed molecular modeling; A.H.L. and M.F.M. contributed to data interpretation; P.E.K. provided reagents; T.M.R. carried out LTP studies.

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Correspondence to Sarah Spiegel.

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Integrated supplementary information

Supplementary Figure 1 FTY720 is phosphorylated by nuclear SphK2 and enhances histone acetylations in HeLa cells.

(a-c) HeLa cells transfected with vector, SphK2, or catalytically inactive SphK2G212E (ciSphK2) were treated without or with FTY720 (5 μM) for 6 h (N = 3 per group). Nuclear and cytoplasmic levels of FTY720-P (a) and S1P (b) were determined by LC-ESI-MS/MS. Data are means ± s.d. *P < 0.01 compared to vector. #P < 0.01 compared to untreated (unpaired two-tail t test). (c) Histone acetylations in nuclear extracts were detected by immunoblotting with the indicated antibodies. (d) Purified nuclei from HeLa cells were incubated for the indicated times with vehicle, S1P (1 μM), or FTY720-P (1 μM) and histone acetylations determined. (e) Naïve HeLa cells were treated with vehicle, S1P (100 nM), or SAHA (1 μM) for the indicated times and proteins were analyzed by western blotting.

Supplementary Figure 2 FTY720-P does not affect HAT activity.

HAT activity in nuclear extracts was determined with a colorimetric HAT activity assay in the presence of vehicle, FTY720-P (0.5 or 5 μM) or FTY720 (5 μM). HAT activities are averages of triplicate determinations ± s.d. and expressed as OD at 440 nm per mg.

Supplementary Figure 3 High-affinity binding of S1P and FTY720-P to HDAC1.

Recombinant His-tagged HDAC1 immobilized to Ni-NTA-resin was incubated with [32P]S1P (0.1 nM) in the absence or presence of increasing concentrations of unlabeled S1P or FTY720-P. Beads were washed extensively and [32P]S1P bound to HDAC1 was eluted with 500 mM imidazole and radioactivity detected by scintillation counting. Data were fit to a one-site Scatchard model using GraphPad Prism. S1P and FTY720-P bind to HDAC1 with apparent Kd values of 7.5 and 6.2 nM, respectively. R2 values for displacement by S1P and FTY720-P were 0.9581 and 0.9610, respectively.

Supplementary Figure 4 Docking of FTY720-P into the active site of HDAC2.

(a) Binding mode between FTY720-P and the substrate-binding pocket of HADC2. Active site residues that are involved in the interaction with small molecules are shown as sticks and the Zn atom is shown as a sphere. The polar contacts between active site residues, Zn, and small molecules are represented by black dashed lines. (b) Schematic representation of the interaction of FTY720-P with HDAC2 calculated by Ligplot. Thatched semi-circles indicate van der Waals contacts between hydrophobic amino acid residues and FTY720-P. Hydrogen bonds are shown as green dashed lines.

Supplementary Figure 5 Formation of FTY720-P, inhibition of HDACs, and enhanced histone acetylation and gene expression in the hippocampus following oral administration of FTY720 to C57Bl/6 mice.

(a-e) FTY720 was administered by gavage to C57Bl/6 mice at the indicated doses. 24 h later, lymphocytes in blood were counted (a), FTY720-P levels in serum (b) and hippocampal nuclei (c) measured by LCESI-MS/MS, and hippocampus HDAC activity (d) and H3K9 acetylation (e) were determined. (f-i) QPCR analysis of Vegfd, Bdnf, and cFos expression after treatment of mice with different doses of FTY720 for 24 h (f,g) or with 0.1 mg/kg for the indicated times (h,i). *P < 0.01, compared to untreated (unpaired two-tailed t test).

Supplementary Figure 6 Accumulation of FTY720-P in various brain regions.

SCID mice were treated daily with FTY720 as described in Fig. 5d. Blood was collected and brain regions were isolated and levels of FTY720 and FTY720-P were measured by LC-ESI-MS/MS. Note: Assuming brain is 75% water, the concentration of FTY720-P in hippocampus for example is ∼9 μM compared to 0.27 μM in blood.

Supplementary Figure 7 FTY720 does not affect performance of SCID mice in the Morris water maze, marble burying, exploratory activity and cued fear conditioning test.

SCID mice were treated orally each day by gavage with saline or FTY720 (1 mg/kg) starting 16 h prior to the (a-c) MWM paradigm, (d-f) behavioral testing, and (g) tone-shock paired conditioned fear testing. (a-c) FTY720 did not affect the performance of SCID mice in MWM. (a) Escape latencies (two-way repeated measures ANOVA; interaction: F(4,60) = 1.43; P = 0.23; day: F(4,60) = 1.96; P =0.11; treatment: F(1,60) = 0.64; P = 0.44) and (b) swim speeds (two-way ANOVA; interaction: F(4,60) = 0.26; P = 0.90; day: F(4,60) = 1.68; P = 0.17; treatment: F(1,60) = 0.33; P = 0.57) of FTY720 and saline treated mice did not significantly differ on the five days of fixed platform training. (c) The percentage of time spent in the in each quadrant (T, target quadrant; L, left quadrant; O, opposite quadrant; R, right quadrant) during a 120 s probe trial. 24 h after the fifth day of training also did not differ in mice treated with FTY720 or saline (two-way repeated measures ANOVA; interaction: F(3,45) = 0.10; P = 0.96; quadrant: F(3,45) = 1.15; P = 0.34; treatment: F(1,45) = 0.93; P = 0.35). T, target quadrant; L, left quadrant; O, opposite quadrant; R, right quadrant. (d) In the marble burying test, SCID mice buried a similar number of marbles regardless of FTY720 treatment (t-test: t (1,18) = 0.63; P = 0.54). (e-f) FTY720 did not affect (e) distance traveled (t-test: t(1,18) = 0.32; P = 0.75) or (f) time thigmotaxic during a 3 min exposure to a novel environment (t-test: t(1,18) = 1.61; P = 0.13). (g) SCID mice treated with FTY720 or saline displayed similar freezing and extinction in a tone-shock paired conditioning paradigm. Data represent percent time freezing during the 3 min before tone-shock pairings (preshock), 60 s after tone-shock pairings (post-shock), and during 200 s tone on extinction days. *P < 0.001, vs. pre-shock (Bonferroni post hoc test). # P < 0.001, compared to day 2, the first day of extinction (Bonferroni post hoc test). Both treatment groups had similar levels of freezing preshock, post-shock, and on extinction day 1 (two-way ANOVA; interaction: F(2,36) = 0.51; P = 0.61; time: F(2,36) = 146.54; P <0.0001; treatment: F(1,36) = 0.67; P = 0.42) and exhibited similar rates of extinction (two-way repeated measures ANOVA, interaction: F(4,72) = 0.28; P = 0.89; extinction session: F(4,72) = 8.33; P <0.0001; treatment: F(1,72) = 0.23; P = 0.64). Tests in (d-g) were performed in the same cohort of mice. N = 7-10 mice/group. Data are mean ± s.e.m.

Supplementary Figure 8 Sphk2–/– mice show decreased fear extinction in the contextual fear paradigm.

(a) SphK2-/- and WT mice do not forget the association between the context and footshock 48 h after conditioning. Mice were subjected to three footshocks on the conditioning day and received a 2.5 min test 48 h later. SphK2-/- and WT mice exhibited similar levels of freezing pre-shock, post-shock, and 48 h after conditioning (i.e., no extinction sessions) (two-way repeated-measures ANOVA; interaction: F(2,30) = 0.77; P = 0.47; time: F(2,30) = 100.95; P < 0.0001; genotype: F(1,30) = 0.87; P = 0.37). Percent time freezing in both genotypes was significantly increased post-shock and at 48 h compared to pre-shock (*P < 0.001, Bonferroni post hoc test). N = 8 for WT; N =9 for SphK2-/- mice. (b) SphK2-/- mice display extinction learning deficits in the contextual fear paradigm. SphK2-/- and WT mice were subjected to three footshocks on day 1 and received one extinction session (10 min) 24 h later (E1, consecutive 2.5 min bins are indicated by 1, 2, 3, 4). Mice were then evaluated on day 3 in a 2.5 min test. Both genotypes exhibited similar levels of freezing pre-shock, post-shock, and 24 h after conditioning (two-way repeated-measures ANOVA; interaction: F(2,32) = 1.44; P = 0.26; time: F(2,32) = 110; P < 0.0001; genotype: F(1,32) = 1.87; P = 0.19). Significant increases in freezing were found post-shock (P < 0.001) and in the first 2.5 min bin of the extinction session (E1) (P < 0.001) as compared with pre-shock levels of freezing (Bonferroni post hoc test). In the 10 min extinction session both genotypes exhibited similar freezing behavior (two-way repeated measures ANOVA; interaction: F(3,48) = 2.01; p = 0.13); extinction time: F(3,48) = 6.22; P < 0.0012; genotype: F(1,48) = 1.6; P = 0.22). # P < 0.01 indicates a significant difference from the first bin of the extinction session for both genotypes (Bonferroni post hoc test), †P < 0.05 indicates a significant difference between WT and SphK2-/- mice on test day 3 (Bonferroni post hoc test). N = 9 per group. Data are presented as mean ± s.e.m.

Supplementary Figure 9 Anxiety-like behavior, exploratory activity, and performance in the cued fear conditioned tests were similar in WT and Sphk2–/– mice.

(a-c) WT and SphK2-/- mice show similar responses in the marble-burying test and for exploratory behavior in a novel environment. (a) No genotype differences were found for the number of marbles buried (unpaired t-test: t(1,17) = 0.73; P = 0.48), as well as (b) for the distance traveled (unpaired t-test: t(1,17) = 0.19; P = 0.86) and (c) time spent thigmotaxic in a novel environment (unpaired t-test: t(1,17) = 1.73; P = 0.10). (d) WT and SphK2-/- mice were evaluated in the Light/Dark Box test to assess anxiety-like behavior. Both genotypes spent significantly more time in the dark compartment than the light compartment (paired Student's t test: WT: t(1,8) = 3.91; P < 0.01; SphK2-/-: t(1,8) = 5.69; P< 0.01), but did not differ between each other in the dark (unpaired t-test: t(1,16) = 1.43; P = 0.17) or light (unpaired Student's t test: t(1,16) = 1.43; P = 0.17) compartments. (e) WT and SphK2-/- mice displayed similar freezing behavior and extinction in tone-paired conditioning paradigm. WT and SphK2-/- mice had similar levels of freezing during the 3 min prior to tone-shock pairing (pre-shock), 60 s after tone-shock paring (post-shock), and during 200 s tone presentation without shock 24 h after conditioning (day 2) (two-way repeated measures ANOVA; interaction: F(2,34) = 2.03; P = 0.15; time: F(2,34) = 15.8; P < 0.0001; genotype: F(1,34) = 3.0; P = 0.10). *P < 0.001, vs. pre-shock (Bonferroni post hoc test). Both genotypes exhibited similar decreases in freezing across the six days of extinction training (two-way ANOVA; interaction: F(5,85) = 0.52; P = 0.76; genotype: F(1,85) = 1.52; P = 0.23; test day: F(5,85) = 13.8; P < 0.0001). P < 0.001 for days 6 and 7 as compared to day 2 (Bonferroni post hoc test). Tests in (a-d) were performed in the same cohort of mice. N = 9-10 mice/group. Data are expressed as mean ± s.e.m.

Supplementary Figure 10 Full-length images of the blots presented in the main figures.

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Hait, N., Wise, L., Allegood, J. et al. Active, phosphorylated fingolimod inhibits histone deacetylases and facilitates fear extinction memory. Nat Neurosci 17, 971–980 (2014). https://doi.org/10.1038/nn.3728

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