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Effects of the Cc2d1a/Freud-1 Knockdown in the Hippocampus of BTBR Mice on the Autistic-Like Behavior, Expression of Serotonin 5-HT1A and D2 Dopamine Receptors, and CREB and NF-kB Intracellular Signaling

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Abstract

The mechanisms of autism are of extreme interest due to the high prevalence of this disorder in the human population. In this regard, special attention is given to the transcription factor Freud-1 (encoded by the Cc2d1a gene), which regulates numerous intracellular signaling pathways and acts as a silencer for 5-HT1A serotonin and D2 dopamine receptors. Disruption of the Freud-1 functions leads to the development of various psychopathologies. In this study, we found an increase in the expression of the Cc2d1a/Freud-1 gene in the hippocampus of BTBR mice (model of autistic-like behavior) in comparison with C57Bl/6J mice and examined how restoration of the Cc2d1a/Freud-1 expression in the hippocampus of BTBR mice affects their behavior, expression of 5-HT1A serotonin and D2 dopamine receptors, and CREB and NF-κB intracellular signaling pathways in these animals. Five weeks after administration of the adeno-associated viral vector (AAV) carrying the pAAV_H1-2_shRNA-Freud-1_Syn_EGFP plasmid encoding a small hairpin RNA (shRNA) that suppressed expression of the Cc2d1a/Freud-1 gene, we observed an elevation in the anxiety levels, as well as the increase in the escape latency and path length to the platform in the Morris water maze test, which was probably associated with a strengthening of the active stress avoidance strategy. However, the Cc2d1a/Freud-1 knockdown did not affect the spatial memory and phosphorylation of the CREB transcription factor, although such effect was found in C57Bl/6J mice in our previous study. These results suggest the impairments in the CREB-dependent effector pathway in BTBR mice, which may play an important role in the development of the autistic-like phenotype. The knockdown of Cc2d1a/Freud-1 in the hippocampus of BTBR mice did not affect expression of the 5-HT1A serotonin and D2 dopamine receptors and key NF-κB signaling genes (Nfkb1 and Rela). Our data suggest that the transcription factor Freud-1 plays a significant role in the pathogenesis of anxiety and active stress avoidance in autism.

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Abbreviations

AAV:

adeno-associated viral vector

ASD:

autism spectrum disorder

Cc2d1a/Freud-1:

Cc2d1a gene encoding the transcription factor Freud-1

EGFP:

enhanced green fluorescent protein

References

  1. Christensen, D. L., Baio, J., Van Naarden Braun, K., Bilder, D., Charles, J., et al. (2016) Prevalence and characteristics of autism spectrum disorder among children aged 8 years – autism and developmental disabilities monitoring network, 11 Sites, United States, 2012, Morbid. Mortal. Weekly Rep. Surveill. Summaries, 65, 1-23, https://doi.org/10.15585/mmwr.ss6503a1.

    Article  Google Scholar 

  2. Alexander, A. L., Lee, J. E., Lazar, M., Boudos, R., DuBray, M. B., et al. (2007) Diffusion tensor imaging of the corpus callosum in autism, NeuroImage, 34, 61-73, https://doi.org/10.1016/j.neuroimage.2006.08.032.

    Article  PubMed  Google Scholar 

  3. Ecker, C., Suckling, J., Deoni, S. C., Lombardo, M. V., Bullmore, E. T., et al. (2012) Brain anatomy and its relationship to behavior in adults with autism spectrum disorder: a multicenter magnetic resonance imaging study, Arch. Gen. Psychiatry, 69, 195-209, https://doi.org/10.1001/archgenpsychiatry.2011.1251.

    Article  PubMed  Google Scholar 

  4. Hoischen, A., Krumm, N., and Eichler, E. E. (2014) Prioritization of neurodevelopmental disease genes by discovery of new mutations, Nat. Neurosci., 17, 764-772, https://doi.org/10.1038/nn.3703.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Lainhart, J. E. (2006) Advances in autism neuroimaging research for the clinician and geneticist, Am. J. Med. Genet. C Semin. Med. Genet., 142C, 33-39, https://doi.org/10.1002/ajmg.c.30080.

    Article  PubMed  Google Scholar 

  6. Basel-Vanagaite, L., Attia, R., Yahav, M., Ferland, R. J., Anteki, L., et al. (2006) The CC2D1A, a member of a new gene family with C2 domains, is involved in autosomal recessive non-syndromic mental retardation, J. Med. Genet., 43, 203-210, https://doi.org/10.1136/jmg.2005.035709.

    Article  CAS  PubMed  Google Scholar 

  7. Nakamura, A., Naito, M., Tsuruo, T., and Fujita, N. (2008) Freud-1/Aki1, a novel PDK1-interacting protein, functions as a scaffold to activate the PDK1/Akt pathway in epidermal growth factor signaling, Mol. Cell. Biol., 28, 5996-6009, https://doi.org/10.1128/MCB.00114-08.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Zamarbide, M., Mossa, A., Munoz-Llancao, P., Wilkinson, M. K., Pond, H. L., et al. (2019) Male-specific cAMP signaling in the hippocampus controls spatial memory deficits in a mouse model of autism and intellectual disability, Biol. Psychiatry, 85, 760-768, https://doi.org/10.1016/j.biopsych.2018.12.013.

    Article  CAS  PubMed  Google Scholar 

  9. Oeckinghaus, A., and Ghosh, S. (2009) The NF-kappaB family of transcription factors and its regulation, Cold Spring Harb. Perspect. Biol., 1, a000034, https://doi.org/10.1101/cshperspect.a000034.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Liao, X., and Li, Y. (2020) Nuclear factor kappa B in autism spectrum disorder: a systematic review, Pharmacol. Res., 159, 104918, https://doi.org/10.1016/j.phrs.2020.104918.

    Article  CAS  PubMed  Google Scholar 

  11. El-Ansary, A., and Al-Ayadhi, L. (2012) Neuroinflammation in autism spectrum disorders, J. Neuroinflamm., 9, 265, https://doi.org/10.1186/1742-2094-9-265.

    Article  CAS  Google Scholar 

  12. Matta, S. M., Hill-Yardin, E. L., and Crack, P. J. (2019) The influence of neuroinflammation in autism spectrum disorder, Brain Behav. Immun., 79, 75-90, https://doi.org/10.1016/j.bbi.2019.04.037.

    Article  PubMed  Google Scholar 

  13. Theoharides, T. C., Asadi, S., and Patel, A. B. (2013) Focal brain inflammation and autism, J. Neuroinflamm., 10, 815, https://doi.org/10.1186/1742-2094-10-46.

    Article  CAS  Google Scholar 

  14. Young, A. M., Chakrabarti, B., Roberts, D., Lai, M. C., Suckling, J., et al. (2016) From molecules to neural morphology: understanding neuroinflammation in autism spectrum condition, Mol. Autism, 7, 9, https://doi.org/10.1186/s13229-016-0068-x.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Impey, S., McCorkle, S. R., Cha-Molstad, H., Dwyer, J. M., Yochum, G. S., et al. (2004) Defining the CREB regulon: a genome-wide analysis of transcription factor regulatory regions, Cell, 119, 1041-1054, https://doi.org/10.1016/j.cell.2004.10.032.

    Article  CAS  PubMed  Google Scholar 

  16. Zamarbide, M., Oaks, A. W., Pond, H. L., Adelman, J. S., and Manzini, M. C. (2018) Loss of the intellectual disability and autism gene Cc2d1a and its homolog Cc2d1b differentially affect spatial memory, anxiety, and hyperactivity, Front. Genet., 9, 65, https://doi.org/10.3389/fgene.2018.00065.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ou, X. M., Lemonde, S., Jafar-Nejad, H., Bown, C. D., Goto, A., et al. (2003) Freud-1: A neuronal calcium-regulated repressor of the 5-HT1A receptor gene, J. Neurosci., 23, 7415-7425.

    Article  CAS  Google Scholar 

  18. Al-Tawashi, A., Jung, S. Y., Liu, D., Su, B., and Qin, J. (2012) Protein implicated in nonsyndromic mental retardation regulates protein kinase A (PKA) activity, J. Biol. Chem., 287, 14644-14658, https://doi.org/10.1074/jbc.M111.261875.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhao, M., Raingo, J., Chen, Z. J., and Kavalali, E. T. (2011) Cc2d1a, a C2 domain containing protein linked to nonsyndromic mental retardation, controls functional maturation of central synapses, J. Neurophysiol., 105, 1506-1515, https://doi.org/10.1152/jn.00950.2010.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Chen, K. R., Chang, C. H., Huang, C. Y., Lin, C. Y., Lin, W. Y., et al. (2012) TBK1-associated protein in endolysosomes (TAPE)/CC2D1A is a key regulator linking RIG-I-like receptors to antiviral immunity, J. Biol. Chem., 287, 32216-32221, https://doi.org/10.1074/jbc.C112.394346.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Vahid-Ansari, F., Daigle, M., Manzini, M. C., Tanaka, K. F., Hen, R., et al. (2017) Abrogated Freud-1/Cc2d1a repression of 5-HT1A autoreceptors induces fluoxetine-resistant anxiety/depression-like behavior, J. Neurosci., 37, 11967-11978, https://doi.org/10.1523/JNEUROSCI.1668-17.2017.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kondaurova, E. M., Plyusnina, A. V., Ilchibaeva, T. V., Eremin, D. V., Rodnyy, A. Y., et al. (2021) Effects of a Cc2d1a/Freud-1 knockdown in the hippocampus on behavior, the serotonin system, and BDNF, Int. J. Mol. Sci., 22, 13319 https://doi.org/10.3390/ijms222413319.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Farook, M. F., DeCuypere, M., Hyland, K., Takumi, T., LeDoux, M. S., et al. (2012) Altered serotonin, dopamine and norepinepherine levels in 15q duplication and Angelman syndrome mouse models, PLoS One, 7, e43030, https://doi.org/10.1371/journal.pone.0043030.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Faraji, J., Karimi, M., Lawrence, C., Mohajerani, M. H., and Metz, G. A. S. (2018) Non-diagnostic symptoms in a mouse model of autism in relation to neuroanatomy: the BTBR strain reinvestigated, Translat. Psychiatry, 8, 234, https://doi.org/10.1038/s41398-018-0280-x.

    Article  Google Scholar 

  25. Lemonde, S., Turecki, G., Bakish, D., Du, L., Hrdina, P. D., et al. (2003) Impaired repression at a 5-hydroxytryptamine 1A receptor gene polymorphism associated with major depression and suicide, J. Neurosci., 23, 8788-8799, https://doi.org/10.1523/JNEUROSCI.23-25-08788.2003.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Rogaeva, A., Ou, X. M., Jafar-Nejad, H., Lemonde, S., and Albert, P. R. (2007) Differential repression by freud-1/CC2D1A at a polymorphic site in the dopamine-D2 receptor gene, J. Biol. Chem., 282, 20897-20905, https://doi.org/10.1074/jbc.M610038200.

    Article  CAS  PubMed  Google Scholar 

  27. Ford, C. P. (2014) The role of D2-autoreceptors in regulating dopamine neuron activity and transmission, Neuroscience, 282, 13-22, https://doi.org/10.1016/j.neuroscience.2014.01.025.

    Article  CAS  PubMed  Google Scholar 

  28. Missale, C., Nash, S. R., Robinson, S. W., Jaber, M., and Caron, M. G. (1998) Dopamine receptors: from structure to function, Physiol. Rev., 78, 189-225, https://doi.org/10.1152/physrev.1998.78.1.189.

    Article  CAS  PubMed  Google Scholar 

  29. Stephenson, D. T., O’Neill, S. M., Narayan, S., Tiwari, A., Arnold, E., et al. (2011) Histopathologic characterization of the BTBR mouse model of autistic-like behavior reveals selective changes in neurodevelopmental proteins and adult hippocampal neurogenesis, Mol. Autism, 2, 7, https://doi.org/10.1186/2040-2392-2-7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Gould, G. G., Burke, T. F., Osorio, M. D., Smolik, C. M., Zhang, W. Q., et al. (2014) Enhanced novelty-induced corticosterone spike and upregulated serotonin 5-HT1A and cannabinoid CB1 receptors in adolescent BTBR mice, Psychoneuroendocrinology, 39, 158-169, https://doi.org/10.1016/j.psyneuen.2013.09.003.

    Article  CAS  PubMed  Google Scholar 

  31. Gould, G. G., Hensler, J. G., Burke, T. F., Benno, R. H., Onaivi, E. S., et al. (2011) Density and function of central serotonin (5-HT) transporters, 5-HT1A and 5-HT2A receptors, and effects of their targeting on BTBR T+tf/J mouse social behavior, J. Neurochem., 116, 291-303, https://doi.org/10.1111/j.1471-4159.2010.07104.x.

    Article  CAS  PubMed  Google Scholar 

  32. Guo, Y. P., and Commons, K. G. (2017) Serotonin neuron abnormalities in the BTBR mouse model of autism, Autism Res., 10, 66-77, https://doi.org/10.1002/aur.1665.

    Article  PubMed  Google Scholar 

  33. Wirth, A., Chen-Wacker, C., Wu, Y. W., Gorinski, N., Filippov, M. A., et al. (2013) Dual lipidation of the brain-specific Cdc42 isoform regulates its functional properties, Biochem. J., 456, 311-322, https://doi.org/10.1042/BJ20130788.

    Article  CAS  PubMed  Google Scholar 

  34. Grimm, D., Kay, M. A., and Kleinschmidt, J. A. (2003) Helper virus-free, optically controllable, and two-plasmid-based production of adeno-associated virus vectors of serotypes 1 to 6, Mol. Ther., 7, 839-850, https://doi.org/10.1016/s1525-0016(03)00095-9.

    Article  CAS  PubMed  Google Scholar 

  35. Slotnick, B. M., and Leonard, C. M. (1975) A Stereotaxic Atlas of the Albino Mouse Forebrain, U.S. Dept. of Health, Education and Welfare, Rockville, Maryland.

  36. Khotskin, N. V., Plyusnina, A. V., Kulikova, E. A., Bazhenova, E. Y., Fursenko, D. V., et al. (2019) On association of the lethal yellow (A(Y)) mutation in the agouti gene with the alterations in mouse brain and behavior, Behav. Brain Res., 359, 446-456, https://doi.org/10.1016/j.bbr.2018.11.013.

    Article  CAS  PubMed  Google Scholar 

  37. Kulikov, A. V., Fursenko, D. V., Khotskin, N. V., Bazovkina, D. V., Kulikov, V. A., et al. (2014) Spatial learning in the Morris water maze in mice genetically different in the predisposition to catalepsy: the effect of intraventricular treatment with brain-derived neurotrophic factor, Pharmacol. Biochem. Behav., 122, 266-272, https://doi.org/10.1016/j.pbb.2014.04.009.

    Article  CAS  PubMed  Google Scholar 

  38. Kulikov, A. V., Tikhonova, M. A., and Kulikov, V. A. (2008) Automated measurement of spatial preference in the open field test with transmitted lighting, J. Neurosci. Methods, 170, 345-351, https://doi.org/10.1016/j.jneumeth.2008.01.024.

    Article  PubMed  Google Scholar 

  39. Kulikov, A. V., Naumenko, V. S., Voronova, I. P., Tikhonova, M. A., and Popova, N. K. (2005) Quantitative RT-PCR assay of 5-HT1A and 5-HT2A serotonin receptor mRNAs using genomic DNA as an external standard, J. Neurosci. Methods, 141, 97-101, https://doi.org/10.1016/j.jneumeth.2004.06.005.

    Article  CAS  PubMed  Google Scholar 

  40. Naumenko, V. S., and Kulikov, A. V. (2006) Quantitative assay of 5-HT(1A) serotonin receptor gene expression in the brain, Mol. Biol. (Mosk), 40, 37-44.

    Article  CAS  Google Scholar 

  41. Naumenko, V. S., Osipova, D. V., Kostina, E. V., and Kulikov, A. V. (2008) Utilization of a two-standard system in real-time PCR for quantification of gene expression in the brain, J. Neurosci. Methods, 170, 197-203, https://doi.org/10.1016/j.jneumeth.2008.01.008.

    Article  CAS  PubMed  Google Scholar 

  42. Grove, J., Ripke, S., Als, T. D., Mattheisen, M., Walters, R. K., et al. (2019) Identification of common genetic risk variants for autism spectrum disorder, Nat. Genet., 51, 431-444, https://doi.org/10.1038/s41588-019-0344-8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Manzini, M. C., Xiong, L., Shaheen, R., Tambunan, D. E., Di Costanzo, S., et al. (2014) CC2D1A regulates human intellectual and social function as well as NF-kappaB signaling homeostasis, Cell Rep., 8, 647-655, https://doi.org/10.1016/j.celrep.2014.06.039.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Oaks, A. W., Zamarbide, M., Tambunan, D. E., Santini, E., Di Costanzo, S., et al. (2017) Cc2d1a loss of function disrupts functional and morphological development in forebrain neurons leading to cognitive and social deficits, Cereb. Cortex, 27, 1670-1685, https://doi.org/10.1093/cercor/bhw009.

    Article  PubMed  Google Scholar 

  45. Sener, E. F., Uytun, M. C., Bayramov, K. K., Zararsiz, G., Oztop, D. B., et al. (2016) The roles of CC2D1A and HTR1A gene expressions in autism spectrum disorders, Metab. Brain Dis., 31, 613-619, https://doi.org/10.1007/s11011-016-9795-0.

    Article  CAS  PubMed  Google Scholar 

  46. White, S. W., Oswald, D., Ollendick, T., and Scahill, L. (2009) Anxiety in children and adolescents with autism spectrum disorders, Clin. Psychol. Rev., 29, 216-229, https://doi.org/10.1016/j.cpr.2009.01.003.

    Article  PubMed  PubMed Central  Google Scholar 

  47. Defensor, E. B., Pearson, B. L., Pobbe, R. L., Bolivar, V. J., Blanchard, D. C., et al. (2011) A novel social proximity test suggests patterns of social avoidance and gaze aversion-like behavior in BTBR T+ tf/J mice, Behav. Brain Res., 217, 302-308, https://doi.org/10.1016/j.bbr.2010.10.033.

    Article  PubMed  Google Scholar 

  48. Kida, S., Josselyn, S. A., Pena de Ortiz, S., Kogan, J. H., Chevere, I., et al. (2002) CREB required for the stability of new and reactivated fear memories, Nat. Neurosci., 5, 348-355, https://doi.org/10.1038/nn819.

    Article  CAS  PubMed  Google Scholar 

  49. Sun, S. C. (2011) Non-canonical NF-kappaB signaling pathway, Cell Res., 21, 71-85, https://doi.org/10.1038/cr.2010.177.

    Article  CAS  PubMed  Google Scholar 

  50. Gavalda, N., Gutierrez, H., and Davies, A. M. (2009) Developmental switch in NF-kappaB signalling required for neurite growth, Development, 136, 3405-3412, https://doi.org/10.1242/dev.035295.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Gutierrez, H., O’Keeffe, G. W., Gavalda, N., Gallagher, D., and Davies, A. M. (2008) Nuclear factor kappa B signaling either stimulates or inhibits neurite growth depending on the phosphorylation status of p65/RelA, J. Neurosci., 28, 8246-8256, https://doi.org/10.1523/JNEUROSCI.1941-08.2008.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

The study was conducted at the Center for Genetic Resources of Laboratory Animals; Institute of Cytology and Genetics (RFMEFI62119X0023).

Funding

This article was supported by the Russian Science Foundation (project no. 22-15-00028). Animal maintenance was provided by the Budget Project FWNR-2022-0023.

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Authors and Affiliations

  1. Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 630090, Novosibirsk, Russia

    Irina I. Belokopytova, Elena M. Kondaurova, Elizabeth A. Kulikova, Tatiana V. Ilchibaeva, Vladimir S. Naumenko & Nina K. Popova

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  1. Irina I. Belokopytova
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  2. Elena M. Kondaurova
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  3. Elizabeth A. Kulikova
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  4. Tatiana V. Ilchibaeva
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Contributions

V. S. Naumenko, N. K. Popova, and E. M. Kondaurova developed the study concept and supervised the study; E. A. Kulikova, T. V. Ilchibaeva, V. S. Naumenko, and I. I. Belokopytova conducted the experiments; V. S. Naumenko and E. M. Kondaurova discussed the results; I. I. Belokopytova and V. S. Naumenko wrote the article; V. S. Naumenko, N. K. Popova, and E. M. Kondaurova edited the manuscript.

Corresponding author

Correspondence to Vladimir S. Naumenko.

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The authors declare no conflict of interest. All procedures with experimental animals were conducted in compliance with the international protocols on the treatment of laboratory animals (Directive 2010/63/EU EC) and the Order of the Ministry of Health of the Russian Federation from 01.04.2016 no. 199n “On the establishment of Rules for appropriate laboratory practice” (registered 15.08.2016, no. 43232).

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Belokopytova, I.I., Kondaurova, E.M., Kulikova, E.A. et al. Effects of the Cc2d1a/Freud-1 Knockdown in the Hippocampus of BTBR Mice on the Autistic-Like Behavior, Expression of Serotonin 5-HT1A and D2 Dopamine Receptors, and CREB and NF-kB Intracellular Signaling. Biochemistry Moscow 87, 1206–1218 (2022). https://doi.org/10.1134/S0006297922100145

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