Crybb2 Mutations Consistently Affect Schizophrenia Endophenotypes in Mice

  • Tamara Heermann
  • Lillian Garrett
  • Wolfgang Wurst
  • Helmut Fuchs
  • Valerie Gailus-Durner
  • Martin Hrabě de Angelis
  • Jochen Graw
  • Sabine M. HölterEmail author


As part of the βγ-superfamily, βB2-crystallin (CRYBB2) is an ocular structural protein in the lens, and mutation of the corresponding gene can cause cataracts. CRYBB2 also is expressed in non-lens tissue such as the adult mouse brain and is associated with neuropsychiatric disorders such as schizophrenia. Nevertheless, the robustness of this association as well as how CRYBB2 may contribute to disease-relevant phenotypes is unknown. To add further clarity to this issue, we performed a comprehensive analysis of behavioral and neurohistological alterations in mice with an allelic series of mutations in the C-terminal end of the Crybb2 gene. Behavioral phenotyping of these three βB2-mutant lines Crybb2O377, Crybb2Philly, and Crybb2Aey2 included assessment of exploratory activity and anxiety-related behavior in the open field, sensorimotor gating measured by prepulse inhibition (PPI) of the acoustic startle reflex, cognitive performance measured by social discrimination, and spontaneous alternation in the Y-maze. In each mutant line, we also quantified the number of parvalbumin-positive (PV+) GABAergic interneurons in selected brain regions that express CRYBB2. While there were allele-specific differences in individual behaviors and affected brain areas, all three mutant lines exhibited consistent alterations in PPI that paralleled alterations in the PV+ cell number in the thalamic reticular nucleus (TRN). The direction of the PPI change mirrored that of the TRN PV+ cell number thereby suggesting a role for TRN PV+ cell number in modulating PPI. Moreover, as both altered PPI and PV+ cell number are schizophrenia-associated endophenotypes, our result implicates mutated Crybb2 in the development of this neuropsychiatric disorder.


Crybb2 Schizophrenia Parvalbumin Prepulse inhibition (PPI) Thalamic reticular nucleus (TRN) 



anterior cingulate cortex


acoustic startle response


cornu ammonis area 1–3






dentate gyrus


open field




prepulse inhibition


granular retrosplenial cortex


social discrimination


thalamic reticular nucleus


quantitative trait loci



The authors thank Jan Einicke and Bettina Sperling as well as Erika Bürkle and Monika Stadler for expert technical assistance.

Authors’ Contributions

TH made contributions to conceptualization, methodology, formal analysis, writing (original draft), and visualization. LG made contributions to conceptualization, methodology, formal analysis, supervision, and writing (original draft). JG made contributions to conceptualization, resources, and writing (review and editing). VGD, HF, and MHdA contributed to conceptualization, methodology, and supervision of experiments at the German Mouse Clinic. WW and SMH contributed to conceptualization, resources, supervision, formal analysis, writing (original draft), and funding acquisition.


This work has been funded by the German Federal Ministry of Education and Research to the GMC (Infrafrontier grant 01KX1012), to the German Center for Diabetes Research (DZD e.V.), the German Federal Ministry of Education and Research (BMBF) through the Integrated Network MitoPD (Mitochondrial endophenotypes of Morbus Parkinson), under the auspices of the e:Med Programme (grant 031A430E) as well as by the DFG grant ‘DJ-1 Linked Neurodegeneration Pathways in New Mouse Models of Parkinson’s Disease’ (WU 164/5-1) to WW.

Compliance with Ethical Standards

Ethics Approval and Consent to Participate

This animal work was approved ethically by the Regierung von Oberbayern in Germany.

Competing Interests


Supplementary material

12035_2018_1365_MOESM1_ESM.docx (58 kb)
ESM 1 (DOCX 58 kb)


  1. 1.
    Ganguly K, Favor J, Neuhauser-Klaus A, Sandulache R, Puk O, Beckers J, Horsch M, Schadler S et al (2008) Novel allele of crybb2 in the mouse and its expression in the brain. Invest Ophthalmol Vis Sci 49(4):1533–1541. CrossRefPubMedGoogle Scholar
  2. 2.
    Magabo KS, Horwitz J, Piatigorsky J, Kantorow M (2000) Expression of βB(2)-crystallin mRNA and protein in retina, brain, and testis. Invest Ophthalmol Vis Sci 41(10):3056–3060PubMedPubMedCentralGoogle Scholar
  3. 3.
    Andley UP (2007) Crystallins in the eye: function and pathology. Prog Retin Eye Res 26(1):78–98. CrossRefPubMedGoogle Scholar
  4. 4.
    Sun M, Holter SM, Stepan J, Garrett L, Genius J, Kremmer E, Hrabe de Angelis M, Wurst W et al (2013) Crybb2 coding for βB2-crystallin affects sensorimotor gating and hippocampal function. Mamm Genome 24(9–10):333–348. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Graw J (2009) Genetics of crystallins: cataract and beyond. Exp Eye Res 88(2):173–189. CrossRefPubMedGoogle Scholar
  6. 6.
    Jobby MK, Sharma Y (2007) Calcium-binding to lens βB2- and βA3-crystallins suggests that all β-crystallins are calcium-binding proteins. FEBS J 274(16):4135–4147. CrossRefPubMedGoogle Scholar
  7. 7.
    Srivastava SS, Mishra A, Krishnan B, Sharma Y (2014) Ca2+-binding motif of βγ-crystallins. J Biol Chem 289(16):10958–10966. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Zhou Y, Zhai Y, Huang L, Gong B, Li J, Hao F, Wu Z, Shi Y et al (2016) A novel CRYBB2 Stopgain mutation causing congenital autosomal dominant cataract in a Chinese family. Am J Ophthalmol 2016:4353957. CrossRefGoogle Scholar
  9. 9.
    Weisschuh N, Aisenbrey S, Wissinger B, Riess A (2012) Identification of a novel CRYBB2 missense mutation causing congenital autosomal dominant cataract. Mol Vis 18:174–180PubMedPubMedCentralGoogle Scholar
  10. 10.
    Kador PF, Fukui HN, Fukushi S, Jernigan HM Jr, Kinoshita JH (1980) Philly mouse: a new model of hereditary cataract. Exp Eye Res 30(1):59–68CrossRefGoogle Scholar
  11. 11.
    Bateman JB, von-Bischhoffshaunsen FR, Richter L, Flodman P, Burch D, Spence MA (2007) Gene conversion mutation in crystallin, βB2 (CRYBB2) in a Chilean family with autosomal dominant cataract. Ophthalmology 114(3):425–432. CrossRefPubMedGoogle Scholar
  12. 12.
    Graw J, Loster J, Soewarto D, Fuchs H, Reis A, Wolf E, Balling R, Hrabe de Angelis M (2001) Aey2, a new mutation in the βB2-crystallin-encoding gene of the mouse. Invest Ophthalmol Vis Sci 42(7):1574–1580PubMedGoogle Scholar
  13. 13.
    Pauli S, Soker T, Klopp N, Illig T, Engel W, Graw J (2007) Mutation analysis in a German family identified a new cataract-causing allele in the CRYBB2 gene. Mol Vis 13:962–967PubMedPubMedCentralGoogle Scholar
  14. 14.
    Santhiya ST, Kumar GS, Sudhakar P, Gupta N, Klopp N, Illig T, Soker T, Groth M et al (2010) Molecular analysis of cataract families in India: new mutations in the CRYBB2 and GJA3 genes and rare polymorphisms. Mol Vis 16:1837–1847PubMedPubMedCentralGoogle Scholar
  15. 15.
    Geyer MA, Krebs-Thomson K, Braff DL, Swerdlow NR (2001) Pharmacological studies of prepulse inhibition models of sensorimotor gating deficits in schizophrenia: a decade in review. Psychopharmacology 156(2–3):117–154CrossRefGoogle Scholar
  16. 16.
    Swerdlow NR, Braff DL, Taaid N, Geyer MA (1994) Assessing the validity of an animal model of deficient sensorimotor gating in schizophrenic patients. Arch Gen Psychiatry 51(2):139–154CrossRefGoogle Scholar
  17. 17.
    Kumari V, Soni W, Mathew VM, Sharma T (2000) Prepulse inhibition of the startle response in men with schizophrenia: effects of age of onset of illness, symptoms, and medication. Arch Gen Psychiatry 57(6):609–614CrossRefGoogle Scholar
  18. 18.
    Mena A, Ruiz-Salas JC, Puentes A, Dorado I, Ruiz-Veguilla M, De la Casa LG (2016) Reduced prepulse inhibition as a biomarker of schizophrenia. Front Behav Neurosci 10:202. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Hammer TB, Oranje B, Fagerlund B, Bro H, Glenthøj BY (2011) Stability of prepulse inhibition and habituation of the startle reflex in schizophrenia: a 6-year follow-up study of initially antipsychotic-naive, first-episode schizophrenia patients. Int J Neuropsychopharmacol 14(7):913–925. CrossRefPubMedGoogle Scholar
  20. 20.
    Nakazawa K, Zsiros V, Jiang Z, Nakao K, Kolata S, Zhang S, Belforte JE (2012) GABAergic interneuron origin of schizophrenia pathophysiology. Neuropharmacol 62(3):1574–1583. CrossRefGoogle Scholar
  21. 21.
    Heckers S, Konradi C (2010) Hippocampal pathology in schizophrenia. Curr Top Behav Neurosci 4:529–553CrossRefGoogle Scholar
  22. 22.
    Konradi C, Yang CK, Zimmerman EI, Lohmann KM, Gresch P, Pantazopoulos H, Berretta S, Heckers S (2011) Hippocampal interneurons are abnormal in schizophrenia. Schizophr Res 131(1–3):165–173. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Kalus P, Senitz D, Beckmann H (1997) Altered distribution of parvalbumin-immunoreactive local circuit neurons in the anterior cingulate cortex of schizophrenic patients. Psychiatry Res 75(1):49–59CrossRefGoogle Scholar
  24. 24.
    Borkowska M, Millar JK, Price DJ (2016) Altered disrupted-in-schizophrenia-1 function affects the development of cortical parvalbumin interneurons by an indirect mechanism. PLoS One 11(5):e0156082. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Cotter D, Landau S, Beasley C, Stevenson R, Chana G, MacMillan L, Everall I (2002) The density and spatial distribution of GABAergic neurons, labelled using calcium binding proteins, in the anterior cingulate cortex in major depressive disorder, bipolar disorder, and schizophrenia. Biol Psychiatry 51(5):377–386CrossRefGoogle Scholar
  26. 26.
    Kim Y, Xia K, Tao R, Giusti-Rodriguez P, Vladimirov V, van den Oord E, Sullivan PF (2014) A meta-analysis of gene expression quantitative trait loci in brain. Transl Psychiatry 4:e459. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Powell CM, Miyakawa T (2006) Schizophrenia-relevant behavioral testing in rodent models: a uniquely human disorder? Biol Psychiatry 59(12):1198–1207. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    DuPrey KM, Robinson KM, Wang Y, Taube JR, Duncan MK (2007) Subfertility in mice harboring a mutation in βB2-crystallin. Mol Vis 13:366–373PubMedPubMedCentralGoogle Scholar
  29. 29.
    Holter SM, Garrett L, Einicke J, Sperling B, Dirscherl P, Zimprich A, Fuchs H, Gailus-Durner V et al (2015) Assessing cognition in mice. Curr Protoc Mouse Biol 5(4):331–358. CrossRefPubMedGoogle Scholar
  30. 30.
    Wall PM, Blanchard RJ, Yang M, Blanchard DC (2003) Infralimbic D2 receptor influences on anxiety-like behavior and active memory/attention in CD-1 mice. Prog Neuro-Psychopharmacol Biol Psychiatry 27(3):395–410. CrossRefGoogle Scholar
  31. 31.
    Garrett L, Zhang J, Zimprich A, Niedermeier KM, Fuchs H, Gailus-Durner V, Hrabě de Angelis M, Vogt Weisenhorn D et al (2015) Conditional reduction of adult born doublecortin-positive neurons reversibly impairs selective behaviors. Front Behav Neurosci 9:302. CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    West MJ, Slomianka L, Gundersen HJ (1991) Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator. Anat Rec 231(4):482–497. CrossRefPubMedGoogle Scholar
  33. 33.
    Schmitz C, Hof PR (2005) Design-based stereology in neuroscience. Neuroscience 130(4):813–831. CrossRefPubMedGoogle Scholar
  34. 34.
    Franklin K, Paxinos G (1997) The mouse brain in stereotaxic coordinates. Academic PressGoogle Scholar
  35. 35.
    Roy A, Kucukural A, Zhang Y (2010) I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5(4):725–738. CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y (2015) The I-TASSER suite: protein structure and function prediction. Nat Methods 12(1):7–8. CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9:40. CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Crawley JN (2008) Behavioral phenotyping strategies for mutant mice. Neuron 57(6):809–818. CrossRefPubMedGoogle Scholar
  39. 39.
    Beauquis J, Roig P, Homo-Delarche F, De Nicola A, Saravia F (2006) Reduced hippocampal neurogenesis and number of hilar neurones in streptozotocin-induced diabetic mice: reversion by antidepressant treatment. Eur J Neurosci 23(6):1539–1546. CrossRefPubMedGoogle Scholar
  40. 40.
    Popelář J, Rybalko N, Burianová J, Schwaller B, Syka J (2013) The effect of parvalbumin deficiency on the acoustic startle response and prepulse inhibition in mice. Neurosci Lett 553:216–220. CrossRefPubMedGoogle Scholar
  41. 41.
    Ferrarelli F, Tononi G (2011) The thalamic reticular nucleus and schizophrenia. Schizophr Bull 37(2):306–315. CrossRefPubMedGoogle Scholar
  42. 42.
    Vann SD, Aggleton JP, Maguire EA (2009) What does the retrosplenial cortex do? Nat Rev Neurosci 10(11):792–802CrossRefGoogle Scholar
  43. 43.
    Wright NF, Erichsen JT, Vann SD, O’Mara S, Aggleton JP (2010) Parallel but separate inputs from limbic cortices to the mammillary bodies and anterior thalamic nuclei in the rat. J Comp Neurol 518(12):2334–2354. CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Brown JA, Ramikie TS, Schmidt MJ, Baldi R, Garbett K, Everheart MG, Warren LE, Gellert L et al (2015) Inhibition of parvalbumin-expressing interneurons results in complex behavioral changes. Mol Psychiatry 20(12):1499–1507. CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Leppä E, Linden A-M, Vekovischeva OY, Swinny JD, Rantanen V, Toppila E, Höger H, Sieghart W et al (2011) Removal of GABA(A) receptor γ2 subunits from parvalbumin neurons causes wide-ranging behavioral alterations. PLoS One 6(9):e24159. CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Wells MF, Wimmer RD, Schmitt LI, Feng G, Halassa MM (2016) Thalamic reticular impairment underlies attention deficit in Ptchd1(Y/−) mice. Nature 532(7597):58–63. CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Maren S, Holt WG (2004) Hippocampus and Pavlovian fear conditioning in rats: muscimol infusions into the ventral, but not dorsal, hippocampus impair the acquisition of conditional freezing to an auditory conditional stimulus. Behav Neurosci 118(1):97–110. CrossRefPubMedGoogle Scholar
  48. 48.
    Hartings JA, Temereanca S, Simons DJ (2003) State-dependent processing of sensory stimuli by thalamic reticular neurons. J Neurosci 23(12):5264–5271CrossRefGoogle Scholar
  49. 49.
    McAlonan K, Cavanaugh J, Wurtz RH (2006) Attentional modulation of thalamic reticular neurons. J Neurosci 26(16):4444–4450. CrossRefPubMedGoogle Scholar
  50. 50.
    Koch M, Kungel M, Herbert H (1993) Cholinergic neurons in the pedunculopontine tegmental nucleus are involved in the mediation of prepulse inhibition of the acoustic startle response in the rat. Exp Brain Res 97(1):71–82CrossRefGoogle Scholar
  51. 51.
    Beierlein M (2014) Synaptic mechanisms underlying cholinergic control of thalamic reticular nucleus neurons. J Physiol 592(19):4137–4145. CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Sokhadze G, Campbell PW, Guido W (2018) Postnatal development of cholinergic input to the thalamic reticular nucleus of the mouse. Eur J Neurosci.
  53. 53.
    Harris KD, Thiele A (2011) Cortical state and attention. Nat Rev Neurosci 12(9):509–523. CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Schmitt LI, Halassa MM (2017) Interrogating the mouse thalamus to correct human neurodevelopmental disorders. Mol Psychiatry 22(2):183–191. CrossRefPubMedGoogle Scholar
  55. 55.
    Steullet P, Cabungcal JH, Bukhari SA, Ardelt MI, Pantazopoulos H, Hamati F, Salt TE, Cuenod M et al (2017) The thalamic reticular nucleus in schizophrenia and bipolar disorder: role of parvalbumin-expressing neuron networks and oxidative stress. Mol Psychiatry.
  56. 56.
    Schmalbach B, Lepsveridze E, Djogo N, Papashvili G, Kuang F, Leshchyns’ka I, Sytnyk V, Nikonenko AG et al (2015) Age-dependent loss of parvalbumin-expressing hippocampal interneurons in mice deficient in CHL1, a mental retardation and schizophrenia susceptibility gene. J Neurochem 135(4):830–844. CrossRefPubMedGoogle Scholar
  57. 57.
    Wu YC, Du X, van den Buuse M, Hill RA (2014) Sex differences in the adolescent developmental trajectory of parvalbumin interneurons in the hippocampus: a role for estradiol. Psychoneuroendocrinology 45:167–178. CrossRefPubMedGoogle Scholar
  58. 58.
    Ravenelle R, Berman AK, La J, Mason B, Asumadu E, Yelleswarapu C, Donaldson ST (2018) Sex matters: females in proestrus show greater diazepam anxiolysis and brain-derived neurotrophin factor- and parvalbumin-positive neurons than males. Eur J Neurosci 47(8):994–1002. CrossRefPubMedGoogle Scholar
  59. 59.
    Rowniak M, Bogus-Nowakowska K, Robak A (2015) The densities of calbindin and parvalbumin, but not calretinin neurons, are sexually dimorphic in the amygdala of the guinea pig. Brain Res 1604:84–97. CrossRefPubMedGoogle Scholar
  60. 60.
    Wischhof L, Irrsack E, Osorio C, Koch M (2015) Prenatal LPS-exposure--a neurodevelopmental rat model of schizophrenia--differentially affects cognitive functions, myelination and parvalbumin expression in male and female offspring. Prog Neuro-Psychopharmacol Biol Psychiatry 57:17–30. CrossRefGoogle Scholar
  61. 61.
    Holland FH, Ganguly P, Potter DN, Chartoff EH, Brenhouse HC (2014) Early life stress disrupts social behavior and prefrontal cortex parvalbumin interneurons at an earlier time-point in females than in males. Neurosci Lett 566:131–136. CrossRefPubMedPubMedCentralGoogle Scholar
  62. 62.
    Leussis MP, Freund N, Brenhouse HC, Thompson BS, Andersen SL (2012) Depressive-like behavior in adolescents after maternal separation: sex differences, controllability, and GABA. Dev Neurosci 34(2–3):210–217. CrossRefPubMedPubMedCentralGoogle Scholar
  63. 63.
    Vendra VPR, Agarwal G, Chandani S, Talla V, Srinivasan N, Balasubramanian D (2013) Structural integrity of the Greek key motif in βγ-crystallins is vital for central eye lens transparency. PLoS One 8(8):e70336. CrossRefPubMedPubMedCentralGoogle Scholar
  64. 64.
    Moreau KL, King JA (2012) Protein misfolding and aggregation in cataract disease and prospects for prevention. Trends Mol Med 18(5):273–282. CrossRefPubMedPubMedCentralGoogle Scholar
  65. 65.
    Liu BF, Liang JJ (2006) Domain interaction sites of human lens βB2-crystallin. J Biol Chem 281(5):2624–2630. CrossRefPubMedGoogle Scholar
  66. 66.
    Lee SH, Schwaller B, Neher E (2000) Kinetics of Ca2+ binding to parvalbumin in bovine chromaffin cells: implications for [Ca2+] transients of neuronal dendrites. J Physiol 525(Pt 2):419–432CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Tamara Heermann
    • 1
    • 2
  • Lillian Garrett
    • 1
    • 3
  • Wolfgang Wurst
    • 1
    • 4
    • 5
    • 6
  • Helmut Fuchs
    • 3
  • Valerie Gailus-Durner
    • 3
  • Martin Hrabě de Angelis
    • 3
    • 7
    • 8
  • Jochen Graw
    • 1
  • Sabine M. Hölter
    • 1
    • 3
    Email author
  1. 1.Institute of Developmental GeneticsHelmholtz Zentrum München, German Research Centre for Environmental HealthNeuherbergGermany
  2. 2.Max Planck Institute of BiochemistryMunichGermany
  3. 3.German Mouse Clinic, Institute of Experimental GeneticsHelmholtz Zentrum München, German Research Centre for Environmental HealthNeuherbergGermany
  4. 4.Developmental GeneticsTechnische Universität München- Weihenstephan, c/o Helmholtz Zentrum MünchenMunichGermany
  5. 5.German Centre of Neurodegenerative Diseases (DZNE)MunichGermany
  6. 6.Munich Cluster of Systems Neurology (SyNergy)Adolf-Butenandt-Institut, Ludwig-Maximilians-Universität MünchenMunichGermany
  7. 7.Experimental Genetics, School of Life Science WeihenstephanTechnische Universität MünchenFreisingGermany
  8. 8.German Center for Diabetes Research (DZD)NeuherbergGermany

Personalised recommendations