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Cited by 65Genetics and Neurodevelopmental DisordersNeuroscience and Neuropharmacology ResearchNeurogenesis and neuroplasticity mechanisms

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Neural cell adhesion molecule Negr1 deficiency in mouse results in structural brain endophenotypes and behavioral deviations related to psychiatric disorders

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Singh, Katyayani, Jayaram, Mohan, Kaare, Maria, Leidmaa, Este, Jagomäe, Toomas, Heinla, Indrek, Hickey, Miriam A., Kaasik, Allen, Schäfer, Michael K., Innos, Jürgen, Lilleväli, Kersti, Philips, Mari-Anne, Vasar, Eero·2019·Scientific Reports
DOI 10.1038/s41598-019-41991-8PMC PMC6443666
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Abstract

Neuronal growth regulator 1 (NEGR1) belongs to the immunoglobulin (IgLON) superfamily of cell adhesion molecules involved in cortical layering. Recent functional and genomic studies implicate the role of NEGR1 in a wide spectrum of psychiatric disorders, such as major depression, schizophrenia and autism. Here, we investigated the impact of Negr1 deficiency on brain morphology, neuronal properties and social behavior of mice. In situ hybridization shows Negr1 expression in the brain nuclei which are central modulators of cortical-subcortical connectivity such as the island of Calleja and the reticular nucleus of thalamus. Brain morphological analysis revealed neuroanatomical abnormalities in Negr1−/− mice, including enlargement of ventricles and decrease in the volume of the whole brain, corpus callosum, globus pallidus and hippocampus. Furthermore, decreased number of parvalbumin-positive inhibitory interneurons was evident in Negr1−/− hippocampi. Behaviorally, Negr1−/− mice displayed hyperactivity in social interactions and impairments in social hierarchy. Finally, Negr1 deficiency resulted in disrupted neurite sprouting during neuritogenesis. Our results provide evidence that NEGR1 is required for balancing the ratio of excitatory/inhibitory neurons and proper formation of brain structures, which is prerequisite for adaptive behavioral profiles. Therefore, Negr1−/− mice have a high potential to provide new insights into the neural mechanisms of neuropsychiatric disorders.

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Figure 1 Neuroanatomy of Negr1 in adult mouse brain. Expression of Negr1 by in situ hybridisation in adult sagittal brain sections (a,b). OB olfactory bulb, Pir piriform cortex, OT olfactory tubercle, iSI island of Calleja, Th thalamus, Amy amygdala, Sub subiculum, DG dentate gyrus, IGL inter geniculate lateral geniculate complex, CP caudate putamen, MO somatomotor cortex, SS somatosensory cortex, PTLP posterior parietal association area, VIS visual area, Epd endopiriform cortex, HA hypothalamus, Cb cerebellum, RsPD retrosplenial cortex, CLA claustrum, RT reticular nucleus of thalamus, LGD/V dorsal/ventral part of lateral geniculate complex, MG medial geniculate nucleus. Gross cytoarchitecture shown by neurofilament immunostaining in Wt (c), enlarged ventricles in Negr1−/− mice brain (d), LV lateral ventricles. A 3D reconstruction of the Wt (e) and Negr1−/− (f) mice brains and ventricles (green: lateral ventricles, yellow: third ventricle and purple: fourth ventricle) in Wt (g) and Negr1−/− (h) brains. Ventricle enlargement is observed in the Negr1−/− mice. Quantitative analysis of effects of Negr1 deletion in mice on the volume of total brain (i), lateral ventricles (j), third ventricles (k), corpus callosum (l), globus pallidus (m) and in hippocampus (n). Data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, Mann-Whitney U test (Wilcoxon rank sum test).
Figure 1 Neuroanatomy of Negr1 in adult mouse brain. Expression of Negr1 by in situ hybridisation in adult sagittal brain sections (a,b). OB olfactory bulb, Pir piriform cortex, OT olfactory tubercle, iSI island of Calleja, Th thalamus, Amy amygdala, Sub subiculum, DG dentate gyrus, IGL inter geniculate lateral geniculate complex, CP caudate putamen, MO somatomotor cortex, SS somatosensory cortex, PTLP posterior parietal association area, VIS visual area, Epd endopiriform cortex, HA hypothalamus, Cb cerebellum, RsPD retrosplenial cortex, CLA claustrum, RT reticular nucleus of thalamus, LGD/V dorsal/ventral part of lateral geniculate complex, MG medial geniculate nucleus. Gross cytoarchitecture shown by neurofilament immunostaining in Wt (c), enlarged ventricles in Negr1−/− mice brain (d), LV lateral ventricles. A 3D reconstruction of the Wt (e) and Negr1−/− (f) mice brains and ventricles (green: lateral ventricles, yellow: third ventricle and purple: fourth ventricle) in Wt (g) and Negr1−/− (h) brains. Ventricle enlargement is observed in the Negr1−/− mice. Quantitative analysis of effects of Negr1 deletion in mice on the volume of total brain (i), lateral ventricles (j), third ventricles (k), corpus callosum (l), globus pallidus (m) and in hippocampus (n). Data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, Mann-Whitney U test (Wilcoxon rank sum test).CC BY · PubMed Central
Figure 2 Reduced number of parvalbumin (PV) positive interneurons in the hippocampus of Negr1−/− mice. Representative confocal images of the sagittal section of brains of Wt (a–d) and Negr1−/− (e–j) mice for NeuN (red), PV (green) and DAPI (blue) immunostaining. Scale bar is 250 μm for a–c, e–g and 100 μm for d and j. Graph represents mean number of NeuN positive cells (k) and PV positive cells (l) in total hippocampus, DG and in CA region. N = 3 mice for both genotype with 8–9 sections per brain. Data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, Mann–Whitney U test.
Figure 2 Reduced number of parvalbumin (PV) positive interneurons in the hippocampus of Negr1−/− mice. Representative confocal images of the sagittal section of brains of Wt (a–d) and Negr1−/− (e–j) mice for NeuN (red), PV (green) and DAPI (blue) immunostaining. Scale bar is 250 μm for a–c, e–g and 100 μm for d and j. Graph represents mean number of NeuN positive cells (k) and PV positive cells (l) in total hippocampus, DG and in CA region. N = 3 mice for both genotype with 8–9 sections per brain. Data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, Mann–Whitney U test.CC BY · PubMed Central
Figure 3 Barbering behavior and direct social interaction test. Percent of whisker size in mice at 8–9 weeks (a), at 20–22 weeks (b) of age. Illustrative examples used for scoring direct social interactions such as sniffing other body parts (c), anogenital sniffing (d), active contact (e), passive contact (f), rearings (g), digging (h) and self-grooming (i). Graph represents total number of bouts (j–o), time spent (p–u) and bout duration (v–z’) during each parameter of social interaction. Data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, Mann-Whitney U test (Wilcoxon rank sum test).
Figure 3 Barbering behavior and direct social interaction test. Percent of whisker size in mice at 8–9 weeks (a), at 20–22 weeks (b) of age. Illustrative examples used for scoring direct social interactions such as sniffing other body parts (c), anogenital sniffing (d), active contact (e), passive contact (f), rearings (g), digging (h) and self-grooming (i). Graph represents total number of bouts (j–o), time spent (p–u) and bout duration (v–z’) during each parameter of social interaction. Data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, Mann-Whitney U test (Wilcoxon rank sum test).CC BY · PubMed Central
Figure 4 Resident intruder (RI) and Open field (OF) test. Graph represents total number of rearing bouts (a), time spent in rearing (b), digging (c) and in anogenital sniffing (d) during RIT. Total number of rearing bouts (e), total distance travelled (f) and distance travelled in the center zone of the open field by 10 min period (g) during OFT. Data represent mean ± SEM, *p < 0.05, **; ##p < 0.01, by two-way ANOVA, followed by Newman-Keuls post hoc test (RI) and Mann–Whitney U test (OF).
Figure 4 Resident intruder (RI) and Open field (OF) test. Graph represents total number of rearing bouts (a), time spent in rearing (b), digging (c) and in anogenital sniffing (d) during RIT. Total number of rearing bouts (e), total distance travelled (f) and distance travelled in the center zone of the open field by 10 min period (g) during OFT. Data represent mean ± SEM, *p < 0.05, **; ##p < 0.01, by two-way ANOVA, followed by Newman-Keuls post hoc test (RI) and Mann–Whitney U test (OF).CC BY · PubMed Central
Figure 5 Neuritogenesis is impaired in Negr1−/− hippocampal neurons. Confocal images of DIV 0.25 hippocampal neurons derived from P0–1 Wt (a–d) and Negr1−/− (e–h) mice. Phalloidin staining marks F-actin (red), MAP2 for neurons (green), and DAPI stains nuclei (blue). Scale bar is 10 μm. Graph (i) represents quantification of F-actin intensity for actin aggregates at the neurite initiation site (μm2). Scanning electron microscopy images of Wt (j,l), Negr1−/− (k,m), hippocampal neurons at DIV0.25. Scale bar in (j,k) is 5 μm, in (l,m) 2 μm. Representative images of pAAV-hSyn-RFP transfected hippocampal neurons derived from Wt (n) and Negr1−/− (o) mice. Scale bar is 50 μm. Graph (p–r) represents the number, length, and branch points of the neurites per neuron obtained by neuron tracing Neuroleucida. N = 75–82 neurons evaluated per genotype from three independent experiments. Data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, Mann–Whitney U test (Wilcoxon rank sum test).
Figure 5 Neuritogenesis is impaired in Negr1−/− hippocampal neurons. Confocal images of DIV 0.25 hippocampal neurons derived from P0–1 Wt (a–d) and Negr1−/− (e–h) mice. Phalloidin staining marks F-actin (red), MAP2 for neurons (green), and DAPI stains nuclei (blue). Scale bar is 10 μm. Graph (i) represents quantification of F-actin intensity for actin aggregates at the neurite initiation site (μm2). Scanning electron microscopy images of Wt (j,l), Negr1−/− (k,m), hippocampal neurons at DIV0.25. Scale bar in (j,k) is 5 μm, in (l,m) 2 μm. Representative images of pAAV-hSyn-RFP transfected hippocampal neurons derived from Wt (n) and Negr1−/− (o) mice. Scale bar is 50 μm. Graph (p–r) represents the number, length, and branch points of the neurites per neuron obtained by neuron tracing Neuroleucida. N = 75–82 neurons evaluated per genotype from three independent experiments. Data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, Mann–Whitney U test (Wilcoxon rank sum test).CC BY · PubMed Central

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Authors
Singh, K., Jayaram, M., Kaare, M., Leidmaa, E., Jagomäe, T., Heinla, I., Hickey, M. A., Kaasik, A., Schäfer, M. K., Innos, J., Lilleväli, K., Philips, M.-A., & Vasar, E.
Affiliations
  • University of Tartu (EE)
  • University of Bonn (DE)
  • UiT The Arctic University of Norway (NO)
  • Johannes Gutenberg University Mainz (DE)
Corresponding author
Singh, Katyayani
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2019-04-01
Volume
9
Issue
1
Pages
5457-5457
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-019-41991-8
PMC
PMC6443666
OpenAlex
W2930826460
Cited by
65
References
105
Citations by year
2026: 5 · 2025: 13 · 2024: 12 · 2023: 9 · 2022: 7 · 2021: 9 · 2020: 8 · 2019: 1
FWCI
3.76
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • NORA - Norwegian Open Research Archives (submittedVersion, other-oa)
Funders
  • European Commission (IUT20-41, 2014‐2020, 2020 ‘, 2014‐2020.4.01.15‐0012, HORIZON2020, 15-0012, 692202)
  • Eesti Teadusagentuur (2014-2020, 2014-2020.4.01.15-0012, IUT20-41)
  • European Regional Development Fund (IUT20-41, 2014-2020’, 2014-2020.4.01.15-0012, (Project No. 2014-2020.4.01.15-0012), 15-0012, No. 2014-2020.4.01.15-0012)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetics and Neurodevelopmental Disorders
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
Keywords
NEGR1, adhesion molecules, brain structure, neuroanatomical abnormalities, parvalbumin interneurons, excitation-inhibition balance, social behavior, hyperactivity, social hierarchy, neurite sprouting, neurite outgrowth, thalamic reticular nucleus, mental illness, schizophrenia, autism spectrum disorder, major depressive disorder
MeSH terms
Animals, Behavior, Animal, Mental Disorders, Brain, Cell Adhesion Molecules, Neuronal, Mice, Knockout, Mice, Endophenotypes

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