A Jacob/Nsmf Gene Knockout Results in Hippocampal Dysplasia and Impaired BDNF Signaling in Dendritogenesis

Jacob, the protein encoded by the Nsmf gene, is involved in synapto-nuclear signaling and docks an N-Methyl-D-Aspartate receptor (NMDAR)-derived signalosome to nuclear target sites like the transcription factor cAMP-response-element-binding protein (CREB). Several reports indicate that mutations in NSMF are related to Kallmann syndrome (KS), a neurodevelopmental disorder characterized by idiopathic hypogonadotropic hypogonadism (IHH) associated with anosmia or hyposmia. It has also been reported that a protein knockdown results in migration deficits of Gonadotropin-releasing hormone (GnRH) positive neurons from the olfactory bulb to the hypothalamus during early neuronal development. Here we show that mice that are constitutively deficient for the Nsmf gene do not present phenotypic characteristics related to KS. Instead, these mice exhibit hippocampal dysplasia with a reduced number of synapses and simplification of dendrites, reduced hippocampal long-term potentiation (LTP) at CA1 synapses and deficits in hippocampus-dependent learning. Brain-derived neurotrophic factor (BDNF) activation of CREB-activated gene expression plays a documented role in hippocampal CA1 synapse and dendrite formation. We found that BDNF induces the nuclear translocation of Jacob in an NMDAR-dependent manner in early development, which results in increased phosphorylation of CREB and enhanced CREB-dependent Bdnf gene transcription. Nsmf knockout (ko) mice show reduced hippocampal Bdnf mRNA and protein levels as well as reduced pCREB levels during dendritogenesis. Moreover, BDNF application can rescue the morphological deficits in hippocampal pyramidal neurons devoid of Jacob. Taken together, the data suggest that the absence of Jacob in early development interrupts a positive feedback loop between BDNF signaling, subsequent nuclear import of Jacob, activation of CREB and enhanced Bdnf gene transcription, ultimately leading to hippocampal dysplasia

Subregion- and age-dependent changes in <i>Bdnf</i> mRNA expression and BDNF protein levels in <i>Jacob/Nsmf</i> ko mice.

<p>(A) Transcript levels of <i>Bdnf</i> exon IV in CA1 and CA3 regions of hippocampus in P10 <i>Jacob/Nsmf</i> ko (-/-) mice exhibit a decrease compared to wt (+/+) mice. (B, C) BDNF-ELISA analysis of CA1 and CA3 tissue samples from P10 and 8–10 weeks old (B) <i>Jacob/Nsmf</i> ko (-/-) and wt (+/+) mice revealed significant lower levels of BDNF only in CA1 region of P10 mice. (C) The expression levels for both regions in mice at the age of 8–10 weeks are similar. (*p < 0.05, **p < 0.01, two-tailed unpaired t-test, values represent mean ± SEM).</p

<i>Jacob/Nsmf</i> ko mouse hippocampal neurons display a simplified dendritic tree which is rescued by chronic BDNF (100 ng/ml) application (at DIV2 and at DIV6).

<p>(A, B) Representative micrographs of wt and <i>Jacob/Nsmf</i> ko hippocampal neurons immunostained with MAP2 at (A) DIV5 and (B) DIV10. For Sholl analysis the number of dendritic intersections of wt and <i>Jacob/Nsmf</i> ko hippocampal neurons was plotted against the distance. (A) At DIV5 wt and <i>Jacob/Nsmf</i> ko hippocampal neurons display no difference in the arborization (n = 40 for wt and n = 40 for <i>Jacob/Nsmf</i> ko). (B) At DIV10 <i>Jacob/Nsmf</i> ko hippocampal neurons display a simplified dendritic tree as compared to wt neurons. A two-way repeated measures ANOVA revealed that there was both a main effect of genotype F(1,102) = 16.41, p<0.001, ηp2 = 3.5, as well as a main effect of distance F(1,18) = 152.2, p<0.001, ηp2 = 44.37. In addition, the interaction of these two variables was also significant (F(1,18) = 2.27, p = 0.002, ηp2 = 0.66). Post hoc Bonferroni tests showed that there are significant differences in dendritic arborization regarding distances at 60 μm away from the soma p<0.05; at 70 μm, p<0.01; at 80 μm, p<0.05; at 90 μm, p<0.001; at 100 μm, p<0.001; at 110, p<0.01; at 120, p<0.05. (C) Chronic application of BDNF (100 ng/ml, DIV2 and DIV6) rescues the dendritic defect of <i>Jacob/Nsmf</i> ko hippocampal neurons, as there is no significant difference when compared to wt neurons. (D) <i>Jacob/Nsmf</i> ko neurons display a reduced number of synaptic contacts as compared to wt controls. Representative micrographs of DIV15 wt and <i>Jacob/Nsmf</i> ko hippocampal neurons primary distal dendrites immunostained with MAP2 (blue), Homer1 (green) and Synaptophysin (Syn, red), following treatment with BDNF (100 ng/ml) at DIV 2 and DIV 6. Co-localization of synaptic puncta per 10 μm was quantified. Chronic BDNF treatment of <i>Jacob/Nsmf</i> ko neurons rescues the synaptic phenotype observed, as no significant differences appear between wt controls and the BDNF-treated groups. Two-way repeated measures ANOVA with Bonferroni posttest revealed that there was both a main effect of genotype F(1,77) = 15.48, p<0.001 as well as a main effect of treatment F(1,77) = 17.10, p<0.001. ***p<0.001. Scale bars in A, B, C = 50μm. Dendritic segment in D is 20μM.</p

Organization of the olfactory bulb and hypothalamus in wt and Jacob-deficient mice.

<p>(A-C) Microphotographs showing the morphology of the olfactory bulb (BO) of wt (+/+) and <i>Jacob/Nsmf</i> ko (-/-) mice. (A) Nissl stained sections display the general morphology and subregions of the olfactory bulb (Bregma 2.8 mm, 50x magnification, scale bar 500 μm; Gl, stratum glomerulosum; EPl, stratum plexiforme externum, Mi, mitral cell layer, IPl, internal plexiform layer, AON, anterior olfactory nucleus, aci, anterior commissure, intrabulbar part, lo, lateral olfactory tract). (B) shows sections of both genotypes stained against GnRH (50x magnification, scale bar 500 μm). The frames in (B) indicate the image sections shown in (C). In (C) (200x magnification, scale bar 100 μm) the sample fields are marked, which were used to analyze the fiber densities in Gl and EPl. There are no differences in general morphology between wt and Jacob-deficient mice. (D, E) Quantification of GnRH-IR neurons (D) and fiber densities (E) in brains of wt (+/+) and <i>Jacob/Nsmf</i> ko (-/-) mice. (D) The number of GnRH-positive neurons was estimated in the olfactory bulb (Gl, EPl), in the medial septum (MS), ventral diagonal (VDB) and horizontal diagonal band (HDB) of Broca and preoptic area (PA). (E) The density of GnRH-IR fibers was analyzed in the same regions and furthermore in anteroventral paraventricular hypothalamic nucleus (AVPe) and nucleus arcuatus hypothalami (Arc). No significant differences were found between wt and <i>Jacob/Nsmf</i> ko mice (data are reported as mean ± SEM, two-way repeated measures ANOVAs were performed using REGION as within-subject factor and MOUSE LINE as between-subject factor. Post hoc analyses were performed using unpaired t-tests (Welch’s test) with Bonferroni-Holm adjustment). (F-H) Microphotographs showing the distribution of GnRH-IR neurons and fibers in the hypothalamic area of wt and <i>Jacob/Nsmf</i> ko mice. (F) and (G) display the periventricular (Pe) and arcuate hypothalamic nuclei (Arc) around the third ventricle (III) (Bregma -1.70 mm, scale bars are 100 μm). (H) shows exemplary GnRH-IR neurons in the medial preoptic area (PA) of wt and <i>Jacob/Nsmf</i> ko mice (Bregma 0.5 mm, scale bars are 20 μm).</p

Fertility parameters of male <i>Jacob/Nsmf</i> ko mice.

<p>(A, B) Testicular morphology in adult wt (A, +/+) and <i>Jacob/Nsmf</i> ko (B, -/-) mice testes stained by hematoxylin and eosin protocol. WT and ko mice showed no differences in appearance of different stages of differentiation from spermatogonia to sperm cells and comparable lumina. (C) Testicular weight of wt (+/+), heterozygous (+/-) and <i>Jacob/Nsmf</i> ko (-/-) mice. (D) Testosteron serum levels were determined in mice of all genotypes by ELISA. Unpaired t-test; *p<0.05. Scale bar in B is 100μm.</p

Morphology of Golgi-stained pyramidal neurons in the hippocampal CA1 region of wt and <i>Jacob/Nsmf</i> ko mice.

<p>(A) Dendritic branching was examined using Sholl analysis in wt (+/+) and ko (-/-) pyramidal neurons (n = 12 neurons, 2–4 slices of 4 adult, 12 weeks old animals were analyzed). (B) Apical dendrites of ko mice seem to be less branched compared to wt mice although the entire dendritic lengths do not differ significantly. (C) Basal dendrites of <i>Jacob/Nsmf</i> ko mice are significantly less branched, i.e. have fewer intersections per Sholl-segment in particular 10–110 μm apart from the soma compared to wt mice. Also, basal dendrites of ko mice are significantly shorter in CA1. Arrowheads in A correspond to enlarged images D and F. (D, F) Enlarged areas showing the dendritic spine distribution and morphology of Golgi-stained pyramidal neurons in the CA1 region (apical dendrites D, E, and basal F, G) in wt (+/+) and <i>Jacob/Nsmf</i> ko mice (-/-). Both second-order apical (E) and basal (G) dendrites of ko mice bear less spines compared to wt mice. Scale bar in A is 50 μm, scale bar in D is 20 μm. Data are represented as mean ± SEM (ANOVA or Student´s t-test, * p<0.05, **p<0.01, ***p<0.001).</p

BDNF induces the nuclear import of pJacob and increase in pCREB in a NMDAR-dependent manner.

<p>Rat dissociated hippocampal cultures were treated at DIV10 with BDNF, AP5 or both, fixed and stained for panJacob (A), pJacob (B), panCREB (C), and pCREB (D). The BDNF-induced accumulation of pJacob in the nucleus is abolished by NMDAR blocking with AP5. BDNF application caused an increase in nuclear pCREB levels, which is partially abolished by NMDAR blocking (C) with total unchanged levels of nuclear CREB (D). Confocal images averaged from two confocal sections of the nucleus. Lookup table indicates original pixel intensities from 0 to 255. Graphs represent mean +/-SEM staining intensity within nuclear plane normalized to control. One-way ANOVA with Tukey posttest ***p<0.001; **p<0.01, *p<0.05. Scale bar indicate 20μm.</p