Heterozygous Reeler Mice Research Articles

Assessment of cognitive function in the heterozygous reeler mouse

The heterozygous reeler mouse has been proposed as a genetic mouse model of schizophrenia based on several neuroanatomical and behavioral similarities between these mice and patients with schizophrenia. However, the effect of reelin haploinsufficiency on one of the cardinal symptoms of schizophrenia, the impairment of prefrontal-cortex-dependent cognitive function, has yet to be determined. Here, we investigated multiple aspects of cognitive function in heterozygous reeler mice that are known to be impaired in schizophrenic patients. Heterozygous reeler mice were assessed for (1) cognitive flexibility in an instrumental reversal learning task, (2) impulsivity in an inhibitory control task, (3) attentional function in a three-choice serial reaction time task, and (4) working memory in a delayed matching-to-position task. No differences were found between heterozygous reeler mice and wild-type littermate controls in any prefrontal-related cognitive measures. However, heterozygous reeler mice showed deficits in the acquisition of two operant tasks, consistent with a role for reelin in certain forms of learning. These findings suggest that heterozygous reeler mice may not be an appropriate model for the core prefrontal-dependent cognitive deficits observed in schizophrenia, but may model more general learning deficits that are associated with many psychiatric disorders.

NMDA-receptor proteins are upregulated in the hippocampus of postnatal heterozygous reeler mice

Reelin is a large glycoprotein that is secreted into the extracellular matrix. In the embryonic brain, the binding of Reelin to its receptors ApoER2 and VLDLR induces subcellular events that include the activation Fyn tyrosine kinase, and plays a crucial role in cortical formation. Reelin signaling is also involved in postnatal brain functions such as dendrite development and synaptic plasticity. However, the molecular events involved in Reelin signaling in the postnatal brain remain to be elucidated. Here, we evaluated the proteins downstream of Reelin signaling by comparing the tyrosine-phosphorylated proteins in the postnatal hippocampus of heterozygous and homozygous reeler and wild-type mice, by Western blot analyses. We found that the levels of several phosphoproteins were highest in the hippocampus of the heterozygous reeler mice. The most prominent increase was of two 180-kDa phosphoproteins, which were identified as the NR2A and NR2B subunits of NMDA-R. The amounts of these proteins also increased in the hippocampus of heterozygous reeler mice. However, the mRNA levels of the NMDA-R subunits, determined by quantitative RT-PCR, were the same as in wild-type mice. We also found that the increase in NR2A and NR2B proteins in heterozygous reeler was dependent on Fyn, because this change was absent in heterozygous reeler/homozygous Fyn-deficient double-mutant mice. Thus, the NMDA-R protein level is regulated by the Reelin protein level in a Fyn-dependent manner in the mouse brain.

Executive functions in the heterozygous reeler mouse model of schizophrenia.

Deficits in working memory and executive functions are now considered among the most reliable endophenotypes for schizophrenia. To determine whether cognitive deficits exist in mouse models of the disease, the authors trained heterozygous reeler (+/rl) mice on a series of visual discriminations similar to those used to test executive abilities in primates. These mice resemble schizophrenia patients in that both have reduced levels of reelin protein and altered gamma aminobutyric acid neurotransmission in the prefrontal cortex. The +/rl mice showed a selective deficit in reversal learning, with a pattern of errors that suggested impaired visual attention rather than a deficiency in perseveration and inhibitory control. These results show that cognitive dysfunction may serve as a useful biomarker in mouse models of neuropsychiatric disease.

Psychostimulant Alters Expression of DNA Methyltransferase mRNA in the Rat Brain

Methamphetamine (MAP), the most frequently abused substance in Japan, causes severe drug dependence and psychosis, similar to schizophrenia. It is suggested that long-term alterations in gene expression is related to MAP-induced brain dysfunction, including dependence and psychosis. DNA (cytosine-5) methyltransferase (Dnmt), a methylating enzyme of cytosine residues on CpG-dinucleotides, plays an important role in X chromosome inactivation, genomic imprinting, and gene expression. Reelin is an extracellular matrix protein secreted by GABAergic interneurons. Heterozygous reeler mice that exhibit a 50% downregulation of reelin expression replicate the dendritic spine and GABAergic defects described in schizophrenia. DNA methylation plays an important role in the epigenetic modification of reelin expression. We previously found that MAP could alter expression of Dnmt1 mRNA in the rat brain. In this study, we examined the brain mRNA for Dnmt2 and reelin in MAP-treated Wistar rats. Acute MAP (4 mg/kg) treatment significantly decreased Dnmt2 mRNA by 27% to 39% in hippocampus dentate gyrus, CA1, and CA3 24 h after treatment, and significantly decreased reelin mRNA by 28% in frontal cortex 3 h after treatment. These results suggest that (1) MAP can alter DNA methylation as well as expression of genes in these brain regions, and (2) decrease in reelin mRNA in the frontal cortex is similar to heterozygous reeler mice, which might be related to schizophrenia-like psychotic symptoms of MAP psychosis.

Enhanced dizocilpine efficacy in heterozygous reeler mice relates to GABA turnover downregulation

Reelin synthesized by cortical GABAergic interneurons throughout the telencephalon is secreted into the extracellular matrix (ECM) and binds with nM affinity to integrin receptors located at dendritic spine postsynaptic densities and positively modulates Arc and other dendritic resident mRNAs translation, thereby facilitating the onset of synaptic plasticity and LTP consolidation.Accordingly, the reelin haploinsufficient heterozygous reeler mice (HRM) express a marked decrease of cortical thickness, of cortical and hippocampal dendritic spine density, and of cortical GAD67 expression. Behaviorally, HRM manifest a sensorimotor deficit, an exaggerated response to fear, and a deficit in olfactory discrimination learning. HRM and wild-type mice (WTM) were trained to retrieve to criterion palatable chocolate-flavored food pellets in an eight-arm radial maze. In 9–14 days of training HRM and WTM learned the task equally well committing only a few errors. However, HRM, when compared with WTM, show a greater cognitive impairment following the administration of dizocilpine. Also, HRM are more susceptible to the increased locomotion and stereotypic behavior elicited by dizolcipine. The enhanced dizocilpine susceptibility of HRM is not due to differences in pharmacokinetics because the levels of dizocilpine in cortices of HRM and WTM were virtually equal. We also failed to detect differences between HRM and WTM in glutamate brain content and in the rate of 13C-glucose incorporation into the glutamate brain pools. In contrast we found that the conversion index of glutamate into GABA (an indirect measurement of GABA turnover rate) is decreased in cortex, hippocampus and striatum of HRM when compared to WTM. Thus, HRM recapitulate several neurochemical and behavioral endophenotypes reminiscent of schizophrenia and these mice can be proposed as a relevant animal model for the study of pharmacological treatments aimed at alleviating the sensory-motor and cognitive dysregulation associated with schizophrenia.

The heterozygous reeler mouse: behavioural phenotype

The aim of this study was to investigate whether heterozygous reeler mice ( +/ rl) could be used as a genetic mouse model of schizophrenia, as previously suggested [J. Med. Chem. 44 (2001) 477]. The behavioural phenotype of male and female +/ rl mice (young adult: 50–70 days old, and fully adult: >75 days old) was compared to their wild type (+/+) littermates. A complex behavioural test battery was employed: Irwin test, rotarod, spontaneous locomotor activity, social behaviour, light–dark transition test, startle response and prepulse inhibition, and hot-plate test. Overall, +/ rl mice did not differ from their +/+ littermates at either age, although fully adult +/ rl male mice spent more time engaged in social investigation. Some of the behavioural measures investigated were influenced by gender. Young female mice were more active in the light/dark transition test than males, while males were more aggressive than females during social interaction. In addition, performance on the rotarod was shown to deteriorate with age. Our data are in agreement with previous findings [Soc. Neurosci. Abst. 27 (2001) 238; J. Psychopharmacol. 17 (2003) A43], but contrary to those of Costa et al. [Curr. Opin. Pharmacol. 2 (2002) 56], although mice used in the present and previous studies were derived from the same genetic stock at Jackson Laboratories, USA. The present study clearly shows that, compared to its +/+ littermates, the +/ rl mouse (young/fully adult) exhibits normal behaviour in a wide range of behavioural measures and suggest that these mice may not be suitable for use as a genetic animal model of schizophrenia.

Immunocytochemical localization of reelin in the olfactory bulb of the heterozygous reeler mouse: An animal model for schizophrenia

Because heterozygous reeler (HR) mice share some abnormal traits with schizophrenic patients, and schizophrenia is often accompanied by impairment of olfactory function, this study examines reelin in the olfactory bulb of the HR mouse. In the WT mouse, reelin immunoreactivity is found in the extracellular matrix, and in the cytoplasm of olfactory nerve fibers, GABAergic interneurons, and glutamatergic mitral cells. Western blot analysis reveals that reelin immunoreactivity in the HR mouse is reduced by 45% compared to WT mouse. This is especially evident in the glomerular GABAergic interneurons. In WT mitral cells, reelin is found in discrete clumps near the axon hillock and within the axon. In the HR mouse, reelin axonal staining is diffuse and densely packed. In the rostral migratory stream of the HR mouse, immunolabeling shows an accumulation of reelin-containing neuronal precursors, apparently unable to shift from tangential to radial migration. These observations indicate that there is a downregulation of reelin in the HR mouse and suggest that secretion of reelin may be compromised. Further studies of the HR mouse may provide a new basis for understanding the role of reelin in the adult CNS, especially as it may relate to schizophrenia.

A reelin-integrin receptor interaction regulates Arc mRNA translation in synaptoneurosomes.

Reelin is synthesized and secreted into extracellular matrix by cortical gamma-aminobutyric acid (GABA)ergic interneurons and binds with high affinity to the extracellular domain of integrin receptors expressed in dendritic shaft and spine postsynaptic densities (DSPSD) of pyramidal neurons. In heterozygous reeler mice, reelin bound to DSPSD, and the expression of Arc (activity-regulated cytoskeletal protein) is lower than in wild-type mice. We studied the effect of reelin on Arc and total protein synthesis in synaptoneurosomes (SNSs) prepared from mouse neocortex. Recombinant full-length mouse reelin displaces the high affinity (K(D) = 60 fM) binding of [(125)I]echistatin (a competitive integrin receptor antagonist) to integrin receptors with a K(i) of 22 pM and with a Hill slope close to 1. Echistatin (50-100 nM) competitively antagonizes and abates reelin binding. The addition of reelin (2-40 pM) to SNSs enhances the incorporation of [(35)S]methionine into Arc and other rapidly translated proteins in a concentration-dependent manner. This incorporation is virtually abolished by 50-100 nM echistatin or by 5-10 nM rapamycin, a blocker of the mammalian target of rapamycin kinase. We conclude that reelin binds with high affinity to integrin receptors expressed in SNSs and thereby activates Arc protein synthesis.

Cortical-subcortical dysconnectivity expressed by attenuated cortical c-fos induction by acute amphetamine treatment or acute swim stress in mice with reduced NMDA receptor1 expression

Reelin is a large glycoprotein that is secreted into the extracellular matrix. In the embryonic brain, the binding of Reelin to its receptors ApoER2 and VLDLR induces subcellular events that include the activation Fyn tyrosine kinase, and plays a crucial role in cortical formation. Reelin signaling is also involved in postnatal brain functions such as dendrite development and synaptic plasticity. However, the molecular events involved in Reelin signaling in the postnatal brain remain to be elucidated. Here, we evaluated the proteins downstream of Reelin signaling by comparing the tyrosine-phosphorylated proteins in the postnatal hippocampus of heterozygous and homozygous reeler and wild-type mice, by Western blot analyses. We found that the levels of several phosphoproteins were highest in the hippocampus of the heterozygous reeler mice. The most prominent increase was of two 180-kDa phosphoproteins, which were identified as the NR2A and NR2B subunits of NMDA-R. The amounts of these proteins also increased in the hippocampus of heterozygous reeler mice. However, the mRNA levels of the NMDA-R subunits, determined by quantitative RT-PCR, were the same as in wild-type mice. We also found that the increase in NR2A and NR2B proteins in heterozygous reeler was dependent on Fyn, because this change was absent in heterozygous reeler/homozygous Fyn-deficient double-mutant mice. Thus, the NMDA-R protein level is regulated by the Reelin protein level in a Fyn-dependent manner in the mouse brain.

Preferential alterations in the mesolimbic dopamine pathway of heterozygous reeler mice: an emerging animal-based model of schizophrenia.

Based on a number of neuroanatomical and behavioural similarities, recent evidence suggests that heterozygous reeler mice, haploinsufficient for reelin expression, represent a useful model of psychosis vulnerability. As brain mesolimbic dopamine pathways have been proposed to be associated with the pathophysiology of psychotic disorders, we thought it would be of interest to examine whether these animals present disturbances in the mesolimbic dopamine system. To this end we studied by immunocytochemical, in situ hybridization procedures and receptor autoradiography, several markers of the mesotelencephalic dopamine pathway in heterozygous reeler mice and controls. We report that heterozygous reeler mice exhibit a reduction in the number of tyrosine hydroxylase-immunoreactive cell bodies and tyrosine hydroxylase mRNA levels in the ventral tegmental area, as well as a reduction of tyrosine hydroxylase and dopamine transporter immunoreactivity in the dopamine terminal fields of the limbic striatum. In these areas we also observed a reduction of dopamine D2 receptor mRNA. Finally, a marked increase in D3 receptor mRNA levels was observed concomitant with a significant increase in D3 binding sites. On the contrary, the nigrostriatal pathway did not show any significant alteration in heterozygous reeler mice with regards to the dopaminergic markers examined in substantia nigra cell bodies and dorsal striatum dopamine terminal fields. These results suggest a specific link between reelin-related neuronal pathology and dopamine involvement in the pathophysiology of psychotic disorders.

REELIN and schizophrenia: a disease at the interface of the genome and the epigenome.

The downregulation of the Reelin gene (RELN) that occurs in schizophrenic brains, which are characterized by pyramidal neurons with shortened dendrites and by reduced expression densities of dendritic spines, may well result from hypermethylation of the RELN promoter. In the adult mammalian brain, gamma-aminoburytic acid-secreting (GABAergic) interneurons release RELN into the extracellular matrix, where it binds with high affinity to the integrin receptors present at dendritic spine postsynaptic densities and likely plays a role, elaborated in this article, in synaptic plasticity. In heterozygous reeler mice, which are haploinsufficient in RELN, inhibitors of histone deacetylase increase DNA demethylase activity and restore RELN expression. Such inhibitors could thus be of therapeutic value in mitigating vulnerability to schizophrenia among high-risk individuals.

The heterozygote reeler mouse as a model for the development of a new generation of antipsychotics.

Neurochemical and structural prefrontal cortex abnormalities, including decreased reelin and glutamic acid decarboxylase (GAD)(67) expression, decreased thickness, increased neuronal packing density and decreased neuropil and dendritic spine number, are characteristics of schizophrenia neuropathology. Reelin is an extracellular matrix protein secreted by GABAergic interneurons that, acting through pyramidal neuron integrin receptors, provides a signal for dendritic spine plasticity. Heterozygous reeler mice that exhibit a 50% downregulation of reelin expression (mRNA and protein) replicate the dendritic spine and GABAergic defects described in schizophrenia. This genetic mouse model may be of value to reveal those GABAergic and integrin receptor signal transduction mechanisms that are likely to be downregulated by reelin deficiency in the brain of schizophrenia patients. An understanding of the epigenetic regulation of reelin gene expression and of the possible pathogenetic role of reelin deficiency in schizophrenia, may become a major focus that will open new avenues for the treatment of this disease.

Down-regulation of dendritic spine and glutamic acid decarboxylase 67 expressions in the reelin haploinsufficient heterozygous reeler mouse.

Heterozygous reeler mice (HRM) haploinsufficient for reelin express approximately 50% of the brain reelin content of wild-type mice, but are phenotypically different from both wild-type mice and homozygous reeler mice. They exhibit, (i) a down-regulation of glutamic acid decarboxylase 67 (GAD(67))-positive neurons in some but not every cortical layer of frontoparietal cortex (FPC), (ii) an increase of neuronal packing density and a decrease of cortical thickness because of neuropil hypoplasia, (iii) a decrease of dendritic spine expression density on basal and apical dendritic branches of motor FPC layer III pyramidal neurons, and (iv) a similar decrease in dendritic spines expressed on the basal dendrite branches of CA1 pyramidal neurons of the hippocampus. To establish whether the defect of GAD(67) down-regulation observed in HRM is responsible for neuropil hypoplasia and decreased dendritic spine density, we studied heterozygous GAD(67) knockout mice (HG(67)M). These mice exhibited a down-regulation of GAD(67) mRNA expression in FPC (about 50%), but they expressed normal amounts of reelin and had no neuropil hypoplasia or down-regulation of dendritic spine expression. These findings, coupled with electron-microscopic observations that reelin colocalizes with integrin receptors on dendritic spines, suggest that reelin may be a factor in the dynamic expression of cortical dendritic spines perhaps by promoting integrin receptor clustering. These findings are interesting because the brain neurochemical and neuroanatomical phenotypic traits exhibited by the HRM are in several ways similar to those found in postmortem brains of psychotic patients.

474. Reelin, a protein regulating neural development, is expressed in the blood in adult mammals

Reelin is a regulatory protein for telencephalic and cerebellar lamination during development and is expressed in several structures in the adult brain. However, only trace amounts have previously been reported in peripheral tissues. Since reelin structurally resembles extracellular matrix proteins, and since many matrix proteins have been detected in blood, we hypothesized that reelin might also be detectable in the circulation. Reelin (420 kDa) and two smaller reelin-immunoreactive bands (310 and 160 kDa) were readily observed in serum and platelet-poor plasma of rats, wild-type mice and humans, but were not detectable in homozygous reeler (rl −/−) mice. Plasma levels of reelin were reduced by half in heterozygous reeler (rl +/−) mice. Western blotting and immunocytochemistry using anti-reelin monoclonal antibodies indicated that reelin-like immunoreactivity was expressed in a subset of chromaffin cells within the adrenal medulla, and in a subset of cells co-expressing α-melanocyte stimulating hormone within the pituitary pars intermedia. However, surgical removal of adrenal or pituitary in rats failed to decrease the amount of full-length (420 kDa) reelin expressed in serum. Adult liver expressed an amount of reelin mRNA that is 1/3 of that expressed in cerebral cortex of the adult mouse; full-length reelin protein was detectable in liver extracts in situ and acutely isolated liver cells secreted full-length reelin in vitro. Given its size, liver may play a major role in producing the pool of circulating reelin. It now becomes feasible to study the regulation of reelin gene expression in an accessible bodily fluid in living humans.

The phenotypic characteristics of heterozygous reeler mouse.

Histological and behavioral traits are associated with reelin (Reln) haplo-insufficiency in heterozygous reeler mouse (rl+/-). These phenotypic traits are an approximately 50% decrease of brain Reln mRNA and Reln protein, an accumulation of nicotinamide-adenine dinucleotide phosphate-diaphorase (NADPH-d)-positive neurons in subcortical white matter, an age-dependent decrease in prepulse inhibition of startle (PPI), and neophobic behavior on the elevated plus-maze. Possible analogies between these rl+/- phenotypic traits and signs of psychosis vulnerability are discussed.

Tyrosine phosphorylation of the NMDA receptor: Mechanism and implication

The extracellular protein Reelin regulates radial neuronal migration in the embryonic brain, promotes dendrite outgrowth in the developing postnatal forebrain, and strengthens synaptic transmission in the adult brain. Heterozygous reeler mice expressing reduced levels of Reelin are grossly normal but exhibit behavioral and physiological abnormalities. We previously demonstrated that dendritic spine density is reduced in the developing hippocampus of these mice. In this study, we investigated the consequence of Reelin deficiency on synapse formation in adult heterozygous reeler mice using imaging and biochemical approaches. Using a reeler colony that expresses yellow fluorescent protein in selected neurons, we analyzed spine density in hippocampal area CA1 by confocal microscopy and found modest abnormalities in heterozygous reeler mice. However, biochemical analysis of synaptic composition revealed specific postsynaptic defects in scaffolding proteins, neurotransmitter receptors, and signaling proteins. Using whole brain homogenates and purified pre- and postsynaptic fractions, we found that the defects were localized to the postsynaptic compartment of heterozygous reeler synapses. Decreased levels of postsynaptic density-95 (PSD-95), the N-methyl d-aspartate (NMDA) receptor subunits NR2A and NR2B, and the phosphatase PTEN were found specifically in the postsynaptic density fraction obtained from these mice. Furthermore, we found that PSD-95, NR2A, and PTEN interact with each other at the synapse. Finally, we show that levels of NR2A are reduced in conditional Pten knock out mice, demonstrating that the PTEN phosphatase regulates NMDA receptor expression at the synapse in vivo. These studies may provide insights into the etiology of cognitive disorders associated with deficiencies in Reelin signaling and PTEN dysfunction.

Drosophila glia cells missing gene determines glia vs. neuron cell fate by asymmetric segregation of mRNA at stem cell division

Reelin is a large glycoprotein that is secreted into the extracellular matrix. In the embryonic brain, the binding of Reelin to its receptors ApoER2 and VLDLR induces subcellular events that include the activation Fyn tyrosine kinase, and plays a crucial role in cortical formation. Reelin signaling is also involved in postnatal brain functions such as dendrite development and synaptic plasticity. However, the molecular events involved in Reelin signaling in the postnatal brain remain to be elucidated. Here, we evaluated the proteins downstream of Reelin signaling by comparing the tyrosine-phosphorylated proteins in the postnatal hippocampus of heterozygous and homozygous reeler and wild-type mice, by Western blot analyses. We found that the levels of several phosphoproteins were highest in the hippocampus of the heterozygous reeler mice. The most prominent increase was of two 180-kDa phosphoproteins, which were identified as the NR2A and NR2B subunits of NMDA-R. The amounts of these proteins also increased in the hippocampus of heterozygous reeler mice. However, the mRNA levels of the NMDA-R subunits, determined by quantitative RT-PCR, were the same as in wild-type mice. We also found that the increase in NR2A and NR2B proteins in heterozygous reeler was dependent on Fyn, because this change was absent in heterozygous reeler/homozygous Fyn-deficient double-mutant mice. Thus, the NMDA-R protein level is regulated by the Reelin protein level in a Fyn-dependent manner in the mouse brain.

Gender effect on Purkinje cell loss in the cerebellum of the heterozygous reeler mouse.

Homozygous mutant mice such as staggerer (sg/sg) or reeler (rl/rl) exhibit a marked ataxia associated with an atrophic cerebellum during the first postnatal weeks and a reduced number of Purkinje cells, the deficit reaching about 75% in sg/sg and 50% in rl/rl as compared to age- and sex-matched mice from the same strain background. These two mutations are classically viewed as recessive, but we have recently shown that heterozygous staggerer (+/sg) mice exhibit a progressive and age-related loss of Purkinje cells between 3 and 12 months of age, despite their apparent clinical normality (Shojaeian-Zanjani et al., 1992). In the present study, we have investigated whether a similar cell loss exists in the cerebellum of heterozygous +/rl mice. The number of Purkinje cells was counted in serial parasagittal sections of the cerebellum of +/rl and their +/+ littermates at 3, 16 and 26 months of age. Our results reveal a 16% deficit in the number of Purkinje cells in 3-month-old +/rl and a 24% one in 16-month-old animals: surprisingly this deficit is only present in the +/rl males, while the females are spared. These results suggest that the reeler gene (D'Arcangelo et al., 1995) exerts its effect on Purkinje cell number in a gender-specific fashion in heterozygous mutant mice.