Reeler Mutant Mice Research Articles

The role of retinoid-related orphan receptor-α in cigarette smoke-induced autophagic response

BackgroundRetinoid-related orphan receptor-α (RORα) and autophagy dysregulation are involved in the pathophysiology of chronic obstructive pulmonary disease (COPD), but little is known regarding their association. We investigated the role of RORα in COPD-related autophagy.MethodsThe lung tissues and cells from a mouse model were analyzed for autophagy markers by using western blot analysis and transmission electron microscopy.ResultsCigarette smoke increased the LC3-II level and decreased the p62 level in whole lung homogenates of a chronic cigarette smoking mouse model. Although cigarette smoke did not affect the levels of p62 in Staggerer mutant mice (RORαsg/sg), the baseline expression levels of p62 were significantly higher than those in wild type (WT) mice. Autophagy was induced by cigarette smoke extract (CSE) in Beas-2B cells and in primary fibroblasts from WT mice. In contrast, fibroblasts from RORαsg/sg mice failed to show CSE-induced autophagy and exhibited fewer autophagosomes, lower LC3-II levels, and higher p62 levels than fibroblasts from WT mice. Damage-regulated autophagy modulator (DRAM), a p53-induced modulator of autophagy, was expressed at significantly lower levels in the fibroblasts from RORαsg/sg mice than in those from WT mice. DRAM knockdown using siRNA in Beas-2B cells inhibited CSE-induced autophagy and cell death. Furthermore, RORα co-immunoprecipitated with p53 and the interaction increased p53 reporter gene activity.ConclusionsOur findings suggest that RORα promotes autophagy and contributes to COPD pathogenesis via regulation of the RORα-p53-DRAM pathway.

Glutamate released by Cajal-Retzius cells impacts specific hippocampal circuits and behaviors.

SUMMARYThe impact of Cajal-Retzius cells on the regulation of hippocampal circuits and related behaviors is unresolved. Here, we directly address this issue by impairing the glutamatergic output of Cajal-Retzius cells with the conditional ablation of vGluT2, which is their main vesicular glutamate transporter. Although two distinct conditional knockout lines do not reveal major alterations in hippocampal-layer organization and dendritic length of principal neurons or GABAergic cells, we find parallel deficits in specific hippocampal-dependent behaviors and in their putative underlying microcircuits. First, conditional knockout animals show increased innate anxiety and decreased feedforward GABAergic inhibition on dentate gyrus granule cells. Second, we observe impaired spatial memory processing, which is associated with decreased spine density and reduced AMPA/NMDA ratio of postsynaptic responses at the perforant- and entorhino-hippocampal pathways. We conclude that glutamate synaptically released by Cajal-Retzius cells is critical for the regulation of hippocampal microcircuits and specific types of behaviors.

Synaptic and Genetic Bases of Impaired Motor Learning Associated with Modified Experience-Dependent Cortical Plasticity in Heterozygous Reeler Mutants.

Patients with neurodevelopmental disorders show impaired motor skill learning. It is unclear how the effect of genetic variation on synaptic function and transcriptome profile may underlie experience-dependent cortical plasticity, which supports the development of fine motor skills. RELN (reelin) is one of the genes implicated in neurodevelopmental psychiatric vulnerability. Heterozygous reeler mutant (HRM) mice displayed impairments in reach-to-grasp learning, accompanied by less extensive cortical map reorganization compared with wild-type mice, examined after 10days of training by intracortical microstimulation. Assessed by patch-clamp recordings after 3days of training, the training induced synaptic potentiation and increased glutamatergic-transmission of cortical layer III pyramidal neurons in wild-type mice. In contrast, the basal excitatory and inhibitory synaptic functions were depressed, affected both by presynaptic and postsynaptic impairments in HRM mice; and thus, no further training-induced synaptic plasticity occurred. HRM exhibited downregulations of cortical synaptophysin, immediate-early gene expressions, and gene enrichment, in response to 3days of training compared with trained wild-type mice, shown using quantitative reverse transcription polymerase chain reaction, immunohistochemisty, and RNA-sequencing. We demonstrated that motor learning impairments associated with modified experience-dependent cortical plasticity are at least partially attributed by the basal synaptic alternation as well as the aberrant early experience-induced gene enrichment in HRM.

Comparative gene expression analysis of the engulfment and cell motility (ELMO) protein family in the mouse brain

Engulfment and cell motility (ELMO) proteins bind to Dock180, a guanine nucleotide exchange factor (GEF) of the Rac family, and regulate GEF activity. The resultant ELMO/Dock180/Rac module regulates cytoskeletal reorganization responsible for the engulfment of apoptotic cells, cell migration, and neurite extension. The expression and function of Elmo family proteins in the nervous system, however, are not yet fully understood. Here, we characterize the comparative gene expression profiles of three Elmo family members (Elmo1, Elmo2, and Elmo3) in the brain of C57BL/6J mice, a widely used inbred strain, together with reeler mutant mice to understand gene expression in normal laminated brain areas compared with abnormal areas. Although all three Elmo genes showed widespread mRNA expression over various mouse tissues tested, Elmo1 and Elmo2 were the major types expressed in the brain, and three Elmo genes were up-regulated between the first postnatal week (infant stage) and the third postnatal week (juvenile, weaning stage). In addition, the mRNAs of Elmo genes showed distinct distribution patterns in various brain areas and cell-types; such as neurons including inhibitory interneurons as well as some non-neuronal cells. In the cerebral cortex, the three Elmo genes were widely expressed over many cortical regions, but the predominant areas of Elmo1 and Elmo2 expression tended to be distributed unevenly in the deep (a lower part of the VI) and superficial (II/III) layers, respectively, which also changed depending on the cortical areas and postnatal stages. In the dentate gyrus of the hippocampus, Elmo2 was expressed in dentate granule cells more in the mature stage rather than the immature-differentiating stage. In the thalamus, Elmo1 but not the other members was highly expressed in many nuclei. In the medial habenula, Elmo2 and Elmo3 were expressed at intermediate levels. In the cerebellar cortex, Elmo1 and Elmo2 were expressed in differentiating-mature granule cells and mature granule cells, respectively. In the Purkinje cell layer, Elmo1 and Elmo2 were expressed in Purkinje cells and Bergmann glia, respectively. Disturbed cellular distributions and laminar structures caused by the reeler mutation did not severely change expression in these cell types despite the disturbed cellular distributions and laminar structures, including those of the cerebrum, hippocampus, and cerebellum. Taken together, these results suggested that these three Elmo family members share their functional roles in various brain regions during prenatal-postnatal development.

Reelin regulates male mouse reproductive capacity via the sertoli cells.

Reelin plays important roles in brain development. Reeler mutant mice that lack the protein reelin (RELN) suffer from cell type- and region-dependent changes in their neocortical layers, and adult reeler mutant mice have dilated seminiferous tubules. Meanwhile, the mechanism by which Reelin regulates the spermatogenic cell development in mice and their reproductive abilities remains unclear. In the present study, we used reeler mutant mice to investigate the effects of Reelin on reproduction in mice. The results indicated variations in sex hormone expression among the reeler mice, indicating that they produce few offspring and their spermatogenic cells are irregularly developed. Moreover, glial cell line-derived neurotrophic factor (GDNF)/GDNF family receptor alpha 1, Ras/extracellular regulated protein kinases (ERK), and promyelocytic leukemia zinc finger (PLZF)/chemokine (C-X-C motif) receptor 4 (CXCR4) serve as potential regulatory pathways that respond to the changes in sertoli cells and the niche of male germ cells. Our findings provided valuable insights into the role of reeler in the reproductive abilities of male mice and development of their spermatogonia stem cells.

The Functioning of a Cortex without Layers.

A major hallmark of cortical organization is the existence of a variable number of layers, i.e., sheets of neurons stacked on top of each other, in which neurons have certain commonalities. However, even for the neocortex, variable numbers of layers have been described and it is just a convention to distinguish six layers from each other. Whether cortical layers are a structural epiphenomenon caused by developmental dynamics or represent a functionally important modularization of cortical computation is still unknown. Here we present our insights from the reeler mutant mouse, a model for a developmental, “molecular lesion”-induced loss of cortical layering that could serve as ground truth of what an intact layering adds to the cortex in terms of functionality. We could demonstrate that the reeler neocortex shows no inversion of cortical layers but rather a severe disorganization that in the primary somatosensory cortex leads to the complete loss of layers. Nevertheless, the somatosensory system is well organized. When exploring an enriched environment with specific sets of whiskers, activity-dependent gene expression takes place in the corresponding modules. Precise whisker stimuli lead to the functional activation of somatotopically organized barrel columns as visualized by intrinsic signal optical imaging. Similar results were obtained in the reeler visual system. When analyzing pathways that could be responsible for preservation of tactile perception, lemniscal thalamic projections were found to be largely intact, despite the smearing of target neurons across the cortical mantle. However, with optogenetic experiments we found evidence for a mild dispersion of thalamic synapse targeting on layer IV-spiny stellate cells, together with a general weakening in thalamocortical input strength. This weakening of thalamic inputs was compensated by intracortical mechanisms involving increased recurrent excitation and/or reduced feedforward inhibition. In conclusion, a layer loss so far only led to the detection of subtle defects in sensory processing by reeler mice. This argues in favor of a view in which cortical layers are not an essential component for basic perception and cognition. A view also supported by recent studies in birds, which can have remarkable cognitive capacities despite the lack of a neocortex with multiple cortical layers. In conclusion, we suggest that future studies directed toward understanding cortical functions should rather focus on circuits specified by functional cell type composition than mere laminar location.

Molecular signatures of neural connectivity in the olfactory cortex.

The ability to target subclasses of neurons with defined connectivity is crucial for uncovering neural circuit functions. The olfactory (piriform) cortex is thought to generate odour percepts and memories, and odour information encoded in piriform is routed to target brain areas involved in multimodal sensory integration, cognition and motor control. However, it remains unknown if piriform outputs are spatially organized, and if distinct output channels are delineated by different gene expression patterns. Here we identify genes selectively expressed in different layers of the piriform cortex. Neural tracing experiments reveal that these layer-specific piriform genes mark different subclasses of neurons, which project to distinct target areas. Interestingly, these molecular signatures of connectivity are maintained in reeler mutant mice, in which neural positioning is scrambled. These results reveal that a predictive link between a neuron's molecular identity and connectivity in this cortical circuit is determined independent of its spatial position.

Reelin and cofilin cooperate during the migration of cortical neurons: a quantitative morphological analysis.

In reeler mutant mice, which are deficient in reelin (Reln), the lamination of the cerebral cortex is disrupted. Reelin signaling induces phosphorylation of LIM kinase 1, which phosphorylates the actin-depolymerizing protein cofilin in migrating neurons. Conditional cofilin mutants show neuronal migration defects. Thus, both reelin and cofilin are indispensable during cortical development. To analyze the effects of cofilin phosphorylation on neuronal migration we used in utero electroporation to transfect E14.5 wild-type cortical neurons with pCAG-EGFP plasmids encoding either a nonphosphorylatable form of cofilin 1 (cofilin(S3A)), a pseudophosphorylated form (cofilin(S3E)) or wild-type cofilin 1 (cofilin(WT)). Wild-type controls and reeler neurons were transfected with pCAG-EGFP. Real-time microscopy and histological analyses revealed that overexpression of cofilin(WT) and both phosphomutants induced migration defects and morphological abnormalities of cortical neurons. Of note, reeler neurons and cofilin(S3A)- and cofilin(S3E)-transfected neurons showed aberrant backward migration towards the ventricular zone. Overexpression of cofilin(S3E), the pseudophosphorylated form, partially rescued the migration defect of reeler neurons, as did overexpression of Limk1. Collectively, the results indicate that reelin and cofilin cooperate in controlling cytoskeletal dynamics during neuronal migration.

RORα Regulates Multiple Aspects of Dendrite Development in Cerebellar Purkinje Cells In Vivo.

The establishment of cell-type-specific dendritic arbors is fundamental for proper neural circuit formation. Here, using temporal- and cell-specific knock-down, knock-out, and overexpression approaches, we show that multiple aspects of the dendritic organization of cerebellar Purkinje cells (PCs) are controlled by a single transcriptional factor, retinoic acid-related orphan receptor-alpha (RORα), a gene defective in staggerer mutant mice. As reported earlier, RORα was required for regression of primitive dendrites before postnatal day 4 (P4). RORα was also necessary for PCs to form a single Purkinje layer from P0 to P4. The knock-down of RORα from P4 impaired the elimination of perisomatic dendrites and maturation of single stem dendrites in PCs at P8. Filopodia and spines were also absent in these PCs. The knock-down of RORα from P8 impaired the formation and maintenance of terminal dendritic branches of PCs at P14. Finally, even after dendrite formation was completed at P21, RORα was required for PCs to maintain dendritic complexity and functional synapses, but their mature innervation pattern by single climbing fibers was unaffected. Interestingly, overexpression of RORα in PCs at various developmental stages did not facilitate dendrite development, but had specific detrimental effects on PCs. Because RORα deficiency during development is closely related to the severity of spinocerebellar ataxia type 1, delineating the specific roles of RORα in PCs in vivo at different time windows during development and throughout adulthood would facilitate our understanding of the pathogenesis of cerebellar disorders. Significance statement: The genetic programs by which each neuron subtype develops and maintains dendritic arbors have remained largely unclear. This is partly because dendrite development is modulated dynamically by neuronal activities and interactions with local environmental cues in vivo. In addition, dendrites are formed and maintained by the balance between their growth and regression; the effects caused by the disruption of transcription factors during the early developmental stages could be masked by dendritic growth or regression in the later stages. Here, using temporal- and cell-specific knock-down, knock-out, and overexpression approaches in vivo, we show that multiple aspects of the dendritic organization of cerebellar Purkinje cells are controlled by a single transcriptional factor, retinoic acid-related orphan receptor alpha.

Abstract 2382: Exploring the relationship between reelin signaling and breast cancer metastasis

Abstract We have investigated the role of reelin signaling in the progression of metastatic breast cancer. Reelin signaling serves to position neurons in the developing central nervous system, but functions of this signaling pathway in other tissues have not been studied until recently. Our laboratory discovered that the reelin pathway is an important regulator of mammary gland development. We showed that reelin and Dab1, the intracellular adaptor protein for reelin, are required for timely development and proper morphology of the mammary gland. We also found that normal mammary epithelial cells (MECs) can respond to exogenous reelin by slowing their migration rate. Since aberration of developmental pathways is often implicated in breast cancer progression, we studied the possible roles of the reelin pathway in this process. We found that reelin signaling pathway components are differentially expressed across a variety of human breast cancer cell lines. We also showed that mouse mammary carcinoma 4T1 cells do not respond to reelin by slowing their migration, likely through downregulation of Dab1 mRNA. Orthotopic injections of 4T1 cells into Balb/C mice are used to model stage IV triple negative breast cancer, and resulted in aggressive tumors that produced numerous metastases in the lungs by 4 weeks post-injection. In order to find whether reelin signaling functions in the tumor microenvironment, we injected 4T1 cells into mice that lack reelin or Dab1 (reeler and Dab1 mutant mice). We found that 4T1 tumor growth was delayed in reeler mutants, and we are currently in the process of assessing the metastatic burden in these mice. We did not see differences in tumor growth or metastatic lung colonization in Dab1 mutant mice. These results indicate that the reelin signaling pathway may promote tumor growth via Dab1-independent, non-canonical signaling events. Citation Format: Elvira Khialeeva, Joan Chou, Ellen M. Carpenter. Exploring the relationship between reelin signaling and breast cancer metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2382. doi:10.1158/1538-7445.AM2015-2382

Abstract B26: Reelin signaling in tumor microenvironment is required for metastasis of mammary carcinoma cells.

Abstract We have investigated the role of reelin signaling in the microenvironment of breast cancer and found that reelin is essential for progression of mammary tumors to metastasis. Reelin signaling serves to position neurons in the developing central nervous system, but functions of this signaling pathway in other tissues have not been studied until recently. Our laboratory discovered that the reelin pathway is an important regulator of mammary gland development. We showed that reelin and Dab1, the intracellular adaptor protein for reelin, are required for timely development and proper morphology of the mammary gland. We also found that normal mammary epithelial cells (MECs) can respond to exogenous reelin by slowing their migration rate, while mouse mammary carcinoma 4T1 cells lose this response, likely through downregulation of Dab1 mRNA. Orthotopic injections of 4T1 cells into wild type Balb/C mice resulted in aggressive tumors that produced numerous metastases in the lungs by 4 weeks post-injection. However, when these cells were injected into mice that lack reelin or Dab1 (reeler and Dab1 mutant mice), we saw a dramatic reduction in lung metastases despite the presence of primary tumors comparable in size to the tumors formed in wild type mice. We show that this phenotype is dependent upon loss of reelin in the host environment, since tumor cells isolated from reeler mutant mice produce primary tumors and lung metastases when injected into Balb/C mice. Using soft agar colony assays and Western blotting, we show that the inability of 4T1 cells to metastasize in mutant animals might be due to a reduced capacity of mammary carcinoma cells to undergo epithelial to mesenchymal transition (EMT) in an environment deficient for reelin signaling. In addition, tumors from reeler and Dab1 mutant mice appear to have more differentiated cells as shown by anti-α smooth muscle Actin and anti-K14 immunostaining. Ki-67 staining showed no differences in proliferation between tumors from wild type, reeler or Dab1 mutant mice. Finally, tail vein injections of 4T1 cells into reeler mutant mice also fail to produce metastases, indicating that reelin signaling in the host environment might have important functions at several steps of the metastatic process. Our results implicate reelin signaling as an essential component of transition from the primary tumor to a metastatic carcinoma. Citation Format: Elvira Khialeeva, Joan W. Chou, Ellen M. Carpenter. Reelin signaling in tumor microenvironment is required for metastasis of mammary carcinoma cells.. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B26. doi:10.1158/1538-7445.CHTME14-B26

Inversion of layer‐specific cadherin expression profiles and maintenance of cytoarchitectonic areas in the allocortex of the reeler mutant mouse

Cadherins are calcium-depending cell adhesion proteins that play critical roles in brain morphogenesis and wiring. They provide an adhesive code for the development of cortical layers, due to their homophilic interactions and their restricted spatiotemporal expression patterns. In the adult organism, cadherins are involved in the maintenance and plasticity of neuronal circuits that play a role in learning. A well-known model for studying corticogenesis is the reeler mouse model. Numerous investigations of neocortical development suggest that, in the reeler mutant mouse, the lack of the protein Reelin results in cell-type and region-dependent changes of the neocortical layers. To investigate in detail how layer formation and regionalization is perturbed in the phylogenetically older archicortex of the adult reeler mutant mouse, we studied the expression of 11 different cadherins (Cdh4, Cdh7, Cdh8, Cdh11, Pcdh1, Pcdh7, Pcdh8, Pcdh9, Pcdh10, Pcdh17, and Pcdh19) and of the transcription factors ER81 and Cux2 by in situ hybridization in the (peri-)archicortex. All cadherins studied show a layer-specific expression in the (peri-)archicortex of the wildtype brain. In the archicortex of the reeler mutant, the cadherin-expressing cell layers are dispersed in the radial dimension, whereas in the periarchicortex the superficial and deep layers are inverted, both in the adult and during development. Possibly, this inversion relates to the histoarchitectural division of the reeler entorhinal cortex into an external and an internal zone. The regionalized, gradient-like expression of the cadherins is preserved in the reeler mutant mouse.

Characterization of Neonatal Vocal and Motor Repertoire of Reelin Mutant Mice

Reelin is a large secreted extracellular matrix glycoprotein playing an important role in early neurodevelopment. Several genetic studies found an association between RELN gene and increased risk of autism suggesting that reelin deficiency may be a vulnerability factor in its etiology. Moreover, a reduced reelin expression has been observed in several brain regions of subjects with Autism Spectrum Disorders. Since a number of reports have documented presence of vocal and neuromotor abnormalities in patients with autism and suggested that these dysfunctions predate the onset of the syndrome, we performed a fine-grain characterization of the neonatal vocal and motor repertoire in reelin mutant mice to explore the developmental precursors of the disorder. Our findings evidence a general delay in motor and vocal development in heterozygous (50% reduced reelin) and reeler (lacking reelin gene) mutant mice. As a whole, an increased number of calls characterized heterozygous pup's emission. Furthermore, the typical ontogenetic peak in the number of calls characterizing wild-type pups on postnatal day 4 appeared slightly delayed in heterozygous pups (to day 6) and was quite absent in reeler littermates, which exhibited a flat profile during development. We also detected a preferential use of a specific call category (two-components) by heterozygous and reeler mice at postnatal days 6 and 8 as compared to their wild-type littermates. With regard to the analysis of spontaneous movements, a differential profile emerged early in development among the three genotypes. While only slight coordination difficulties are exhibited by heterozygous pups, all indices of motor development appear delayed in reeler mice. Overall, our results evidence a genotype-dependent deviation in ultrasonic vocal repertoire and a general delay in motor development in reelin mutant pups.

Mapping remodeling of thalamocortical projections in the living reeler mouse brain by diffusion tractography.

A major challenge in neuroscience is to accurately decipher in vivo the entire brain circuitry (connectome) at a microscopic level. Currently, the only methodology providing a global noninvasive window into structural brain connectivity is diffusion tractography. The extent to which the reconstructed pathways reflect realistic neuronal networks depends, however, on data acquisition and postprocessing factors. Through a unique combination of approaches, we designed and evaluated herein a framework for reliable fiber tracking and mapping of the living mouse brain connectome. One important wiring scheme, connecting gray matter regions and passing fiber-crossing areas, was closely examined: the lemniscal thalamocortical (TC) pathway. We quantitatively validated the TC projections inferred from in vivo tractography with correlative histological axonal tracing in the same wild-type and reeler mutant mice. We demonstrated noninvasively that changes in patterning of the cortical sheet, such as highly disorganized cortical lamination in reeler, led to spectacular compensatory remodeling of the TC pathway.

Reelin and the Cdc42/Rac1 guanine nucleotide exchange factor αPIX/Arhgef6 promote dendritic Golgi translocation in hippocampal neurons

In the cerebral cortex of reeler mutant mice lacking reelin expression, neurons are malpositioned and display misoriented apical dendrites. Neuronal migration defects in reeler have been studied in great detail, but how misorientation of apical dendrites is related to reelin deficiency is poorly understood. In wild-type mice, the Golgi apparatus transiently translocates into the developing apical dendrite of radially migrating neurons. This dendritic Golgi translocation has recently been shown to be promoted by reelin. However, the underlying signalling mechanisms are largely unknown. Here, we show that the Cdc42/Rac1 guanine nucleotide exchange factor αPIX/Arhgef6 promoted translocation of Golgi cisternae into developing dendrites of hippocampal neurons. Reelin treatment further increased the αPIX-dependent effect. In turn, overexpression of exchange activity-deficient αPIX or dominant-negative (dn) Cdc42 or dn-Rac1 impaired dendritic Golgi positioning, an effect that was not compensated by reelin treatment. Together, these data suggest that αPIX may promote dendritic Golgi translocation, as a downstream component of a reelin-modulated signalling pathway. Finally, we found that reelin promoted the translocation of the Golgi apparatus into the dendrite that was most proximal to the reelin source. The distribution of reelin may thus contribute to the selection of the process that becomes the apical dendrite.

2012 Late-Breaking Basic Science Oral Session

### Cardiac Myosin Binding Protein C is an Ultra-early and Cardiac-specific Biomarker of Myocardial Necrosis Diederik W Kuster1, Adriana Cardenas-Ospina2, Lawson Miller2, Christian Troidl3, Holger M Nef3, Christoph Liebetrau3, Mollmann Helge3, Karen S Pieper4, Kenneth W Mahaffey4, Neal S Kleiman5, Bruno D Stuyvers2, Ali J Marian6, Sakthivel Sadayappan1; 1Loyola Univ Chicago, Maywood, IL, 2Memorial Univ, St John’s, Canada, 3Kerckhoff Heart and Thorax Cntr, Bad Neuheim, Germany, 4Duke Univ Med Cntr, Durham, NC, 5The Methodist DeBakey Heart and Vascular Cntr, Houston, TX, 6Univ of Texas Health Sciences, Houston, TX Rationale: We recently demonstrated that proteolytic cleavage fragments of cardiac myosin binding protein-C (cMyBP-C) can be detected in the serum after myocardial infarction (MI) in a rat model and in patients with MI. The findings implied that serum cMyBP-C might be useful as cardiac-specific biomarker for MI. The release kinetics of cMyBP-C in the circulation post-MI remains to be elucidated. Objective: Determine the release kinetics of cMyBP-C as an ultra-early and cardiac-specific biomarker of myocardial necrosis. Method and Results: To determine the exact timing of cMyBP-C release in the bloodstream post-acute MI, left anterior descending (LAD) coronary artery was ligated in adult swine (n=6). ECG showed significant ST elevation. Infarct size represented 12.4 ± 1.9% of total ventricular mass. Blood samples were collected before and at predetermined time points between 30 min and 14 days after LAD ligation. Plasma cMyBP-C level was quantified using a highly sensitive and rapid sandwich enzyme-linked immunosorbent assay. Compared with baseline, cMyBP-C levels were increased in post-MI serum within 45 min (0.64 ± 0.52 ng/ml) after LAD ligation and declined after 16 hrs to the baseline level (0.01 ± 0.00 ng/ml). In contrast, cardiac troponin I (cTnI) level peaked after …

Vldlr overexpression causes hyperactivity in rats

BackgroundReelin regulates neuronal positioning in cortical brain structures and neuronal migration via binding to the lipoprotein receptors Vldlr and Lrp8. Reeler mutant mice display severe brain morphological defects and behavioral abnormalities. Several reports have implicated reelin signaling in the etiology of neurodevelopmental and psychiatric disorders, including autism, schizophrenia, bipolar disorder, and depression. Moreover, it has been reported that VLDLR mRNA levels are increased in the post-mortem brain of autistic patients.MethodsWe generated transgenic (Tg) rats overexpressing Vldlr, and examined their histological and behavioral features.ResultsSpontaneous locomotor activity was significantly increased in Tg rats, without detectable changes in brain histology. Additionally, Tg rats tended to show performance deficits in the radial maze task, suggesting that their spatial working memory was slightly impaired. Thus, Vldlr levels may be involved in determining locomotor activity and memory function.ConclusionsUnlike reeler mice, patients with neurodevelopmental or psychiatric disorders do not show striking neuroanatomical aberrations. Therefore, it is notable, from a clinical point of view, that we observed behavioral phenotypes in Vldlr-Tg rats in the absence of neuroanatomical abnormalities.

Selective and regulated gene expression in murine Purkinje cells by in utero electroporation

Cerebellar Purkinje cells, which convey the only output from the cerebellar cortex, play an essential role in cerebellar functions, such as motor coordination and motor learning. To understand how Purkinje cells develop and function in the mature cerebellum, an efficient method for molecularly perturbing them is needed. Here we demonstrate that Purkinje cell progenitors at embryonic day (E)11.5 could be efficiently and preferentially transfected by spatially directed in utero electroporation (IUE) with an optimized arrangement of electrodes. Electrophysiological analyses indicated that the electroporated Purkinje cells maintained normal membrane properties, synaptic responses and synaptic plasticity at postnatal days 25-28. By combining the L7 promoter and inducible Cre/loxP system with IUE, transgenes were expressed even more specifically in Purkinje cells and in a temporally controlled manner. We also show that three different fluorescent proteins could be simultaneously expressed, and that Bassoon, a large synaptic protein, could be expressed in the electroporated Purkinje cells. Moreover, phenotypes of staggerer mutant mice, which have a deletion in the gene encoding retinoid-related orphan receptor α (RORα1), were recapitulated by electroporating a dominant-negative form of RORα1 into Purkinje cells at E11.5. Together, these results indicate that this new IUE protocol, which allows the selective, effective and temporally regulated expression of multiple foreign genes transfected into Purkinje cell progenitors in vivo, without changing the cells' physiological characteristics, is a powerful tool for elucidating the molecular mechanisms underlying early Purkinje cell developmental events, such as dendritogenesis and migration, and synaptic plasticity in mature Purkinje cells.

Reelin, a Marker of Stress Resilience in Depression and Psychosis

Reelin protein is an extracellular matrix protease responsible for normal lamination of the brain during embryogenesis, and is involved in cell signaling and synaptic plasticity in adult life. The Reelin gene (RELN) is localized to chromosome 7 in humans and chromosome 5 in mice, and produces a protein product with relative molecular mass of 388 kDa. Reelin protein is localized to a number of brain sites, specifically Cajal–Retzius cells located in layer 1 of neocortex, GABAergic interneurons, and cerebellar granule cells. Activation of the Reelin signaling pathway leads to various, important functions such as enhancement of long-term potentiation, cell proliferation, cell migration, and more importantly dendritic spine morphogenesis. Spontaneous mutation of the RELN gene in the reeler homozygous mutant mouse leads to the development of ataxia and a reeling gait and reversal of the normal layering of the brain (Goffinet, 1979). The reeler cerebellum is hypoplastic and the Purkinje cell number is diminished potentially explaining the ataxic gait. Historically, comparative studies of the homozygous and heterozygous reeler mutant mice and the similarities in their cognitive, anatomic, and biochemical abnormalities to mental diseases caused an upsurge in generation of new information about Reelin's involvement in various neuropsychiatric disorders. Multiple postmortem studies have implicated a pathologic involvement of RELN gene in several neuropsychiatric disorders, namely, schizophrenia, bipolar disorder, autism, major depression, lissencephaly, and Alzheimer's disease. Erminio Costa and coworkers reported on deficits in brain levels of Reelin mRNA and the protein in patients suffering from schizophrenia and psychotic bipolar disorder (Impagnatiello et al, 1998). Later reports by Fatemi (2005) confirmed and extended these findings to patients with non-psychotic bipolar disorder, major depression, and autism. Additional reports by other investigators showed that Reelin deficits are common phenomena subserving cognitive deficits in multiple disorders including lissencephaly, Alzheimer's disease, and temporal lobe epilepsy. The causative factors may include a mutation in the RELN gene (lissencephaly) to variable expression of the molecule due to hypermethylation of the promoter region of the RELN gene or other unknown mechanisms. Moreover, several non-CNS disorders have been associated with changes in expression of Reelin including several forms of cancers and otosclerosis. In the current issue, Teixeira and colleagues evaluated the effects of overexpression of Reelin in a transgenic mouse model as compared with wild-type mice and to heterozygous reeler mutant mice using a battery of behavioral tests (Texeira et al, 2011). These investigators tested the hypothesis that if downregulation of Reelin production could lead to various behavioral abnormalities, upregulation of this molecule could lead to reversal of these deficits. The effects of Reelin overexpression on (1) anxiety (open field, novelty-suppressed feeding, black and white box), (2) depression (forced-swim test in presence or absence of the stress-associated hormone corticosterone), (3) mania and positive symptoms of schizophrenia (cocaine sensitization and cocaine self-administration), and (4) gating abnormalities in schizophrenia (prepulse inhibition) were assessed at 8 weeks of age in presence or absence of the NMDA antagonist MK-801. Additionally, all animals were subjected to electrophysiological recordings to evaluate for functionality of the NMDA-NR2B glutamatergic transmission. The results demonstrated that although heterozygousreeler mice did not show any behavioral abnormalities, mice overexpressing Reelin exhibited several salient and novel findings ie, (1) increased brain levels of Reelin protected the transgenic mice from developing depression-like behavior in the forced-swim test, (2) stress induced NMDA NR2B—mediated synaptic transmission (a marker of depression) was reduced in transgenic mice, and lastly (3) evidence for positive symptoms of schizophrenia ie, behavioral sensitization to cocaine and gating deficits (PPI abnormalities) were reduced in transgenic mice. These findings are both positive and potentially important as they point to Reelin as a therapeutic agent to treat some aspects of depression, psychosis, and autistic behavior. Previous findings of Reelin production induced by various psychotropic medications including citalopram support these results (Jaako et al, 2011). Additionally, in a previous work by the same laboratory, it was demonstrated that Reelin overexpression in the hippocampus of the transgenic mice resulted in potentiation of synaptic strength and hypertrophy of dendritic spine morphology, albeit with no change in dendritic spine density (Pujadas et al, 2010). These changes in the hippocampus reflect Reelin's ability to act as an antidepressant by both increasing neurogenesis and improving learning and memory. As Reelin abnormalities are pervasive and span a range of neuropsychiatric disorders, future studies should focus on and expand the scope of genetic, biochemical, and clinical studies to better define the etiopathologic and therapeutic roles of this important and evolutionary conserved molecule.

Impairment of mGluR signaling at cerebellar parallel fiber–Purkinje cell synapses in staggerer mutant mice

Glutamate activates peripheral group I metabotropic glutamate receptors (mGluRs) and contributes to inflammatory pain. However, it is still not clear the mechanisms are involved in group I mGluR-mediated peripheral sensitization. Herein, we report that group I mGluRs signaling sensitizes acid-sensing ion channels (ASICs) in dorsal root ganglion (DRG) neurons and contributes to acidosis-evoked pain. DHPG, a selective group I mGluR agonist, can potentiate the functional activity of ASICs, which mediated the proton-induced events. DHPG concentration-dependently increased proton-gated currents in DRG neurons. It shifted the proton concentration⿿response curve upwards, with a 47.3 ± 7.0% increase of the maximal current response to proton. Group I mGluRs, especially mGluR5, mediated the potentiation of DHPG via an intracellular cascade. DHPG potentiation of proton-gated currents disappeared after inhibition of intracellular Gq/11 proteins, PLCβ, PKC or PICK1 signaling. Moreover, DHPG enhanced proton-evoked membrane excitability of rat DRG neurons and increased the amplitude of the depolarization and the number of spikes induced by acid stimuli. Finally, peripherally administration of DHPG dose-dependently exacerbated nociceptive responses to intraplantar injection of acetic acid in rats. Potentiation of ASIC activity by group I mGluR signaling in rat DRG neurons revealed a novel peripheral mechanism underlying group I mGluRs involvement in hyperalgesia.