Developmental expression of EphA4-tyrosine kinase receptor in the mouse brain and spinal cord

Eph receptor tyrosine kinases and their ephrin ligands are involved in some of the most important steps during the development of the central nervous system, including cell migration, axon guidance, topographic mapping and synapse formation. Moreover, in the adult, they have been implicated in plasticity and regulation of neural stem cell function. One member of the Eph family, EphA4, can bind to both classes of ephrins and may have multiple functions in nervous system development. In order to look for potential sites of EphA4 action during central nervous system development, we conducted a spatio-temporal analysis of EphA4 protein expression. We used immunohistochemistry in preference to in situ hybridization because of the high likelihood that EphA4 protein is expressed on axon tracts, long distances from EphA4 mRNA. In the telencephalon, EphA4 was expressed in the developing cortex from embryonic day 11 (E11) and, later, on major cortical tracts including the corpus callosum and cortico-spinal tract. Robust EphA4 expression was also found in the hippocampus and fornix, and cells and tracts in the striatum. In the diencephalon, the thalamus, the hypothalamus and thalamo-cortical projection were strongly positive. In the mesencephalon, a number of different nuclei were weakly positive, most prominently the red nucleus. In the rhombencephalon, many nuclei were strongly positive including the cerebellum and one of its afferent paths, the inferior cerebellar peduncle, as well as the olivary region. In the spinal cord, there was a dynamic pattern of expression through development, with persistent expression in the dorsal funiculus and ventral grey matter.     

Differential gene expression in the EphA4 knockout spinal cord and analysis of the inflammatory response following spinal cord injury

Mice lacking the axon guidance molecule EphA4 have been shown to exhibit extensive axonal regeneration and functional recovery following spinal cord injury. To assess mechanisms by which EphA4 may modify the response to neural injury a microarray was performed on spinal cord tissue from mice with spinal cord injury and sham injured controls. RNA was purified from spinal cords of adult EphA4 knockout and wild-type mice four days following lumbar spinal cord hemisection or laminectomy only and was hybridised to Affymetrix All-Exon Array 1.0 GeneChips?. While subsequent analyses indicated that several pathways were altered in EphA4 knockout mice, of particular interest was the attenuated expression of a number of inflammatory genes, including Arginase 1, expression of which was lower in injured EphA4 knockout compared to wild-type mice. Immunohistological analyses of different cellular components of the immune response were then performed in injured EphA4 knockout and wildtype spinal cords. While numbers of infiltrating CD3+ T cells were low in the hemisection model, a robust CD11b+ macrophage/microglial response was observed post-injury. There was no difference in the overall number or spread of macrophages/activated microglia in injured EphA4 knockout compared to wild-type spinal cords at 2, 4 or 14 days post-injury, however a lower proportion of Arginase-1 immunoreactive macrophages/activated microglia was observed in EphA4 knockout spinal cords at 4 days post-injury. Subtle alterations in the neuroinflammatory response in injured EphA4 knockout spinal cords may contribute to the regeneration and recovery observed in these mice following injury.     

Proteolysis in quaking mouse brain and spinal cord

Six proteolytic enzymes were assayed for activity in quaking CNS in examining the hypothesis that increased proteolytic activity contributes to the hypomyelination characteristic of this mutant. Cathepsin B-like enzyme, cathepsin D, neutral proteinase, calcium-activated neutral proteinase, prolyl endopeptidase, and diaminopeptidase II were assayed in whole homogenate of brain or spinal cord and each was found to have activity similar to that in normal mice. These results do not support a relationship between proteolysis and the genetic defect and suggest that other factors should be investigated to delineate the pathogenesis of this mutant.     

High-resolution gene expression atlases for adult and developing mouse brain and spinal cord

Knowledge of the structure, genetics, circuits, and physiological properties of the mammalian brain in both normal and pathological states is ever increasing as research labs worldwide probe the various aspects of brain function. Until recently, however, comprehensive cataloging of gene expression across the central nervous system has been lacking. The Allen Institute for Brain Science, as part of its mission to propel neuroscience research, has completed several large gene-mapping projects in mouse, nonhuman primate, and human brain, producing informative online public resources and tools. Here we present the Allen Mouse Brain Atlas, covering ~20,000 genes throughout the adult mouse brain; the Allen Developing Mouse Brain Atlas, detailing expression of approximately 2,000 important developmental genes across seven embryonic and postnatal stages of brain growth; and the Allen Spinal Cord Atlas, revealing expression for ~20,000 genes in the adult and neonatal mouse spinal cords. Integrated data-mining tools, including reference atlases, informatics analyses, and 3-D viewers, are described. For these massive-scale projects, high-throughput industrial techniques were developed to standardize and reliably repeat experimental goals. To verify consistency and accuracy, a detailed analysis of the 1,000 most viewed genes for the adult mouse brain (according to website page views) was performed by comparing our data with peer-reviewed literature and other databases. We show that our data are highly consistent with independent sources and provide a comprehensive compendium of information and tools used by thousands of researchers each month. All data and tools are freely available via the Allen Brain Atlas portal (www.brain-map.org).     

Developmental expression of MNAR mRNA in the mouse brain

During the development of the central nervous system, estrogen influences cellular differentiation and determines the functional connectivity of distinct neural networks. Estrogens generally act through nuclear estrogen receptors (ERs). Recent research has additionally revealed rapid estrogen effects requiring the binding of estrogen to membrane/cytoplasmic ERs and the activation of intracellular signaling systems such as the Src/MAPK cascade. The scaffold protein MNAR/PELP1 appears to be the designated functional mediator of such non-genomic estrogen effects between non-nuclear ERs and Src/MAPKs. In this study, we demonstrate the expression and differential regulation of MNAR mRNA in the developing male and female mouse brain by quantitative polymerase chain reaction. In the midbrain and hypothalamus, a gradual decline in MNAR mRNA levels has been observed prenatally with the highest values at embryonic day 15 and lowest at postnatal day 15. In the cortex, mRNA levels do not fluctuate until postnatal day 7 but decrease thereafter. No differences in MNAR expression between sexes have been detected. Analysis of neuronal and astroglia-enriched cell cultures has revealed the presence of MNAR in both cell types.     

EphA4 blockers promote axonal regeneration and functional recovery following spinal cord injury in mice

Upregulation and activation of developmental axon guidance molecules, such as semaphorins and members of the Eph receptor tyrosine kinase family and their ligands, the ephrins, play a role in the inhibition of axonal regeneration following injury to the central nervous system. Previously we have demonstrated in a knockout model that axonal regeneration following spinal cord injury is promoted in the absence of the axon guidance protein EphA4. Antagonism of EphA4 was therefore proposed as a potential therapy to promote recovery from spinal cord injury. To further assess this potential, two soluble recombinant blockers of EphA4, unclustered ephrin-A5-Fc and EphA4-Fc, were examined for their ability to promote axonal regeneration and to improve functional outcome following spinal cord hemisection in wildtype mice. A 2-week administration of either of these blockers following spinal cord injury was sufficient to promote substantial axonal regeneration and functional recovery by 5 weeks following injury. Both inhibitors produced a moderate reduction in astrocytic gliosis, indicating that much of the effect of the blockers may be due to promotion of axon growth. These studies provide definitive evidence that soluble inhibitors of EphA4 function offer considerable therapeutic potential for the treatment of spinal cord injury and may have broader potential for the treatment of other central nervous system injuries.     

Analysis of EphA4 receptor tyrosine kinase substrate specificity using peptide-based arrays

Eph receptor tyrosine kinases regulate many important biological processes. In the present study, we explored the substrate specificity of the EphA4 receptor tyrosine kinase using peptide arrays. We define a consensus substrate motif for EphA4 and go on to identify and test a number of potential EphA4 substrates and map their putative site(s) of phosphorylation. Cotransfection studies validate two of the predicted substrates: Nck2 and Dok1. Our findings identify several potential EphA4 substrates and demonstrate the general utility of using peptide arrays to rapidly identify and map protein kinase phosphorylation sites.     

4-Substituted quinazoline derivatives as novel EphA2 receptor tyrosine kinase inhibitors

Erythropoietin-producing hepatocellular receptor tyrosine kinase subtype A2 (EphA2) is an attractive therapeutic target for suppressing tumor progression. In our efforts to discover novel small molecules to inhibit EphA2, a class of compound based on 4-substituted quinazoline containing 7-(morpholin-2-ylmethoxy) group was identified as a novel hit by high throughput screening campaign. Structural modification of parent quinazoline scaffolds by introducing substituents on aniline displayed potent inhibitory activities toward EphA2.     

Developmental regulation of myelin basic protein expression in mouse brain

Developmental regulation of myelin basic protein expression in mouse brain has been examined by comparing the myelin basic protein coding potential of mRNA in vitro with the accumulation of myelin basic protein-related polypeptides in vivo. In vitro translation of mRNA isolated from mouse brain generated eight myelin basic protein-related polypeptides with apparent molecular weights of 34K, 30K, 29K, 26K, 21.5K, 18.5K, 17K, and 14K. A similar set of eight myelin basic protein-related polypeptides with corresponding molecular weights was identified in vivo when total brain proteins were analyzed by immunoblotting. Each of the myelin basic protein-related polypeptides shows a characteristic developmental profile in terms of mRNA level and rate of accumulation implying a complex developmental program of myelin basic protein gene expression with regulation and modulation at several different biosynthetic levels.     

Developmental regulation of GABA(B) receptor function in rat spinal cord

GABA(B) receptors are heterodimers coupled to G-proteins. The present study was undertaken to investigate activation of GABA(B) receptors in cerebral cortex and spinal cord using [35S]GTPgammaS binding assays, a direct measure of G-protein activity. The results revealed that the GABA(B) agonist baclofen stimulates GTPgammaS binding in cerebral cortex, with an ED50 of 50microM. This response is blocked by the GABA(B) receptor antagonist CGP 55845A (100nM). In contrast, baclofen-stimulated GTPgammaS binding was not observed in adult spinal cord tissue under similar incubation conditions, or after varying magnesium, calcium, GDP, [35S]GTPgammaS, or membrane concentrations in the assay medium. Stimulation of adult rat spinal cord muscarinic receptors did result in a concentration-related increase in [35S]GTPgammaS binding. Baclofen-stimulated GTPgammaS binding in adult spinal cord did not appear after peripheral inflammation, despite significant increases in GABA(B) subunit mRNA levels. As opposed to adult, appreciable GTPgammaS binding was observed in membranes prepared from spinal cords of rats within the first 14 days of postnatal development, suggesting that GABA(B) receptor function in the rat spinal cord is developmentally regulated. The results indicate that GABA(B) receptors may not be coupled to G-proteins in the adult rat spinal cord, or couple in a way that differs from that in newborns or adult cerebral cortex.     

Regulation of gene expression by corticoid hormones in the brain and spinal cord

Glucocorticoids (GC) and mineralocorticoids (MC) have profound regulatory effects upon the central nervous system (CNS). Hormonal regulation affects several molecules essential to CNS function. First, evidences are presented that mRNA expression of the 3 and β1-subunits of the Na,K-ATPase are increased by GC and physiological doses of MC in a region-dependent manner. Instead, high MC doses reduce the β1 isoform and enzyme activity in amygdaloid and hypothalamic nuclei, an effect which may be related to MC control of salt appetite. The 3-subunit mRNA of the Na,K-ATPase is also stimulated by GC in motoneurons of the injured spinal cord, suggesting a role for the enzyme in GC neuroprotection. Second, we provide evidences for hormonal effects on the expression of mRNA for the neuropeptide arginine vasopressin (AVP). Our data show that GC inhibition of AVP mRNA levels in the paraventricular nucleus is sex-hormone dependent. This sexual dimorphism may explain sex differences in the hypothalamic–pituitary–adrenal axis function between female and male rats. Third, steroid effects on the astrocyte marker glial fibrillary acidic protein (GFAP) points to a complex regulatory mechanism. In an animal model of neurodegeneration (the Wobbler mouse) showing pronounced astrogliosis of the spinal cord, in vivo GC treatment down-regulated GFAP immunoreactivity, whereas the membrane-active steroid antioxidant U-74389F up-regulated this protein. It is likely that variations in GFAP protein expression affect spinal cord neurodegeneration in Wobbler mice. Fourth, an interaction between neurotrophins and GC is shown in the injured rat spinal cord. In this model, intensive GC treatment increases immunoreactive low affinity nerve growth factor (NGF) receptor in motoneuron processes. Because GC also increases immunoreactive NGF, this mechanism would support trophism and regeneration in damaged tissues. In conclusion, evidences show that some molecules regulated by adrenal steroids in neurons and glial cells are not only involved in physiological control, but additionally, may play important roles in neuropathology.     

Developmental regulation of PSD-95 and nNOS expression in lumbar spinal cord of rats

Postsynaptic density (PSD)-95 is originally isolated from glutamatergic synapse where it serves as a physical tether to allow neuronal nitric oxide synthase (nNOS) signaling by N-methyl-D-aspartate receptor (NMDAR) activity. Considering the physiological importance of glutamate receptor and nitric oxide (NO) during development, we examined the spatiotemporal expression of PSD-95 and nNOS in the lumbar spinal cord at a postnatal stage. Temporally, both gene and protein levels of them gradually increased with age after birth, peaked at the postnatal day 14 (P14), and then decreased to an adult level. In addition, the enhanced coimmunoprecipitations between PSD-95 and nNOS were detected in developing spinal cord. Spatially, PSD-95 staining codistributed with nNOS in NeuN-positive motor neurons and sensory neurons at P14. These findings indicate that PSD-95 and nNOS might collectively participate in spinal cord development.     

Detection of phosphorylated mitogen-activated protein kinase in the developing spinal cord of the mouse embryo

Global understanding of the proteome is a major research topic. The comprehensive visualization of the distribution of proteins in vivo or the construction of in situ protein atlases may be a valuable strategy for proteomic researchers. Information about the distribution of various proteins under physiological and pathological conditions should be extremely valuable for the basic and clinical sciences.The mitogen-activated protein kinase (MAPK) cascade plays an essential role in intracellular signaling in organisms. This cascade also regulates biological processes involving development, differentiation, and proliferation. Phosphorylation and dephosphorylation are integral reactions in regulating the activity of MAPKs. Changes in the phosphorylation state of MAPKs are rapid and reversible; therefore, the localizations of physiologically phosphorylated MAPKs in vivo are difficult to accurately detect. Furthermore, phosphorylated MAPKs are likely to change phosphorylated states through commonly used experimental manipulations.In the present study, as a step toward the construction of in situ phosphoprotein atlases, we attempted to detect physiologically phosphorylated MAPKs in vivo in developing spinal cords of mice. We previously reported an improved immunohistochemical method for detecting unstable phosphorylated MAPKs. The distribution patterns of phosphorylated MAPKs in the spinal cords of embryonic mice from embryonic day 13 (E13) to E17 were observed with an improved immunohistochemical method. Phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and phosphorylated c-Jun N-terminal kinase 1/2 (p-JNK1/2) were strongly observed in the marginal layer and the dorsal horn from E13 to E17. Our results suggest that p-ERK1/2 and p-JNK1/2 play critical roles in the developing spinal cord. Constructing phosphoprotein atlases will be possible in the future if this work is systematically developed on a larger scale than we presented here.     

Protein kinase and protein phosphatase expression in amyotrophic lateral sclerosis spinal cord

The Kinetworks trade mark multi-immunoblotting technique was used to evaluate the expressions of 78 protein kinases, 24 protein phosphatases and phosphorylation states of 31 phosphoproteins in thoracic spinal cord tissue from control subjects and patients having the sporadic form of amyotrophic lateral sclerosis (ALS). In both the cytosolic (C) and particulate (P) fractions of spinal cord from ALS patients as compared with controls, there were increased levels of calcium/calmodulin-dependent protein kinase kinase (CaMKK; C = 120% increase/P = 580% increase;% change, compared with control), extracellular regulated kinase 2 (ERK2; C = 120% increase/P = 170% increase), G protein-coupled receptor kinase 2 (GRK2; C = 140% increase/P = 140% increase), phospho-Y279/216 glycogen synthase kinase 3 alpha/beta (GSK3alpha/beta; C = 90% increase/P = 220% increase), protein kinase B alpha (PKBalpha; C = 360% increase/P = 200% increase), phospho-T638 PKCalpha/beta (C = 630% increase/P = 170% increase), cGMP-dependent protein kinase (PKG; C = 100% increase/P = 75% increase), phospho-T451 dsRNA-dependent protein kinase (PKR; C = 2600% increase/P = 3330% increase), ribosomal S6 kinase 1 (RSK1; C = 750% increase/P = 630% increase), phospho-T389 p70 S6 kinase (S6K; C = 1000% increase/P = 460% increase), and protein-tyrosine phosphatase 1 delta (PTP1delta; C = 43% increase/P = 70% increase). Cytosolic increases in phospho-alpha-S724/gamma-S662 adducin (C = 15650% increase), PKCalpha (C = 100% increase) and PKCzeta (C = 190% increase) were found in ALS patients as compared with controls, while particulate increases in cAMP-dependent protein kinase (PKA; 43% increase), protein kinase C beta (PKCbeta; 330% increase), and stress-activated protein kinase beta (SAPKbeta; 34% increase) were also observed. Cyclin-dependent kinase-associated phosphatase (KAP) was apparently translocated, as it was reduced (31% decrease) in cytosolic fractions but elevated (100% increase) in particulate fractions of ALS spinal cord tissue. Our observations indicate that ALS is associated with the elevated expression and/or activation of many protein kinases, including PKCalpha, PKCbeta, PKCzeta and GSK3alpha/beta, which may augment neural death in ALS, and CaMKK, PKBalpha, Rsk1, S6K, and SAPK, which may be a response to neuronal injury that potentially can mitigate cell death.     

丝氨酸/苏氨酸蛋白激酶31(STK31)基因在小鼠脊髓损伤后的差异表达

目的:研究丝氨酸/苏氨酸蛋白激酶31(STK31)基因与脊髓损伤修复的潜在关联。方法:以半定量RT-PCR方法检测STK31在小鼠脊髓损伤后的差异表达。结果:STK31在脊髓、肾脏、胃、心脏、大脑、脾、胸腺、肺等器官中均有表达且在脊髓中表达明显;在脊髓损伤后4 h及1 d,STK31表达量显著低于对照组(P<0.01)。结论:STK31基因与脊髓损伤及修复有着潜在的关联。     

Unaltered D1, D2, D4, and D5 dopamine receptor mRNA expression and distribution in the spinal cord of the D3 receptor knockout mouse

Dopamine (DA) acts through five receptor subtypes (D1–D5). We compared expression levels and distribution patterns of all DA mRNA receptors in the spinal cord of wild-type (WT) and loss of function D3 receptor knockout (D3KO) animals. D3 mRNA expression was increased in D3KO, but no D3 receptor protein was associated with cell membranes, supporting the previously reported lack of function. In contrast, mRNA expression levels and distribution patterns of D1, D2, D4, and D5 receptors were similar between WT and D3KO animals. We conclude that D3KO spinal neurons do not compensate for the loss of function of the D3 receptor with changes in the other DA receptor subtypes. This supports use of D3KO animals as a model to provide insight into D3 receptor dysfunction in the spinal cord.