Distribution of Kiaa0319-like immunoreactivity in the adult mouse brain--a novel protein encoded by the putative dyslexia susceptibility gene KIAA0319-like

Kiaa0319L is a novel protein encoded by a recently discovered gene KIAA0319-like(L) that may be associated with reading disability. Little is known about the characteristics of this protein and its distribution in the brain. We investigated here expression of this protein in adult mice, using an antibody specific for human and rodent Kiaa0319L. In the brain, Kiaa0319L was localized strongly in the olfactory bulb, and strong expression was found in other regions, including hippocampus, cerebellum, diencephalon and the cerebral cortex. Immunohistochemistry confirmed expression in these brain regions, and showed further that the protein was expressed preferentially in neurons in layer IV and VI of the neocortex, CA1 and CA2 subfields of the hippocampus and a subpopulation of neurons in CA3 and dentate gyrus. Furthermore, the protein was confined to dendrites of CA1 neurons in the stratum radiatum, but not those in the stratum oriens, and in astrocytes within the hippocampus. In the cerebellum, the protein was observed in the molecular layer and a fraction of Purkinje neurons. These findings confirmed expression of Kiaa0319L in brain regions that are involved in reading performance, supporting its possible involvement in reading disability. The specific patterns of localization in the neocortex, hippocampus and cerebellum suggest further that this protein may be related to other biological processes in a subpopulation of neurons within these regions, eg. formation and maintenance of polarity in the neuron.     

Ultrastructural co-localization of TFF3-peptide and oxytocin in the neural lobe of the porcine pituitary

TFF-peptides (formerly P-domain peptides, trefoil factors) are typical secretory products of many mucous epithelial cells. TFF3 is also synthesized in oxytocinergic neurons in the paraventricular and supraoptic nuclei of the human hypothalamus. Here, TFF3 and oxytocin are shown to be co-localized within the same secretory vesicles in the neural (posterior) lobe of the procine pituitary by means of immunoelectron microscopy. Relatively large amounts of TFF3, but not TFF1 and TFF2, are present in the neural lobe of the porcine pituitary, where it is probably released into the bloodstream.     

Expression of cyclin E in postmitotic neurons during development and in the adult mouse brain

Cyclin E, a member of the G1 cyclins, is essential for the G1/S transition of the cell cycle in cultured cells, but its roles in vivo are not fully defined. The present study characterized the spatiotemporal expression profile of cyclin E in two representative brain regions in the mouse, the cerebral and cerebellar cortices. Western blotting showed that the levels of cyclin E increased towards adulthood. In situ hybridization and immunohistochemistry showed the distributions of cyclin E mRNA and protein were comparable in the cerebral cortex and the cerebellum. Immunohistochemistry for the proliferating cell marker, proliferating cell nuclear antigen (PCNA) revealed that cyclin E was expressed by both proliferating and non-proliferating cells in the cerebral cortex at embryonic day 12.5 (E12.5) and in the cerebellum at postnatal day 1 (P1). Subcellular localization in neurons was examined using immunofluorescence and western blotting. Cyclin E expression was nuclear in proliferating neuronal precursor cells but cytoplasmic in postmitotic neurons during embryonic development. Nuclear cyclin E expression in neurons remained faint in newborns, increased during postnatal development and was markedly decreased in adults. In various adult brain regions, cyclin E staining was more intense in the cytoplasm than in the nucleus in most neurons. These data suggest a role for cyclin E in the development and function of the mammalian central nervous system and that its subcellular localization in neurons is important. Our report presents the first detailed analysis of cyclin E expression in postmitotic neurons during development and in the adult mouse brain.     

Co-localization of TFF3 peptide and oxytocin in the human hypothalamus.

TFF-peptides (formerly P domain peptides, trefoil factors) are typical secretory products of many mucous epithelial cells. TFF3 is also synthesized in the hypothalamus and has anxiolytic or anxiogenic activities when injected into the rat amygdala. Here we show by immunohistochemistry that TFF3 is localized to a distinct population of neurons of the human hypothalamic paraventricular and supraoptic nuclei. Generally, TFF3-positive cells are co-localized in oxytocin-producing cells and not in vasopressin-producing cells. Relatively large amounts of TFF3-but not TFF1 and TFF2-are present in the posterior lobe of the human pituitary, where it is probably released into the bloodstream. Furthermore, TFF3 was also detectable in human postmortem cerebrospinal fluid.     

Notch1 and its ligands Delta-like and Jagged are expressed and active in distinct cell populations in the postnatal mouse brain

Notch signaling plays a pivotal role in the regulation of vertebrate neurogenesis. However, in vitro experiments suggest that Notch1 may also be involved in the regulation of later stages of brain development. We have addressed putative roles in the central nervous system by examining the expression of Notch signaling cascade components in the postnatal mouse brain. In situ mRNA hybridization revealed that Notch1 is associated with cells in the subventricular zone, the dentate gyrus and the rostromigratory stream, all regions of continued neurogenesis in the postnatal brain. In addition, Notch1 is expressed at low levels throughout the cortex and olfactory bulb and shows striking expression in the cerebellar Purkinje cell layer. The Notch ligands, including Delta-like1 and 3 and Jagged1 and Jagged2, show distinct expression patterns in the developing and adult brain overlapping that of Notch1. In addition, the downstream targets of the Notch signaling cascade Hes1, Hes3, Hes5 and the intrinsic Notch regulatory proteins Numb and Numblike also show active signaling in distinct brain regions. Hes5 coincides with the majority of Notch1 expression and can be detected in the cerebral cortex, cerebellum and putative germinal zones. Hes3, on the other hand, shows a restricted expression in cerebellar Purkinje cells. The distribution of Notch1 and its putative ligands suggest distinct roles in specific subsets of cells in the postnatal brain including putative stem cells and differentiated neurons.     

Mouse erythrocyte tropomodulin in the brain reported by lacZ knocked-in downstream from the E1 promoter

Erythrocyte tropomodulin (E-Tmod, Tmod1) is a tropomyosin-binding protein that caps the slow-growing end of actin filaments. In erythrocytes, it may favor the formation of short actin protofilaments needed for elastic cell deformation. Previously we created a knockout mouse model in which lacZ was knocked-in downstream of the E1 promoter to report the expression of full length E-Tmod. Here we utilize E-Tmod(+/lacZ) mice to study E-Tmod expression patterns in the CNS. X-gal staining and in situ hybridization of adults revealed its restricted expression in the olfactory bulb, hippocampus, cerebral cortex, basal ganglia, nuclei of brain stem and cerebellum. In neonates, signals in the cortex and caudate putamen increased from days 15 to 40. Immunohistochemistry also revealed that signals for beta-galactosidase coincided with that of NeuN, a post-mitotic nuclear marker for neurons, but not that for GFAP+ astrocytes or APC+ oligodendrocytes, suggesting E-Tmod/lacZ-positive cells in the CNS were neurons. Large neurons, e.g., mitral cells in olfactory bulb and mossy cells in hilus of the dentate gyrus are among those that expressed very high levels of E-Tmod in the CNS.     

Spastin in the human and mouse central nervous system with special reference to its expression in the hippocampus of mouse pilocarpine model of status epilepticus and temporal lobe epilepsy

In the present in situ hybridization and immunocytochemical studies in the mouse central nervous system (CNS), a strong expression of spastin mRNA and protein was found in Purkinje cells and dentate nucleus in the cerebellum, in hippocampal principal cells and hilar neurons, in amygdala, substantia nigra, striatum, in the motor nuclei of the cranial nerves and in different layers of the cerebral cortex except piriform and entorhinal cortices where only neurons in layer II were strongly stained. Spastin protein and mRNA were weakly expressed in most of the thalamic nuclei. In selected human brain regions such as the cerebral cortex, cerebellum, hippocampus, amygdala, substania nigra and striatum, similar results were obtained. Electron microscopy showed spastin immunopositive staining in the cytoplasma, dendrites, axon terminals and nucleus. In the mouse pilocarpine model of status epilepticus and subsequent temporal lobe epilepsy, spastin expression disappeared in hilar neurons as early as at 2h during pilocarpine induced status epilepticus, and never recovered. At 7 days and 2 months after pilocarpine induced status epilepticus, spastin expression was down-regulated in granule cells in the dentate gyrus, but induced expression was found in reactive astrocytes. The demonstration of widespread distribution of spastin in functionally different brain regions in the present study may provide neuroanatomical basis to explain why different neurological, psychological disorders and cognitive impairment occur in patients with spastin mutation. Down-regulation or loss of spastin expression in hilar neurons may be related to their degeneration and may therefore initiate epileptogenetic events, leading to temporal lobe epilepsy.     

Down syndrome cell adhesion molecule is conserved in mouse and highly expressed in the adult mouse brain

Down Syndrome (DS) is a major cause of mental retardation and is associated with characteristic well-defined although subtle brain abnormalities, many of which arise after birth, with particular defects in the cortex, hippocampus and cerebellum. The neural cell adhesion molecule DSCAM (Down syndrome cell adhesion molecule) maps to 21q22.2-->q22.3, a region associated with DS mental retardation, and is expressed largely in the neurons of the central and peripheral nervous systems during development. In order to evaluate the contribution of DSCAM to postnatal morphogenetic and cognitive processes, we have analyzed the expression of the mouse DSCAM homolog, Dscam, in the adult mouse brain from 1 through 21 months of age. We have found that Dscam is widely expressed in the brain throughout adult life, with strongest levels in the cortex, the mitral and granular layers of the olfactory bulb, the granule cells of the dentate gyrus and the pyramidal cells of the CA1, CA2 and CA3 regions, the ventroposterior lateral nuclei of the thalamus, and in the Purkinje cells of the cerebellum. Dscam is also expressed ventrally in the adult spinal cord. Given the homology of DSCAM to cell adhesion molecules involved in development and synaptic plasticity, and its demonstrated role in axon guidance, we propose that DSCAM overexpression contributes not only to the structural defects seen in these regions of the DS brain, but also to the defects of learning and memory seen in adults with DS.     

Circadian oscillators in the mouse brain: molecular clock components in the neocortex and cerebellar cortex

The circadian timekeeper of the mammalian brain resides in the suprachiasmatic nucleus of the hypothalamus (SCN), and is characterized by rhythmic expression of a set of clock genes with specific 24-h daily profiles. An increasing amount of data suggests that additional circadian oscillators residing outside the SCN have the capacity to generate peripheral circadian rhythms. We have recently shown the presence of SCN-controlled oscillators in the neocortex and cerebellum of the rat. The function of these peripheral brain clocks is unknown, and elucidating this could involve mice with conditional cell-specific clock gene deletions. This prompted us to analyze the molecular clockwork of the mouse neocortex and cerebellum in detail. Here, by use of in situ hybridization and quantitative RT-PCR, we show that clock genes are expressed in all six layers of the neocortex and the Purkinje and granular cell layers of the cerebellar cortex of the mouse brain. Among these, Per1, Per2, Cry1, Arntl, and Nr1d1 exhibit circadian rhythms suggesting that local running circadian oscillators reside within neurons of the mouse neocortex and cerebellar cortex. The temporal expression profiles of clock genes are similar in the neocortex and cerebellum, but they are delayed by 5 h as compared to the SCN, suggestively reflecting a master–slave relationship between the SCN and extra-hypothalamic oscillators. Furthermore, ARNTL protein products are detectable in neurons of the mouse neocortex and cerebellum, as revealed by immunohistochemistry. These findings give reason to further pursue the physiological significance of circadian oscillators in the mouse neocortex and cerebellum.     

Glucose transporter expression in brain. cDNA sequence of mouse GLUT3, the brain facilitative glucose transporter isoform, and identification of sites of expression by in situ hybridization.

Complementary DNAs encoding the mouse GLUT3/brain facilitative glucose transporter have been isolated and sequenced. The predicted amino acid sequence indicates that mouse GLUT3 is composed of 493 amino acids and has 83 and 89% identity and similarity, respectively, to the sequence of human GLUT3. In contrast to human GLUT3 mRNA, which can be readily detected by RNA blotting in all human tissues that have been examined, mouse GLUT3 mRNA was only present at significant levels in brain. In situ hybridization showed differential expression of GLUT3 mRNA in several regions of adult mouse brain. Specific expression was observed in the hippocampus, with GLUT3 mRNA levels being higher in areas CA1 to CA3 than in the dentate gyrus. It was also detected in the Purkinje cell layer of the cerebellum and in the cerebral cortex, with higher expression in the piriform cortex than in other regions of the cortex. Antisera to mouse GLUT3 immunoblotted a series of proteins of 45-50 kDa in mouse brain plasma membranes. These results are consistent with GLUT3 being a neuronal glucose transporter.     

Distribution of PINK1 and LRRK2 in rat and mouse brain

Mutations in two kinases, PTEN induced kinase 1 (PINK1) and leucine-rich repeat kinase 2 (LRRK2), have been shown to segregate with familial forms of Parkinson's disease. Although these two genes are expected to be involved in molecular mechanisms relevant to Parkinson's disease, their precise anatomical localization in mammalian brain is unknown. We have mapped the expression of PINK1 and LRRK2 mRNA in the rat and mouse brain via in situ hybridization histochemistry using riboprobes. We found that both genes are broadly expressed throughout the brain with similar neuroanatomical distribution in mouse compared to rat. PINK1 mRNA abundance was rather uniform throughout the different brain regions with expression in cortex, striatum, thalamus, brainstem and cerebellum. LRRK2, on the other hand, showed strong regional differences in expression levels with highest levels seen in the striatum, cortex and hippocampus. Weak LRRK2 expression was seen in the hypothalamus, olfactory bulb and substantia nigra. We confirmed these distributions for both genes using quantitative RT-PCR and for LRRK2 by western immunoblot. As their broad expression patterns contrast with localized neuropathology in Parkinson's disease, the pathogenicity of clinical mutant forms of PINK1 and LRRK2 may be mediated by nigrostriatal-specific mechanisms.     

A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons

GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS) of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP) in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca²⁺-imaging using the superior green Ca²⁺-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65). TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type.     

Developmental expression and localization of IA-2 mRNA in mouse neuroendocrine tissues

Islet antigen (IA)-2 is a novel autoantigen of insulin-dependent diabetes mellitus (IDDM), and belongs to a new class within the receptor-type protein tyrosine phosphatase (PTP) family characterized by lack of PTP enzymatic activity with conventional substrates. Its expression is restricted primarily to the pancreas, pituitary, and brain with the highest level in the brain. IA-2 mRNA expressions in the brain, pituitary and pancreas of 1-, 4-, and 8-week-old mice were examined. In situ hybridization of the brain revealed that IA-2 mRNA was expressed in the cerebral cortex, hippocampus, thalamus, choroid plexus, hypothalamus, Purkinje cells, and granular layer of the cerebellum. In the pituitary, IA-2 mRNA was located in the anterior and posterior pituitary by in situ hybridization. The pattern of IA-2 mRNA expression in normal male mouse brain at 1, 4, and 8 weeks of age by the Northern blot analysis was similar to that in the pituitary by RT-PCR analysis. The expression level was higher at 4 weeks and lower at 1 week of age. In the pancreas, IA-2 mRNA expressions detected by RT-PCR were highest at 8 weeks of age. These results indicated that the amount of mRNA expression increased in accordance to development in brain, pituitary, and pancreas.