Expression of the presenilin-like signal peptide peptidase (SPP) in mouse adult brain and during development

Recently, a new member of the presenilin family was identified as an aspartyl protease that cleaves signal peptides within hydrophobic domains, and was, therefore, named signal peptide peptidase (SPP). We isolated cDNAs coding for mouse and human orthologues of SPP. The human gene spans 55 kilobases on chromosome 20q11.21. The SPP-protein is encoded in mouse and man by 12 exons. The highly conserved intron/exon-structure in the SPP/presenilin family hints at a common precursor. Northern blot and in situ hybridization analysis revealed a widespread expression of SPP in many tissues. A distinct pattern of expression in the mature murine brain and during development indicates that SPP plays an important role in the establishment and maintenance of the nervous system. We prepared an antiserum against the carboxy-terminal domain of SPP, which is highly conserved between species. It reacted specifically, both in western blots and in immunocytochemical preparations, with SPP from various mammalian origins. The antiserum was used to demonstrate that SPP is oriented in the membrane of the endoplasmic reticulum with its carboxy-terminal tail extending into the cytosol.     

Increased cyclin E expression may obviate the role of cyclin D1 during brain development in cyclin D1 knockout mice

Cyclins D and E play critical roles during the G1 phase of mammalian cell division. Cyclin D1 expression is high and expected to play an important role during mouse brain development. However, in the present study, we found no difference in CNS morphology between cyclin D1 knockout (KO) and control wild-type mice at the ages of 1, 4 and 12 months. Analysis of protein expression in embryonic brains revealed that cyclin E is obviously increased in cyclin D1 KO mice at 13.5 days post coitum. At the same age a high level of cyclin D1 expression is detected in the embryonic brain of wild-type mice. The data indicate that enhanced cyclin E protein expression in cyclin D1 KO mice may obviate the role of cyclin D1 and contribute to the normal brain development of cyclin D1 KO mice.     

Expression of the cystathionine beta synthase (CBS) gene during mouse development and immunolocalization in adult brain.

Hyperhomocysteinemia, caused by a lack of cystathionine beta synthase (CBS), leads to elevated plasma concentrations of homocysteine. This is a common risk factor for atherosclerosis, stroke, and possibly neurodegenerative diseases. However, the mechanisms that link hyperhomocysteinemia due to CBS deficiency to these diseases are still unknown. Early biochemical studies describe developmental and adult patterns of transsulfuration and CBS expression in a variety of species. However, there is incomplete knowledge about the regional and cellular expression pattern of CBS, notably in the brain. To complete the previous data, we used in situ hybridization and Northern blotting to characterize the spatial and temporal patterns of Cbs gene expression during mouse development. In the early stages of development, the Cbs gene was expressed only in the liver and in the skeletal, cardiac, and nervous systems. The expression declined in the nervous system in the late embryonic stages, whereas it increased in the brain after birth, peaking during cerebellar development. In the adult brain, expression was strongest in the Purkinje cell layer and in the hippocampus. Immunohistochemical analyses showed that the CBS protein was localized in most areas of the brain but predominantly in the cell bodies and neuronal processes of Purkinje cells and Ammon's horn neurons.     

Expression of the mitotic kinesin Kif15 in postmitotic neurons: Implications for neuronal migration and development

Kif15 is a kinesin-related protein whose mitotic homologues are believed to crosslink and immobilize spindle microtubules. We have obtained rodent sequences of Kif15, and have studied their expression and distribution in the developing nervous system. Kif15 is indeed expressed in actively dividing fibroblasts, but is also expressed in terminally postmitotic neurons. In mitotic cells, Kif15 localizes to spindle poles and microtubules during prometaphase to early anaphase, but then to the actin-based cleavage furrow during cytokinesis. In interphase fibroblasts, Kif15 localizes to actin bundles but not to microtubules. In cultured neurons, Kif15 localizes to microtubules but shows no apparent co-localization with actin. Localization of Kif15 to microtubules is particularly good when the microtubules are bundled, and there is a notable enrichment of Kif15 in the microtubule bundles that occupy stalled growth cones and dendrites. Studies on developing rodent brain show a pronounced enrichment of Kif15 in migratory neurons compared to other neurons. Notably, migratory neurons have a cage-like configuration of microtubules around their nucleus that is linked to the microtubule array within the leading process, such that the entire array moves in unison as the cell migrates. Since the capacity of microtubules to move independently of one another is restricted in all of these cases, we propose that Kif15 opposes the capacity of other motors to generate independent microtubule movements within key regions of developing neurons.     

Expression of multiple formins in adult tissues and during developmental stages of mouse brain

Formins are highly conserved heterogeneous family of proteins with several isoforms having significant contribution in multiple cellular functions. Formins play crucial role in remodelling of actin cytoskeleton and thus play important role in cell motility. Formins are also involved in many cellular activities like determining cell polarity, cytokinesis and morphogenesis. Formins are multi domain protein with characteristic homodimeric formin homology 2 (FH2) domain. It nucleates the actin filaments and its activity is regulated by the presence of characteristic formin homology 1 (FH1) domain. In higher mammals like human and mouse fifteen different formin isoforms are present. However the function and expression pattern of each and every formin in different adult tissues are not well characterized. Here we have found that multiple formins are expressing in each adult tissue of mouse, irrespective of their origin from the germ layer. Formins are also expressing from early stage of development to the adulthood in brain. The expression of many formins in a single tissue of adult mouse indicates that regulation of actin cytoskeleton dynamics by formins may be crucial for physiological processes like wound healing, tissue repairing, exocytosis, endocytosis, synapse formation and maintenance. Expression of FMNL2 and Fhdc1 are high in adult mouse brain as compare to embryonic stages. Higher expression of FMNL2 and Fhdc1 indicates that FMNL2 and Fhdc1 might be very important for the adult brain functions.     

Expression pattern of ACK1 tyrosine kinase during brain development in the mouse

Ack1 is a non-receptor tyrosine kinase that is highly expressed in the adult central nervous system (CNS). Here, we studied the distribution of Ack1 mRNA throughout the development of mouse CNS. Expression was detected in all areas of the brain but especially high levels were observed in the neocortex, hippocampus, and cerebellum. Interestingly, expression levels were prominent in areas of proliferation such as the subventricular zone and areas that originate other structures such the pontine nucleus and the ganglionic eminence. During development, several areas showed an increase in Ack1 expression, especially the dentate gyrus and CA3 in the hippocampus, layer V in the neocortex, and the Purkinje cell layer in the cerebellum. These results demonstrate that this kinase is up-regulated during development and that it is expressed in proliferative areas and in migratory pathways in the developing brain.     

Expression of cellular retinoc acid-binding protein I is specific to neurons in adult transgenic mouse brain

Cellular retinoic acid binding protein I (CRABP-I) plays a role in retinoic acid (RA) metabolism or transport. This report shows specific neuronal expression of CRABP-I in adult transgenic mouse brain using CRABP-I promotor-driven lac-Z and neuron- and astrocyte-markers. Double staining indicates that CRABP-I is expressed in neurons and large cells (>12 μm) but to much lesser degree the astrocytes. CRABP-I-lac-Z⁺ neurons were distributed throughout the brain, but in a very discreet pattern in each brain region. CRABP-I expression in specific populations of brain neurons suggests that RA is extensively metabolized in mature brains, mostly in neurons. Additionally, the genetic basis of its specific expression in these brain areas is located in the 5′ regulatory region of this gene.     

Expression of cellular retinoic acid-binding protein I is specific to neurons in adult transgenic mouse brain

Cellular retinoic acid binding protein I (CRABP-I) plays a role in retinoic acid (RA) metabolism or transport. This report shows specific neuronal expression of CRABP-I in adult transgenic mouse brain using CRABP-I promotor-driven lac-Z and neuron- and astrocyte-markers. Double staining indicates that CRABP-I is expressed in neurons and large cells (>12 microm) but to much lesser degree the astrocytes. CRABP-I-lac-Z(+) neurons were distributed throughout the brain, but in a very discreet pattern in each brain region. CRABP-I expression in specific populations of brain neurons suggests that RA is extensively metabolized in mature brains, mostly in neurons. Additionally, the genetic basis of its specific expression in these brain areas is located in the 5' regulatory region of this gene.     

Heterogeneity of Tau proteins during mouse brain development and differentiation of cultured neurons.

Tau microtubule-associated proteins constitute a group of developmentally regulated neuronal proteins. Using the high-resolution two-dimensional polyacrylamide gel electrophoresis system, we have resolved more than 60 distinct Tau isoforms in the adult mouse brain. Tau protein heterogeneity increases drastically during the second week of brain development. In neuronal primary cell cultures, some of these developmental changes can be observed. The increase of Tau heterogeneity in culture is more limited and reaches a plateau after a period corresponding to the second week of development. Most, if not all, of the vast Tau heterogeneity can be attributed to intensive post-translational phosphorylation, which may affect the structure of the proteins.     

Expression of Dbn1 during mouse brain development and neural stem cell differentiation

Dbn1 is a newly discovered gene in the drebrin gene family of mice. Previous studies have reported that Dbn1 is specifically expressed in the mouse brain suggesting its potential role in brain development. However, a detailed analysis of Dbn1 expression during mouse brain development has not been demonstrated. Here, we describe the expression pattern of Dbn1 and the coexpression of Dbn1 and actin during the development of the mouse brain from embryonic day 14 (E14) to adulthood and during the differentiation of neural stem cells (NSCs), as determined using immunohistochemistry, double-labeling immunofluorescence, and quantitative real-time polymerase chain reaction. During mouse brain development, Dbn1 expression level was high at E14, attenuated postnatally, reached its highest point at postnatal day 7 (P7), and showed a very low level at adulthood. Imaging data showed that Dbn1 was mainly expressed in the hippocampus, ventricular zone, and cortex, where NSCs are densely distributed, and that the intracellular distribution of Dbn1 was predominantly located in the cytoplasm edges and neurites. Moreover, the signal for colocalization of Dbn1 with actin was intense at E14, P0, and P7, but it was weak at adulthood. During NSC differentiation, Dbn1 mRNA expression increased after the onset of differentiation and reached its highest point at 3 days, followed by a decrease in expression. The imaging data showed that Dbn1 was increasingly expressed in the extending neurites in accordance with the cell morphological changes that occur during differentiation. Furthermore, obvious colocalization signals of Dbn1 with actin were found in the neurites and dendritic spines. Collectively, these results suggest that Dbn1 may play a key role in mouse brain development and may regulate NSC differentiation by filamentous actin.     

Expression and regulation of the Fkbp5 gene in the adult mouse brain

Background

Chronic stress has been found to be a major risk factor for various human pathologies. Stress activates the hypothalamic-pituitary-adrenal (HPA) axis, which is tightly regulated via, among others, the glucocorticoid receptor (GR). The activity of the GR is modulated by a variety of proteins, including the co-chaperone FK506 binding protein 51 (FKBP5). Although FKBP5 has been associated with risk for affective disorders and has been implicated in GR sensitivity, previous studies focused mainly on peripheral blood, while information about basal distribution and induction in the central nervous system are sparse.

Methodology/Principal Findings

In the present study, we describe the basal expression pattern of Fkbp5 mRNA in the brain of adult male mice and show the induction of Fkbp5 mRNA via dexamethasone treatment or different stress paradigms. We could show that Fkbp5 is often, but not exclusively, expressed in regions also known for GR expression, for example the hippocampus. Furthermore, we were able to induce Fkbp5 expression via dexamethasone in the CA1 and DG subregions of the hippocampus, the paraventricular nucleus (PVN) and the central amygdala (CeA). Increase of Fkbp5 mRNA was also found after restrained stress and 24 hours of food deprivation in the PVN and the CeA, while in the hippocampus only food deprivation caused an increase in Fkbp5 mRNA.

Conclusions/Significance

Interestingly, regions with a low basal expression showed higher increase in Fkbp5 mRNA following induction than regions with high basal expression, supporting the hypothesis that GR sensitivity is, at least partly, mediated via Fkbp5. In addition, this also supports the use of Fkbp5 gene expression as a marker for GR sensitivity. In summary, we were able to give an overview of the basal expression of fkbp5 mRNA as well as to extend the findings of induction of Fkbp5 and its regulatory influence on GR sensitivity from peripheral blood to the brain.     

Expression of low-molecular-weight neurofilament (NF-L) mRNA during postnatal development of the mouse brain

A regional Northern blot analysis demonstrated that the highest levels of NF-L mRNA in the adult mouse brain are present in brain stem followed by mid-brain, with lower levels found in neocortex, cerebellum, and hippocampus. The study was extended to the cellular level over the time course of postnatal development using in situ hybridization. This developmental analysis revealed that the expression of NF-L mRNA closely follows the differentiation pattern of many large neurons during postnatal neurogenesis. Neurons which differentiate early such as Purkinje, mitral, pyramidal, and large neurons of brain stem and thalamic nuclei, expressed high levels of NF-L mRNA at postnatal day 1. Early expression of NF-L mRNA may be required for the maintenance of the extensive neurofilament protein networks that are detected within the axons of larger neurons. Smaller neurons which differentiate later, such as dentate gyrus granule cells, small pyramidal and granule cells of the neocortex, and granule cells of the cerebellum, exhibit a delayed expression of NF-L mRNA.To whom to address reprint requests.     

Larval optic nerve and adult extra-retinal photoreceptors sequentially associate with clock neurons during Drosophila brain development

The visual system is one of the input pathways for light into the circadian clock of the Drosophila brain. In particular, extra-retinal visual structures have been proposed to play a role in both larval and adult circadian photoreception. We have analyzed the interactions between extra-retinal structures of the visual system and the clock neurons during brain development. We first show that the larval optic nerve, or Bolwig nerve, already contacts clock cells (the lateral neurons) in the embryonic brain. Analysis of visual system-defective genotypes showed that the absence of the afferent Bolwig nerve resulted in a severe reduction of the lateral neurons dendritic arborization, and that the inhibition of nerve activity induced alterations of the dendritic morphology. During wild-type development, the loss of a functional Bolwig nerve in the early pupa was also accompanied by remodeling of the arborization of the lateral neurons. Approximately 1.5 days later, visual fibers that came from the Hofbauer-Buchner eyelet, a putative photoreceptive organ for the adult circadian clock, were seen contacting the lateral neurons. Both types of extra-retinal photoreceptors expressed rhodopsins RH5 and RH6, as well as the norpA-encoded phospholipase C. These data strongly suggest a role for RH5 and RH6, as well as NORPA, signaling in both larval and adult extra-retinal circadian photoreception. The Hofbauer-Buchner eyelet therefore does not appear to account for the previously described norpA-independent light input to the adult clock. This supports the existence of yet uncharacterized photoreceptive structures in Drosophila.     

Expression of Apc2 during mouse development

APC2 (previously known as APCL), a molecule closely related to the adenomatous polyposis coli (APC) tumor suppressor, can deplete cytoplasmic beta-catenin, like APC itself. Recently, it has been shown that APC2 may regulate the localization of p53 and the microtubule stability and/or extension. Although it has been reported that APC2 mRNA is expressed in human brain, the anatomical and ontogenic expression patterns remain unclear. The purpose of this study was to investigate the distribution of mouse Apc2 during mouse development. In the adult brain, Apc2 is expressed predominantly in neurons and throughout the brain. Northern blot analysis demonstrated a high level of Apc2 expression in embryonic and early postnatal brain. Ontogenic analysis has indicated that Apc2 is expressed in neural tissue, including the peripheral nervous system. During development of cortex, retina and cerebellum, Apc2 is expressed in post-mitotic cells. These findings suggest that Apc2 may contribute to the development of neuronal cells.