Spatiotemporal expression pattern of non-clustered protocadherin family members in the developing rat brain

Protocadherins (PCDHs) consist of the largest subgroup of the cadherin superfamily, and most PCDHs are expressed dominantly in the CNS. Because PCDHs are involved in the homophilic cell-cell adhesion, PCDHs in the nervous system have been suggested to play roles in the formation and maintenance of the synaptic connections. Although many PCDHs (>50) are in tandem arranged as a cluster in a specific chromosome locus, there are also considerable numbers of non-clustered PCDH members (approximately 20). In this study, we examined the spatiotemporal distribution of mRNAs for 12 non-clustered PCDHs in rat brain using in situ hybridization. Some of them (PCDH1, PCDH7, PCDH9, PCDH10, PCDH11, PCDH17, and PCDH20) exhibited region-dependent expression pattern in the cerebral cortex during the early postnatal stage (P3), which is a critical period for the establishment of specific synaptic connections: PCDH7 and PCDH20 mRNAs were predominantly expressed in the somatosensory (parietal) and visual (occipital) cortices, whereas PCDH11 and PCDH17 mRNAs were preferentially expressed in the motor (forelimb and hindlimb areas) and auditory (temporal) cortices, and PCDH9 mRNA was highly expressed in the motor and main somatosensory cortices. These PCDHs were also expressed in the specific regions of the connecting thalamic nuclei. These cortical regionalization and thalamic nuclei-specificity appeared to be most distinct in P3 compared with those of embryonic and adult stages. Taken together, these results suggest that PCDHs may play specific roles in the establishment of selective synaptic connections of specific modality of cerebral cortex with other communicating brain regions such as the thalamus.     

Expression of the neuronal calcium sensor protein family in the rat brain

The neuronal calcium sensor proteins are members of the calcium-binding protein superfamily. They control localized calcium signalling on membranes and may make G-protein cascades sensitive to cytosolic calcium. The family members are recoverin (visinin, S-modulin), neuronal calcium sensor-1 (frequenin), hippocalcin, neuronal visinin-like protein-1 (visinin-like protein, neurocalcin-alpha), neuronal visinin-like protein-2 and neuronal visinin-like protein-3. Recoverin is expressed only in the retina and pineal gland. Using in situ hybridization, we mapped the expression of the other neuronal calcium sensor protein genes in the adult rat brain. Neuronal visinin-like protein-1 messenger RNA has a widespread distribution and is abundant in all brain areas except the caudate-putamen. Neuronal calcium sensor-1 gene expression is pan-neuronal. Neuronal calcium sensor-1 messenger RNA is present in the dendrites of hippocampal pyramidal and granule cells, suggesting a specific role in dendritic function. Hippocalcin and neuronal visinin-like protein-2 are mainly expressed in the forebrain and have similar expression patterns (neocortex, hippocampus and caudate-putamen). Neuronal visinin-like protein-3 has the most restricted expression; its highest expression level is in the cerebellum (Purkinje and granule cells). However, the neuronal visinin-like protein-3 gene is also expressed in many ventral nuclei throughout the fore- and midbrain, in the medial habenulae, and in the superior and inferior colliculi.The neuronal calcium sensor proteins are a relatively unexplored family of Ca(2+)-binding proteins. They are likely to be involved in many diverse areas of neuronal signalling. In this paper, we describe their expression in the rat brain as determined by in situ hybridization. As all five neuronal calcium sensor protein genes have distinctive expression patterns, they probably perform specific functions.     

Release of mitochondrial cytochrome c and DNA fragmentation after cold injury-induced brain trauma in mice: possible role in neuronal apoptosis.

Recent studies have shown that release of mitochondrial cytochrome c is a critical step in the apoptosis process. In this study, we examined the subcellular distribution of the cytochrome c protein after cold injury (CI), in which apoptosis is assumed to participate. Western blotting and immunohistochemistry showed cytosolic cytochrome c as early as 1 h after CI, and correspondingly, there was a reduction in mitochondrial cytochrome c after injury. Neuronal distribution of cytosolic cytochrome c was shown by double staining with a neuronal nuclear marker by immunohistochemistry. A significant amount of DNA laddering was detected 4 h after CI, and increased in a time-dependent manner. These data suggest that early cytochrome c release from mitochondria may contribute to apoptosis induction after traumatic brain injury.     

GDNF family ligand RET receptor in the brain of adult zebrafish

RET is a tyrosine kinase receptor, and transduces signaling by family of glial cell line-derived neurotrophic factor ligands (GFLs). RET is involved in the development of enteric nervous system, of sympathetic, parasympathetic, motor and sensory neurons. RET exists in two main isoforms originated by differential splicing, RET9 and RET51; phylogenetic studies have shown that the RET gene is conserved across vertebrates. The aim of this study was to investigate the RET expression within the brain of zebrafish, using immunohistochemistry, western blotting and RT-PCR. In homogenate brains both RET protein and mRNA were observed. RET immunoreactivity was widespread in neurons and neural processes of all the major regions of the brain. These results demonstrate the occurrence of RET and suggest an involvement of GDNF family ligands in the brain of adult zebrafish.     

Comparative DNA sequence analysis of mouse and human protocadherin gene clusters

The genomic organization of the human protocadherin alpha, beta, and gamma gene clusters (designated Pcdh alpha [gene symbol PCDHA], Pcdh beta [PCDHB], and Pcdh gamma [PCDHG]) is remarkably similar to that of immunoglobulin and T-cell receptor genes. The extracellular and transmembrane domains of each protocadherin protein are encoded by an unusually large "variable" region exon, while the intracellular domains are encoded by three small "constant" region exons located downstream from a tandem array of variable region exons. Here we report the results of a comparative DNA sequence analysis of the orthologous human (750 kb) and mouse (900 kb) protocadherin gene clusters. The organization of Pcdh alpha and Pcdh gamma gene clusters in the two species is virtually identical, whereas the mouse Pcdh beta gene cluster is larger and contains more genes than the human Pcdh beta gene cluster. We identified conserved DNA sequences upstream of the variable region exons, and found that these sequences are more conserved between orthologs than between paralogs. Within this region, there is a highly conserved DNA sequence motif located at about the same position upstream of the translation start codon of each variable region exon. In addition, the variable region of each gene cluster contains a rich array of CpG islands, whose location corresponds to the position of each variable region exon. These observations are consistent with the proposal that the expression of each variable region exon is regulated by a distinct promoter, which is highly conserved between orthologous variable region exons in mouse and human.     

Roles for the pro-neurotrophin receptor sortilin in neuronal development, aging and brain injury

Neurotrophins are essential for development and maintenance of the vertebrate nervous system. Paradoxically, although mature neurotrophins promote neuronal survival by binding to tropomyosin receptor kinases and p75 neurotrophin receptor (p75(NTR)), pro-neurotrophins induce apoptosis in cultured neurons by engaging sortilin and p75(NTR) in a death-signaling receptor complex. Substantial amounts of neurotrophins are secreted in pro-form in vivo, yet their physiological significance remains unclear. We generated a sortilin-deficient mouse to examine the contribution of the p75(NTR)/sortilin receptor complex to neuronal viability. In the developing retina, Sortilin 1 (Sort1)(-/-) mice showed reduced neuronal apoptosis that was indistinguishable from that observed in p75(NTR)-deficient (Ngfr(-/-)) mice. To our surprise, although sortilin deficiency did not affect developmentally regulated apoptosis of sympathetic neurons, it did prevent their age-dependent degeneration. Furthermore, in an injury protocol, lesioned corticospinal neurons in Sort1(-/-) mice were protected from death. Thus, the sortilin pathway has distinct roles in pro-neurotrophin-induced apoptotic signaling in pathological conditions, but also in specific stages of neuronal development and aging.     

大鼠皮质N-甲基-D-天冬氨酸受体在脑损伤后的时相变化

目的；观测Ｎ－甲基－Ｄ－天冬氨酸（ＮＭＤＡ）受体在脑损伤后的变化规律以及与继发性脑水肿发生和发展的关系。方法：用放射性配基结合分析法对伤后不同时间的大鼠伤侧大脑皮质ＮＭＤＡ受体活性进行测定干湿法测伤后伤测皮质水含量。     

Olfactory bulbectomy induces neuronal rearrangement in the entorhinal cortex in the rat

In humans, depression has been associated with disturbances in olfactory circuitry. Symptoms of depression can be mimicked in animals after olfactory bulbectomy (OBX). Animal models of depression-like behavior produce similar neuronal rearrangements in various brain regions as seen in patients affected by depression. We have recently observed that OBX produces neuronal hypotrophy in the piriform cortex (PirC) and CA1 hippocampus as well as decreased adult cell proliferation in the dentate gyrus (DG) of hippocampus. Thus we further evaluated the effects of OBX in neuronal arborization and spine density in brain regions involved in the control of circadian circle, emotion and memory processing such as the prefrontal cortex (PFC), nucleus accumbens (NAcc), infralimbic cortex (ILC), orbitolateral cortex (OLC) and entorhinal cortex (EC). Our present results show that along with severe behavioral deficits observed in these animals, OBX considerably decreased dendritic branching and the total dendritic length in the EC, a major interface of the hippocampus and neocortical regions. The remaining cortices and NAcc were not affected by OBX. Thus, we propose that the lack of input from the olfactory bulbs resulted in serial neuronal rearrangements in the PirC, EC, and hippocampus leading, at least partially, to behavioral deficits in emotion and memory processes.     

Diversity of potassium channels in neuronal dendrites

Complex computations in the nervous system begin with electrical signals generated in single neurons. Such signals include action potentials mediated by the opening of voltage-dependent ion channels, and synaptic potentials arising from neurotransmitter receptor activation. The amplitude, waveform, and propagation of action potentials and synaptic potentials influence cellular signaling in profound ways, and are largely determined by activities of ion channels in the cell membrane. The location and properties of ion channels therefore play critical roles in shaping electrical signaling in the neuron, which is the foundation for more complex computations at network levels. This review summarizes what we know about the great diversity of K⁺ channels found in neuronal dendrites, the subcellular compartment where synaptic signals integrate and where various forms of plasticity occur. Specifically, we discuss the molecular identity, the distribution, kinase modulation, biophysical properties, and functional roles of a variety of K⁺ channels including voltage-gated, calcium-activated, and ligand-gated/G-protein coupled K⁺ channels. One emerging theme from recent literature is the recognition that K⁺ channels are powerful regulators of the function of dendrites. A second theme indicates that this K⁺ channel regulation depends on their unique subcellular distribution. In particular, the mechanisms underlying the establishment and maintenance of non-uniform distributions of ion channels are beginning to be understood in greater detail. An especially intriguing aspect of above mechanisms is that they are achieved through protein kinase phosphorylation and may thus be activity-dependent. In parts of this review, we choose to focus on CA1 pyramidal neurons of the rodent hippocampus and the K⁺ channels in their dendrites. Being one of the best-characterized cell types in the nervous system, the CA1 pyramidal neuron has long been studied as a prototypic neuron from which general rules of neuronal computation and synaptic plasticity emerge. A great deal of what we know about dendritic K⁺ channels comes from studies on CA1 pyramidal neurons. Where available, we also include up-to-date findings on dendritic K⁺ channels in other cell types.     

Distribution of neuronal cannabinoid receptor in the adult rat brain: a comparative receptor binding radioautography and in situ hybridization histochemistry.

The neuronal distribution of cannabinoid receptor in the adult rat brain is reported, combining receptor binding radioautography using the synthetic psychoactive cannabinoid ligand CP55,940 with in situ hybridization histochemistry using oligonucleotide probes complementary to rat cannabinoid receptor cDNA. In the cerebral cortex, especially in the frontal and cingulate cortex, dense binding was found in layers I and VI together with slight mRNA levels in a majority of both pyramidal and non-pyramidal-shaped neurons and of high mRNA levels in a moderate number of non-pyramidal-shaped neurons especially in layers II-III and V-VI. In the hippocampal dentate gyrus, very dense staining was found in the molecular layer together with high mRNA levels in a moderate number of hilar neurons close to the granular layer. In Ammon's horn, especially in the CA3 sector, very dense binding was found in the dendritic layers together with slight mRNA levels in the majority of the pyramidal cells and high mRNA levels in a moderate number of interneurons. In the basal ganglia, binding was very dense in the lateral putamen, substantia nigra pars reticulata, globus pallidus and entopeduncular nucleus, moderate in the medial putamen and caudate; and slight in the accumbens, together with slight to moderate mRNA levels in the striatal medium-sized neurons. Together with slight binding, slight to moderate mRNA levels were found in the majority of the neurons in the subthalamic nucleus. No binding and mRNA were found in the substantia nigra pars compacta and ventral tegmental area. Slight to moderate binding was found together with slight to moderate mRNA levels in the majority of neurons in the anterior olfactory nucleus; septum, especially medial septum and diagonal band of Broca; amygdala, especially basolateral amygdala; lateral habenula; ventromedial hypothalamic nucleus; lateral interpeduncular nucleus; central gray, dorsal cochlear nucleus; parabrachial nucleus; dorsal pontine tegmentum; pontine nuclei; commissural part of the nucleus tractus solitarius; inferior olive and dorsal horn of the spinal cord. In the cerebellum, very dense binding was found in the molecular layer together with slight mRNA levels in the majority of the granule cells and moderate mRNA levels in the basket and stellate cells. In conclusion, this study provides, for the first time, indirect assessment of the neurons containing cannabinoid receptor in the entire adult rat brain and will serve as a basis for future direct morphological confirmation using receptor immunohistochemistry and for functional studies.     

Major rearrangement of cellular DNA in the vicinity of integrated polyomavirus DNA

The Cyp cell line was produced by transforming mouse embryo cells at the restrictive temperature with an early thermosensitive mutant of polyomavirus (Py). Transfer of Cyp cells to the nonrestrictive temperature causes excision to occur at a single chromosomal site carrying viral DNA, and leads to the production of infectious virus. We have attempted to elucidate the recombination event that occurred during the integration of Py DNA in this inducible line. Physical characterization of two recombinant DNAs-one selected from a genomic library of normal mouse DNA and the other constructed from the unoccupied allele of the Cyp integration site-indicates that generation of the Cyp line has involved the joining of not only viral DNA to a cellular alpha site, but also the cellular alpha site to a cellular alpha site to cellular beta site. Hence, previously described hybrid excision products from the Cyp line were made of mouse DNA segments representing two distinct cellular sites. The alpha-beta joining may play a role in the expression of integrated Py DNA.     

Diversity of the mammalian sodium/proton exchanger SLC9 gene family

Sodium/proton antiporters or exchangers (NHE) are integral membrane proteins present in most, if not all, living organisms. In mammals, these transporters chiefly catalyze the electroneutral exchange of Na(+) and H(+) down their respective concentration gradients and are crucial for numerous physiological processes, ranging from the fine control of intracellular pH and cell volume to systemic electrolyte, acid-base and fluid volume homeostasis. NHE activity also facilitates the progression of other cellular events such as adhesion, migration, and proliferation. Thus far, eight distinct NHE genes (NHE1/SLC9A1-NHE8/SLC9A8) and several pseudogenes have been identified in the human genome. The functional genes encode proteins of varying primary sequence identity (25-70%), but share a common predicted secondary structure comprising 12 conserved membrane-spanning segments at the amino-terminus and a more divergent, cytoplasmically-oriented, carboxy-terminus. They show considerable heterogeneity in their patterns of tissue/cell expression and membrane localization. Functional studies have revealed further differences in their kinetic properties, sensitivity to pharmacological antagonists, and regulation by diverse hormonal and mechanical stimuli. Altered NHE activity has been linked to the pathogenesis of several diseases, including essential hypertension, congenital secretory diarrhea, diabetes, and tissue damage caused by ischemia/reperfusion. Further characterization of their functional properties should lead to a better understanding of their unique contributions to human health and disease.     

The EGF/ErbB receptor family and apoptosis

The ErbB receptor family can activate a multitude of cell signaling pathways that involve many aspects of cellular function. The four members of the ErbB receptor family interact with diverse ligands and substrates, as well as with each other through cell surface heterodimerization. The sum of these diverse interactions is a signaling network that is complex but also finely regulated. Among the cellular functions influenced by ErbB signaling is cell survival. ErbB receptor signaling has been demonstrated to interact with all of the major mechanisms of cell death signaling in a manner that promotes cell survival. Survival factors such as Ras, PI3-K, Akt, and Bcl-x/-2 all have been shown to be activated by ErbB signaling (Fig. 5). ErbB abrogation of apoptotic signals has been shown to play an important role during embryonic tissue development, in normal adult tissue maintenance (e.g. mammary tissue, wound healing), and also in tumor development and progression. Although the majority of studies suggest that ErbB receptor family members are mediators of cell survival, there have been occasional reports suggesting that ErbB receptors can induce cell death under selected experimental conditions. While this apparent discrepancy remains unresolved, in many of these reports, cell death may be the result of anoikis in response to changes in the cytoskeleton associated with hyperstimulation of ErbB signaling. The notion that ErbB receptor family members function to promote cell survival is not a recent observation. However, how this family functions to prevent apoptosis is an area that only recently has been considered. The understanding of ErbB receptor signaling as it relates to the avoidance of apoptosis had profound implications for the treatment of solid tumors originating in multiple tissues, as well as for the treatment of neurodegenerative disease. Further elucidation of the complex relationships between ErbB receptor signaling networks and the apoptotic machinery is certain to yield biologically important and potentially life-saving information.     

Diversity and evolution of the rhoph1/clag multigene family of Plasmodium falciparum

A complex of high-molecular-mass proteins (PfRhopH) of the human malaria parasite Plasmodium falciparum induces host protective immunity and therefore is a candidate for vaccine development. Understanding the level of polymorphism and the evolutionary processes is important for advancements in both vaccine design and knowledge of the evolution of cell invasion in this parasite. In the present study, we sequenced the entire open reading frames of seven genes encoding the proteins of the PfRhopH complex (rhoph2, rhoph3, and five rhoph1/clag gene paralogs). We found that four rhoph1/clag genes (clag2, 3.1, 3.2, and 8) were highly polymorphic. Amino acid substitutions and indels are predominantly clustered around amino acid positions 1000-1200 of these four rhoph1/clag genes. An excess of nonsynonymous substitutions over synonymous substitutions was detected for clag8 and 9, indicating positive selection. The McDonald-Kreitman test with a Plasmodium reichenowi orthologous sequence also supports positive selection on clag8. Based on the ratio of interspecific genetic distance to intraspecific distance, the time to the most recent common ancestor of the clag2 and 8 polymorphisms was estimated to be 1.89 and 0.87 million years ago, respectively, assuming divergence of P. falciparum and P. reichenowi 6 million years ago. In addition to a copy number polymorphism, gene conversion events were detected for the rhoph1/clag genes on chromosome 3, which likely play a role in increasing the diversity of each locus. Our results indicate that a high diversity of the PfRhopH1/Clag multigene family is maintained by diversifying selection forces over a considerably long period.     

Down-regulation of Bcl-2 in the fetal brain of the Gaucher disease mouse model: a possible role in the neuronal loss

Gaucher disease is a lysosomal storage disorder resulting from an inborn deficiency of glucocerebrosidase. To investigate the genes responsible for the neuronal symptoms of Gaucher disease, gene expression profiles were analyzed in brains of the Gaucher disease mouse model using a cDNA microarray, and it was found that the bcl-2 gene is down-regulated. Immunoblotting and apoptosis assay were performed to study the relationship between the decreased expression of Bcl-2 and neuronal death on the brains of Gaucher mice fetuses at embryonic day 17.5 (E17.5) and E19.5. Decreased expression of Bcl-2 was observed in the brain stem and cerebellum but not in cortex by immunoblotting. In situ labeling of DNA fragmentation using terminal transferase-mediated dUTP nick-end-labeling (TUNEL) assay confirmed that apoptosis occurred in the brain stem and cerebellum. More apoptotic cells were detected in the brains of Gaucher mice fetuses at E19.5 than at E17.5. These results suggest that the accumulation of either glucocerebroside or glucosylsphingosine, as a result of glucocerebrosidase deficiency, affects gene expression and could be responsible for neuronal cell death.     

Aryl hydrocarbon receptor-mediated apoptosis of neuronal cells: A possible interaction with estrogen receptor signaling

Activation of aryl hydrocarbon receptors (AhRs) induces neuronal damage, but the mechanism by which this occurs is largely unknown. This study evaluated the effects of an AhR agonist, β-naphthoflavone, on apoptotic pathways in mouse primary neuronal cell cultures. β-Naphthoflavone (0.1–100 μM) enhanced caspase-3 activity and lactate dehydrogenase (LDH) release in neocortical and hippocampal cells. These data were supported at the cellular level with Hoechst 33342 and calcein AM staining. α-Naphthoflavone inhibited the action of β-naphthoflavone, thus confirming specific activation of AhRs. A high-affinity estrogen receptor (ER) antagonist, ICI 182,780, and a selective estrogen receptor modulator (SERM), tamoxifen, enhanced β-naphthoflavone-mediated apoptosis. Another SERM, raloxifene, and an ERα antagonist, methyl-piperidino-pyrazole, did not affect β-naphthoflavone-induced caspase-3 activity. However, they inhibited β-naphthoflavone-induced LDH release at a late hour of treatment, thus suggesting delayed control of AhR-mediated neuronal cell death. The apoptotic effects of β-naphthoflavone were accompanied by increased levels of AhRs, and these receptors colocalized with ERβ as demonstrated by confocal microscopy. These data strongly support apoptotic effects of AhR activation in neocortical and hippocampal tissues. Moreover, this study provides evidence for direct interaction of the AhR-mediated apoptotic pathway with estrogen receptor signaling, which provides insight into new strategies to treat or prevent AhR-mediated neurotoxicity.     

Isolation of surface membranes from neuronal and non-neuronal cell populations from embryonic chick brain. Opiate receptor activity of neuronal membranes

Surface membranes were isolated from neuronal and non-neuronal cell populations prepared from embryonic chick brain. Sodium dodecyl sulfate gel electrophoresis showed that membranes from the two cell populations have distinctly different protein compositions. The neuronal cell membranes have opiate receptor activity with high- and low-affinity binding sites similar to those determined for intact neuronal cells.