The paralemmin protein family: identification of paralemmin-2, an isoform differentially spliced to AKAP2/AKAP-KL, and of palmdelphin, a more distant cytosolic relative

Paralemmin is a protein implicated in plasma membrane dynamics. Here we describe the identification of two new paralemmin-related proteins. A partial paralemmin homolog, palmdelphin, is predominantly cytosolic, unlike paralemmin which is lipid-anchored to the plasma membrane through a C-terminal CaaX motif. We have mapped the mouse palmdelphin gene to distal chromosome 3 between Amy2 and Abcd3, in a region homologous to human chromosome 1p22-p21 where the human palmdelphin gene is located. We have also identified a second paralemmin isoform, paralemmin-2. It is expressed from a gene on human chromosome 9q31-q33 which ends only 33 kb upstream of the gene encoding the protein kinase A-binding protein,AKAP2/AKAP-KL. The closely adjacent paralemmin-2 and AKAP2 genes are functionally linked in a very unusual manner. Chimeric mRNAs are expressed, apparently by RNA readthrough and differential splicing, that encode natural fusion proteins in which either the N-terminal coiled-coil region or nearly the complete sequence of paralemmin-2 except its C-terminal CaaX motif is fused to AKAP2/AKAP-KL. The N-terminal coiled-coil region is conserved in paralemmin-1, paralemmin-2/AKAP2, palmdelphin and a fourth, uncharacterized gene, suggesting that it is a modular functional domain.     

Evolution of the vertebrate paralemmin gene family: ancient origin of gene duplicates suggests distinct functions

Paralemmin-1 is a protein implicated in plasma membrane dynamics, the development of filopodia, neurites and dendritic spines, as well as the invasiveness and metastatic potential of cancer cells. However, little is known about its mode of action, or about the biological functions of the other paralemmin isoforms: paralemmin-2, paralemmin-3 and palmdelphin. We describe here evolutionary analyses of the paralemmin gene family in a broad range of vertebrate species. Our results suggest that the four paralemmin isoform genes (PALM1, PALM2, PALM3 and PALMD) arose by quadruplication of an ancestral gene in the two early vertebrate genome duplications. Paralemmin-1 and palmdelphin were further duplicated in the teleost fish specific genome duplication. We identified a unique sequence motif common to all paralemmins, consisting of 11 highly conserved residues of which four are invariant. A single full-length paralemmin homolog with this motif was identified in the genome of the sea lamprey Petromyzon marinus and an isolated putative paralemmin motif could be detected in the genome of the lancelet Branchiostoma floridae. This allows us to conclude that the paralemmin gene family arose early and has been maintained throughout vertebrate evolution, suggesting functional diversification and specific biological roles of the paralemmin isoforms. The paralemmin genes have also maintained specific features of gene organisation and sequence. This includes the occurrence of closely linked downstream genes, initially identified as a readthrough fusion protein with mammalian paralemmin-2 (Palm2-AKAP2). We have found evidence for such an arrangement for paralemmin-1 and -2 in several vertebrate genomes, as well as for palmdelphin and paralemmin-3 in teleost fish genomes, and suggest the name paralemmin downstream genes (PDG) for this new gene family. Thus, our findings point to ancient roles for paralemmins and distinct biological functions of the gene duplicates.     

The interaction of Bex and OMP reveals a dimer of OMP with a short half-life

Olfactory marker protein (OMP) participates in the olfactory signal transduction pathway. This is evident from the behavioral and electrophysiological deficits of OMP-null mice, which can be reversed by intranasal infection of olfactory sensory neurons with an OMP-expressing adenovirus. Bex, brain expressed X-linked protein, has been identified as a protein that interacts with OMP. We have now further characterized the interaction of OMP and Bex1/2 by in vitro binding assays and by immuno-coprecipitation experiments. OMP is a 19 kDa protein but these immunoprecipitation studies have revealed the unexpected presence of a 38 kDa band in addition to the expected 19 kDa band. Furthermore, the 38 kDa form was preferentially co-immunoprecipitated with Bex from cell extracts. In-gel tryptic digestion, mass spectrometry, and two-dimensional gel electrophoresis indicate that the 38 kDa protein behaves as a covalently cross-linked OMP-homodimer. The 38 kDa band was also identified in western blots of olfactory epithelium demonstrating its presence in vivo. The stabilities and subcellular localizations of the OMP-monomer and -dimer were studied in transfected cells. These results demonstrated that the OMP-dimer is much less stable than the monomer, and that while the monomer is present both in the nuclear and cytosolic compartments, the dimer is preferentially located in a Triton X-100 insoluble cytoskeletal fraction. These novel observations led us to hypothesize that regulation of the level of the rapidly turning-over OMP-dimer and its interaction with Bex1/2 is critical for OMP function in sensory transduction.     

Palmdelphin,a novel target of p53 with Ser46 phosphorylation,controls cell death in response to DNA damage

The tumor suppressor gene p53 regulates apoptosis in response to DNA damage. Promoter selectivity of p53 depends on mainly its phosphorylation. Particularly, the phosphorylation at serine-46 of p53 is indispensable in promoting pro-apoptotic genes that are, however, poorly determined. In the current study, we identified palmdelphin as a pro-apoptotic gene induced by p53 in a phosphorylated serine-46-specific manner. Upregulation of palmdelphin was observed in wild-type p53-transfected cells, but not in serine-46-mutated cells. Expression of palmdelphin was induced by p53 in response to DNA damage. In turn, palmdelphin induced apoptosis. Intriguingly, downregulation of palmdelphin resulted in necroptosis-like cell death via ATP depletion. Upon DNA damage, palmdelphin dominantly accumulated in the nucleus to induce apoptosis. These findings define palmdelphin as a target of serine-46-phosphorylated p53 that controls cell death in response to DNA damage.     

Ontogenesis of Adenosine Deaminase Activity in Rat Brain

The activity of adenosine deaminase (ADA) was determined in whole brain of rats at the embryonic age of 15 days through to adulthood and in nine brain regions in rats 1 day old through to adulthood. In 1-day-old rats, the highest activity was seen in olfactory bulbs (550 +/- 15 nmol/mg protein/30 min) and this was 4.5-fold higher than that in the pons, which was the lowest. In adult animals, olfactory bulb still contained the greatest activity, which was about eightfold higher than hippocampus, which had the lowest. Except for hypothalamus, where ADA activity increased nearly twofold in rats between the ages of 1 and 50 days, significant decreases of as much as fivefold were found in whole brain, superior colliculus, cortex, hippocampus, cerebellum, olfactory bulbs, and olfactory nucleus. In contrast, ADA activity in pons and subcortex remained relatively constant throughout the developmental period. The Km values for ADA in whole brain at 18 days gestation (48 +/- 5 microM) were not significantly different from that observed in adult rats (38 +/- 7 microM), whereas the Vmax values decreased significantly from 339 +/- 9 to 108 +/- 8 nmol/mg protein/30 min. Taken together, the developmental patterns observed in the various brain regions appear not to correspond to any one particular process such as periods of rapid cell proliferation, cell death, synaptogenesis, or myelination. Nor do they correspond to known developmental profiles of transmitters, their receptors, or their metabolic enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular and subcellular localization of paralemmin-1, a protein involved in cell shape control,in the rat brain,adrenal gland and kidney

Paralemmin-1 is a phosphoprotein, lipid-anchored to the cytoplasmic face of membranes and implicated in plasma membrane dynamics and cell process formation. We report an immunoperoxidase histochemical analysis of the cellular and subcellular localization of paralemmin-1 in the rat tissues where its expression is highest: the brain, the adrenal gland and the kidney. Paralemmin-1 is detected throughout the brain, in neuronal perikarya, axons and dendrites including dendritic spines and also in glial processes. In the adrenal gland, paralemmin-1 is highly expressed in the medulla. The kidney displays a pattern of differential paralemmin-1 expression in various structures and cell types, with high concentrations in cells of the parietal epithelium of Bowman’s capsule, intermediate tubules, distal tubules and principal cells of outer medullary collecting ducts. Mosaics of paralemmin-positive and paralemmin-negative cells are observed in proximal tubules, the parietal epithelium of Bowman’s capsule and the endothelium of many blood vessels. Plasma membrane association in epithelia is often polarized: paralemmin-1 concentrates at the apical membranes of adrenal chromaffin cells, but at the basolateral plasma membranes of proximal and distal tubule cells in the kidney. Paralemmin-1 immunoreactivity exhibits a spotted pattern and can be seen both at plasma membranes and within the cytoplasm, where it is often associated with endomembranes. This discontinuous distribution and the detergent extraction properties of paralemmin-1 suggest an association with lipid microdomains. The findings are consistent with a role for paralemmin-1 in the formation and stabilization of plasma membrane elaborations, in neurons as well as in other cell types.     

Primary structure of a dynamin-related mouse mitochondrial GTPase and its distribution in brain, subcellular localization, and effect on mitochondrial morphology

A new member of the dynamin GTPase family (OPA1) was recently identified in humans and shown to be mutated in patients with dominant optic atrophy. To understand better the function of mammalian OPA1, we isolated a mouse ortholog (mOPA1) from brain and raised a specific antibody against its C terminus. The subcellular distribution of mOPA1 overexpressed in COS-7 cells largely overlapped that of endogenous cytochrome c, a well known mitochondrial marker, and dramatically affected mitochondrial morphology, altering it from tubular to vesicular. Mitochondrial targeting was mediated by the N-terminal region of mOPA1 as follows: deletion of the 124 N-terminal amino acids eliminated mitochondrial targeting, although fusion of the N-terminal 60 or 90 amino acids of mOPA1 with green fluorescent protein resulted in its mitochondrial targeting. mOPA1 was expressed widely in the mouse brain, especially in neurons of olfactory bulb, cerebral cortex, piriform cortex, hypothalamus, hippocampus, red nucleus, cochlear nucleus, motor trigeminal nucleus, facial nucleus, cerebellar nucleus, and Purkinje cells. Within dissociated cerebellar cells, mOPA1 protein was clearly observed in the dendrites and somas of neuronal cells, as well as in astrocytes and meningeal cells. In each case, it was distributed in the vesicular pattern seen in other cell types.     

Adenovirus-mediated WGA gene delivery for transsynaptic labeling of mouse olfactory pathways

Detailed knowledge of neuronal connectivity patterns is indispensable for studies of various aspects of brain functions. We previously established a genetic strategy for visualization of multisynaptic neural pathways by expressing wheat germ agglutinin (WGA) transgene under the control of neuron type-specific promoter elements in transgenic mice and Drosophila. In this paper, we have developed a WGA-expressing recombinant adenoviral vector system and applied it for analysis of the olfactory system. When the WGA-expressing adenovirus was infused into a mouse nostril, various types of cells throughout the olfactory epithelium were infected and expressed WGA protein robustly. WGA transgene products in the olfactory sensory neurons were anterogradely transported along their axons to the olfactory bulb and transsynaptically transferred in glomeruli to dendrites of the second-order neurons, mitral and tufted cells. WGA protein was further conveyed via the lateral olfactory tract to the olfactory cortical areas including the anterior olfactory nucleus, olfactory tubercle, piriform cortex and lateral entorhinal cortex. In addition, transsynaptic retrograde labeling was observed in cholinergic neurons in the horizontal limb of diagonal band, serotonergic neurons in the median raphe nucleus, and noradrenergic neurons in the locus coeruleus, all of which project centrifugal fibers to the olfactory bulb. Thus, the WGA-expressing adenovirus is a useful and powerful tool for tracing neural pathways and could be used in animals that are not amenable to the transgenic technology.     

Expression of dihydropyridine-sensitive brain calcium channels in the rat central nervous system.

We have localized dihydropyridine (DHP-sensitive calcium channels in rat brain by in situ hybridization and immunohistochemistry. The mRNA for the dihydropyridine-sensitive calcium channel alpha 1 subunit (DHPR-B) is prominently localized in neuronal cells in the olfactory bulb, dentate gyrus, hippocampus, arcuate nucleus, paraventricular nucleus, ventromedial nucleus, cerebral cortex, superior colliculus and the cerebellar Purkinje cell layer. Strong expression of DHPR-B mRNA was also found in the pituitary and pineal glands. DHP-sensitive calcium channel alpha 1 subunit distribution has also been examined immunohistochemically with polyclonal antibodies raised against synthetic peptides specific for the DHPR-B alpha 1 subunit protein. The results from immunohistochemistry were in good agreement with those from in situ hybridization. Thus, regional distribution and localization of DHPR-B mRNA and alpha 1 subunit protein in rat brain suggest that this type of DHP-sensitive brain calcium channel may play an important role in excitation-secretion coupling functions in the neuroendocrine system.     

Distribution of calcium-independent phospholipase A<Subscript>2</Subscript> (iPLA<Subscript>2</Subscript>) in monkey brain

The present study was carried out to elucidate the distribution of calcium-independent phospholipase A₂ (iPLA₂) in the normal monkey brain. iPLA₂ immunoreactivity was observed in structures derived from the telencephalon, including the cerebral neocortex, amygdala, hippocampus, caudate nucleus, putamen, and nucleus accumbens, whereas structures derived from the diencephalon, including the thalamus, hypothalamus and globus pallidus were lightly labeled. The midbrain, vestibular, trigeminal and inferior olivary nuclei, and the cerebellar cortex were densely labeled. Immunoreactivity was observed on the nuclear envelope of neurons, and dendrites and axon terminals at electron microscopy. Western blot analysis showed higher levels of iPLA₂ protein in the cytosolic, than the nuclear fraction, but little or no protein in the membrane fraction. Similarly, subcellular fractionation studies of iPLA₂ activity in rat brain cortical cell cultures showed greater enzymatic activity in the cytosolic, than the nuclear fraction, and the least activity in non-nuclear membranes. The association of iPLA₂ with the nuclear envelope suggests a role of the enzyme in nuclear signaling, such as during neuronal proliferation and differentiation or death. In addition, the localization of iPLA₂ in dendrites and axon terminals suggests a role of the enzyme in neuronal signaling.     

The Embryonic Septum and Ventral Pallium, New Sources of Olfactory Cortex Cells

The mammalian olfactory cortex is a complex structure located along the rostro-caudal extension of the ventrolateral prosencephalon, which is divided into several anatomically and functionally distinct areas: the anterior olfactory nucleus, piriform cortex, olfactory tubercle, amygdaloid olfactory nuclei, and the more caudal entorhinal cortex. Multiple forebrain progenitor domains contribute to the cellular diversity of the olfactory cortex, which is invaded simultaneously by cells originating in distinct germinal areas in the dorsal and ventral forebrain. Using a combination of dye labeling techniques, we identified two novel areas that contribute cells to the developing olfactory cortices, the septum and the ventral pallium, from which cells migrate along a radial and then a tangential path. We characterized these cell populations by comparing their expression of calretinin, calbindin, reelin and Tbr1 with that of other olfactory cell populations.     

Heterogeneous Expression of Transketolase in Rat Brain

Abstract: Transketolase (TK; EC 2.2.1.1) is a key pentose phosphate shunt enzyme that plays an important role in the production of reducing equivalents and pentose sugars. TK activity declines in the brains of patients with Alzheimer's disease or Wernicke-Korsakoff syndrome, as well as in thiamine-deficient rats. Understanding the role of TK in the pathophysiology of these neurodegenerative conditions requires knowledge of its regional, cellular, and subcellular distribution within the brain. The current study employed in situ hybridization and immunocytochemistry to examine the distribution of TK mRNA and its encoded protein in adult rat brain. TK mRNA and protein were widely distributed throughout the brain. However, they were enriched in selective perikarya in the piriform cortex, nucleus of the diagonal band, red nucleus, dorsal raphe, pontine nucleus, locus coeruleus, trapezoid, inferior olive, and several cranial nerve nuclei. Lower expression of TK mRNA and protein occurred in layer V of cortex, olfactory tubercle, ventral pallidum, medial septal nucleus, hippocampus, thalamic and hypothalamic nuclei, mammillary body, central gray, and the substantia nigra. TK immunoreactivity also occurred in the nuclei of ubiquitously distributed glial cells, as well as ependymal cells. The heterogeneous distribution of TK may reflect a variety of metabolic activities among different brain regions but does not provide a simple molecular explanation for selective cell death in either thiamine deficiency or other conditions where TK is reduced.     

Regional and Temporal Alterations in DNA Fragmentation Factor (DFF)-Like Proteins Following Experimental Brain Trauma in the Rat

DNA fragmentation, an early event in neuronal death following traumatic brain injury, may be triggered by the 40-kDa subunit of DNA fragmentation factor (DFF40). DFF40 is typically bound to the 45-kDa subunit of DFF (DFF45), and activation of DFF40 may occur as a result of caspase-3-mediated cleavage of DFF45 into 30- and 11-kDa fragments. In this study, the intracellular distribution of DFF45 and DFF40 was examined following lateral fluid percussion brain injury of moderate severity (2.4-2.7 atm) in male Sprague-Dawley rats. In the cytosolic fraction (S1) of the injured cortex at 2 and 24 h postinjury, significant decreases in the intensities of DFF45-like proteins at 45- and 32-kDa bands and a concomitant increase in the 11-kDa bands were observed (p < 0.05 vs. uninjured controls). A significant decrease in the intensities of the 32-kDa band in the nuclear (P1) fraction of the injured cortex was observed at 30 min and 2 h postinjury (p < 0.01). Concomitantly, a decrease in DFF40 was observed in the cortical S1 fraction at 2 and 24 h (p < 0.05) and in the P1 fraction at 30 min and 2 h postinjury (p < 0.01). In the hippocampus, DFF45 decreased at 30 min in the P1 and 2 h in the S1 fraction (p < 0.05) and recovered by 24 h postinjury, whereas DFF40 was significantly decreased in the S1 and increased in the P1 fraction at both 2 and 24 h (p < 0.01), which indicated a translocation of DFF40 from cytosol to nucleus. These data are the first to demonstrate that changes in DFF proteins occur after brain trauma and suggest that these changes may play a role in apoptotic cell death via caspase-3-DFF45/DFF40-DNA cleavage observed following traumatic brain injury.     

Differential expression of isoforms of the regulatory subunit of type II cAMP-dependent protein kinase in rat neurons, astrocytes, and oligodendrocytes

The RII-B isoform of the regulatory subunit (R) of cAMP-dependent protein kinase II is abundantly and selectively expressed in cerebral cortex (Erlichman, J., Sarkar, D., Fleischer, N., and Rubin, C. S. (1980) J. Biol. Chem. 255, 8179-8184). In contrast to the cytosolic RII-H isoform from heart and other non-neural tissues, a substantial fraction of cerebral cortex RII-B is tightly associated with cell organelles. In order to study the cellular basis for the localization and abundance of RII-B in this complex and heterogeneous tissue, rat cerebral cortex was fractionated into highly purified populations of neurons, astrocytes, and oligodendrocytes. In neurons and astrocytes more than 80% of the total cAMP-binding activity is contributed by RII subunits, whereas the myelin-producing oligodendrocytes contain nearly equal proportions of RI (from protein kinase I) and RII. Approximately 70% of RII and RI subunits are associated with the particulate fraction in each of the three types of brain cells. The nature of the RII isoforms expressed in the cytosolic and particulate fractions of the purified brain cells was established by performing Western immunoblot and indirect immunoprecipitation analyses with selective and sensitive polyclonal antibodies directed against RII-B. Astrocytes and neurons exhibit high levels of RII-B, whereas oligodendrocytes contain the RII-H isoform. Thus, the expression of RII isoforms is not uniform among brain cells that are anatomically and developmentally related. Rather, it appears that RII-B and RII-H are expressed in a cell-specific fashion within cerebral cortex and this might reflect an RII-mediated adaptation of protein kinase II to the specialized metabolic and functional roles of neurons, astrocytes, and oligodendrocytes.     

Neuronal expression, cytosolic localization, and developmental regulation of the organic solute carrier partner 1 in the mouse brain

Organic solute carrier partner 1 (OSCP1) is a mammalian, transporter-related protein that is able to facilitate the uptake of structurally diverse organic compounds into the cell when expressed in Xenopus laevis oocytes. This protein has been implicated in testicular handling of organic solutes because its mRNA expression is almost exclusive in the testis. However, in this study, we demonstrated significant expression of OSCP1 protein in mouse brain, the level of which was rather higher than that in the testis, although the corresponding mRNA expression was one-tenth of the testicular level. Immunohistochemistry revealed that OSCP1 was broadly distributed throughout the brain, and various neuronal cells were immunostained, including pyramidal cells in the cerebral cortex and hippocampus. However, there was no evidence of OSCP1 expression in glia. In primary cultures of cerebral cortical neurons, double-labeling immunofluorescence localized OSCP1 to the cytosol throughout the cell body and neurites including peri-synaptic regions. This was consistent with the subcellular fractionation of brain homogenates, in which OSCP1 was mainly recovered after centrifugation both in the cytosolic fraction and the particulate fraction containing synaptosomes. Immunoelectron microscopy of brain sections also demonstrated OSCP1 in the cytosol near synapses. In addition, it was revealed that changes in the expression level of OSCP1 correlated with neuronal maturation during postnatal development of mouse brain. These results indicate that OSCP1 may have a role in the brain indirectly mediating substrate uptake into the neurons in adult animals.     

Interactions of Phencyclidine Receptor Agonist MK-801 with Dopaminergic System: Regional Studies in the Rat

Interactions of the potent phencyclidine receptor agonist MK-801 with the dopaminergic system were examined in various brain regions in the rat. MK-801 increased dopamine (DA) metabolism in the pyriform cortex, entorhinal cortex, prefrontal cortex, striatum, olfactory tubercle, amygdala, and septum without affecting DA metabolism in the cingulate cortex and nucleus accumbens. In pyriform cortex and amygdala, MK-801 was more potent than phencyclidine at increasing DA metabolism. Local injections of MK-801 into ventral tegmental area and into the amygdala/pyriform cortex interface indicated that MK-801 may act at the cell body as well as the nerve terminal level to increase DA metabolism and that ongoing dopaminergic neuronal activity is a prerequisite for full drug action.     

AVP免疫阳性反应在无蹼壁虎全脑的分布

目的为研究精氨酸加压素（Arginine Vasopressin）免疫阳性反应在无蹼壁虎全脑的分布。方法我们采用苏木素染色和免疫组织化学ABC法。结果发现AVP免疫阳性细胞在无蹼壁虎脑部的鲁卡斜角带核（nucleus of thedi—agonalband of Broca）、视上核（supraopticnucleus）、室旁核（paraventricular nucleus）分布较多,在室周视前区（periventricular preoptic area）、终纹床核（bed nucleusof the stria terminalis）、下丘脑外侧区（area lateralis hypothalami）、前脑外侧束（1ateral forebrain bundle）、室周核（periventricular nucleus）分布较少;AVP免疫阳性纤维除在嗅球、嗅束、大脑皮层、视叶、小脑皮层未见外,其它脑区均可见或多或少的阳性纤维;AVP阳性纤维在全脑投射广泛,且均与哺乳动物相似;AVP免疫阳性细胞数在PVN的6个部位呈中间阳性细胞数较多,两侧阳性细胞数较少（F=58．02,P〈O．001）。结论上述结果显示AVP免疫阳性反应在无蹼壁虎与哺乳动物脑区间存在相似性,提示AVP在无蹼壁虎脑区执行着类似于哺乳动物的生理功能。