Regional distribution of NPC1 protein in monkey brain

NPC1 is a member of a family of polytopic membrane-bound proteins with sterol-sensing domains. Inactivating mutations of NPC1 are responsible for most cases of Niemann-Pick type C disease, whose hallmark is progressive neurodegeneration. The precise molecular mechanisms whereby defective NPC1 function leads to neurodegeneration are unknown. In the brain, we have previously found NPC1 to localize predominantly within perisynaptic astrocytic processes. Here we have mapped the regional distribution of NPC1 in the monkey brain. Dense NPC1 immunoreactivity was observed in telencephalic structures, including the cerebral neocortex, hippocampus, caudate nucleus and putamen, whilst light immunostaining was observed in diencephalic structures, including the globus pallidus, thalamus and hypothalamus. Light staining was also generally observed in the midbrain, pons, medulla oblongata and cerebellum, except the inferior olive, which was densely stained. By light microscopy, only a few indistinctly labeled cell bodies were observed even within densely labeled regions, where most of the immunoreactivity appeared to be due to the large numbers of labeled cellular processes. On electron microscopy, these processes were identified as glial, and not neuronal. The astrocytic localization of NPC1 was further confirmed by double labeling for NPC1 and GFAP. The regional pattern of NPC1 expression suggests that areas normally expressing low levels of the NPC1 protein are more susceptible to neuronal degeneration in Niemann-Pick type C disease.     

Regional distribution of colchicine-binding (microtubular) protein in the rat brain

The relative concentrations of microtubular protein (tubulin) in regions of rat brain were determined by taking advantage of its specific property of binding colchicine. In comparison with other tissues, all regions of the brain were rich in tubulin. The cerebral cortex, thalamus and hypothalamus had essentially the same concentration. The cerebellum and brain stem had a lower concentration (about 60 per cent of the cortical level). Although the functional significance of these differences is not clear, they may relate to two proposed functions of microtubules–cytoskeleton and cytoplasmic transport. The complex geometry of neurons and many glial cells with ramified processes must require extensive systems for maintaining shape and an active cytoplasmic transport.     

Structural analysis of Mg2+ and Ca2+ binding, myristoylation, and dimerization of the neuronal calcium sensor and visinin-like protein 1 (VILIP-1)

Visinin-like protein 1 (VILIP-1) belongs to the neuronal calcium sensor family of Ca(2+)-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca(2+) and Mg(2+) binding, characterize metal-induced conformational changes, and determine structural effects of myristoylation and dimerization. Mg(2+) binds functionally to VILIP-1 at EF3 (ΔH = +1.8 kcal/mol and K(D) = 20 μM). Unmyristoylated VILIP-1 binds two Ca(2+) sequentially at EF2 and EF3 (K(EF3) = 0.1 μM and K(EF2) = 1-4 μM), whereas myristoylated VILIP-1 binds two Ca(2+) with lower affinity (K(D) = 1.2 μM) and positive cooperativity (Hill slope = 1.5). NMR assignments and structural analysis indicate that Ca(2+)-free VILIP-1 contains a sequestered myristoyl group like that of recoverin. NMR resonances of the attached myristate exhibit Ca(2+)-dependent chemical shifts and NOE patterns consistent with Ca(2+)-induced extrusion of the myristate. VILIP-1 forms a dimer in solution independent of Ca(2+) and myristoylation. The dimerization site is composed of residues in EF4 and the loop region between EF3 and EF4, confirmed by mutagenesis. We present the structure of the VILIP-1 dimer and a Ca(2+)-myristoyl switch to provide structural insights into Ca(2+)-induced trafficking of nicotinic acetylcholine receptors.     

Comparison of VILIP-1 and VILIP-3 Binding to Phospholipid Monolayers

The neuronal calcium sensor proteins Visinin-like Proteins 1 (VILIP-1) and 3 (VILIP-3) are effectors of guanylyl cyclase and acetyl choline receptors, and transduce calcium signals in the brain. The “calcium-myristoyl” switch, which involves a post-translationally added myristoyl moiety and calcium binding, is thought to regulate their membrane binding capacity and therefore, play a critical role in their mechanism of action. In the present study, we investigated the effect of membrane composition and solvent conditions on the membrane binding mechanisms of both VILIPs using lipid monolayers at the air/buffer interface. Results based on comparison of the adsorption kinetics of the myristoylated and non-myristoylated proteins confirm the pivotal role of calcium and the exposed myristol moiety for sustaining the membrane-bound state of both VILIPs. However, we also observed binding of both VILIP proteins in the absence of calcium and/or myristoyl conjugation. We propose a two-stage membrane binding mechanism for VILIP-1 and VILIP-3 whereby the proteins are initially attracted to the membrane surface by electrostatic interactions and possibly by specific interactions with highly negatively charged lipids head groups. The extrusion of the conjugated myristoyl group, and the subsequent anchoring in the membrane constitutes the second stage of the binding mechanism, and ensures the sustained membrane-bound form of these proteins.     

cDNA cloning of a neural visinin-like Ca(2+)-binding protein.

A 21,000-dalton Ca(2+)-binding protein (Walsh, M.P., Valentine, K.A., Ngai, P.K., Carruthers, C.A., and Hollengerg, M.D. (1984) Biochem. J. 224, 117-127) was purified from the rat brain and through the use of oligonucleotide probe based on partial amino acid sequence, cDNA clones were obtained from rat brain cDNA library. The complete amino acid sequence deduced from the cDNA contains 191 residues and has a calculated molecular mass of 22,142 daltons. There are three potential Ca(2+)-binding sites like the EF hands in the sequence. It displays striking sequence homology with visinin and recoverin, retina-specific Ca(2+)-binding proteins. Northern blot analysis revealed that the protein is highly and specifically expressed in the brain.     

Intracellular neuronal calcium sensor (NCS) protein VILIP-1 modulates cGMP signalling pathways in transfected neural cells and cerebellar granule neurones

The family of intracellular neuronal calcium-sensors (NCS) belongs to the superfamily of EF-hand proteins. Family members have been shown by in vitro assays to regulate signal cascades in retinal photoreceptor cells. To study the functions of NCS proteins not expressed in photoreceptor cells we examined Visinin-like protein-1 (VILIP-1) effects on signalling pathways in living neural cells. Visinin-like protein-1 expression increased cGMP levels in transfected C6 and PC12 cells. Interestingly, in transfected PC12 cells stimulation was dependent on the subcellular localization of VILIP-1. In cells transfected with membrane-associated wild-type VILIP-1 particulate guanylyl cyclase (GC) was stimulated more strongly than soluble GC. In contrast, deletion of the N-terminal myristoylation site resulted in cytosolic localization of VILIP-1 and enhanced stimulation of soluble GC. To study the molecular mechanisms underlying GC stimulation VILIP-1 was examined to see if it can physically interact with GCs. A direct physical interaction of VILIP-1 with the recombinant catalytic domain of particulate GCs-A, B and with native GCs enriched from rat brain was observed in GST pull-down as well as in surface plasmon resonance interaction studies. Furthermore, following trituration of recombinant VILIP-1 protein into cerebellar granule cells the protein influenced only signalling by GC-B. Together with the observed colocalization of GC-B, but not GC-A, with VILIP-1 in cerebellar granule cells, these results suggest that VILIP-1 may be a physiological regulator of GC-B.     

Regional distribution, developmental changes, and cellular localization of CNTF-mRNA and protein in the rat brain

Ciliary neurotrophic factor (CNTF) is a potent survival molecule for a variety of embryonic neurons in culture. The developmental expression of CNTF occurs clearly after the time period of the physiological cell death of CNTF-responsive neurons. This, together with the sites of expression, excludes CNTF as a target-derived neuronal survival factor, at least in rodents. However, CNTF also participates in the induction of type 2 astrocyte differentiation in vitro. Here we demonstrate that the time course of the expression of CNTF-mRNA and protein in the rat optic nerve (as evaluated by quantitative Northern blot analysis and biological activity, respectively) is compatible with such a glial differentiation function of CNTF in vivo. We also show that the type 2 astrocyte-inducing activity previously demonstrated in optic nerve extract can be precipitated by an antiserum against CNTF. Immunohistochemical analysis of astrocytes in vitro and in vivo demonstrates that the expression of CNTF is confined to a subpopulation of type 1 astrocytes. The olfactory bulb of adult rats has comparably high levels of CNTF to the optic nerve, and here again, CNTF-immunoreactivity is localized in a subpopulation of astrocytes. However, the postnatal expression of CNTF in the olfactory bulb occurs later than in the optic nerve. In other brain regions both CNTF-mRNA and protein levels are much lower.     

Evidence for different functional properties of the neuronal calcium sensor proteins VILIP-1 and VILIP-3: from subcellular localization to cellular function

The visinin-like-proteins VILIP-1 and -3 are EF-hand calcium-binding proteins and belong to the family of neuronal calcium sensor (NCS) proteins. Members of this family are involved in the calcium-dependent regulation of signal transduction cascades mainly in the nervous system. VILIP-1 and VILIP-3 are expressed in different populations of neuronal cells. To gain insights into the different functional characteristics of VILIP-1 and -3, we have compared the localization of the proteins in intact cells and the calcium-dependent membrane association in subcellular fractions. Furthermore, we have investigated the different functional properties of the two proteins in activating cGMP signal pathways and have defined different sets of protein interaction partners. Our data indicate that VILIP-3, which is mainly expressed in Purkinje cells, and VILIP-1, which is expressed in granule cells in the cerebellum, show a different calcium-dependent subcellular localization, may activate different cellular signaling pathways, and thus have signaling functions which seem to be cell-type specific.     

The distribution of melanopsin (OPN4) protein in the human brain

Until now, melanopsin (OPN4) – a specialized photopigment being responsive especially to blue light wavelengths – has not been found in the human brain at protein level outside the retina. More specifically, OPN4 has only been found in about 2% of retinal ganglion cells (i.e. in intrinsically photosensitive retinal ganglion cells), and in a subtype of retinal cone-cells. Given that Allen Institute for Brain Science has described a wide distribution of OPN4 mRNA in two human brains, we aimed to investigate whether OPN4 is present in the human brain also at protein level. Western blotting and immunohistochemistry, as well as immunoelectron microscopy, were used to analyse the existence and distribution of OPN4 protein in 18 investigated areas of the human brain in samples obtained in forensic autopsies from 10 male subjects (54 ± 3.5 years). OPN4 protein expression was found in all subjects, and, furthermore, in 5 out of 10 subjects in all investigated brain areas localized in membranous compartments and cytoplasmic vesicles of neurons. To our opinion, the wide distribution of OPN4 in central areas of the human brain evokes a question whether ambient light has important straight targets in the human brain outside the retinohypothalamic tract (RHT). Further studies are, however, needed to investigate the putative physiological phototransductive actions of inborn OPN4 protein outside the RHT in the human brain.     

Regional distributions of somatostatin and cholecystokinin-like immunoreactivities in rat and bovine brain

The distribution of somatostatin- and cholecystokinin octapeptide (CCK-8)-like immunoreactivities in the central nervous system of bovine and rat has been studied using a sensitive and specific radioimmunoassay. The most interesting information is the high concentration of CCK-8 in the various cortical areas in rat and bovine. The nuclei of amygdala contain the highest amount of octapeptide in rat while in bovine brain, this structure contains the second highest concentration after polus frontalis.     

Regional distribution of anchorless prion protein,PrP226*, in the human brain

It was shown previously that truncated molecules of prion protein can be found in brains of patients with some types of transmissible spongiform encephalopathy. One such molecule, PrP226*, is a fragment of prion protein, truncated at Tyr226. It was found to be present in aggregates, from which it can be released using chaotropic salts. In this study we investigated the distribution of PrP226* in Creutzfeldt–Jakob disease affected human brain, employing the mAb V5B2, specifically recognizing this fragment. The results show that PrP226* is not evenly distributed among different regions of human brain. Among brain regions analyzed, the fragment was found most likely to be accumulated in the cerebellum. Its distribution correlates with the distribution of PrP^Sc.     

Developmental regulation and cellular distribution of human cytosolic malate dehydrogenase (MDH1)

Human cyotsolic malate dehydrogenase (MDH1) is important in transporting NADH equivalents across the mitochondrial membrane, controlling tricarboxylic acid (TCA) cycle pool size and providing contractile function. Cellular localization studies indicate that MDH1 mRNA expression has a strong tissue-specific distribution, being expressed primarily in cardiac and skeletal muscle and in the brain, at intermediate levels in the spleen, kidney, intestine, liver, and testes and at low levels in lung and bone marrow. The observed MDH1 localizations reflect the role of NADH in the support of a variety of functions in different organs. These functions are primarily related to aerobic energy production for muscle contraction, neuronal signal transmission, absorption/resorption functions, collagen-supporting functions, phagocytosis of dead cells, and processes related to gas exchange and cell division. During neonatal development, MDH1 is expressed in human embryonic heart as early as the 3rd month and then is over-expressed from the 5th month until the birth. The expression of MDH1 is maintained in the adult heart but is not present in levels as high as in the fetus. Finally, over-expression of MDH1 is found in left ventricular cardiac muscle of dilated cardiomyopathy (DCM) patients when contrasted to the diseased non-DCM and normal heart muscle by in situ hybridization and Western blot. These observations are compatible with the activation of glucose oxidation in relatively hypoxic environments of fetal and hypertrophied myocardium.     

Differential distribution of orexin-A-like and orexin receptor 1 (OX1R)-like immunoreactivities in the Xenopus pituitary

Immunohistochemical techniques were employed to investigate orexin-A-like and orexin receptor 1 (OX1R)-like immunoreactivities in the Xenopus pituitary gland. Orexin-A-immunoreactive cells were mainly scattered in the posterior half of the pars distalis. They corresponded to thyroid-stimulating hormone (TSH)-containing cells and so far have not corresponded to other types of pituitary adenocytes. On the other hand, OX1R-immunoreactive cells were mainly distributed in the anterior half of the pars distalis and corresponded to prolactin (PRL)-containing cells; however, we found that OX1R-immunoreactive cells did not correspond to other types of adenocytes in the Xenopus pituitary. These results suggest that an orexin-A-like substance secretes with and/or without TSH from TSH-containing cells and that the peptide modulates the functions of PRL-containing cells via OX1R in a paracrine fashion.     

Regional distribution of immunoreactive prolactin-releasing peptide in the human brain

Regional distribution of prolactin-releasing peptide (PrRP) in the human brain was studied by radioimmunoassay. The antiserum raised against human PrRP-31 in a rabbit was used in the assay, which showed 100% cross reaction with PrRP-20 and no significant cross reaction with other peptides. The highest concentrations of immunoreactive-PrRP were found in hypothalamus (912 +/- 519 fmol/g wet weight, n = 6, mean +/- SEM), followed by medulla oblongata (496 +/- 136 fmol/g wet weight) and thalamus (307 +/- 117 fmol/g wet weight). On the other hand, immunoreactive-PrRP was not detected in frontal lobe or temporal lobe (<50 fmol/g wet weight). Sephadex G50 column chromatography of the immunoreactive-PrRP in the hypothalamus and medulla oblongata showed three immunoreactive peaks; one peak eluting in the position of PrRP-20, one eluting in the position of PrRP-31 and one eluting earlier. Reverse phase high-performance liquid chromatography (HPLC) of these brain tissue extracts showed a peak eluting in the position of PrRP-20 and PrRP-31. The present study has shown for the first time the presence of immunoreactive-PrRP in the human brain. The immunoreactive-PrRP levels in the human hypothalamus were, however, lower than the levels of other neuropeptides with prolactin-releasing activity, such as thyrotropin-releasing hormone and vasoactive intestinal polypeptide.     

Regional distribution and in vivo release of tachykinin-like immunoreactivities in rat brain: evidence for regional differences in relative proportions of tachykinins

The regional distribution of various forms of tachykinin-like immunoreactivity (TKLI) was studied in rat brain using radioimmunoassay. TKLI was measured with two different tachykinin-antisera (K12 and E7), which react with neurokinin A (NKA) and neurokinin B (NKB) but not with substance P (SP) and with a specific SP-antiserum. TKLI-K12 and TKLI-E7 were found to have similar regional distributions which were, however, significantly different from that of the substance P-like immunoreactivity (SPLI). Thus, the ratio of the tissue concentrations of TKLI-K12 or TKLI-E7 to that of SPLI was higher in frontal cortex and hippocampus and lower in pons/medulla oblongata than in the other regions studied. Cation-exchange chromatography of neutral water extracts of brain tissue revealed two major immunoreactive components of TKLI-K12 and TKLI-E7, one of which co-eluted with synthetic NKB while the other appeared in the same region as synthetic NKA. The relative quantities of these components varied depending on the brain region studied. No TKLI-K12 or TKLI-E7 co-eluted with synthetic SP. Almost all of the SPLI in acetic acid or water extracts of brain tissue eluted as a single chromatographic component in the same position as synthetic SP. Potassium-stimulated in vivo release of TKLI-K12, TKLI-E7 and SPLI in striatum of rat brain could be demonstrated using intracerebral dialysis. The present results imply that tachykinins, which may serve as neurotransmitters or neuromodulators, are present in different proportions in different regions of rat brain.     

Regional distribution and pharmacological characteristics of [3H]N-acetyl-aspartyl-glutamate (NAAG) binding sites in rat brain

Autoradiographical studies revealed that 10 nM [3H]N-acetyl-aspartyl-glutamate (NAAG) labelled grey matter structures, particularly in the hippocamus, cerebral neocortex, striatum, septal nuclei and the cerebellar cortex. The binding was inhibited by (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)-glycine (DCG IV), an agonist at group II metabotropic glutamate receptors (mGluR II). (RS)-alpha-Methyl-4-tetrazolylphenylglycine (MTPG), (RS)-alpha-cyclopropyl-4-phosphonoglycine (CPPG) and (RS)-alpha-methylserine-O-phosphate monophenyl ester (MSOPPE), all antagonists at mGluR II and mGluR III, also inhibited [3H]NAAG binding. Other inhibitors were (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD), a broad-spectrum mGluR agonist with preference for groups I and II and the mGluR I agonists/mGluR II antagonists (S)-3-carboxy-4-hydroxyphenylglycine (3,4-CHPG) and (S)-4-carboxy-3-hydroxyphenylglycine (4,3-CHPG). Neither the mGluR I specific agonist (S)-dihydroxyphenylglycine nor any of the ionotropic glutamate receptor ligands such as kainate, AMPA and MK-801 had strong effects (except for the competitive NMDA antagonist CGS 19755, which produced 20-40% inhibition at 100 microM) suggesting that, at low nM concentrations, [3H]NAAG binds predominantly to metabotropic glutamate receptors, particularly those of the mGluR II type. Several studies have indicated that NAAG can interact with mGluR II and the present study supports this notion by demonstrating that sites capable of binding NAAG at low concentrations and displaying pharmacological characteristics of mGluR II exist in the central nervous tissue. Furthermore, the results show that autoradiography of [3H]NAAG binding can be used to quantify the distribution of such sites in distinct brain regions and study their pharmacology at the same time.     

WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) delays cellular senescence by promoting p27(Kip1) degradation in human diploid fibroblasts

WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) plays an important role in the proliferation of tumor cells and the lifespan of Caenorhabditis elegans. However, the role of WWP1 in cellular senescence is still unknown. Here, we show that the expression patterns of p27(Kip1) and WWP1 are inversely correlated during cellular senescence. Moreover, the overexpression of WWP1 delayed senescence, whereas the knockdown of WWP1 led to premature senescence in human fibroblasts. Furthermore, we demonstrate that WWP1 repressed endogenous p27(Kip1) expression through ubiquitin-proteasome-mediated degradation. Additionally, WWP1 had a strong preference for catalyzing the Lys-48-linked polyubiquitination of p27(Kip1) in vitro. Finally, we demonstrate that WWP1 markedly inhibited the replicative senescence induced by p27(Kip1) by promoting p27(Kip1) degradation. Therefore, our study provides a new molecular mechanism for the regulation of cellular senescence.