CB2 cannabinoid receptors promote neural progenitor cell proliferation via mTORC1 signaling

The endocannabinoid system is known to regulate neural progenitor (NP) cell proliferation and neurogenesis. In particular, CB(2) cannabinoid receptors have been shown to promote NP proliferation. As CB(2) receptors are not expressed in differentiated neurons, CB(2)-selective agonists are promising candidates to manipulate NP proliferation and indirectly neurogenesis by overcoming the undesired psychoactive effects of neuronal CB(1) cannabinoid receptor activation. Here, by using NP cells, brain organotypic cultures, and in vivo animal models, we investigated the signal transduction mechanism involved in CB(2) receptor-induced NP cell proliferation and neurogenesis. Exposure of hippocampal HiB5 NP cells to the CB(2) receptor-selective agonist HU-308 led to the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway, which, by inhibiting its downstream target p27Kip1, induced NP proliferation. Experiments conducted with the CB(2) receptor-selective antagonist SR144528, inhibitors of the PI3K/Akt/mTORC1 axis, and CB(2) receptor transient-transfection vector further supported that CB(2) receptors control NP cell proliferation via activation of mTORC1 signaling. Likewise, CB(2) receptor engagement induced cell proliferation in an mTORC1-dependent manner both in embryonic cortical slices and in adult hippocampal NPs. Thus, HU-308 increased ribosomal protein S6 phosphorylation and 5-bromo-2'-deoxyuridine incorporation in wild-type but not CB(2) receptor-deficient NPs of the mouse subgranular zone. Moreover, adult hippocampal NP proliferation induced by HU-308 and excitotoxicity was blocked by the mTORC1 inhibitor rapamycin. Altogether, these findings provide a mechanism of action and a rationale for the use of nonpsychotomimetic CB(2) receptor-selective ligands as a novel strategy for the control of NP cell proliferation and neurogenesis.     

Serological reactivity of baculovirus-expressed Ebola virus VP35 and nucleoproteins

Ebola virus (EBOV) is a member of the family Filoviridae and is classified as a biosafety level 4 virus. This classification makes the preparation of antigen and performance of diagnostic assays time-consuming and complicated. The objective of this study was to evaluate the value of EBOV immunoassays based on recombinant nucleoprotein (r-NP) and recombinant VP35 (r-VP35) using large serum panels of African origin and from primates. Furthermore, we investigated whether the results obtained with EBOV r-VP35 enzyme-linked immunosorbent assay (ELISA) could improve on the findings obtained with the EBOV r-NP ELISA. The full-length EBOV NP and VP35 of the EBOV subtype Zaire were expressed as histidine-tagged recombinant proteins in the baculovirus expression system. The antigenic reactivity and specificity of these recombinant proteins were determined by Western blotting and ELISA using EBOV specific monoclonal antibodies. The results obtained with the r-NP and r-VP35 ELISAs were compared with the results obtained in an indirect immunofluorescence assay based on native EBOV subtype Zaire. EBOV specific monoclonal antibodies reacted specifically with the respective proteins in both Western blot and ELISA. Five hundred and twenty six samples from humans and primates were tested with r-NP and r-VP35 ELISAs. Monkey serum samples positive for EBOV subtype Reston and Zaire were both positive in the EBOV r-NP ELISA, whereas only the EBOV Zaire infected monkeys were positive in the r-VP35 ELISA. The sensitivity and specificity values of the EBOV recombinants' ELISAs compared to those of the immunofluorescence assay were 92% and 99% for r-NP and 44% and 100% for r-VP35. r-NP ELISA proved to be a sensitive and specific assay for EBOV diagnosis and for epidemiological studies for both EBOV subtypes Reston and Zaire. The use of r-VP35 in an ELISA format has no additional value for EBOV serodiagnosis.     

Analysis of the stability and function of nucleoplasmin through cysteine mutants

Xenopus laevis nucleoplasmin is a pentameric nuclear chaperone. The relation between the structure and the multifunctional aspects of the molecule has not yet been clearly established. In the course of analysing a C-terminally His-tagged recombinant version of the region equivalent to the trypsin resistant core (r-NP142) of the molecule, we found that this domain exhibited a substantially decreased oligomerization potential. To better understand the role of the three cysteines of nucleoplasmin on its pentameric functional structure, we have selectively mutated these residues to serine and generated three mutants (C15S, C35S, and C45S) both for the complete recombinant nucleoplasmin (r-NP) and the truncated r-NP142 non-tagged forms. We demonstrate that there are no disulphide bridges stabilizing either the monomer or the pentamer. Neither C15S nor C35S has any structural effects, while the mutation C45S abolishes the ability of r-NP142 to pentamerize. This structural impairment suggests that hydrophobic interactions of Cys 45 are critical for the stability of the protein. Our studies allow to analyse for the first time the structural and functional properties of nucleoplasmin in its monomeric form.     

Distribution of serine proteinase inhibitor, clade B, member 6 (Serpinb6) in the adult mouse brain

In the brain, Serpinb6 was identified as an endogenous inhibitor of neuropsin, a member of the S1 (clan SA) family of serine proteases [J. Biol. Chem. 276 (2001) 14562]. In the present study, we investigated the localization of Serpinb6 in the adult mouse brain using in situ hybridization histochemistry and immunohistochemistry. Region-specific patterns of expression were observed and two characteristics were recognized. First, the forebrain limbic area that expressed neuropsin mRNA contained Serpinb6 mRNA at moderate levels but not the lateral septum. On the other hand, Serpinb6 mRNA was also expressed moderately in the substantia nigra-ventral tegmental area system, whose fibers projected to the lateral septum. Additionally, Serpinb6 protein was detected in the lateral septum. Together, it was suggested that the expression of neuropsin in the brain is regulated entirely by Serpinb6. Second, Serpinb6 mRNA and the protein were strongly expressed in most somatic and visceral motoneurons among cranial nerve nuclei. This suggests that another serine protease is regulated by Serpinb6 in motoneurons and/or fibers.     

Differential effects of chronic treatment with haloperidol and clozapine on the level of preprosomatostatin mRNA in the striatum,nucleus accumbens,and frontal cortex of the rat

1. The goal of this work was to determine the effects of typical and atypical neuroleptics on the level of preprosomatostatin messenger RNA (mRNA) in regions of the rat brain innervated by dopaminergic neurons. 2. Quantitative in situ hybridization histochemistry was used to measure the levels of mRNA encoding preprosomatostatin in neurons of the striatum, the nucleus accumbens, and the medial and lateral agranular areas of the frontal cortex in adult rats treated with either haloperidol or clozapine. 3. In untreated animals, the density of neurons containing preprosomatostatin mRNA was higher in the nucleus accumbens than in the striatum and frontal cortex. The intensity of labeling per neuron, however, was higher in the striatum than in the two other areas examined, suggesting that the expression of preprosomatostatin mRNA is differentially regulated in these brain regions. Chronic administration of haloperidol (1 mg/kg for 28 days) induced a significant decrease in the labeling for preprosomatostatin mRNA in neurons of the nucleus accumbens, frontal cortex, and medial but not lateral striatum. Treatment with clozapine (20 mg/kg for 28 days) increased the levels of preprosomatostatin mRNA in the nucleus accumbens but not in the striatum or the frontal cortex. 4. These results support a role for dopamine in the regulation of central somatostatinergic neurons. The differences in the effects of haloperidol, a neuroleptic which induces extrapyramidal side effects, and clozapine, which does not, suggest that somatostatinergic neurons may play an important role in the regulation of motor behavior.     

Class 3 Semaphorin Mediates Dendrite Growth in Adult Newborn Neurons through Cdk5/FAK Pathway

Class 3 semaphorins are well-known axonal guidance cues during the embryonic development of mammalian nervous system. However, their activity on postnatally differentiated neurons in neurogenic regions of adult brains has not been characterized. We found that silencing of semaphorin receptors neuropilins (NRP) 1 or 2 in neural progenitors at the adult mouse dentate gyrus resulted in newly differentiated neurons with shorter dendrites and simpler branching in vivo. Tyrosine phosphorylation (Tyr 397) and serine phosphorylation (Ser 732) of FAK were essential for these effects. Semaphorin 3A and 3F mediate serine phosphorylation of FAK through the activation of Cdk5. Silencing of either Cdk5 or FAK in newborn neurons phenocopied the defects in dendritic development seen upon silencing of NRP1 or NRP2. Furthermore, in vivo overexpression of Cdk5 or FAK rescued the dendritic phenotypes seen in NRP1 and NRP2 deficient neurons. These results point to a novel role for class 3 semaphorins in promoting dendritic growth and branching during adult hippocampal neurogenesis through the activation of Cdk5-FAK signaling pathway.     

Nucleoplasmin interaction with protamines. Involvement of the polyglutamic tract

Different recombinant forms of nucleoplasmin including truncations at the carboxyl-terminal end of the molecule (r-NP121, r-NP142) have been expressed and purified. All of them were found to oligomerize, forming pentameric complexes which, according to their hydrodynamic properties, can be modeled by oblate ellipsoids of constant width. In this model, the highly charged carboxyl ends appear to be arranged around a pentameric core along the plane defined by the major axes of the ellipsoid. Importantly, all the recombinant forms appear to be able to decondense protamine-containing sperm nuclei. However, although the stoichiometry with which protamines bind to these forms appears to be constant (2.5 mol of protamine/mol of nucleoplasmin pentamer), the efficiency with which they remove protamines from the sperm DNA decreases in the following order: o-NP > r-NP142 > or = r-NP > r-NP121. Therefore, the main polyglutamic tract of nucleoplasmin (which is absent in r-NP121), while enhancing the efficiency of protamine removal, is not indispensable for sperm chromatin decondensation in the biological model we have used.     

UCP2 induced by natural birth regulates neuronal differentiation of the hippocampus and related adult behavior

Mitochondrial uncoupling protein 2 (UCP2) is induced by cellular stress and is involved in regulation of fuel utilization, mitochondrial bioenergetics, cell proliferation, neuroprotection and synaptogenesis in the adult brain. Here we show that natural birth in mice triggers UCP2 expression in hippocampal neurons. Chemical inhibition or genetic ablation of UCP2 lead to diminished neuronal number and size, dendritic growth and synaptogenezis in vitro and impaired complex behaviors in the adult. These data reveal a critical role for Ucp2 expression in the development of hippocampal neurons and circuits and hippocampus-related adult behaviors.     

Region-specific expression of mitochondrial complex I genes during murine brain development

Mutations in the nuclear encoded subunits of mitochondrial complex I (NADH:ubiquinone oxidoreductase) may cause circumscribed cerebral lesions ranging from degeneration of the striatal and brainstem gray matter (Leigh syndrome) to leukodystrophy. We hypothesized that such pattern of regional pathology might be due to local differences in the dependence on complex I function. Using in situ hybridization we investigated the relative expression of 33 nuclear encoded complex I subunits in different brain regions of the mouse at E11.5, E17.5, P1, P11, P28 and adult (12 weeks). With respect to timing and relative intensity of complex I gene expression we found a highly variant pattern in different regions during development. High average expression levels were detected in periods of intense neurogenesis. In cerebellar Purkinje and in hippocampal CA1/CA3 pyramidal neurons we found a second even higher peak during the period of synaptogenesis and maturation. The extraordinary dependence of these structures on complex I gene expression during synaptogenesis is in accord with our recent findings that gamma oscillations--known to be associated with higher cognitive functions of the mammalian brain--strongly depend on the complex I activity. However, with the exception of the mesencephalon, we detected only average complex I expression levels in the striatum and basal ganglia, which does not explain the exquisite vulnerability of these structures in mitochondrial disorders.     

ANEPIII, a new recombinant neurotoxic polypeptide derived from scorpion peptide, inhibits delayed rectifier, but not A-type potassium currents in rat primary cultured hippocampal and cortical neurons

A new recombinant neurotoxic polypeptide ANEPIII (BmK ANEPIII) derived from Scorpion peptide, which was demonstrated with antineuroexcitation properties in animal models, was examined for its action on K+ currents in primary cultured rat hippocampal and cortical neurons using the patch clamp technique in the whole-cell configuration. The delayed rectifier K+ current (I(k)) was inhibited by externally applied recombinant BmK ANEPIII, while the transient A-current (I(A)) remained virtually unaffected. BmK ANEPIII 3 microM, reduced the delayed rectifier current by 28.2% and 23.6% in cultured rat hippocampal and cortical neurons, respectively. The concentration of half-maximal block was 155.1 nM for hippocampal neurons and 227.2 nM for cortical neurons, respectively. These results suggest that BmK ANEPIII affect K+ currents, which may lead to a reduction in neuronal excitability.     

Distribution of Equilibrative, Nitrobenzylthioinosine-Sensitive Nucleoside Transporters (ENT1) in Brain

Nucleoside transport processes may play a role in regulating endogenous levels of the inhibitory neuromodulator adenosine in brain. The cDNAs encoding species homologues of one member of the equilibrative nucleoside transporter (ENT) gene family have recently been isolated from rat (rENT1) and human (hENT1) tissues. The current study used RT-PCR, northern blot, in situ hybridization, and [3H]nitrobenzylthioinosine autoradiography to determine the distribution of mRNA and protein for ENT1 in rat and human brain. Northern blot analysis indicated that hENT1 mRNA is widely distributed in adult human brain. 35S-labeled sense and antisense riboprobes, transcribed from a 153-bp segment of rENT1, were hybridized to fresh frozen coronal sections from adult rat brain and revealed widespread rENT1 mRNA in pyramidal neurons of the hippocampus, granule neurons of the dentate gyrus, Purkinje and granule neurons of the cerebellum, and cortical and striatal neurons. Regional localization in rat brain was confirmed by RT-PCR. Thus, ENT1 mRNA has a wide cellular and regional distribution in brain, indicating that this nucleoside transporter subtype may be important in regulating intra- and extracellular levels of adenosine in brain.     

Mitochondria are More Resistant to Hypoxic Depolarization in the Newborn than in the Adult Brain

Hypoxic-ischemic brain injury subsequent to asphyxia represents a major cause of morbidity and death in the newborn. The newborn brain has been considered more resistant to hypoxia than the adult brain because of lower energy demand. The mechanisms underlying hypoxic brain injury, in particular the age-related vulnerability, are still only partially understood. The mitochondrial function is pivotal for the function and survival of neurons. Acutely isolated CA1 neurons from neonatal (3-6 days) and adult rats (5-6 weeks) were loaded with Rh 123, and the effect of hypoxia on the inner mitochondrial membrane potential (Delta psi(m)) was compared. During prolonged hypoxia (15 min), Delta psi(m) was lost in a majority of the neonatal neurons (83%) and in all the adult neurons. During hypoxia (5 min) followed by reoxygenation the mitochondria in 23% of the neonatal neurons were completely depolarized, whereas 85% of the adult neurons demonstrated a complete loss of Delta psi(m). In conclusion hippocampal CA1 mitochondria in the newborn rat are more resistant to hypoxic depolarization than in the adult rat.     

Protection by 3'-methoxypuerarin of rat hippocampal neurons against ischemia/reperfusion injury

3'-Methoxypuerarin (3'-MOP) is an isoflavone extracted from radix puerariae. The aim of this study was to investigate the role and the mechanism of 3'-MOP in the protection of hippocampal neurons against cerebral ischemia/reperfusion (I/R) injury in rats. I/R injury was induced by a modified four-vessel occlusion model. Rats were randomly divided into an I/R group, an I/R + 3'-MOP group and a control group. Histological changes in the neurons of the hippocampal CA1 region were observed with hematoxylin and eosine (H&E) staining. The apoptotic neurons in the hippocampal CA1 area were counted with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The results showed that compared with the I/R group, 3'-MOP increased the number of surviving neurons in the hippocampal CA1 region (P < 0.001) and markedly reduced the number of apoptotic pyramidal neurons (P < 0.001) after I/R injury. In conclusion, 3'-MOP can protect hippocampal neurons against I/R injury by inhibiting apoptosis.     

Neural stem cells and the regulation of adult neurogenesis

Presumably, the 'hard-wired' neuronal circuitry of the adult brain dissuades addition of new neurons, which could potentially disrupt existing circuits. This is borne out by the fact that, in general, new neurons are not produced in the mature brain. However, recent studies have established that the adult brain does maintain discrete regions of neurogenesis from which new neurons migrate and become incorporated into the functional circuitry of the brain. These neurogenic zones appear to be vestiges of the original developmental program that initiates brain formation. The largest of these germinal regions in the adult brain is the subventricular zone (SVZ), which lines the lateral walls of the lateral ventricles. Neural stem cells produce neuroblasts that migrate from the SVZ along a discrete pathway, the rostral migratory stream, into the olfactory bulb where they form mature neurons involved in the sense of smell. The subgranular layer (SGL) of the hippocampal dentate gyrus is another neurogenic region; new SGL neurons migrate only a short distance and differentiate into hippocampal granule cells. Here, we discuss the surprising finding of neural stem cells in the adult brain and the molecular mechanisms that regulate adult neurogenesis.     

SDS-stable complex formation between native apolipoprotein E3 and beta-amyloid peptides

Extracellular senile plaques composed predominantly of fibrillar amyloid-beta (Abeta) are a major neuropathological feature of Alzheimer's disease (AD). Genetic evidence and in vivo studies suggest that apolipoprotein E (apoE) may contribute to amyloid clearance and/or deposition. In vitro studies demonstrate that native apoE2 and E3 form an SDS-stable complex with Abeta(1-40), while apoE4 forms little such complex. Our current work extends these observations by presenting evidence that apoE3 also binds to Abeta(1-42) and with less avidity to modified species of the peptide found in senile plaque cores. These modified peptides include a form that originates at residue 3-Glu as pyroglutamyl and another with isomerization at the 1-Asp and 7-Asp positions. In addition, we used binding reactions between apoE3 and various Abeta fragments, as well as binding reactions with apoE3 and Abeta(1-40) plus Abeta fragments as competitors, to identify the domain(s) of Abeta involved in the formation of an SDS-stable complex with apoE3. Residues 13-28 of Abeta appear to be necessary, while complex formation is further enhanced by the presence of residues at the C-terminus of the peptide. These results contribute to our understanding of the biochemical basis for the SDS-stable apoE3/Abeta complex and support the hypothesis that Abeta can be transported in vivo complexed with apoE. This complex may then be cleared from the interstitial space by apoE receptors in the brain or become part of an extracellular amyloid deposit.     

Regional distribution of brain-derived neurotrophic factor mRNA in the adult mouse brain.

Brain-derived neurotrophic factor (BDNF) is a protein that allows the survival of specific neuronal populations. This study reports on the distribution of the BDNF mRNA in the adult mouse brain, where the BDNF gene is strongly expressed, using quantitative Northern blot analysis and in situ hybridization. All brain regions examined were found to contain substantial amounts of BDNF mRNA, the highest levels being found in the hippocampus followed by the cerebral cortex. In the hippocampus, which is also the site of highest nerve growth factor (NGF) gene expression in the central nervous system (CNS), there is approximately 50-fold more BDNF mRNA than NGF mRNA. In other brain regions, such as the granule cell layer of the cerebellum, the differences between the levels of BDNF and NGF mRNAs are even more pronounced. The BDNF mRNA was localized by in situ hybridization in hippocampal neurons (pyramidal and granule cells). These data suggest that BDNF may play an important role in the CNS for a wide variety of adult neurons.     

Distribution and characterisation of somatostatin receptor mRNA and binding sites in the brain and periphery.

The distribution and nature of (somatostatin) SRIF receptors and receptor mRNAs was studied in the brain and periphery of various laboratory animals using in situ hybridisation, autoradiography and radioligand binding. The messenger RNA (mRNA) expression of SRIF receptors msst1, msst2, msst3, msst4 and msst5 was studied in the adult mouse brain by in situ hybridisation histochemistry using specific oligonucleotide probes and compared to that of adult rats. As observed in rat brain, sst3 receptor mRNA is prominently expressed across the mouse brain, although equivalent binding has not yet been identified in situ. Sst1 and sst2 receptor mRNA expression, was prominent and again comparable to that observed in rat brain, whereas sst4 and especially sst5 receptor mRNA show comparatively low levels, although the former appears to be widely distributed while the latter could only be identified in a few nuclei. Altogether, the data are compatible with current knowledge, i.e. sst1 and sst2 receptor mRNA is prominent (both receptors have been functionally identified in the brain and for sst2 in the periphery), sst3 mRNA is highly expressed but in the absence of any functional correlate remains elusive. The expression of sst4 mRNA is comparatively low (especially when compared to what is seen in the lung, where high densities of sst4 receptors are present) and it remains to be seen whether sst5 receptor mRNA, which is confined to a few nuclei, will play a role in the brain, keeping in mind that high levels are found in the pituitary. Radioligand binding studies were performed in CCL39 cells expressing the five human recombinant receptors and compared to binding in membranes of rat cerebral cortex with [125I]Tyr11-SRIF14 which in the presence of 120 mM labels primarily sst1 receptor as suggested by the better correlation hsst1 and similar rank order of potency. The profile of [125I]Tyr3-octreotide labelled sites in rat cortex correlates better with recombinant sst2 than sst3 or sst5 binding profiles. Finally, [125I]LTT-SRIF28-labelled sites in rat lung express a sst4 receptor profile in agreement with previous findings. SRIF receptor autoradiography was performed in the brain and peripheral tissue of rat and/or guinea-pig using a number of ligands known to label recombinant SRIF receptors: [125I]LTT-SRIF28, [125I]CGP 23996, [125I]Tyr10-CST, or [125I]Tyr3-octreotide. Although, [125I]Tyr10-CST has been shown to label all five recombinant SRIF receptors, it is apparent that this radioligand is not useful for autoradiographic studies. By contrast, the other three ligands show good signal to noise ratios in rat or guinea-pig brain, rat lung, rat pancreas, or guinea-pig ileum. In most tissues, [125I]Tyr3-octreotide represents a prominent part of the binding (when compared to [125I]LTT-SRIF28 and [125I]CGP 23996), suggesting that sst2 receptors are strongly expressed in most tissues; it is only in rat lung that [125I]LTT-SRIF28 and [125I]CGP 23996 show marked binding, whereas [125I]Tyr3-octreotide does apparently label no sites, in agreement with the sole presence of sst4 receptors in this tissue.