Cheng Ji Zhou

PACAP protects hippocampal neurons against apoptosis: Involvement of JNK/SAPK signaling pathway

Abstract: We have demonstrated that the ischemia‐induced apoptosis of neurons in the CA1 region of the rat hippocampus was prevented by either intracerebroventricular or intravenous infusion of pituitary adenylate cyclase‐activating polypeptide (PACAP). However, the molecular mechanisms underlying the anti‐apoptotic effect of PACAP remain to be determined. Within 3–6 h after ischemia, the activities of members of the mitogen‐activated protein (MAP) kinase family, including extracellular signal‐regulated kinase (ERK), Jun N‐terminal kinase (JNK)/stress‐activated protein kinase (SAPK), and p38 were increased in the hippocampus. The ischemic stress had a potent influence on the MAP kinase family, especially on JNK/SAPK. PACAP inhibited the activation of JNK/SAPK after ischemic stress. Secretion of interleukin‐6 (IL‐6) into the cerebrospinal fluid was intensely stimulated after PACAP infusion. IL‐6 inhibited the activation of JNK/SAPK, while it activated ERK. These observations suggest that PACAP and IL‐6 act to inhibit the JNK/SAPK signaling pathway, thereby protecting neurons against apoptosis.

Delayed neuronal cell death in the rat hippocampus is mediated by the mitogen-activated protein kinase signal transduction pathway

Transient global ischemia caused by 5 min of cardiac arrest induced delayed neuronal cell death (apoptosis) in the CA1 region of the rat hippocampus. To characterize the molecular mechanisms that regulate apoptosis in vivo, the contributions to cell death of mitogen-activated protein kinase family members were examined in the hippocampal region after brain ischemia-reperfusion. Ischemia-reperfusion led to a strong activation of the JNK/SAPK (c-Jun NH2-terminal protein kinase/stress activated protein kinase), ERK (extracellular signal-regulated kinase), and p38 enzymes. These results with other previous studies suggest that the activation of JNK/SAPK in accordance with p38 contributes to the induction of apoptosis in CA1 neurons.

ATP-Binding Cassette Transporter ABC2/ABCA2 in the Rat Brain: A Novel Mammalian Lysosome-Associated Membrane Protein and a Specific Marker for Oligodendrocytes But Not for Myelin Sheaths

We recently cloned a full-length cDNA of the rat ATP-binding cassette transporter 2 (ABC2, or ABCA2) protein, a member of the ABC1 (or ABCA) subfamily (-ABC1/ABCA1 is a causal gene for Tangier disease) and found it to be strongly expressed in the rat brain. In this study, we identified ABC2 as a lysosome-associated membrane protein that is being localized specifically in oligodendrocytes. The ABC2-immunolabeled cells were detected mainly in the white matter but were also scattered in gray matter throughout the whole brain. In addition, these cells were found to be colocalized with 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) immunoreactivity when the marker antibody for oligodendrocytes was used. However, no such colocalization was observed with markers for other kinds of glial cells. Unlike the CNP antibody, which also intensely stains myelin sheaths in the white matter, ABC2 immunoreactivity was detected only in the cell bodies of oligodendrocytes. At the ultrastructural level, ABC2 immunoreactivity was detected mostly around lysosome and partly in Golgi apparatus by electron microscopy. This was confirmed by immunocolocalization of ABC2 and lysosomal markers in a neuroblastoma cell line. Immunoblotting analysis of ABC2 from the whole brain and the ABC2-transfected cell line revealed bands at ∼260 kDa. The result ofin situ hybridization with a riboprobe for ABC2 matched the results obtained from immunostaining. These findings strongly suggest that ABC2 is a specific marker for oligodendrocytes but not for myelinsheaths and that it is as a novel mammalian lysosome-associated membrane protein involved in myelinization or other kinds of metabolism in the CNS.

Cloning, characterization and tissue distribution of the rat ATP-binding cassette (ABC) transporter ABC2/ABCA2.

The ABC1 (ABCA) subfamily of the ATP-binding cassette (ABC) transporter superfamily has a structural feature that distinguishes it from other ABC transporters. Here we report the cloning, molecular characterization and tissue distribution of ABC2/ABCA2, which belongs to the ABC1 subfamily. Rat ABC2 is a protein of 2434 amino acids that has 44.5%, 40.0% and 40.8% identity with mouse ABC1/ABCA1, human ABC3/ABCA3 and human ABCR/ABCA4 respectively. Immunoblot analysis showed that proteins of 260 and 250 kDa were detected in COS-1 cells transfected with ABC2 having a haemagglutinin tag, while no band was detected in mock-transfected cells. After incubation with N-glycosidase F, the mobilities of the two proteins increased and a single band was detected, suggesting that ABC2 is a glycoprotein. Photoaffinity labelling with 8-azido-[alpha-(32)P]ATP confirmed that ATP binds to the ABC2 protein in the presence of Mg(2+). RNA blot analysis showed that ABC2 mRNA is most abundant in rat brain. Examination of brain by in situ hybridization determined that ABC2 is expressed at high levels in the white matter, indicating that it is expressed in the oligodendrocytes. ABC2, therefore, is a glycosylated ABC transporter protein, and may play an especially important role in the brain. In addition, the N-terminal 60-amino-acid sequence of the human ABC1, which was missing from previous reports, has been determined.

Temporal and spatial profiles of ABCA2-expressing oligodendrocytes in the developing rat brain

ABCA2 protein belongs to the ABCA subclass of ATP‐binding cassette (ABC) transporters proposed to exert critical functions in transmembrane transport of endogenous lipids. In this study, we found by immunoblot analyses that approximately 260 kDa of ABCA2 protein is expressed predominantly in oligodendrocytes, and that the expression of the protein is upregulated in the brain during maturation, especially between postnatal days 6 and 19. Parallel to the changes in expression of ABCA2, immunohistochemical analyses showed rapid spatial spread of ABCA2‐immunolabeled oligodendrocytes in the brain during this period. These temporal and spatial changes in ABCA2 expression were in good agreement with findings in myeloarchitectonics reported previously. Further, double immunolabeling with ABCA2 and a major structural protein of myelin, myelin basic protein, demonstrated that onset of ABCA2 expression in oligodendrocytes coincides with the appearance of thick myelin segments immunolabeled with myelin basic protein. Because ABCA2 was abundantly expressed in adult cortex in white matter and gray matter, coexpression of ABCA2 and a marker for the oligodendroglial progenitors NG2 or platelet‐derived growth factor α receptor was investigated. No cells coexpressing ABCA2 and the marker were observed, suggesting that ABCA2 is expressed predominantly in myelin‐forming oligodendrocytes distinct from the adult oligodendroglial progenitors tested. These results suggested a role for ABCA2 in membrane transport of substrates such as the lipids that are closely linked to myelination processes. J. Comp. Neurol. 455:353–367, 2003.

Gene Expression for PACAP Receptor mRNA in the Rat Retina by in Situ Hybridization and in Situ RT‐PCR

Expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor (PAC 1 -R) are localized in the rat retina by in situ hybridization and in situ RT-PCR. PAC 1 -R is distributed in the rat brain and eye. 1,2 PAC 1 -R exists in at least eight splice variants that are differentially coupled to signal transduction pathways, 3 but the distribution and localization of these splice variants in the retina remain to be determined. There are at least eight isoforms of the PAC 1 -R, and the signal transduction system is different for each subtype. We studied gene expression of PAC 1 -R mRNA in the rat retina by in situ hybridization. In addition, we tried to identify PAC 1 -R subtypes in the rat retina by in situ RT-PCR.

PACAP activates PKA, PKC and Ca2+ signaling cascades in rat neuroepithelial cells

Several studies have reported that the PAC(1) receptor (PAC1-R), the specific receptor for PACAP, is expressed at early developmental stages. Here, we describe that the cytosolic Ca(2+) concentration ([Ca(2+)](i)) was increased by PACAP, but not VIP, in a concentration range from 10(-12) to 10(-8) M via the PAC(1)-R in isolated single cells from the rat neural fold. This activation of the cells by PACAP was mimicked by agonists and inhibited by antagonists of the cAMP/PKA and PLC/PKC cascades. These data indicate that PACAP/PAC(1)-R is linked to [Ca(2+)](i) signaling via two G-protein-coupled protein kinase pathways and may thereby play an important role in early neurodevelopment.

Molecular cloning and characterization of a novel developmentally regulated gene, Bdm1, showing predominant expression in postnatal rat brain.

Postnatal development, such as synapse refinement, is necessary for the establishment of a mature and functional central nervous system (CNS). Using differential display analysis, we identified a novel gene, termed Bdm1, that is more abundantly expressed in the adult brain than in the embryonic brain. The full-length Bdm1 cDNA is 2718 base pairs long and contains an open reading frame of 1059 base pairs encoding a 38-kDa protein. Northern blot analysis revealed that expression of Bdm1 mRNA in the brain was weak on embryonic days and increased in the early postnatal period. Bdm1 mRNA was significantly expressed in the brain and heart, but there was no or little expression in other tissues. During the differentiation of mouse carcinoma cells P19 to neuron-like cells by retinoic acid, Bdm1 mRNA was up-regulated almost parallel to neurofilament mRNA. Expression of Bdm1 mRNA was observed appreciably in PC12 cells after neuronal differentiation but not in the nonneural cell lines examined. In situ hybridization demonstrated that Bdm1 was expressed widely in the olfactory bulb, cerebral cortex, hippocampus, cerebellum, thalamus, and medulla oblongata. Taken together, these data suggest that Bdm1 gene plays a role in the early postnatal development and function of neuronal cells.

Effect of Prolactin and Androgen on the Expression of the Female-Attracting Pheromone Silefrin in the Abdominal Gland of the Newt, Cynops ensicauda

Abstract Silefrin is a sodefrin-like, female-attracting pheromone comprising 10 amino acids that was isolated from the abdominal gland of the sword-tailed newt, Cynops ensicauda. Hormonal effects on the silefrin precursor mRNA expression and silefrin content in the abdominal gland were investigated in the present study by using Northern blot analysis and radioimmunoassay, respectively. In the abdominal gland of newts treated with prolactin (PRL) plus testosterone propionate (TP), silefrin precursor mRNA expression was markedly enhanced as compared with that in the newts injected with saline, PRL, or TP. Values for radioimmunoassayable silefrin content in the abdominal gland paralleled those for the silefrin precursor mRNA levels. Moreover, silefrin precursor mRNA signals, as revealed by in situ hybridization, as well as stainability of immunoreactive silefrin were much more intense in the epithelial cells of the abdominal gland of the PRL-plus-TP-treated animals than in those of controls. We thus conclude that PRL and androgen are important factors for enhancing silefrin synthesis.

Distribution of PACAP receptor and its splice variants in the rat brain by in situ RT-PCR.

but the distribution and localization of these splice variants in the brain remain to be determined. The PAC 1 -R mRNA is detected intensely in the olfactory bulb, hippocampal formation, cerebellum by in situ hybridization histochemistry. 2,3 Using the initial specific primer pairs that correspond to the hip or hop types of receptors for the solution-phase RT-PCR, we have demonstrated that the major splice variants of PAC 1 -R in various regions of the rat brain and we have applied an innovative molecular histochemical technique, in situ RT-PCR, to detect these major splice variants in brain sections with the same primer pairs that are used in solution phase RT-PCR. Two-month-old male Sprague-Dawley rats were used in this study. To identify the PAC 1 -R mRNA with solution phase RT-PCR, two oligonucleotide primers were synthesized based on the reported sequence that is present in the all splice variants of the rat PAC 1 -R cDNA. 2,3 To discriminate between PAC 1 -Rhop and PAC 1 -Rhip , since they have the same size (84-bp) cassette, two initial forward primers were designed to correspond to the hop or hip cassette sequences, respectively. The total RNAs were extracted from different regions of the brain tissues and purified. They were then reverse transcribed and amplified using the different primer pairs. There are two splice variants of the PAC 1 -R, the short form PAC 1 -R-s, which does not contain either the hip or hop cassette, and another splice isoform PAC 1 -R-hop, which contains the hop cassette, found in the olfactory bulb, cerebellum, hippocampal formation, hypothalamus, and thalamus, as well as in the neocortex. Frozen brain sections were thaw mounted on amino alkylsilane-coated in situ PCR glass slides (Perkin-Elmer). After being fixed with 4 % neutral buffered formaldehyde and permeabilized with 0.25 % Triton X-100 buffer, the sections were blocked in hydrogen peroxide. The genomic DNA of the tissue section was digested with deoxyribonucleas (DNase) I, and the no DNase-treatment section was used as a positive control. A single step in situ RT-PCR was performed with the rTth enzyme