Reduced Cholinergic Activity in the Hippocampus of Hippocampal Cholinergic Neurostimulating Peptide Precursor Protein Knockout Mice.

The cholinergic efferent network from the medial septal nucleus to the hippocampus has an important role in learning and memory processes. This cholinergic projection can generate theta oscillations in the hippocampus to efficiently encode novel information. Hippocampal cholinergic neurostimulating peptide (HCNP) induces acetylcholine synthesis in medial septal nuclei. HCNP is processed from the N-terminal region of a 186 amino acid, 21 kD HCNP precursor protein called HCNP-pp (also known as Raf kinase inhibitory protein (RKIP) and phosphatidylethanolamine-binding protein 1 (PEBP1)). In this study, we generated HCNP-pp knockout (KO) mice and assessed their cholinergic septo-hippocampal projection, local field potentials in CA1, and behavioral phenotypes. No significant behavioral phenotype was observed in HCNP-pp KO mice. However, theta power in the CA1 of HCNP-pp KO mice was significantly reduced because of fewer cholineacetyltransferase-positive axons in the CA1 stratum oriens. These observations indicated disruption of cholinergic activity in the septo-hippocampal network. Our study demonstrates that HCNP may be a cholinergic regulator in the septo-hippocampal network.

Hippocampal Cholinergic Neurostimulating Peptide as a Possible Modulating Factor against Glutamatergic Neuronal Disability by Amyloid Oligomers.

Despite having pathological changes in the brain associated with Alzheimer's disease (AD), some patients have preserved cognitive function. A recent epidemiological study has shown that diet, exercise, cognitive training, and vascular risk monitoring interventions may reduce cognitive decline in at-risk elderly people in the general population. However, the details of molecular mechanisms underlying this cognitive function preservation are still unknown. Previous reports have demonstrated that enriched environments prevent the impairment of hippocampal long-term potentiation (LTP) through ß2-adrenergic signals, when LTP is incompletely suppressed by synthetic amyloid-ß (Aß) oligomers. The cholinergic network from the medial septal nucleus (MSN) is also a main modulating system for hippocampal glutamatergic neural activation through nicotinergic and/or muscarinergic acetylcholine receptors. Previously, we reported the importance of a cholinergic regulator gene in the MSN, hippocampal cholinergic neurostimulating peptide (HCNP). By using hippocampal sections from mice, we here demonstrated that the cholinergic neural activation from the MSN enhanced the glutamatergic neuronal activity during unsaturated LTP but not during saturated LTP. Synthetic Aß oligomers suppressed the hippocampal glutamatergic activity in a concentration-dependent manner. Furthermore, HCNP, as well as a cholinergic agonist acting through the muscarinic M1 receptor, prevented the suppression of hippocampal glutamatergic neuronal activity induced by synthetic Aß oligomers. This result suggests that the persisting cholinergic activation might be a potential explanation for the individual differences in cognitive effects of AD pathological changes.

Phosphorylation of collapsin response mediator protein-2 regulates its localization and association with hippocampal cholinergic neurostimulating peptide precursor in the hippocampus.

Hippocampal cholinergic neurostimulating peptide (HCNP) induces the synthesis of acetylcholine in the medial septal nucleus in vitro and in vivo. The precursor, HCNP-pp, is a multifunctional protein participating in important signaling pathways, such as MAPK/ERK kinase (MEK) and G-protein-coupled receptor kinase 2 (GRK2). We recently demonstrated that HCNP-pp colocalizes with collapsin response mediator protein-2 (CRMP-2) at presynaptic terminals in the hippocampus, suggesting that HCNP-pp may play an important role in presynaptic function in association with CRMP-2. To clarify the involvement of phosphorylation in regulating the interaction between HCNP-pp and CRMP-2, we investigated the colocalization of HCNP-pp with unphosphorylated- and/or phosphorylated-CRMP-2 (pCRMP-2) at presynaptic terminals. We further determined if the phosphorylation of CRMP-2 affects the binding between those proteins. Here, we demonstrate that HCNP-pp predominantly colocalizes and associates with unphosphorylated and/or pSer-522-CRMP-2 at presynaptic terminals in the hippocampus. Interestingly, HCNP-pp does not associate with pThr-509/514-CRMP-2, which is primarily localized at postsynaptic terminals. These findings suggest that HCNP-pp, in association with unphosphorylated and/or pSer522-CRMP-2, plays an important role in presynaptic function in the mature hippocampus.

Co-localization of hippocampal cholinergic neurostimulating peptide precursor with collapsin response mediator protein-2 at presynaptic terminals in hippocampus.

Hippocampal cholinergic neurostimulating peptide (HCNP) induces the synthesis of acetylcholine in medial septal nucleus in vitro and in vivo. HCNP precursor protein (HCNP-pp) is a multifunctional protein that participates in a number of signaling pathways, including MAPK/extracellular signal and G-protein-coupled receptor kinase 2. We recently demonstrated that the amount of collapsin response mediator protein-2 (CRMP-2) is increased in hippocampus of HCNP-pp transgenic mice. To clarify the interaction between HCNP/HCNP-pp and CRMP-2 and its role in synaptic function, we investigated whether HCNP-pp is localized to the synapse and if it affects protein expression. Here, we demonstrate that HCNP-pp co-localizes with CRMP-2 at presynaptic terminals. Furthermore, HCNP-pp overexpression increases synaptophysin levels. These findings suggest that HCNP-pp, in association with CRMP-2, plays an important role in presynaptic function in the hippocampus.

Suppression of astrocyte lineage in adult hippocampal progenitor cells expressing hippocampal cholinergic neurostimulating Peptide precursor in an in vivo ischemic model.

Hippocampal cholinergic neurostimulating peptide (HCNP) is known to promote differentiation of septohippocampal cholinergic neurons. The HCNP precursor protein (HCNP-pp) may play several roles, for example, as an ATP-binding protein, a Raf kinase inhibitor protein, and a phosphatidylethanolamine-binding protein, as well as a precursor for HCNP. This study therefore aimed to elucidate the involvement of HCNP-pp in specific neural lineages after stroke using a hypoxic-ischemic (HI) rat model of brain ischemia. The specific neural lineages in the hippocampus were investigated 14 days after ischemia. Some bromodeoxyuridine (BrdU)(+) neural progenitor cells in the hippocampus of hypoxic, HI, or sham-operated rats expressed HCNP-pp. Almost half of the BrdU(+)/HCNP-pp(+) cells also expressed the oligodendrocyte lineage marker 2',3'-cyclic nucleotide 3'-phosphodiesterase, whereas only a few BrdU(+)/HCNP-pp(+) cells in the hippocampus in HI brains expressed the neuronal lineage marker, doublecortin (DCX). Interestingly, no BrdU(+)/HCNP-pp(+) progenitor cells in hypoxic, HI, or sham-operated brains expressed the astrocyte lineage marker, glial fibrillary acidic protein. Together with previous in vitro data, the results of this study suggest that the expression level of HCNP-pp regulates the differentiation of neural progenitor cells into specific neural lineages in the HI hippocampus, indicating that neural stem cell fate can be controlled via the HCNP-pp mediating pathway.

Suppressed phosphorylation of collapsin response mediator protein-2 in the hippocampus of HCNP precursor transgenic mice.

We previously reported a novel peptide, Hippocampal Cholinergic Neurostimulating Peptide (HCNP), which induces acetylcholine synthesis by increasing the amount of choline acetyltransferase (ChAT) in medial septal nuclei. The HCNP precursor protein (HCNP-pp), composed of 186 amino acids, is an inhibitory factor of the c-Raf/MEK cascade and may be involved in fetal rat brain development via the inhibition of phosphorylation of Erk. To clarify the involvement of HCNP in hippocampal cholinergic circuitry, we previously generated HCNP-pp transgenic (HCNP-pp Tg) mice using the promoter of the α subunit of Ca(2+) calmodulin-dependent protein kinase II (CaMKIIα). These mice showed increased levels of ChAT in medial septal nuclei at 12 weeks of age, and the phenotype of depressive mood at 30 weeks of age. Here, through proteomic analysis we investigated the alteration of protein expression in the hippocampus of HCNP-pp Tg mice compared with wild-type littermate mice. We demonstrate that the activation of collapsin response mediator protein-2 (CRMP-2) is increased in the transgenic mice at 12 weeks of age when compared with wild-type littermate mice.

Directed neural lineage differentiation of adult hippocampal progenitor cells via modulation of hippocampal cholinergic neurostimulating peptide precursor expression.

Hippocampal cholinergic neurostimulating peptide (HCNP), originally purified from young rat hippocampus, has been known to promote the differentiation of septo-hippocampal cholinergic neurons. Recently, the precursor protein of HCNP (HCNP-pp) has also received attention as a multifunctional protein with roles, in addition to serving as the HCNP precursor, such as acting as an ATP-binding protein, a Raf kinase inhibitor protein (RKIP), and phosphatidylethanolamine-binding protein (PEBP). In particular, the function of RKIP has attracted attention over several years for its role in controlling cellular proliferation and metastasis in cancer cells. HCNP-pp is also thought to be important in regulating the proliferation and differentiation of neuronal cells in vitro and in vivo by modification of the MAPK cascade. In the present study, we used cultured adult rat hippocampal progenitor cells (AHPs), which are thought to be important for memory formation, and focused on the role of HCNP-pp in adult neurogenesis, namely, the production of new neurons from neural stem/progenitor cells. We found that HCNP-pp expression in AHPs was closely associated with differentiation into MAP2ab-positive neurons and RIP-positive oligodendrocytes, but not into GFAP-positive astrocytes. By contrast, a down-regulated HCNP-pp expression in AHPs accompanied differentiation into GFAP-positive astrocytes. Direct manipulations of HCNP-pp via viral over-expression or siRNA downregulation further confirmed the HCNP-pp contribution to specific neural lineage commitment of AHPs. Our results show that the expression level of HCNP-pp acts as a key regulator for differentiation of cultured AHPs into specific neural lineages, indicating that the control of neural stem cell fate can be achieved via the HCNP-pp pathway.

Overexpression of hippocampal cholinergic neurostimulating peptide in heterozygous transgenic mice increases the amount of ChAT in the medial septal nucleus.

Acetylcholine modulates neural activity in the hippocampal glutamatergic pathway via the induction of phosphorylated Erk and may act as a novel transmitter in septohippocampal memory formation. However, how acetylcholine synthesis in the septal nucleus is regulated is unknown. We have purified a peptide from the hippocampus of the young adult rat, named hippocampal cholinergic neurostimulating peptide (HCNP) that induces acetylcholine synthesis in vitro in the septal nucleus. Previously, levels of this peptide and/or precursor protein were reported to be decreased, and the protein to be nitrated in the brains of patients with Alzheimer's disease. Here, to clarify the involvement in the regulation of acetylcholine synthesis in vivo in the medial septal nucleus, we generated HCNP precursor transgenic mice, using a Ca2+ calmodulin-dependent protein kinase II genomic promoter. The amount of cholineacetyltransferase (ChAT) in the medial septal nucleus was increased in heterozygous HCNP transgenic mice, compared with non-transgenic littermates. This result suggests that HCNP is involved in regulating acetylcholine synthesis in vivo in the medial septal nucleus and, as such, is important for memory function.

Novel carbohydrate-binding activity of bovine liver beta-glucuronidase toward lactose/N-acetyllactosamine sequences.

Beta-glucuronidase is a lysosomal enzyme that plays an essential role in normal turnover of glycosaminoglycans and remodeling of the extracellular matrix components in both physiological and inflammatory states. The regulation mechanisms of enzyme activity and protein targeting of beta-glucuronidase have implications for the development of a variety of therapeutics. In this study, the effectiveness of various carbohydrate-immobilized adsorbents for the isolation of bovine liver beta-glucuronidase (BLG) from other glycosidases was tested. Beta-glucuronidase and contaminating glycosidases in commercial BLG preparations bound to and were coeluted from adsorbents immobilized with the substrate or an inhibitor of beta-glucuronidase, whereas beta-glucuronidase was found to bind exclusively with lactamyl-Sepharose among the adsorbents tested and to be effectively separated from other enzymes. Binding and elution studies demonstrated that the interaction of beta-glucuronidase with lactamyl-Sepharose is pH dependent and carbohydrate specific. BLG was purified to homogeneity by lactamyl affinity chromatography and subsequent anion-exchange high-performance liquid chromatography (HPLC). Lactose was found to activate beta-glucuronidase noncompetitively, indicating that the lactose-binding site is different from the substrate-binding site. Binding studies with biotinyl glycoproteins, lipids, and synthetic sugar probes revealed that beta-glucuronidase binds to N-acetyllactosamine/lactose-containing glycoconjugates at neutral pH. The results indicated the presence of N-acetyllactosamine/lactose-binding activity in BLG and provided an effective purification method utilizing the novel carbohydrate binding activity. The biological significance of the carbohydrate-specific interaction of beta-glucuronidase, which is different from the substrate recognition, is discussed.