The neuroprotective peptide NAP inhibits the aggregation of the beta-amyloid peptide

Alzheimer's disease (AD) is characterized by brain plaques containing the beta-amyloid peptide (Abeta). One approach for treating AD is by blocking Abeta aggregation. Activity-dependent neuroprotective protein contains a peptide, NAP that protects neurons in culture against Abeta toxicity. Here, NAP was shown to inhibit Abeta aggregation using: (1) fluorimetry; (2) electron microscopy; (3) high-throughput screening of Abeta deposition onto a synthetic template (synthaloid); and (4) Congo Red staining of neurons. Further assays showed biotin-NAP binding to Abeta. These results suggest that part of the neuroprotective mechanism exerted by NAP is through modulation of toxic protein folding in the extracellular milieu.     

Cytokine Regulation of Neuronal Survival

Interleukin-1 is a cytokine involved in the immune response to infection and inflammation as well as a growth promotor for several cell types. Interleukin-1-like immunoreactive material has been found in the nervous system. We now show that antisera, which blocked the T-cell proliferative effects of interleukin-1 alpha, decreased neuronal cell counts (to 40% of control) in dissociated spinal cord cultures derived from fetal mice. This neuronal loss was prevented by addition of interleukin-1 alpha, and to a lesser extent by interleukin-1 beta. Exogenous interleukin-1 alpha increased the survival of neurons when added to cultures in which the electrical activity was blocked with tetrodotoxin, whereas no such cytokine-related increase in neuronal survival was observed in electrically active cultures. The antiserum-induced death could also be prevented by cotreatment of the cultures with 0.1 nM vasoactive intestinal peptide, a substance that induces the secretion of neuronal trophic factors from nonneuronal spinal cord cells and thereby increases neuronal survival in electrically inactive cultures. These studies indicate that the cytokine interleukin-1, or an immunologically cross-reactive protein, can increase neuronal survival.     

Subcellular localization and secretion of activity-dependent neuroprotective protein in astrocytes

Activity-dependent neuroprotective protein (ADNP, approximately 123562.8 Da), is synthesized in astrocytes and expression of ADNP mRNA is regulated by the neuroprotective peptide vasoactive intestinal peptide (VIP). The gene that encodes ADNP is conserved in human, rat and mouse, and contains a homeobox domain profile that includes a nuclear-export signal and a nuclear-localization signal. ADNP is essential for embryonic brain development, and NAP, an eight-amino acid peptide that is derived from ADNP, confers potent neuroprotection. Here, we investigate the subcellular localization of ADNP through cell fractionation, gel electrophoresis, immunoblotting and immunocytochemistry using alpha-CNAP, an antibody directed to the neuroprotective NAP fragment that constitutes part of an N-terminal epitope of ADNP. Recombinant ADNP was used as a competitive ligand to measure antibody specificity. ADNP-like immunoreactivity was found in the nuclear cell fraction of astrocytes and in the cytoplasm. In the cytoplasm, ADNP-like immunoreactivity colocalized with tubulin-like immunoreactivity and with microtubular structures, but not with actin microfilaments. Because microtubules are key components of developing neurons and brain, possible interaction between tubulin and ADNP might indicate a functional correlate to the role of ADNP in the brain. In addition, ADNP-like immunoreactivity in the extracellular milieu of astrocytes increased by approximately 1.4 fold after incubation of the astrocytes with VIP. VIP is known to cause astrocytes to secrete neuroprotective/neurotrophic factors, and we suggest that ADNP constitutes part of this VIP-stimulated protective milieu.     

NAP protects hippocampal neurons against multiple toxins

The femtomolar-acting protective peptide NAP (NAPVSIPQ), derived from activity-dependent neuroprotective protein (ADNP), is broadly neuroprotective in vivo and in vitro in cerebral cortical cultures and a variety of cell lines. In the present study, we have extended previous results and examined the protective potential of NAP in primary rat hippocampal cultures, using microtubule-associated protein 2 (MAP2) as a measure for neuroprotection. Results showed that NAP, at femtomolar concentrations, completely protected against oxygen-glucose deprivation, and cyanide poisoning. Furthermore, NAP partially protected against kainic acid excitotoxicity. In summary, we have significantly expanded previous findings in demonstrating here direct neuroprotective effects for NAP on vital hippocampal neurons that are key participants in cognitive function in vivo.     

Novel extended and branched N-terminal analogs of VIP

The effects of vasoactive intestinal peptide (VIP) are primarily mediated through VPAC1 and VPAC2, receptors that are preferentially coupled to adenylate cyclase activation. As a large majority of the potent VIP antagonists have modifications in the N-terminal domain of the peptide, the effect of multiplication of this domain on VIP was examined with the aim of possibly amplifying peptide-receptor (VPAC1) activation. Several VIP analogs were designed and synthesized, each carrying multiplication of the N-terminal domain that was obtained by either linear tandem extension or by parallel branching. Circular dichorism (CD) analysis revealed that these extended/branched peptides maintained an alpha helical structure in organic environment, similar to VIP. A specific branched VIP analog was found to be slightly more potent towards VPAC1-related cAMP production as compared to VIP. This analog could have potential therapeutic value in several disorders, similar to VIP. Two branched N-terminal VIP sequences demonstrated superior receptor binding and activation as compared to two N-terminals in tandem. The results suggest that correct alignment of the VIP N-terminal region is important for receptor binding and activation. However, increased receptor binding was not directly associated with increased cAMP production suggesting steric dynamic interactions.     

Protection against developmental retardation in apolipoprotein E-deficient mice by a fatty neuropeptide: Implications for early treatment of Alzheimer's disease

Stearyl-Nle-¹⁷-VIP (SNV) is a novel agonist of vasoactive intestinal peptide (VIP) exhibiting a 100-fold greater potency than the parent molecule and specificity for a receptor associated with neuronal survival. Here, mice deficient in apolipoprotein E (ApoE), a molecule associated with the etiology of Alzheimer's disease, served as a model to investigate the developmental and protective effects of SNV. In comparison to control animals, the deficient mice exhibited (a) reduced amounts of VIP messenger RNA; (b) decreased cholinergic activity (c) significant retardation in the acquisition of developmental milestones: forelimb placing behavior and cliff avoidance behavior; and (d) learning and memory impairments. Daily injections of SNV to ApoE-deficient newborn pups resulted in increased cholinergic activity and marked improvements in the time of acquisition of behavioral milestones, with peptide-treated animals developing as fast as control animals and exhibiting improved cognitive functions after cessation of peptide treatment. Specificity was demonstrated in that treatment with a related peptide (PACAP), pituitary adenylate cyclase-activating peptide, produced only limited amelioration. As certain genotypes of ApoE increase the probability of Alzheimer's disease, early counseling and preventive treatments may now offer an important route for therapeutics design. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 329–342, 1997     

Interés medicinal de Poria cocos (= Wolfiporia extensa)

The sclerotium of Poria cocos has been used in the Chinese pharmacopoeia during thousands of years. In this note several aspects about the biology and nomenclature of this fungus are summarized, with emphasis on its composition and therapeutic applications.     

The increased proliferation of cultured neuroblastoma cells treated with vasoactive intestinal peptide is enhanced by simultaneous inhibition of neutral endopeptidase

Vasoactive intestinal peptide (VIP) stimulates the neuroblastoma cell line (NMB) to proliferate. Neuropeptide activity can be inhibited by neutral endopeptidases that function intracellularly and in the extracellular milieu. NMB cells express neutral endopeptidase (NEP) activity that can be specifically inhibited by phosphoramidon (PA). Our data now show that phosphoramidon treatment increases the efficacy of VIP-stimulated neuroblastoma proliferation. These results suggest that membrane endopeptidases modulate VIP-associated cell proliferation and enhancement of endopeptidase activity may serve as a target for cancer therapy.     

In vitro and in vivo treatment of colon cancer by VIP antagonists

Vasoactive intestinal peptide (VIP) is secreted from many cancer lines and VIP binding was observed in many tumors. We have shown before that VIP antagonists are potent inhibitors of neoplastic growth of neuroblastoma, lung and breast cancer cells in vitro. Here, the cultured colon cancer cell line HCT-15 that exhibited VIP receptor expression was treated with the VIP hybrid antagonist neurotensin(6-11)VIP(7-28). The antineoplastic activity was assessed by thymidine incorporation. Neurotensin(6-11)VIP(7-28) efficiently inhibited cancer growth with a maximal effect at nanomolar concentrations. Once the inhibitory properties of the VIP antagonist on colon cancer cells were established, the in vivo curative effects were analyzed. Sprague-Dawley rats were injected with azoxymethane (AOM) (15 mg/kg/week) for 2 weeks, providing artificial induction of colon tumors. The rats were then allocated into four experimental groups: (1) receiving no treatment; (2) receiving treatment with saline; (3, 4) receiving treatment with 10 or 20 microg of neurotensin(6-11)VIP(7-28), respectively. After 10 weeks of daily injections, rats were sacrificed and tumors assessed for stage, volume, location, differentiation and lymphocytic infiltrate. Embedded mucosa was assessed for dysplastic crypts. Results showed that the antagonist treatment reduced the tumor volume, staging, lymphocyte infiltrate and the number of dysplastic crypts. Thus, neurotensin(6-11)VIP(7-28) could serve as an effective cancer treatment and a preventing agent.