Peptide neuroprotection through specific interaction with brain tubulin.

This study aimed to identify the neuronal target for the potent neuroprotective peptide NAP. When added to pheochromocytoma cells (neuronal model), NAP was found in the intracellular milieu and was co-localized with microtubules. NAP induced neurite outgrowth and protected primary neurons against microtubule-associated ZnCl2 toxicity. Rapid microtubule reorganization into distinct microtubules ensued after NAP addition to both pheochromocytoma cells and primary cerebral cortical neurons, but not to fibrobalsts. While binding neuronal tubulin and protecting pheochromocytoma cells against oxidative stress, NAP did not bind tubulin extracted from fibroblasts, nor did it protect those cells against oxidative stress. Affinity chromatography identified the brain-specific betaIII-tubulin as a major NAP binding protein. Paclitaxel (a microtubule aggregating agent that interacts with beta-tubulin) reduced NAP tubulin binding. Thus, the underlying mechanism for the neuroprotection offered by NAP is targeting neuronal microtubules that are essential for neuronal survival and function.

PolyADP-ribosylation is involved in neurotrophic activity.

PolyADP-ribosylation is a transient posttranslational modification of proteins, mainly catalyzed by poly(ADP-ribose)polymerase-1 (PARP-1). This highly conserved nuclear protein is activated rapidly in response to DNA nick formation and promotes a fast DNA repair. Here, we examine a possible association between polyADP-ribosylation and the activity of neurotrophins and neuroprotective peptides taking part in life-or-death decisions in mammalian neurons. The presented results indicate an alternative mode of PARP-1 activation in the absence of DNA damage by neurotrophin-induced signaling mechanisms. PARP-1 was activated in rat cerebral cortical neurons briefly exposed to NGF-related nerve growth factors and to the neuroprotective peptides NAP (the peptide NAPVSIPQ, derived from the activity-dependent neuroprotective protein ADNP) and ADNF-9 (the peptide SALLRSIPA, derived from the activity-dependent neurotrophic factor ADNF) In addition, polyADP-ribosylation was involved in the neurotrophic activity of NGF-induced and NAP-induced neurite outgrowth in differentiating pheochromocytoma 12 cells as well as in the neuroprotective activity of NAP in neurons treated with the Alzheimer's disease neurotoxin beta-amyloid. A fast loosening of the highly condensed chromatin structure by polyADP-ribosylation of histone H1, which renders DNA accessible to transcription and repair, may underlie the role of polyADP-ribosylation in neurotrophic activity.

The peptides ADNF-9 and NAP increase survival and neurite outgrowth of rat retinal ganglion cells in vitro.

PURPOSE: Recent studies demonstrated that short peptides derived from activity-dependent neurotrophic factor (ADNF) and activity-dependent neuroprotective protein (ADNP) are neuroprotective at femtomolar concentrations. We evaluated these findings in cultures of purified rat retinal ganglion cells (RGCs) using two such peptides: ADNF-9 and NAP. In a second step, the influence of these peptides on neurite outgrowth in retinal explants was investigated. METHODS: Retinal ganglion cells (RGCs) were purified from newborn (postnatal day [P]0-P2) rat retina by immunopanning with antibodies against Thy1.1 and were cultured in serum-free N2 medium for 2 days. RGCs were treated with ADNF-9 and NAP at concentrations ranging from 10(-18) to 10(-10) M. Survival was quantified by counting viable cells by phase-contrast microscopy. Retinal explants from postnatal (P9-P11) rats were cultured in three-dimensional fibrin clots in serum-free medium for 3 days. Explants were treated with 1 microM NAP or 1 microM ADNF-9. Neurite outgrowth was visualized by staining with Sudan black and quantified by measuring axonal length. RESULTS: Both peptides enhanced survival of RGCs in a dose-dependent manner. ADNF-9 showed a maximum effect at 0.1 pM with an increase in survival to 177% (95% confidence interval: 149-204) of the control level. The EC(50) was 10.9 fM. NAP showed a maximum effect at 5 pM with an increase in survival to 167% (146-189) and an EC(50) of 6.1 fM. In the explants, 1 microM ADNF-9 enhanced axonal outgrowth to 126% (118-133) and 1 microM NAP to 117% (98-137) compared with the control. CONCLUSIONS: Both peptides, ADNF-9 and NAP, not only increase RGC survival in vitro but also support neurite outgrowth in retinal explants. These peptides deserve further attention as potential neuroprotective compounds in retinal and optic nerve diseases.

NAP mechanisms of neuroprotection.

An 8-amino-acid peptide, NAPVSIPQ (NAP), was identified as the smallest active element of activity-dependent neuroprotective protein that exhibits potent neuroprotective action. Potential signal transduction pathways include cGMP production and interference with inflammatory mechanisms, tumor necrosis factor-alpha, and MAC1-related changes. Because of its intrinsic structure, NAP might interact with extracellular proteins and also transverse membranes. NAP-associated protection against oxidative stress, glucose deprivation, and apoptotic mechanisms suggests interference with fundamental processes. This paper identifies p53, a key regulator of cellular apoptosis, as an intracellular target for NAP's activity.

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.

Injections of the neuroprotective peptide NAP to newborn mice attenuate head-injury-related dysfunction in adults.

The prophylactic neuroprotective effects of NAP, a femtomolar-acting neuroprotective peptide were tested in a mouse model of head trauma. NAP was injected for the first 3 weeks of life and head injury was initiated at 4 months. After trauma, mice were tested for their performance by evaluating damaged motor ability, balance and alertness. Comparison of the performance 1 h and 1 week after injury indicated that NAP treatment resulted in faster and enhanced recovery. In a 5-day Morris water maze test with mice suffering moderate to severe injuries, only the NAP-treated group learned to find the hidden platform in the maze. Furthermore, NAP treatment resulted in decreased mRNA expression of the inflammation marker, Mac-1. Thus, a potentially new prophylactic treatment against neurodegeneration is suggested.

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.

A vasoactive intestinal peptide receptor analog alters the expression of homeobox genes.

A lipophilic analog of vasoactive intestinal peptide (VIP), stearyl-Nle(17)-neurotensin(6-11)VIP(7-28) (SNH), that inhibited lung cancer growth, has been previously described. The mechanism of SNH inhibition of cancer growth is still being elucidated. The present study examined the effects of SNH on homeobox genes in the colon cancer cell line HT 29 that expresses VIP receptors. Homeobox genes contain a characteristic DNA sequence, coding for a stretch of 61 amino acid homeodomain that binds specific DNA motifs. While the HOX gene family contains a single homeodomain, the POU gene family contains an additional DNA binding homeodomain. HT 29 cells were incubated with SNH; RNA was extracted and subjected to reverse-transcription-polymerase chain reaction (RT-PCR) with primers that matched the conserved area of the various HOX or POU genes. The PCR products that were altered by SNH treatment were sequenced. Three candidate SNH-responsive genes, the HOX A4, the HOX B5 and the PUO V transcription factor I (Oct-3) were identified. Semi-quantitative RT-PCR with specific primers confirmed the increase in HOX A4 and the decrease in Oct-3 expression levels following SNH treatment. Thus, the HOX A4 and the Oct-3 homeobox genes may partially mediate SNH activity on cancer cells.