Activity-dependent Neurotrophic Factor Research Articles

A Novel Microtubule-Tau Association Enhancer and Neuroprotective Drug Candidate: Ac-SKIP.

Activity-dependent neuroprotective protein (ADNP) has been initially discovered through its eight amino acid sequence NAPVSIPQ, which shares SIP motif with SALLRSIPA – a peptide derived from activity-dependent neurotrophic factor (ADNF). Mechanistically, both NAPVSIPQ and SALLRSIPA contain a SIP motif that is identified as a variation of SxIP domain, providing direct interaction with microtubule end-binding proteins (EBs). The peptide SKIP was shown before to provide neuroprotection in vitro and protect against Adnp-related axonal transport deficits in vivo. Here we show, for the first time that SKIP enhanced microtubule dynamics, and prevented Tau-microtubule dissociation and microtubule disassembly induced by the Alzheimer’s related zinc intoxication. Furthermore, we introduced, CH3CO-SKIP-NH2 (Ac-SKIP), providing efficacious neuroprotection. Since microtubule – Tau organization and dynamics is central in axonal microtubule cytoskeleton and transport, tightly related to aging processes and Alzheimer’s disease, our current study provides a compelling molecular explanation to the in vivo activity of SKIP, placing SKIP motif as a central focus for MT-based neuroprotection in tauopathies with axonal transport implications.

Effect of experimentally induced hypothyroidism during gestation period on activity dependent neurotrophic factor (ADNF) in newborn rat brain tissue.

Purpose The aim of the study was to evaluate the effects of prenatal hypothyroidism on neonatal rats by the way of activity-dependent neuroprotective factor (ADNF) expression. Methods Twenty-one Wistar albino neonatal rats were divided into two subgroups; a control group and neonatal rats with experimental maternal hypothyroidism. Hypothyroidism was induced by using propylthiouracil (PTU). Neonatal rats obtained PTU from breast milk continuously for 1 week after birth. The rats from the control group were fed only normal feed and water. After birth, body weight and blood thyroid hormone levels were tested. Glial fibrillary acidic protein (GFAP), Slug, Numb, Notch-1 and ADNF antibodies were used for immunohistochemical analysis. Real-time polymerase chain reaction (RT-PCR) and Western blotting analyses were used to evaluate ADNF gene expression levels from 1-week-old rat's brain. Results There was no difference between the two groups for birth weights. The thyroxine (T4) level from the experimental group was <0.4 ng/mL, and it was 0.8 ng/mL for the control group. It was shown that, the results from the experimental group samples had significantly lower ADNF mRNA levels than control group (p < 0.05). The increase from GFAP and Numb expression and decrease from Slug expression were shown in the experimental group. Local differences were identified for ADNF and a decrease was shown in both sides of brain. There was no difference for Notch-1 expression for both groups. Conclusion In this study, decreasing ADNF expression might contribute to developing neurological problems in congenital hypothyroidism.

Peptides NAP and SAL attenuate human tau granular-shaped oligomers in vitro and in SH-SY5Y cells

Accumulation of human tau protein in the central nervous system is an outstanding feature of Alzheimer's Diseases and other tauopathies. Among the aggregate species, granular-shaped oligomers are recently reported as toxic species and associated with neuronal loss. Many interests have been focused on the reducing of these neurotoxic oligomers in tauopathies. It has been approved that peptides NAP (NAPVSIPQ) and SAL (SALLRSIPA), snipped from activity-dependent neurotrophic protein (ADNP) and activity-dependent neurotrophic factor (ADNF), protect brain against a wide range of toxins in neurodegenerative models. The neuroprotection mechanisms of NAP and SAL are under investigation. We report here that the aggregation pathway of human tau protein, in the presence of NAP or SAL, could be affected and moved toward the less granular-shaped species. Fluorophore binding assays, kinetic parameters and circular dichroism records, showed the presence of unusual aggregated species upon treatment with NAP or SAL, as well as hydrophobicity studies. Also, sizing and morphological investigation of aggregates, using dynamic scattered light and Atomic Force Microscopy, elucidated a significant reduction of granular-shaped oligomers under 50nm. Furthermore, immunospecific detection of oligomers in SH-SY5Y cell line, using flow cytometric assessment supported our results. We conclude that one of the possible mechanisms of neuronal protection by NAP or SAL could be the attenuation of granular-shaped oligomeric tau under stress condition.

D-SAL and NAP: Two Peptides Sharing a SIP Domain.

NAPVSIPQ (NAP) and all D-amino acid SALLRSIPA (D-SAL) are neuroprotective peptides derived from activity-dependent neuroprotective protein (ADNP) and activity-dependent neurotrophic factor (ADNF), respectively. Both proteins were shown to protect against cognitive impairment, using different animal models and to increase neuronal survival following exposure to neurotoxins. NAP was extensively tested and found to increase microtubule stability, protect axonal transport, and inhibit apoptosis. Here, we aimed to further evaluate and correlate effects at the behavioral level, in a rat model of diabetes. Diabetes is primarily a metabolic disorder which presents secondary neurological manifestations. Diabetes induces peripheral nervous system damage which is translated into impaired sensory perception and is termed diabetic neuropathy. Diabetes-related central nervous system damage causes cognitive decline. The behavioral study aimed to evaluate the effect of NAP and D-SAL on peripheral neuropathy and cognitive decline. Peripheral neuropathy was tested by measuring the response to a thermal stimulus, and cognitive ability was measured by a social memory test and a spatial memory test using long- and short-term dependent tasks and a reference memory task. Results indicated an immediate sensory neuropathy in the diabetic model, which was prevented by both peptides and a later neuropathic phase, prevented only by NAP treatment. Cognitive tests revealed impaired performance in both social and spatial memory tests in the diabetes model. Each of the peptides improved different aspects of cognitive behavior, with NAP being more potent than D-SAL. Mechanistically, both NAP and SAL contain a SIP (SxIP) domain that has been shown to interact with microtubule end-binding proteins (EBs). Specifically, we have previously shown a direct interaction of NAP with EB1 and EB3; we have further shown an interaction of the NAP-derived 4 amino acid SKIP peptide with EB3, stimulating axonal transport. Interestingly, the all D-amino acid peptide, D-SKIP, only partially mimicked SKIP activity. Our current results implicate D-SAL activity with potentially reduced potency compared to NAP, partially mimicking the SKIP/D-SKIP results and placing the SIP (SxIP) motif as a central focus for microtubule-based neuroprotection.

Neuritin reverses deficits in murine novel object associative recognition memory caused by exposure to extremely low-frequency (50 Hz) electromagnetic fields.

Animal studies have shown that electromagnetic field exposure may interfere with the activity of brain cells, thereby generating behavioral and cognitive disturbances. However, the underlying mechanisms and possible preventions are still unknown. In this study, we used a mouse model to examine the effects of exposure to extremely low-frequency (50 Hz) electromagnetic fields (ELF MFs) on a recognition memory task and morphological changes of hippocampal neurons. The data showed that ELF MFs exposure (1 mT, 12 h/day) induced a time-dependent deficit in novel object associative recognition memory and also decreased hippocampal dendritic spine density. This effect was observed without corresponding changes in spontaneous locomotor activity and was transient, which has only been seen after exposing mice to ELF MFs for 7-10 days. The over-expression of hippocampal neuritin, an activity-dependent neurotrophic factor, using an adeno-associated virus (AAV) vector significantly increased the neuritin level and dendritic spine density. This increase was paralleled with ELF MFs exposure-induced deficits in recognition memory and reductions of dendritic spine density. Collectively, our study provides evidence for the association between ELF MFs exposure, impairment of recognition memory, and resulting changes in hippocampal dendritic spine density. Neuritin prevented this ELF MFs-exposure-induced effect by increasing the hippocampal spine density.

Preventive, personalized medicine at the level of key regulatory proteins: updates on activity-dependent neuroprotective protein (ADNP) as a case study

Natural ageing, neurodegenerative diseases, acute head (brain) injury and stroke as well as mental disorders are associated with nerve cell damage and death. We have discovered activity-dependent neurotrophic factor (ADNP) that is essential for brain formation, regulating >400 important genes, including the major risk gene for Alzheimer's disease, apolipoprotein E. ADNP constitutes a part of complex essential for proper gene activity [1]. In men, the ADNP gene is mutated in autism, deregulated in terms of brain expression in schizophrenia [2] and significantly reduced in the blood of patients with neurodegenerative disease such as Alzheimer's disease [3] and multiple sclerosis. New results indicate the potential of ADNP monitoring as indicator for disease progression, paving the path to preventive treatment with ADNP replacement therapies. In this respect, the ADNP peptide derivative NAP (davunetide) has shown neuroprotection in multiple animal models of neurodegeneration (protecting against axonal transport deficits) [4] as well as in a recent schizophrenia clinical trial [5]. It is thus of major interest to further investigate ADNP as a predictive marker and ADNP replacement as a personalized, preventive, protecting therapy against neurodegeneration.

Neurotrophic Peptides, ADNF-9 and NAP, Prevent Alcohol-Induced Apoptosis at Midgestation in Fetal Brains of C57BL/6 Mouse

Prenatal alcohol exposure is known to induce fetal brain growth deficits at different embryonic stages. We focused this study on investigating the neuroprotective effects against alcohol-induced apoptosis at midgestation using activity-dependent neurotrophic factor (ADNF)-9, a peptide (SALLRSIPA) derived from activity-dependent neurotrophic factor, and NAP, a peptide (NAPVSIPQ) derived from activity-dependent neuroprotective protein. We used an established fetal alcohol exposure mouse model. On embryonic day 7 (E7), weight-matched pregnant females were assigned to the following groups: (1) ethanol liquid diet (ALC) group with 25 % (4.49 %, v/v) ethanol-derived calories, (2) pair-fed (PF) control group, (3) ALC combined with i.p. injections (1.5 mg/kg) of ADNF-9 (ALC/ADNF-9) group, (4) ALC combined with i.p. injections (1.5 mg/kg) of NAP (ALC/NAP) group, (5) PF liquid diet combined with i.p. injections of ADNF-9 (PF/ADNF-9) group, and (6) PF liquid diet combined with i.p. injections of NAP (PF/NAP) group. On day 15 (E15), fetal brains were collected, weighed, and assayed for TdT-mediated dUTP nick end labeling (TUNEL) staining. ADNF-9 or NAP was administered daily from E7 to E15 alongside PF or ALC liquid diet exposure. Our results show that NAP and ADNF-9 significantly prevented alcohol-induced weight reduction of fetal brains. Apoptosis was determined by TUNEL staining; NAP or ADNF-9 administration alongside alcohol exposure significantly prevented alcohol-induced increase in TUNEL-positive cells in primordium of the cingulate cortex and ganglionic eminence. These findings may pave the path toward potential therapeutics against alcohol intoxication during pregnancy stages.

Cholesteryl‐Functionalized ADNF‐9 Peptide: Enhanced Membrane Transport Through Mouse Neuroblastoma Neuro‐2a Cells

A cholesteryl-functionalized derivative of activity dependent neurotrophic factor-9 peptide (a nine amino acid core peptide of activity-dependent neurotrophic factor, acting against Alzheimer's disease) was synthesized aiming at the improvement of its bioavailability. Therefore, its uptake was comparatively investigated with that of its parent peptide by employing mouse neuroblastoma Neuro-2a cells. Owing to the hydrophobic character of this cholesteryl-functionalized peptide, it exhibited enhanced permeability and intracellular uptake while it also retained its low cytotoxicity at concentrations up to 1 μM. FACS analysis also revealed that when Neuro-2a cells were treated with this activity dependent neurotrophic factor-9 derivative, at a concentration of 50 nM, an almost 100% uptake was obtained. In addition, in vitro biological activity experiments showed that the functionalized peptide retained its neurotrophic activity at femtomolar concentration range.

Activity-dependent neurotrophic factor-derived peptide prevents alcohol-induced apoptosis, in part, through Bcl2 and c-Jun N-terminal kinase signaling pathways in fetal brain of C57BL/6 mouse

Fetal alcohol exposure is known to induce alteration in fetal brain development. In this study, we focused on neuroprotection against the effects of alcohol exposure using ADNF-9, a peptide derived from activity-dependent neurotrophic factor. We used a mouse model of fetal alcohol exposure to identify the intracellular mechanisms underlying the neuroprotective effects of ADNF-9. On embryonic day 7 (E7), weight-matched pregnant females were assigned to the following groups: (1) ethanol liquid diet (ALC) of 25% (4.49%, v/v) ethanol-derived calories; (2) pair-fed control (PF); (3) ALC combined with administration (i.p.) of ADNF-9 (ALC/ADNF-9); and (4) pair-fed combined with administration (i.p.) of ADNF-9 (PF/ADNF-9). On E13, fetal brains were collected, weighed, and apoptosis was determined using TdT-mediated dUTP nick-end labeling (TUNEL) assay. Bcl2 protein and phospho-c-Jun N-terminal kinase (JNK) levels were determined using Western blot and enzyme immunometric assay, respectively. ADNF-9 administration significantly prevented alcohol-induced reductions in fetal brain weight. In addition, ADNF-9 prevented an alcohol-induced increase in cell death in the primordium of the cerebral cortex and ganglionic eminence. Western blot analysis of the mitochondrial protein fractions revealed that ADNF-9 administration prevented an alcohol-induced reduction in the Bcl2 level. Moreover, an analysis of the proteins in the upstream signaling pathway revealed that ADNF-9 downregulated the phosphorylation of JNK. These data indicate that the mitochondrial Bcl2 pathway and JNK upstream signaling pathway are the intracellular targets of ADNF-9. The neuroprotective mechanism of action of ADNF-9 provides a direction for potential therapeutics against alcohol-induced neural damage involving mitochondrial dysfunction.

Protection Against Tauopathy by the Drug Candidates NAP (Davunetide) and D-SAL: Biochemical, Cellular and Behavioral Aspects

Activity-dependent neuroprotective protein (ADNP) is essential for brain formation and partial deficiency in ADNP results in cognitive deficits coupled with tauopathy and neuronal cell death. Our previous results indicated that a peptide snippet from ADNP, NAPVSIPQ (NAP, generic name, davunetide) can restore in part ADNP deficiencies. NAP interacts with tubulin and this interaction is displaced by the NAP related peptide that is derived from activity-dependent neurotrophic factor (ADNF), SALLRSIPA (SAL) and its all D-amino acid peptide derivative (D-SAL, also known as AL-309). Both NAP and D-SAL were shown to protect neurons against amyloid beta toxicity however the mechanism of protection is still under investigation. In addition, NAP protects against tau hyperphosphorylation associated with ADNP deficiency, in vivo. To investigate whether the mechanism of in vitro neuroprotection relates to the in vivo protection against tauopathy and to draw potential additional parallelism between NAP and D-SAL, we asked if: 1]NAP and D-SAL protect against amyloid beta related tau hyperphosphorylation in vitro; and 2] D-SAL protects against haploinsufficiency in ADNP, inhibiting tauopathy in vivo. Assessment of NAP and D-SAL neuroprotection in primary cortical neuro-glial cultures treated with amyloid beta showed that both peptides reduced toxin-related neuronal damage and protected against tau hyperphosphorylation. In vivo, chronic D-SAL administration protected against tau hyperphosphorylation associated with ADNP deficiency (ADNP+/- mice), showing for the first time protection against deficits in odor discrimination and in social recognition. These studies associate neuroprotection in vivo and in vitro and provide a broad base for future drug development based on NAP and D-SAL against multiple neurodegenerative conditions.

Neuroprotective peptide ADNF-9 in fetal brain of C57BL/6 mice exposed prenatally to alcohol

BackgroundA derived peptide from activity-dependent neurotrophic factor (ADNF-9) has been shown to be neuroprotective in the fetal alcohol exposure model. We investigated the neuroprotective effects of ADNF-9 against alcohol-induced apoptosis using TUNEL staining. We further characterize in this study the proteomic architecture underlying the role of ADNF-9 against ethanol teratogenesis during early fetal brain development using liquid chromatography in conjunction with tandem mass spectrometry (LC-MS/MS).MethodsPregnant C57BL/6 mice were exposed from embryonic days 7-13 (E7-E13) to a 25% ethanol-derived calorie [25% EDC, Alcohol (ALC)] diet, a 25% EDC diet simultaneously administered i.p. ADNF-9 (ALC/ADNF-9), or a pair-fed (PF) liquid diet. At E13, fetal brains were collected from 5 dams from each group, weighed, and frozen for LC-MS/MS procedure. Other fetal brains were fixed for TUNEL staining.ResultsAdministration of ADNF-9 prevented alcohol-induced reduction in fetal brain weight and alcohol-induced increases in cell death. Moreover, individual fetal brains were analyzed by LC-MS/MS. Statistical differences in the amounts of proteins between the ALC and ALC/ADNF-9 groups resulted in a distinct data-clustering. Significant upregulation of several important proteins involved in brain development were found in the ALC/ADNF-9 group as compared to the ALC group.ConclusionThese findings provide information on potential mechanisms underlying the neuroprotective effects of ADNF-9 in the fetal alcohol exposure model.

Short neuroprotective peptides, ADNF9 and NAP, are structurally disordered and monomeric in PBS

Activity-dependent neurotrophic factor 9 (ADNF9) and NAP are nine and eight amino acid peptides, which exhibit neuroprotective activity at femtomolar concentrations against cell toxic agents. We have here characterized their structures and interactions with dodecylphosphocholine (DPC) in phosphate-buffered saline (PBS). Circular dichroism analysis showed that ADNF9 and NAP are structurally disordered in PBS independent of peptide concentration and temperature, but appear to assume different secondary structure at increasing temperature. Sedimentation equilibrium analysis showed that both ADNF9 and NAP are monomeric at 37 °C, suggesting no self-association under physiological conditions. No secondary structure changes were observed in the presence of DPC, suggesting that ADNF9 and NAP do not interact with lipids.

Prevention of Developmental Delays in a Down Syndrome Mouse Model

To estimate whether prenatal treatment with neuroprotective peptides prevents the developmental delay and the glial deficit in the Ts65Dn mouse model for Down syndrome and to explore the peptides' effects on achievement of normal development. Pregnant Ts65Dn females were randomly assigned to NAPVSIPQ+SALLRSIPA or control and were treated by investigators blinded to treatment and genotype on gestational days 8-12. Offspring were tested from postnatal day 5 to 21 for motor and sensory milestones with standardized tests by operators blinded to the pup's treatment and genotype. The pup's genotype was determined after completion of all tests. Activity-dependent neurotrophic factor, glial fibrillary acidic protein, and vasoactive intestinal peptide expression were determined using real-time polymerase chain reaction. Trisomic mice achieved milestones with a significant delay in four of five motor and sensory milestones. Trisomic mice that were prenatally exposed to NAPVSIPQ+SALLRSIPA achieved developmental milestones at the same time as the controls in three of four motor and one of four sensory milestones (P<.01). Euploid pups prenatally treated with NAPVSIPQ+SALLRSIPA achieved developmental milestones significantly earlier than the euploid pups prenatally treated with placebo. Activity-dependent neurotrophic factor expression was significantly downregulated in the Ts65Dn brains compared with the controls, prenatal treatment with NAPVSIPQ+ SALLRSIPA prevented the activity-dependent neurotrophic factor decrease in the Ts65Dn brains, and the expression was not different from the controls. The glial marker glial fibrillary acidic protein demonstrated the known glial deficit in the Ts65Dn mice, and treatment with NAPVSIPQ+ SALLRSIPA prevented its downregulation. Lastly, vasoactive intestinal peptide levels were increased in the trisomic brains, whereas treatment with NAPVSIPQ+SALLRSIPA did not prevent its upregulation. Prenatal treatment with NAPVSIPQ and SALLRSIPA prevented developmental delay and the glial deficit in Down syndrome. These findings highlight a possibility for the prevention of developmental sequelae in Down syndrome and suggest a potential intervention during pregnancy that may improve the outcome.

Peptidergic Agonists of Activity‐Dependent Neurotrophic Factor Protect Against Prenatal Alcohol‐Induced Neural Tube Defects and Serotonin Neuron Loss

Prenatal alcohol exposure via maternal liquid diet consumption by C57BL/6 (B6) mice causes conspicuous midline neural tube deficit (dysraphia) and disruption of genesis and development of serotonin (5-HT) neurons in the raphe nuclei, together with brain growth retardation. The current study tested the hypothesis that concurrent treatment with either an activity-dependent neurotrophic factor (ADNF) agonist peptide [SALLRSIPA, (SAL)] or an activity-dependent neurotrophic protein (ADNP) agonist peptide [NAPVSIPQ, (NAP)] would protect against these alcohol-induced deficits in brain development. Timed-pregnant B6 dams consumed alcohol from embryonic day 7 (E7, before the onset of neurulation) until E15. Fetuses were obtained on E15 and brain sections processed for 5-HT immunocytochemistry, for evaluation of morphologic development of the brainstem raphe and its 5-HT neurons. Additional groups were treated either with SAL or NAP daily from E7 to E15 to assess the potential protective effects of these peptides. Measures of incomplete occlusion of the ventral canal and the frequency and extent of the openings in the rhombencephalon were obtained to assess fetal dysraphia. Counts of 5-HT-immunostained neurons were also obtained in the rostral and caudal raphe. Prenatal alcohol exposure resulted in abnormal openings along the midline and delayed closure of ventral canal in the brainstem. This dysraphia was associated with reductions in the number of 5-HT neurons both in the rostral raphe nuclei (that gives rise to ascending 5-HT projections) and in the caudal raphe (that gives rise to the descending 5-HT projections). Concurrent treatment of the alcohol-consuming dams with SAL prevented dysraphia and protected against the alcohol-induced reductions in 5-HT neurons in both the rostral and caudal raphe. NAP was less effective in protecting against dysraphia and did not protect against 5-HT loss in the rostral raphe, but did protect against loss in the caudal raphe. These findings further support the potential usefulness of these peptides for therapeutic interventions in pregnancies at risk for alcohol-induced developmental deficits. Notably, the ascending 5-HT projections of the rostral raphe have profound effects in regulating forebrain development and function, and the descending 5-HT projections of the caudal raphe are critical for regulating respiration. Protection of the rostral 5-HT-system may help prevent structural and functional deficits linked to abnormal forebrain development, and protection of the caudal systems may also reduce the increased risk for sudden infant death syndrome associated with prenatal alcohol exposure.

The multiple T-maze in vivo testing of the neuroprotective effect of humanin analogues

Humanin (HN) and its analogues have been shown to protect cells against death induced by various Alzheimer's disease (AD) genes and amyloid-β-peptides in vitro; the analogues [Gly 14]-HN and colivelin have also been shown to be potent in reversing learning and memory impairment induced by scopolamine or quinuclidinyl benzilate (QNB) in mice or rats in vivo using the Y-maze or multiple T-maze tests. This paper describes the activity of new peptides of the HN family, after i.p. administration, on QNB-induced impairment of spatial memory in the multiple T-maze test in rats. The following peptides have been studied: HN analogues truncated either on the C- or N-terminus, or analogues having a tert-Leu in place of Leu in the central part of the molecule, the active HN core PAGASRLLLLTGEIDLP (RG-PAGA) and its analogues having three or five leucines instead of four, and finally the recently described hybrid peptide colivelin (i.e. a peptide having the activity-dependent neurotrophic factor SALLRSIPA attached to the N-terminus of the active RG-PAGA) and its des-Leu- and plus-Leu-analogues. While the truncated analogues and most of the tert-Leu containing analogues were devoid of activity, the analogues of the RG-PAGA were active, i.e. they reversed the impairment of spatial memory irrespective of the number of Leu present in their sequence. The highest activity was shown by colivelin and its des-Leu-analogue. These results demonstrate the potential of HN analogues in the modulation of the cholinergic system, which plays an important role in the cognitive deficits associated with AD and other neurodegenerative diseases.

6: Prevention of learning deficit in a down syndrome mouse model

Down syndrome (DS) is the most common genetic cause of mental retardation. In DS there is a glial deficit which induces anomalies in neurotrophins, including Activity Dependent Neuroprotective Protein and Activity Dependent Neurotrophic Factor. Previously we have shown that their active fragments NAP+SAL prevented delay in behavioral milestones achievement in the Ts65Dn (Ts) mouse, a DS model. The objective of this study was to test if NAP+SAL prevent the adult DS learning deficit.

Activity‐dependent neurotrophic factor, ADNF, determines the structure characteristics of Colivelin, a fusion protein of ADNF9 and Humanin analog

A 24-amino acid long peptide, Humanin, protects neurons from Alzheimer's disease (AD)-related cell toxicities at sub-nM-uM concentrations. Activity-dependent neurotrophic factor (ADNF) is a glia-derived neurotrophic peptide, which protects neurons from tetrodoxin treatment and AD-related and amyotrophic lateral sclerosis-related insults at fM concentrations. An attempt was made to further improve the activity of Humanin by fusing this peptide to ADNF9, a 9-amino acid long core peptide of the ADNF. This fusion resulted in a novel molecule, termed Colivelin, with the neuroprotective activity at fM range, which is approximately 10(3)-10(7) fold higher than the activity of Humanin and Humanin analogs and follows the activity profile of fM-active ADNF9. We have characterized the structural properties of Colivelin and compared with those of ADNF9 and Humanin in water and phosphate-buffered saline (PBS). The secondary structure of Colivelin was similar to that of ADNF9, but not that of Humanin, and hence was not the average of the contributions of the two peptides fused. Colivelin was stable and monomeric in PBS, consistent with the monomeric property of ADNF9, while Humanin showed strong tendency to self-associate. Thus, it is evident that the structural properties of Colivelin resemble those of ADNF9, rather than those of Humanin.

Concurrent Dietary Administration of D‐SAL and Ethanol Diminishes Ethanol’s Teratogenesis

SAL (SALLRSIPA) is a peptide fragment of activity-dependent neurotrophic factor. Both L- and D-SAL diminish ethanol's pathogenesis, however, the D-peptide is protease resistant, and can therefore be effectively administered in a diet. The present study tested the hypothesis that D-SAL provided in a liquid diet containing ethanol will prevent ethanol-induced teratogenicity in mice. Following an ethanol acclimation period, female C57Bl/6J mice were withdrawn from the ethanol, bred, and then returned during gestational days (GD) 7 and 8 to a control liquid diet or one containing 4.8% ethanol alone or in combination with 5.6 microg/ml D-SAL. At these doses, the mice received approximately 75 microg of D-SAL on each day and achieved peak blood-alcohol concentrations on GD 8 that ranged from 148-162 mg/dl. On GD 14, the fetuses were examined for the presence of ocular abnormalities including microphthalmia and irregularly shaped pupils, teratogenic effects known to result from this ethanol exposure paradigm. Dietary D-SAL reduced the incidence of ocular defects in ethanol-exposed fetuses from 29 to 10% in the right eyes and from 21 to 7.5% in the left eyes; levels similar to those observed in pair-fed controls. In addition to decreasing their incidence, D-SAL also reduced the severity of the ocular defects. These results demonstrate that oral D-SAL can prevent ethanol-induced ocular defects. Because ocular defects are commonly associated with CNS damage, oral D-SAL may also prove valuable in preventing ethanol-induced brain defects.

Structure Analysis of Activity-dependent Neurotrophic Factor 9 by Circular Dichroism and Sedimentation Equilibrium

Activity-dependent neurotrophic factor (ADNF) is a glia-derived neurotrophic peptide, which protects neurons from tetrodoxin treatment and Alzheimer's disease-related and amyotrophic-lateral-sclerosis-related insults at femto-molar concentrations. However, the mechanism of the femto-molar neuroprotection by the peptide has not been elucidated. The characterization of the peptide structure in solution at molecular level should shed light in the mechanism of such extremely high biological activity. From that point of view, the secondary and quaternary structure analysis of ADNF9, an active core fragment peptide of ADNF, was performed by circular dichroism (CD) and sedimentation equilibrium. ADNF9 has also been shown to exhibit a neurotrophic activity in femto-molar concentrations; in this study it showed sub-pM neuroprotective activity against V642I-APP-induced cytotoxity in the mouse primary cortical neuron. CD analysis showed that the secondary structure of ADNF9 is identical in water and phosphate-buffered saline (PBS) and is independent of the peptide concentration. The CD spectra appear to be characterized most likely as disordered. The sedimentation equilibrium experiments demonstrated monomeric structure of the protein over the wide range of peptide concentration. There is a slight enhancement of CD intensity at 37 degrees C relative to 20 degrees C, suggesting a possible hydrophobic association of the peptide. There is no change in the secondary structure in PBS upon freeze-thaw treatment, which has previously been suggested to cause activity loss.

Neuroprotection against neurodegenerative diseases

Neuronal death is directly implicated in the pathogenesis of neurodegenerative diseases (NDDs). NDDs cannot be cured because the mechanisms underlying neuronal death are too complicated to be therapeutically suppressed. Neuroprotective factors, such as neurotrophins, certain growth factors, neurotrophic cytokines, and short neuroprotective peptides, support neuronal survival in both physiological and pathological conditions, suggesting that these factors may be good drug candidates for NDDs. We recently generated a novel neuroprotective peptide named Colivelin by attaching activity-dependent neurotrophic factor (ADNF) to the N-terminus of a potent Humanin derivative, AGA-(C8R)HNG17. HN was originally identified from an Alzheimer's disease (AD) brain as an endogenous neuroprotective peptide that suppresses ADrelevant toxicity. Colivelin protects neurons from death relevant to NDDs by activating two independent prosurvival signals: an ADNF-mediated Ca2+/calmodulin-dependent protein kinase IV pathway and an HN-mediated STAT3 pathway. The neuroprotective effect of Colivelin provides novel insights into therapy for NDDs.