Naltrexone-sensitive behavioral actions of the ACTH 4-9 analog (Org 2766)

The effects of the ACTH 4-9 analog (Org 2766) and the COOH-terminal tripeptide of Org 2766 (Phe-D-Lys-Phe; PDLP) on retrieval of one-trial learning passive avoidance behavior were compared with those of beta-endorphin, [Met5]-enkephalin, [D-Ala2,Met5]-enkephalin, des-Tyr1-[Met5]-enkephalin and des-enkephalin-gamma-endorphin (DE gamma E). Amounts of intracerebroventricularly administered Org 2766, PDLP, [Met5]-enkephalin, [D-Ala2,Met5]-enkephalin and DE gamma E, which induced a comparable attenuation of passive avoidance behavior were determined. Pretreatment with the opiate antagonist naltrexone prevented the attenuating effect of these peptides on passive avoidance behavior except that of DE gamma E. The attenuating effect of Org 2766 and of [Met5]-enkephalin was reversed to facilitation of passive avoidance behavior in the presence of naltrexone. Subcutaneous treatment with Org 2766 and [D-Phe7]-ACTH 4-10 decreased electrical self-stimulation behavior elicited from the medial septal area. Naltrexone prevented the inhibitory effect of Org 2766 on this behavior, but not that of [D-Phe7]-ACTH 4-10. Although the attenuating effect of PDLP on passive avoidance behavior was not reduced by pretreatment with [Met5]-enkephalin- or beta-endorphin-antiserum, and PDLP induced neither analgesia nor excessive grooming, the data suggest that the inhibitory effect of Org 2766 and PDLP on passive avoidance behavior and electrical self-stimulation are mediated by endorphin systems in the brain.     

Binding of a biotinylated neurotrophic ACTH(4–9) analogue, Org 2766, to neurofilament-positive cells in primary or cell line cultures

To study the putative binding sites of the neurotrophic peptide Org 2766, an analogue of ACTH(4–9) [H-Met(O2)-Glu-His-Phe- -Lys-Phe-OH], biotinylated forms of the peptide were used. After fixation, cultures of rat spinal cord and dorsal root ganglia were incubated with 4–10 μM of biotinyl-Org 2766 (b-Org 2766). Binding of both N- and C-terminally biotinylated Org 2766 was seen to phase-bright, round cells with thin processes, but not to flat, orthogonal-shaped cells with tapering processes. The b-Org 2766 binding was displaceable by an excess of nonbiotinylated Org 2766. Light and electron microscopy showed that the biotinylated peptide binds to a cytoplasmatic component as well as to the cell membrane. Double-labeling experiments with b-Org 2766 and an antibody (RT-97) to a high molecular weight neurofilament protein in dorsal root ganglion cultures showed, using fluorescence and confocal scanning laser microscopy, that all b-Org 2766 binding cells were neurofilament positive. Biotinylated Org 2766 did also bind to the neuronally differentiated cells in cultures of the human neuroblastoma cell line SK-N-SH, but not to those differentiated into epithelial cells. The present data suggest that the neurotrophic peptide Org 2766 binds specifically to cell types with neuronal characteristics.     

ACTH4-9类似物对小鼠海马突触体Ca2+水平的调节作用

本工作观察了Ｏｒｇ２７６６（ＡＣＴＨ４－９的类似物）对海马突触体内游离Ｃａ＾２＋水平及对＾４５Ｃａ＾２＋摄取的影响。采用细胞内钙离子荧光探针Ｆｕｒａ－２,通过Ｓｐｅｘ－阳离子测定系统检测突触体内［Ｃａ＾２＋］ｉ的动态变化,并用液闪光谱仪测定突触体对＾４５Ｃａ＾２＋的摄取。结果表明：较低剂量的ＯＲＧ２７６６对突触体内［Ｃａ＾２＋］ｉ的影响不明显,却显著降低突触体对＾４５Ｃａ＾２＋的摄取;高剂量的Ｏｒ     

The influence of neuropeptides related to pro-opiomelanocortin on acquisition of heroin self-administration of rats

The influence of different neuropeptides related to pro-opiomelanocortin were tested on acquisition of heroin self-administration in rats. The animals were allowed to self-administer heroin intravenously on a continuous reinforcement schedule during 6 h daily sessions on 5 consecutive days. Treatment was performed subcutaneously 1 h before each daily session. It was found that the opioid peptides alpha-, gamma- and beta-endorphin hardly influenced acquisition of heroin self-administration, while the non-opioid fragments of alpha- and gamma- endorphin modulated this behavioral response. In fact, beta-endorphin (beta E) 2-9 tended to facilitate the rate of acquisition, while the gamma-type endorphins, des-Tyr1-gamma-endorphin (beta E 2-17) and des-enkephalin-gamma-endorphin (beta E 6-17), decreased heroin intake. Concerning the ACTH/MSH related peptides, a decreasing effect of heroin intake was found following treatment with (D-Phe7)-ACTH 4-10, with a high dose of the ACTH 4-9 analog Org 2766 and with gamma 2-MSH, while ACTH 1-24, ACTH 4-10 and a low dose of Org 2766 did not significantly influence self-injecting behavior. It is concluded that pro-opiomelanocortin serves as a precursor molecule for peptide fragments, which modulate the acquisition of heroin self-administration in rats.     

The ACTH/MSH (4-9) analog Org2766 improves retrieval of information after a fimbria fornix transection

The fimbria fornix of male Wistar rats was transected unilaterally after they had been successfully trained in the Morris maze and the passive avoidance task. Sham-operated and lesioned animals were treated either with Org2766 or saline for two weeks. Subsequently, the performance of all groups was tested again starting two days after the last treatment. The lesioned animals showed a deficit in performance in both tasks, indicating interference of the lesion with retrieval of information. Org2766 improved the poor performance of the lesioned animals in the Morris maze, but not in the passive avoidance task.     

Modeling of pathological traits in Alzheimer's disease based on systemic extracellular signaling proteome

The study of chronic brain diseases including Alzheimer's disease in patients is typically limited to brain imaging or psychometric testing. Given the epidemic rise and insufficient knowledge about pathological pathways in sporadic Alzheimer's disease, new tools are required to identify the molecular changes underlying this disease. We hypothesize that levels of specific secreted cellular signaling proteins in cerebrospinal fluid or plasma correlate with pathological changes in the Alzheimer's disease brain and can thus be used to discover signaling pathways altered in the disease. Here we measured 91 proteins of this subset of the cellular communication proteome in plasma or cerebrospinal fluid in patients with Alzheimer's disease and cognitively normal controls to mathematically model disease-specific molecular traits. We found small numbers of signaling proteins that were able to model key pathological markers of Alzheimer's disease, including levels of cerebrospinal fluid β-amyloid and tau, and classify disease in independent samples. Several of these factors had previously been implicated in Alzheimer's disease supporting the validity of our approach. Our study also points to proteins which were previously unknown to be associated with Alzheimer's disease thereby implicating novel signaling pathways in this disorder.     

Pharmacological aspects of the influence of melanocortins on the formation of regenerative peripheral nerve sprouts

Adrenocorticotropin (ACTH) and alpha-melanocyte-stimulating hormone (alpha-MSH) stimulate the initial sprouting response in the crushed rat sciatic nerve. In this report a detailed analysis of the neurotrophic action of Org.2766 [a degradation resistant ACTH(4-9) analog] and alpha-MSH is described. Org.2766 treatment results in enhanced numbers of outgrowing sprouts in the damaged nerve. The growth velocity of the sprouts is not affected. The peptide effect is dose-dependent. A single peptide injection administered immediately following the crush stimulates the formation of sprouts significantly. Continued high blood levels of Org.2766 are probably not critical for the neurotrophic effect of these peptides, since a more moderate dosing protocol (injections given every 48 hr) was more effective than more frequent injections (injections given every 12 hr). The present results further the understanding of the mode of action of ACTH/alpha-MSH-like peptides and underscore the necessity to test a wide range of doses and injection protocols to avoid false negative results in clinical work being planned to start in the near future.     

Rapid neurotrophic actions of an ACTH/MSH(4–9) analogue after nigrostriatal 6-OHDA lesioning

ACTH peptide fragments demonstrate potent neurotrophic effects on peripheral nerves in situ, central neurons in culture, and have been implicated to have effects on central neurons in vivo. Neurotoxic lesioning of the nigrostriatal system, which depletes the striatum of dopamine, provides a feasible model of central regeneration in which to test these peptides. Male Sprague-Dawley rats were lesioned unilaterally with 6-hydroxydopamine (8 μg/4 μl), infused into the substantia nigra. They were subsequently treated with 10 μg/kg IP of Org 2766 [ACTH/MSH(4–9) analogue] or saline every 24 h starting immediately after the infusion and were observed for 2 weeks. Rotational behavior data indicate that Org 2766 significantly decreases ipsiversive turning (p < 0.05), induced by amphetamine (2 mg/kg), as well as accelerating the onset of denervation supersensitivity induced by apomorphine (0.05 mg/kg). Evaluation of dopamine immunohistochemistry, using an anti-tyrosine hydroxylase antibody, demonstrates an enhanced intensity of staining in the ORG 2766-treated tissue compared to its saline counterpart. This difference is confirmed and quantified through specific high-affinity dopamine uptake. Dopamine uptake is about 17% higher in the striata of animals treated with Org 2766. Higher dopamine uptake levels in these ACTH-treated animals correlate with greater fiber density in this group. Therefore, it appears that treatment with the ACTH/MSH(4–9) analogue Org 2766 (10 μg/kg/24 h) offers a protective effect from 6-OHDA lesions in the substantia nigra as well as accelerating various compensatory mechanisms involved in functional recovery.     

Contrasting effects of Org 2766 and alpha-MSH on social and exploratory behavior in the rat

Intraperitoneal injection of Org 2766 (0.01-0.4 microgram/kg) produced a dose-related increase in the number of social contacts and in the time spent in active social interaction by pairs of male rats tested in arenas with which they were familiar, but had little effect when the rats were tested in unfamiliar arenas. The increased social interaction was not accompanied by any change in motor activity. In contrast, alpha-MSH (20-200 microgram/kg) decreased the time spent in active social dose-related. Both peptides reduced exploratory head-dipping only at high doses (4-8 microgram/kg for Org 2766 and 200 microgram/kg for alpha-MSH); this change was not accompanied by a reduction in motor activity.     

Altered glycosylation of acetylcholinesterase in APP (SW) Tg2576 transgenic mice occurs prior to amyloid plaque deposition

Previous studies have shown that a minor glycoform of acetylcholinesterase (AChE) is increased in Alzheimer's disease brain and cerebrospinal fluid. This glycoform can be distinguished from other AChE species by its lack of binding to concanavalin A (Con A). In this study, the temporal relationship between AChE glycosylation and Abeta deposition was examined in Tg2576 mice. There was a significant (p < 0.05) difference in AChE glycosylation in Tg2576 mice compared with age-matched background strain control mice at 4 months of age. This difference in glycosylation was also observed in 8- and 12-month-old Tg2576 mice. In contrast, Abeta plaques were only seen in the Tg2576 mice at 12 months of age, and were not detected at 4 and 8 months of age. Soluble human-sequence Abeta was detected as early as 4 months of age in the transgenic mice. The altered AChE glycosylation was due to an increase in a minor AChE isoform, which did not bind Con A, similar to that previously observed to be increased in Alzheimer's disease brain and cerebrospinal fluid. The results demonstrate that in transgenic mice altered AChE glycosylation is associated with very early events in the development of AD-like pathology. The study supports the possibility that glycosylation may also be a useful biomarker of AD.     

Clusterin in cerebrospinal fluid: analysis of carbohydrates and quantification of native and glycosylated forms

Clusterin is suggested to be involved in the pathogenesis of Alzheimer's disease. Clusterin expression is increased in brain tissue in affected regions of Alzheimer patients, and intense clusterin staining is found in both senile plaques and in neuronal and glia cells. In contrast, the cerebrospinal fluid level of clusterin in Alzheimer patients has, thus far, been found unchanged. Clusterin is a glycosylated protein, and an alteration of its glycosylation in Alzheimer's disease might influence accurate quantification in cerebrospinal fluid through interference of antibody binding to the protein. Using enzymatic deglycosylation of clusterin isolated from cerebrospinal fluid, we found that the carbohydrates attached to clusterin were of the N-linked type and sialic acids. Based on this finding, cerebrospinal fluid samples from Alzheimer patients (n = 99) and controls (n = 39) were analysed. The samples were treated with peptide: N-glycanase F, cleaving off N-linked carbohydrates, and clusterin was quantified before and after deglycosylation using a new sandwich enzyme-linked immunosorbent assay. Clusterin was significantly increased in Alzheimer patients, in both native (7.17 ± 2.43 AU versus 5.73 ± 2.09 AU; p = 0.002), and deglycosylated samples (12.19 ± 5.00 AU versus 9.68 ± 4.38 AU; p = 0.004). Deglycosylation led to increased measured levels of clusterin by 70% (p < 0.001) in Alzheimer patients and 67% (p < 0.001) in controls. These findings indicate that glycosylation of proteins may interfere with their quantification. The results show that clusterin is significantly increased in cerebrospinal fluid from Alzheimer patients as a group, supporting that clusterin might be involved in the pathogenesis of Alzheimer's disease. However, the individual clusterin levels overlap between the two groups, and thus cerebrospinal fluid clusterin measurement is not suitable as a biochemical marker in the diagnosis of Alzheimer's disease.     

Plasma beta carotene in Alzheimer's disease. Association with cerebrospinal fluid beta-amyloid 1-40, (Abeta40), beta-amyloid 1-42 (Abeta42) and total Tau

We studied the plasma beta carotene concentrations in 40 Alzheimer's disease patients and the association with cerebrospinal fluid beta-amyloid 1-40, (Abeta40), cerebrospinal fluid beta-amyloid 1-42 (Abeta42) and cerebrospinal fluid total Tau. We found that patients with plasma beta carotene levels below the 25th percentile had 55% reduced ratios of Abeta40/Tau and 51% reduced ratios of Abeta 40/Abeta 42 compared with patients in the highest quartile. Mean Tau concentrations in the lowest quartile of plasma beta-carotene levels were 74% higher compared with the highest quartile of plasma beta-carotene levels. Thus, we could demonstrate an statistically significant association between beta carotene levels in plasma and neurochemical markers in the cerebrospinal fluid of Alzheimer's disease patients.     

Neuropeptides,mental performance and aging

The presence of peptidergic neuronal networks in the brain and the modulating action of neuropeptides on brain functions as evidenced by their behavioral influence in particular support the concept that the brain like the peripheral endocrine glands is an endocrine target organ which is as sensitive to treatment with neuropeptides as the peripheral glands are to pituitary hormones. Animal and human data are reviewed showing that neuropeptides related to ACTH/MSH affect motivational and attentional processes and that those related to vasopressin are involved in memory processes. Since these functions decline during aging it is postulated that a decreased bioavailability of neuropeptides in brain of elderly people is associated with specific disturbances in mental performance. Thus, the decreased mental ability of the aged may be restored by treatment with neuropeptides particularly those with little, if any, peripheral, endocrine activity, like the ACTH neuropeptide Org 2766 and the vasopressin neuropeptide DGAVP.     

In cerebrospinal fluid ER chaperones ERp57 and calreticulin bind beta-amyloid

The beta-amyloids (abetas) are the major components of the plaque observed in the brains of patients with Alzheimer's disease. The conundrum is that although they are produced in everyone during the posttranslational processing in the endoplasmic reticulum (ER) of the amyloid precursor protein (APP), deposits are only observed in the elderly. Our work suggests that normals have a carrier protein(s) keeping them in solution. Based on immunoblotting studies of cerebrospinal fluid (CSF) from normals, we find that the bulk of the abetas are bound to the ER chaperones, ERp57 and calreticulin, suggesting that these may be carrier proteins which prevent aggregation of the abetas and that the deposits are due to faulty ER posttranslational processing of APP with the failure to form this complex. If membrane protein synthesis is similarly affected, it could explain the neuronal dysfunction characteristic of Alzheimer's disease.     

N-Acetyl Aspartic Acid (NAA) and N-Acetyl Aspartylglutamic Acid (NAAG) in Human Ventricular,Subarachnoid, and Lumbar Cerebrospinal Fluid

N-Acetylaspartic and N-acetylaspartylglutamic acid concentrations in human ventricular, subarachnoid and lumbar cerebrospinal fluid were measured by combined gas chromatography-mass spectrometry using selected ion monitoring with deuterated internal standards. N-Acetylaspartate concentrations were in the range 55, 9, and 1 M, respectively; N-acetylaspartylglutamate concentrations in the same fluids were in the range 8, 3 and 4 M, respectively. There did not appear to be any difference in lumbar fluid concentrations of either compound between control subjects, schizophrenic patients, Alzheimer's disease patients and a pooled group of patients with neurological degeneration. Ventricular concentrations of both compounds were greatly increased in deceased patients suggesting that maintenance of their intracellular concentrations is probably energy dependent. The concentrations of these compounds in lumbar cerebrospinal fluid from living, and ventricular cerebrospinal fluid from deceased subjects were weakly correlated with one another. In lumbar fluid neither compound appeared to be correlated with age. Analysis of serially collected lumbar samples from two subjects showed a weak concentration gradient for both compounds. Neither antipsychotic medication nor the acid transport inhibitor probenecid had any effect on lumbar concentrations of either compound. Attempts to use anion exchange high pressure liquid chromatography with UV detection for measurement of the low concentrations of N-acetylaspartate found in cerebrospinal fluid from living subjects were unsuccessful.     

Structural modifications of the ACTH-(4-9) analog ORG 2766 yields peptides with high biological activity.

The behavioral effects of two peptides (HOE 427) and ORG 31433) related to the ACTH-(4-9) analog ORG 2766 were investigated in Wistar rats in a number of tests in which Org 2766 is active. Subcutaneous administration of HOE 427 in a dose of 0.5 ng/kg or ORG 31433 in doses of 0.5-5.0 ng/kg facilitated passive avoidance behavior whereas these peptides attenuated the avoidance response in doses of 25 ng/kg and 250 ng/kg respectively. ORG 31433 (0.1 - 1.0 microgram/kg) decreased motor activity of group housed rats tested under low light conditions. Furthermore subcutaneous (1.0- 10.0 ng/kg) or oral (10 microgram/kg) administration of ORG 31433 accelerated functional recovery from 6-hydroxydopamine (6-OHDA)-induced lesions in the nucleus accumbens which cause motor hypoactivity. The experiments show that as compared to ORG 2766 the peptides HOE 427 and ORG 31433 induce qualitatively similar responses but are approximately 10 to 100 times more potent. These data may imply that substitution of the C-terminal COOH group of ORG 2766 yields neuropeptides with increased potency.     

Pituitary-adrenal influences on avoidance behavior of pigs

Pigs submitted to extinction of a signaled conditioned avoidance response were injected daily with various doses of dexamethasone (DX) or ACTH. Pigs treated with 0.2 mg/kg of DX showed a higher number of intertrial crosses, but the extinction rate was not modified by either treatment. The effects of ACTH and DX were further studied on the reaction to a Pavlovian conditioned fear signal presented to pigs having learned a continuous avoidance response in a shuttle-box. DX treatment before both the fear conditioning and the test sessions enhanced the reaction to the fear signal at intermediate doses (0.2 mg/kg) but had little effect at lower (0.1 mg/kg) and higher doses (0.5 and 1 mg/kg). ACTH 1–24 treatment induced the same behavioral changes as intermediate doses of DX. A behaviorally active ACTH 4–9 analog, Org 2766, did not modify behavioral reaction to fear signal presentations when administered before fear conditioning and/or test sessions. These results demonstrate that, in pigs, avoidance performance changes under fear signal presentations are modulated by corticosteroids.     

A novel peptidomics approach to detect markers of Alzheimer's disease in cerebrospinal fluid

Sensitive and specific diagnosis and monitoring of disease progression are of prime importance to develop new therapies for Alzheimer's disease patients. Although the diagnostic accuracy, verified by pathological examination is high, it is currently not possible to diagnose Alzheimer's disease with a high degree of certainty until relatively late in the disease process. Here, we have undertaken a peptidome analysis of postmortem cerebrospinal fluid of neuropathologically confirmed Alzheimer's disease patients and non-demented controls using a combination of methods and technologies. This includes novel sample preparation based on the enrichment of endogenous, proteolytically derived peptides as well as peptides non-covalently bound to abundant proteins. We observed differences in peptide profiles associated with Alzheimer's disease in the endogenous peptide fraction and in the protein-bound peptide fraction. The discriminating peptides in the unbound peptide fraction were identified as VGF nerve growth factor inducible precursor, and complement C4 precursor, whereas the discriminating peptides in the protein-bound fraction were identified as VGF nerve growth factor inducible precursor, and alpha-2-HS-glycoprotein.     

Fish electroreception as a model for vincristine-induced neuropathies and a possible preventive role for Org 2766 treatment

1. Subcutaneous injection of vincristine (more than 3 μg) in the catfish, Ictalurus nebulosus, produces a black spot on the skin with a grey surround. Doses between 1 and 3 μg give a less pronounced discolouration.2. Two to three days after injection of 5 μg vincristine, repetitive activity is detected in primary afferents of unstimulated electroreceptor organs close to the site of injection.3. Vincristine increases the phase-lag of the modulation of afferent activity in electroreceptor organs during electrical stimulation without a clear effect on the sensitivity of catfish electroreceptor organs.4. The amplitude of the action potentials of the primary afferents begins to decrease after 2 days; after3 days they gradually disappear in the background noise.5. Application of Org 2766, a potentially neurotrophic compound, at 2 days, but not 1 day, before vincristine application prevents vincristine effects on the phase shift.6. Preliminary electron-microscopical studies of the synapse shows a severe depletion of glycogen granules in the afferent nerve fibre after vincristine application,7. It is concluded that electroreceptor organs can be used to study neuropathies caused by vincristine, and that Org 2766 may be useful for preventive treatment of such neuropathies.