RESUMEN
This study was designed to assess a new composite implant to induce regeneration of injured spinal cord in paraplegic rats following complete cord transection. Neuronal xenogeneic cells from biopsies of adult nasal olfactory mucosa (NOM) of human origin, or spinal cords of human embryos, were cultured in two consecutive stages: stationary cultures in a viscous semi-solid gel (NVR-N-Gel) and in suspension on positively charged microcarriers (MCs). A tissue-engineered tubular scaffold, containing bundles of parallel nanofibers, was developed. Both the tube and the nanofibers were made of a biodegradable dextran sulphate-gelatin co-precipitate. The suturable scaffold anchored the implant at the site of injury and provided guidance for the regenerating axons. Implants of adult human NOM cells were implanted into eight rats, from which a 4 mm segment of the spinal cord had been completely removed. Another four rats whose spinal cords had also been transected were implanted with a composite implant of cultured human embryonic spinal cord cells. Eight other cord-transected rats served as a control group. Physiological and behavioral analysis, performed 3 months after implantation, revealed partial recovery of function in one or two limbs in three out of eight animals of the NOM implanted group and in all the four rats that were implanted with cultured human embryonic spinal cord cells. Animals of the control group remained completely paralyzed and did not show transmission of stimuli to the brain. The utilization of an innovative composite implant to bridge a gap resulting from the transection and removal of a 4 mm spinal cord segment shows promise, suggesting the feasibility of this approach for partial reconstruction of spinal cord lesions. Such an implant may serve as a vital bridging station in acute and chronic cases of paraplegia.
Asunto(s)
Implantes Experimentales , Traumatismos de la Médula Espinal/cirugía , Animales , Electrofisiología , Humanos , Imagen por Resonancia Magnética , Ratas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/patología , Ingeniería de TejidosRESUMEN
We have recently cloned the mouse activity-dependent neuroprotective protein (ADNP). Here, we disclose the cloning of human ADNP (hADNP) from a fetal brain cDNA library. Comparative sequence analysis of these two ADNP orthologs indicated 90% identity at the mRNA level. Several single nucleotide polymorphic sites were noticed. The deduced protein structure contained nine zinc fingers, a proline-rich region, a nuclear bipartite localization signal, and a homeobox domain profile, suggesting a transcription factor function. Further comparative analysis identified an ADNP paralog (33% identity and 46% similarity), indicating that these genes belong to a novel protein family with a nine-zinc finger motif followed by a homeobox domain. The hADNP gene structure spans approximately 40 kilobases and includes five exons and four introns with alternative splicing of an untranslated second exon. The hADNP gene was mapped to chromosome 20q12-13.2, a region associated with aggressive tumor growth, frequently amplified in many neoplasias, including breast, bladder, ovarian, pancreatic, and colon cancers. hADNP mRNA is abundantly expressed in distinct normal tissues, and high expression levels were encountered in malignant cells. Down-regulation of ADNP by antisense oligodeoxynucleotides up-regulated the tumor suppressor p53 and reduced the viability of intestinal cancer cells by 90%. Thus, ADNP is implicated in maintaining cell survival, perhaps through modulation of p53.
Asunto(s)
Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , División Celular , Cromosomas Humanos Par 20/genética , Clonación Molecular , Secuencia Conservada/genética , Exones/genética , Perfilación de la Expresión Génica , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Intrones/genética , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/química , Oligonucleótidos Antisentido/genética , Mapeo Físico de Cromosoma , Polimorfismo de Nucleótido Simple , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismo , Dedos de ZincRESUMEN
Astrocytes are present in large numbers in the nervous system, are associated with synapses, and propagate ionic signals. Astrocytes influence neuronal physiology by responding to and releasing neurotransmitters, but the mechanisms that establish the close interaction between these cells are not defined. Here we use hippocampal neurons in culture to demonstrate that vasoactive intestinal polypeptide (VIP) promotes neuronal differentiation through activity-dependent neurotrophic factor (ADNF), a protein secreted by VIP-stimulated astroglia. ADNF is produced by glial cells and acts directly on neurons to promote glutamate responses and morphological development. ADNF causes secretion of neurotrophin 3 (NT-3), and both proteins regulate NMDA receptor subunit 2A (NR2A) and NR2B. These data suggest that the VIP-ADNF-NT-3 neuronal-glial pathway regulates glutamate responses from an early stage in the synaptic development of excitatory neurons and may also contribute to the known effects of VIP on learning and behavior in the adult nervous system.
Asunto(s)
Diferenciación Celular/fisiología , Neuroglía/metabolismo , Neuronas/citología , Neuronas/metabolismo , Oligopéptidos/metabolismo , Péptido Intestinal Vasoactivo/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Ácido Glutámico/metabolismo , Ácido Glutámico/farmacología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neuroglía/citología , Neuronas/efectos de los fármacos , Neurotrofina 3/metabolismo , Oligopéptidos/farmacología , Técnicas de Placa-Clamp , Fragmentos de Péptidos/farmacología , Estimulación Física , Ratas , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal/efectos de los fármacos , Estimulación Química , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
Activity dependent neuroprotective protein (ADNP, 828 amino acids, pI 5.99) is a glial-derived protein that contains a femtomolar active neuroprotective peptide, NAPVSIPQ (NAP). VIP induces a two- to threefold increase in ADNP mRNA in astrocytes, suggesting that ADNP is a VIP-responsive gene. ADNP is widely distributed in the mouse hippocampus, cerebellum, and cerebral cortex. VIP has been shown to possess neuroprotective activity that may be exerted through the activation of glial proteins. We suggest that ADNP may be part of the VIP protection pathway through the femtomolar-acting NAP and through putative interaction with other macromolecules.
Asunto(s)
Proteínas de Homeodominio , Proteínas del Tejido Nervioso/genética , Péptido Intestinal Vasoactivo/farmacología , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Encéfalo/metabolismo , Ratones , Modelos Neurológicos , Datos de Secuencia Molecular , Fármacos Neuroprotectores/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Distribución Tisular , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
Activity-dependent neurotrophic factor (ADNF) was recently isolated from conditioned media of astrocytes stimulated with vasoactive intestinal peptide (VIP). ADNF provided neuroprotection at femtomolar concentration against a wide variety of toxic insults. A nine amino acid peptide (ADNF-9) captured with even greater potency the neuroprotective activity exhibited by the parent protein. Utilizing Northern and Western blot analyses, it was now shown that ADNF-9 increased the expression of heat shock protein 60 (hsp60) in rat cerebral cortical cultures. In contrast, treatment with the Alzheimer's toxin, the beta-amyloid peptide, reduced the amount of intracellular hsp60. Treatment with ADNF-9 prevented the reduction in hsp60 produced by the beta-amyloid peptide. The protection against the beta-amyloid peptide-associated cell death provided by ADNF-9 may be mediated in part by intracellular increases in hsp60.
Asunto(s)
Corteza Cerebral/metabolismo , Chaperonina 60/metabolismo , Proteínas del Tejido Nervioso/farmacología , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Péptidos beta-Amiloides/farmacología , Animales , Northern Blotting , Western Blotting , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Chaperonina 60/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Concentración Osmolar , Fragmentos de Péptidos/farmacología , RatasRESUMEN
The vulnerability of neurons and the irreversibility of loss make discoveries of neuroprotective compounds fundamentally important. Here, the complete coding sequence of a novel protein (828 amino acids, pI 5.99), derived from mouse neuroglial cells, is revealed. The sequence contained (1) a neuroprotective peptide, NAPVSIPQ, sharing structural and immunological homologies with the previously reported, activity-dependent neurotrophic factor; (2) a glutaredoxin active site; and (3) a zinc binding domain. Gene expression was enriched in the mouse hippocampus and cerebellum and augmented in the presence of the neuropeptide vasoactive intestinal peptide, in cerebral cortical astrocytes. In mixed neuron-astrocyte cultures, NAPVSIPQ provided neuroprotection at subfemtomolar concentrations against toxicity associated with tetrodotoxin (electrical blockade), the beta-amyloid peptide (the Alzheimer's disease neurotoxin), N-methyl-D-aspartate (excitotoxicity), and the human immunodeficiency virus envelope protein. Daily NAPVSIPQ injections to newborn apolipoprotein E-deficient mice accelerated the acquisition of developmental reflexes and prevented short-term memory deficits. Comparative studies suggested that NAPVSIPQ was more efficacious than other neuroprotective peptides in the apolipoprotein E-deficiency model. A potential basis for rational drug design against neurodegeneration is suggested with NAPVSIPQ as a lead compound. The relative enrichment of the novel mRNA transcripts in the brain and the increases found in the presence of vasoactive intestinal peptide, an established neuroprotective substance, imply a role for the cloned protein in neuronal function.
Asunto(s)
Proteínas de Homeodominio , Proteínas del Tejido Nervioso/química , Fármacos Neuroprotectores/química , Péptidos/química , Secuencia de Aminoácidos , Animales , Apolipoproteínas E/deficiencia , Secuencia de Bases , Northern Blotting , Western Blotting , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Clonación Molecular , Humanos , Aprendizaje por Laberinto/efectos de los fármacos , Memoria/efectos de los fármacos , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/farmacología , Neuronas/efectos de los fármacos , Neuropéptidos , Fármacos Neuroprotectores/farmacología , Oligopéptidos , Péptidos/genética , Péptidos/farmacología , ARN Mensajero/análisis , RatasRESUMEN
The complete coding sequence of a novel protein (828 amino acids, pI 5.99), a potential new mediator of vasoactive intestinal peptide (VIP) activity was recently revealed. The expression of this molecule, activity-dependent neuroprotective protein (ADNP), was augmented in the presence of VIP, in cerebral cortical astrocytes. The mRNA transcripts encoding ADNP were enriched in the mouse hippocampus and cerebellum. The protein deduced sequence contained the following: (1) a unique peptide, NAPVSIPQ, sharing structural and immunological homologies with the previously reported, activity-dependent neurotrophic factor (ADNF) and exhibiting neuroprotection in vitro and in vivo; (2) a glutaredoxin active site; and (3) a classical zinc binding domain. Comparative studies suggested that the peptide, NAPVSIPQ (NAP), was more efficacious than peptides derived from ADNF. ADNP, a potential mediator of VIP-associated neuronal survival, and the new peptide, a potential lead compound for drug design, are discussed below.
Asunto(s)
Encéfalo/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuropéptidos/fisiología , Fármacos Neuroprotectores , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Oligopéptidos , Transcripción Genética , Péptido Intestinal Vasoactivo/fisiologíaRESUMEN
The intracellular stress-induced proteins provide protection against toxic insults. Here, a 60,000-Da heat shock 60 (hsp60)-like protein was detected, with five different antibodies, in conditioned media derived from rat cortical astrocytes and a human neuroblastoma cell line. Extracellular neuroblastoma hsp60-like immunoreactivity was increased 3-fold in the presence of the neuropeptide vasoactive intestinal peptide (VIP) and was augmented 2-fold after temperature elevation. Intracellular hsp60 immunoreactivity was reduced 2-3-fold in the presence of VIP; this reduction was attenuated in the presence of brefeldin A, an inhibitor of protein secretion. In contrast, the activity of lactate dehydrogenase (LDH), an intracellular marker, did not change in the presence of VIP. Essentially no extracellular LDH activity was detected, indicating no cellular damage. A novel aspect for stress proteins having extracellular protective roles is suggested.
Asunto(s)
Chaperonina 60/aislamiento & purificación , Chaperonina 60/metabolismo , Neuroblastoma/metabolismo , Neuroglía/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Células Cultivadas , Humanos , Neuroblastoma/patología , Neuroglía/citología , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
Pituitary stimulating adenylate cyclase (PACAP) is a major regulatory peptide with two active molecular forms: PACAP-27 and PACAP-38. Both molecular forms promote neuronal survival and protect against neurotoxicity. Based on our previous hybrid peptide strategy in designing vasoactive intestinal peptide (VIP) antagonists, novel PACAP analogues were synthesized (neurotensin6-11 PACAP7-27 and neurotensin6-11 PACAP7-38). In addition to the hybrid modification, the methionine in position 17 was replaced by norleucine (Nle). Treatment of rat cerebral cortical cultures for five days with the putative PACAP antagonists (1 nM) resulted in a 35-45% reduction in neuronal cell counts as compared to controls. Neuronal cell death was already obtained at picomolar concentrations for the neurotensin6-11 PACAP7-27 antagonist with 70% death at 10(-8) M. Co-administration of the PACAP hybrid analogue with picomolar amounts of PACAP-27 or Nle17-PACAP-27 attenuated the reduction in neuronal cell counts. While the protective effects of both analogues exhibited a peak at 1 pM concentrations, the Nle-containing agonist displayed a broader range of active concentrations (10(-12)M-10(-9) M). The putative PACAP antagonist also inhibited sperm motility (golden hamster) in a dose-dependent manner as assessed in vitro. Complete inhibition was observed at 10 microM, suggesting a role for PACAP in sperm motility and sexual function. Thus, previous findings of a large number of PACAP and PACAP receptors in the nervous system and the reproductive system are now correlated with a function in neuronal survival and sperm motility. The structure-activity studies suggest that the methionine in position 17 and the first six amino acids are important in the determination of PACAP activity, knowledge that may facilitate PACAP-based drug design.
Asunto(s)
Neuronas/efectos de los fármacos , Neuropéptidos/farmacología , Péptidos/toxicidad , Motilidad Espermática/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/fisiología , Cricetinae , Masculino , Mesocricetus , Datos de Secuencia Molecular , Neuronas/citología , Neuropéptidos/antagonistas & inhibidores , Neuropéptidos/química , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/farmacología , Péptidos/síntesis química , Péptidos/química , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , RatasAsunto(s)
Enfermedad de Alzheimer/prevención & control , Neuronas/efectos de los fármacos , Neuropéptidos/farmacología , Neuropéptidos/fisiología , Administración Intranasal , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Animales , Muerte Celular/efectos de los fármacos , Humanos , Técnicas In Vitro , Aprendizaje/efectos de los fármacos , Memoria/efectos de los fármacos , Neuronas/patología , Ratas , Péptido Intestinal Vasoactivo/administración & dosificación , Péptido Intestinal Vasoactivo/farmacología , Péptido Intestinal Vasoactivo/fisiologíaAsunto(s)
Conducta Animal/efectos de los fármacos , Diseño de Fármacos , Periodicidad , Receptores de Péptido Intestinal Vasoactivo/fisiología , Conducta Sexual Animal/efectos de los fármacos , Péptido Intestinal Vasoactivo/agonistas , Péptido Intestinal Vasoactivo/antagonistas & inhibidores , Animales , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/fisiología , Disfunción Eréctil/tratamiento farmacológico , Humanos , Masculino , Mamíferos , Actividad Motora/efectos de los fármacos , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores de la Hormona Hipofisaria/efectos de los fármacos , Receptores de la Hormona Hipofisaria/fisiología , Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Especificidad por Sustrato , Péptido Intestinal Vasoactivo/síntesis químicaRESUMEN
Neurodegenerative diseases, in which neuronal cell disintegrate, bring about deteriorations in cognitive functions as is evidenced in millions of Alzheimer patients. A major neuropeptide, vasoactive intestinal peptide (VIP), has been shown to be neuroprotective and to play an important role in the acquisition of learning and memory. A potent lipophilic analogue to VIP now has been synthesized, [stearyl-norleucine17]VIP ([St-Nle17]VIP), that exhibited neuroprotection in model systems related to Alzheimer disease. The beta-amyloid peptide is a major component of the cerebral amyloid plaque in Alzheimer disease and has been shown to be neurotoxic. We have found a 70% loss in the number of neurons in rat cerebral cortical cultures treated with the beta-amyloid peptide (amino acids 25-35) in comparison to controls. This cell death was completely prevented by cotreatment with 0.1 pM [St-Nle17]VIP. Furthermore, characteristic deficiencies in Alzheimer disease result from death of cholinergic neurons. Rats treated with a cholinergic blocker (ethylcholine aziridium) have been used as a model for cholinergic deficits. St-Nle-VIP injected intracerebroventricularly or delivered intranasally prevented impairments in spatial learning and memory associated with cholinergic blockade. These studies suggest both an unusual therapeutic strategy for treatment of Alzheimer deficiencies and a means for noninvasive peptide administration to the brain.
Asunto(s)
Enfermedad de Alzheimer/prevención & control , Péptidos beta-Amiloides/antagonistas & inhibidores , Neuronas/citología , Péptido Intestinal Vasoactivo/farmacología , Administración Intranasal , Animales , Aziridinas/farmacología , Muerte Celular/efectos de los fármacos , Células Cultivadas , Colina/análogos & derivados , Colina/farmacología , Aprendizaje/efectos de los fármacos , Lípidos , Masculino , Memoria/efectos de los fármacos , Bloqueantes Neuromusculares , Parasimpatolíticos/farmacología , Ratas , Ratas Wistar , Solubilidad , Relación Estructura-ActividadRESUMEN
309 DNA samples obtained from healthy volunteers were tested for the cystic fibrosis mutations DF508 and W1282X. 14 carriers were identified, 7 of each mutation. Since the 2 mutations account for only 80% of CF mutations, the actual number of carriers is 1 in 18. In spite of the fact that this is only a pilot study, the results suggest that screening for CF carriers is feasible and that it identifies unambiguously those who carry the CF genes. When testing for CF carriers becomes available for the general public, it will undoubtedly contribute in reducing significantly the incidence of children born with the disease.