RAPID COMMUNICATION: Nerve growth factor influences cleavage rate and embryo development in sheep.

Recent information about Nerve growth factor (NGF), a protein traditionally associated to the nervous system that regulates survival and maturation of developing neurons, suggests that it may exert action also on different levels in the reproductive system. The aim of this study was to evaluate the effect of NGF added during in vitro oocyte maturation, fertilization or in vitro embryo development in sheep. Nerve growth factor was supplemented to the culture medium at 0, 100, or 1,000 ng/mL, during either in vitro maturation (Exp. 1), in vitro fertilization (Exp. 2), or in vitro culture (Exp. 3). In addition, NGF mRNA expression was determined in cumulus cells and oocytes. Nerve growth factor induced early cleavage when added during oocyte maturation or fertilization, improved embryo development when added during fertilization, and had no significant effect when added during embryo culture. In general, the effect was more evident with 100 rather than 1,000 ng/mL (P < 0.05). Expression of endogenous NGF was not detected in oocytes, and increased in cumulus cells when 1,000 ng/mL of NGF was added during fertilization, but not during maturation and embryo culture. In conclusion, the addition of NGF during oocyte maturation and fertilization affects in vitro cleavage and embryo development in sheep. We suggest a possible effect of this growth factor on oocyte maturation and mainly on the fertilization process.

IFNγ triggers a LIGHT-dependent selective death of motoneurons contributing to the non-cell-autonomous effects of mutant SOD1.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that primarily affects motoneurons in the brain and spinal cord. Dominant mutations in superoxide dismutase-1 (SOD1) cause a familial form of ALS. Mutant SOD1-damaged glial cells contribute to ALS pathogenesis by releasing neurotoxic factors, but the mechanistic basis of the motoneuron-specific elimination is poorly understood. Here, we describe a motoneuron-selective death pathway triggered by activation of lymphotoxin-ß receptor (LT-ßR) by LIGHT, and operating by a novel signaling scheme. We show that astrocytes expressing mutant SOD1 mediate the selective death of motoneurons through the proinflammatory cytokine interferon-γ (IFNγ), which activates the LIGHT-LT-ßR death pathway. The expression of LIGHT and LT-ßR by motoneurons in vivo correlates with the preferential expression of IFNγ by motoneurons and astrocytes at disease onset and symptomatic stage in ALS mice. Importantly, the genetic ablation of Light in an ALS mouse model retards progression, but not onset, of the disease and increases lifespan. We propose that IFNγ contributes to a cross-talk between motoneurons and astrocytes causing the selective loss of some motoneurons following activation of the LIGHT-induced death pathway.

Mitochondrial DNA HVRI variation in Balearic populations.

The Balearic archipelago (Majorca, Minorca, and Ibiza islands and the Chuetas, a small and inbred community of descendants of Sephardic Jews) and Valencia were studied by means of the sequencing of a 404-bp segment of hypervariable region I (HVRI) mtDNA in 231 individuals. In total, 127 different haplotypes defined by 92 variable positions were identified. The incidence of unique haplotypes was very low, especially in Ibiza and the Chuetas. A remarkable observation in the Chueta community was the high frequency (23%) of preHV-1, a Middle Eastern lineage that is closely related, though not identical, to many others found at high frequencies in different Jewish populations. The presence of this haplogroup convincingly supported the Jewish origin of the Chueta community. The studied populations showed a reduced African contribution, and no individuals were detected with North African haplogroup U6, indicating a lack of maternal contribution from the Moslem settlement to these populations. Only Ibiza showed a lower diversity, indicating a possible genetic drift effect, also supported by the historical information known about this island. The variability in the sequence of mtDNA hypervariable region I correlated well with the existing information from the populations, with the exception of that of the Y-chromosome, which could indicate a differential contribution of the maternal and paternal lineages to the genetic pool of the Balearic Islands. The phylogenetic trees showed the intermediate position of the Chueta population between the Middle Eastern and Majorcan samples, confirming the Jewish origin of this population and their Spanish admixture.

The expression of PEA-15 (phosphoprotein enriched in astrocytes of 15 kDa) defines subpopulations of astrocytes and neurons throughout the adult mouse brain.

Phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) is an abundant phosphoprotein in primary cultures of mouse brain astrocytes. Its capability to interact with members of the apoptotic and mitogen activated protein (MAP) kinase cascades endows PEA-15 with anti-apoptotic and anti-proliferative properties. We analyzed the in vivo cellular sources of PEA-15 in the normal adult mouse brain using a novel polyclonal antibody. Immunohistochemical assays revealed numerous PEA-15-immunoreactive cells throughout the brain of wild-type adult mice while no immunoreactive signal was observed in the brain of PEA-15 -/- mice. Cell morphology and double immunofluorescent staining showed that both astrocytes and neurons could be cellular sources of PEA-15. Closer examination revealed that in a given area only part of the astrocytes expressed the protein. The hippocampus was the most striking example of this heterogeneity, a spatial segregation restricting PEA-15 positive astrocytes to the CA1 and CA3 regions. A PEA-15 immunoreactive signal was also observed in a few cells within the subventricular zone and the rostral migratory stream. In vivo analysis of an eventual PEA-15 regulation in astrocytes was performed using a model of astrogliosis occurring along motor neurons degeneration, the transgenic mouse expressing the mutant G93A human superoxyde-dismutase-1, a model of amyotrophic lateral sclerosis. We observed a marked up-regulation of PEA-15 in reactive astrocytes that had developed throughout the ventral horn of the lumbar spinal cord of the transgenic mice. The heterogeneous cellular expression of the protein and its increased expression in pathological situations, combined with the known properties of PEA-15, suggest that PEA-15 expression is associated with a particular metabolic status of cells challenged with potentially apoptotic and/or proliferative signals.

Secuencias de la HVRI del mtDNA en poblaciones de Valencia y Baleares / HVRI mtDNA in Valencia and Balearic populations

Se ha analizado el DNA mitocondrial (mtDNA) de 231 individuos no relacionados y autóctonos de las islas Baleares y Valencia. La isla de Ibiza resultó la población más homogénea y Valencia fue la más variable. La baja diversidad de haplotipos mitocondriales detectada en Ibiza y en Chuetas indican un efecto fundador, así como diversos cuellos de botella poblacionales que han sufrido las dos poblaciones. Los resultados ponen de manifiesto la necesaria prudencia en el momento de utilizar las secuencias de mtDNA en el análisis de filiación e identificación de restos en estas poblaciones (AU)

The molecular bases of Alzheimer's disease and other neurodegenerative disorders.

Alzheimer's disease, the cause of one of the most common types of dementia, is a brain disorder affecting the elderly and is characterized by the formation of two main protein aggregates: senile plaques and neurofibrillary tangles, which are involved in the process leading to progressive neuronal degeneration and death. Neurodegeneration in Alzheimer's disease is a pathologic condition of cells rather than an accelerated way of aging. The senile plaques are generated by a deposition in the human brain of fibrils of the beta-amyloid peptide (Abeta), a fragment derived from the proteolytic processing of the amyloid precursor protein (APP). Tau protein is the major component of paired helical filaments (PHFs), which form a compact filamentous network described as neurofibrillary tangles (NFTs). Experiments with hippocampal cells in culture have indicated a relationship between fibrillary amyloid and the cascade of molecular signals that trigger tau hyperphosphorylations. Two main protein kinases have been shown to be involved in anomalous tau phosphorylations: the cyclin-dependent kinase Cdk5 and glycogen synthase kinase GSK3beta. Cdk5 plays a critical role in brain development and is associated with neurogenesis as revealed by studies in brain cells in culture and neuroblastoma cells. Deregulation of this protein kinase as induced by extracellular amyloid loading results in tau hyperphosphorylations, thus triggering a sequence of molecular events that lead to neuronal degeneration. Inhibitors of Cdk5 and GSK3beta and antisense oligonucleotides exert protection against neuronal death. On the other hand, there is cumulative evidence from studies in cultured brain cells and on brains that oxidative stress constitutes a main factor in the modification of normal signaling pathways in neuronal cells, leading to biochemical and structural abnormalities and neurodegeneration as related to the pathogenesis of Alzheimer's disease. This review is focused on the main protein aggregates responsible for neuronal death in both sporadic and familial forms of Alzheimer's disease, as well as on the alterations in the normal signaling pathways of functional neurons directly involved in neurodegeneration. The analysis is extended to the action of neuroprotective factors including selective inhibitors of tau phosphorylating protein kinases, estrogens, and antioxidants among other molecules that apparently prevent neuronal degeneration.

Superoxide dismutase and the death of motoneurons in ALS.

Amyotrophic lateral sclerosis (ALS) is a lethal disease that is characterized by the relentless death of motoneurons. Mutations to Cu-Zn superoxide dismutase (SOD), though occurring in just 2-3% of individuals with ALS, remain the only proven cause of the disease. These mutations structurally weaken SOD, which indirectly decreases its affinity for Zn. Zn-deficient SOD induces apoptosis in motoneurons through a mechanism involving peroxynitrite. Importantly, Zn-deficient wild-type SOD is just as toxic as Zn-deficient ALS mutant SOD, suggesting that the loss of Zn from wild-type SOD could be involved in the other 98% of cases of ALS. Zn-deficient SOD could therefore be an important therapeutic target in all forms of ALS.

Liposome-delivered superoxide dismutase prevents nitric oxide-dependent motor neuron death induced by trophic factor withdrawal.

Inhibition of nitric oxide synthesis prevents rat embryonic motor neurons from undergoing apoptosis when initially cultured without brain-derived neurotrophic factor. Using an improved cell culture medium, we found that the partial withdrawal of trophic support even weeks after motor neurons had differentiated into a mature phenotype still induced apoptosis through a process dependent upon nitric oxide. However, nitric oxide itself was not directly toxic to motor neurons. To investigate whether intracellular superoxide contributed to nitric oxide-dependent apoptosis, we developed a novel method using pH-sensitive liposomes to deliver Cu, Zn superoxide dismutase intracellularly into motor neurons. Intracellular superoxide dismutase prevented motor neuron apoptosis from trophic factor withdrawal, whereas empty liposomes, inactivated superoxide dismutase in liposomes or extracellular superoxide dismutase did not. Neither hydrogen peroxide nor nitrite added separately or in combination affected motor neuron survival. Our results suggest that a partial reduction in trophic support induced motor neuron apoptosis by a process requiring the endogenous production of both nitric oxide and superoxide, irrespective of the extent of motor neuron maturation in culture.

Induction of nitric oxide-dependent apoptosis in motor neurons by zinc-deficient superoxide dismutase.

Mutations in copper, zinc superoxide dismutase (SOD) have been implicated in the selective death of motor neurons in 2 percent of amyotrophic lateral sclerosis (ALS) patients. The loss of zinc from either wild-type or ALS-mutant SODs was sufficient to induce apoptosis in cultured motor neurons. Toxicity required that copper be bound to SOD and depended on endogenous production of nitric oxide. When replete with zinc, neither ALS-mutant nor wild-type copper, zinc SODs were toxic, and both protected motor neurons from trophic factor withdrawal. Thus, zinc-deficient SOD may participate in both sporadic and familial ALS by an oxidative mechanism involving nitric oxide.

Nitric oxide-dependent production of cGMP supports the survival of rat embryonic motor neurons cultured with brain-derived neurotrophic factor.

Trophic factor deprivation induces neuronal nitric oxide synthase (NOS) and apoptosis of rat embryonic motor neurons in culture. We report here that motor neurons constitutively express endothelial NOS that helps support the survival of motor neurons cultured with brain-derived neurotrophic factor (BDNF) by activating the nitric oxide-dependent soluble guanylate cyclase. Exposure of BDNF-treated motor neurons to nitro-L-arginine methyl ester (L-NAME) decreased cell survival 40-50% 24 hr after plating. Both low steady-state concentrations of exogenous nitric oxide (<0.1 microM) and cGMP analogs protected BDNF-treated motor neurons from death induced by L-NAME. Equivalent concentrations of cAMP analogs did not affect cell survival. Inhibition of nitric oxide-sensitive guanylate cyclase with 2 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reduced the survival of BDNF-treated motor neurons by 35%. cGMP analogs also protected from ODQ-induced motor neuron death, whereas exogenous nitric oxide did not. In all cases, cell death was prevented with caspase inhibitors. Our results suggest that nitric oxide-stimulated cGMP synthesis helps to prevent apoptosis in BDNF-treated motor neurons.

Nitric oxide and superoxide contribute to motor neuron apoptosis induced by trophic factor deprivation.

Primary cultures of rat embryonic motor neurons deprived of brain-derived neurotrophic factor (BDNF) induce neuronal nitric oxide synthase (NOS) within 18 hr. Subsequently, >60% of the neurons undergo apoptosis between 18 and 24 hr after plating. Nitro-L-arginine and nitro-L-arginine methyl ester (L-NAME) prevented motor neuron death induced by trophic factor deprivation. Exogenous generation of nitric oxide at concentrations lower than 100 nM overcame the protection by L-NAME. Manganese tetrakis (4-benzoyl acid) porphyrin, a cell-permeant superoxide scavenger, also prevented nitric oxide-dependent motor neuron death. Motor neurons cultured without trophic support rapidly became immunoreactive for nitrotyrosine when compared with motor neurons incubated with BDNF, L-NAME, or manganese TBAP. Our results suggest that peroxynitrite, a strong oxidant formed by the reaction of NO and superoxide, plays an important role in the induction of apoptosis in motor neurons deprived of trophic factors and that BDNF supports motor neuron survival in part by preventing neuronal NOS expression.

Role of endogenous nitric oxide and peroxynitrite formation in the survival and death of motor neurons in culture.

Motor neuron survival is highly dependent on trophic factor supply. Deprivation of trophic factors results in induction of neuronal NOS, which is also found in pathological conditions. Growing evidence suggests that motor neuron degeneration involves peroxynitrite formation. Trophic factors modulate peroxynitrite toxicity (Estévez et al., 1995; Shin et al., 1996; Spear et al., 1997). Whether a trophic factor prevents or potentiates peroxynitrite toxicity depends upon when the cells are exposed to the trophic factor (Table 1). These results strongly suggest that a trophic factor that can protect neurons under optimal conditions, but under stressful conditions can increase cell death. In this context, it is possible that trophic factors or cytokines produced as a response to damage may potentiate rather than prevent motor neuron death. A similar argument may apply to the therapeutic administration of trophic factors to treat neurodegenerative diseases. Similarly, the contrasting actions of NO on motor neurons may have important consequences for the potential use of nitric oxide synthase inhibitors in the treatment of ALS and other related neurodegenerative diseases.

Nerve growth factor protects PC12 cells against peroxynitrite-induced apoptosis via a mechanism dependent on phosphatidylinositol 3-kinase.

Nerve growth factor (NGF) prevents apoptosis induced by the oxidant peroxynitrite in undifferentiated PC12 rat pheochromocytoma cells. Previous studies have shown that activation of phosphatidylinositol 3-kinase (PI 3-kinase) by NGF via the TrkA receptor tyrosine kinase protects PC12 cells from serum deprivation-induced apoptosis. We found that two PI 3-kinase inhibitors, wortmannin and LY294002, eliminated the protection NGF provided against peroxynitrite-induced apoptosis at concentrations consistent with their effectiveness as PI 3-kinase inhibitors. When the activity of PI 3-kinase was assayed in phosphotyrosine immunoprecipitates after treatment of PC12 cells with peroxynitrite, PI 3-kinase activity was reduced by 50% of that detected in control cells, whereas PI 3-kinase activity in NGF-treated cells was unaffected by peroxynitrite. If an antibody against PI 3-kinase was used to immunoprecipitate the enzyme, treatment with peroxynitrite had no effect on activity. Therefore, peroxynitrite appeared to disrupt interactions between PI 3-kinase and phosphotyrosine proteins, rather than directly inhibiting the enzyme. NGF also activates p21Ras-dependent pathways, but this did not appear to be required for NGF to exert its protective effect against peroxynitrite. PC12 cells expressing a dominant inhibitory mutant of p21Ras were equally susceptible to peroxynitrite-induced apoptosis, which was prevented by NGF. Wortmannin was also able to block the protective effect of NGF in the p21Ras mutant cell line. Although many signaling pathways are activated by NGF, these results suggest that a PI 3-kinase-dependent pathway is important for inhibiting peroxynitrite-induced apoptosis.

Riluzole promotes survival of rat motoneurons in vitro by stimulating trophic activity produced by spinal astrocyte monolayers.

In the present study we have assessed whether riluzole stimulates the production of trophic activities for motoneurons by spinal astrocyte cultures. Astrocyte monolayers prepared from new-born rats were exposed to vehicle or riluzole (1-10 microM) for 30-36 h, then washed and further incubated without riluzole for 24 h in L15 medium to obtain the astrocyte conditioned media (ACM). Motoneuron-enriched cultures were used to test the ability of the ACM to support motoneuron viability. Astrocyte monolayers exposed to 1 microM riluzole did not show changes in morphology or in DNA or protein synthesis. However, the conditioned medium obtained from astrocyte monolayers after this treatment increased motoneuron survival compared to that from vehicle-treated cultures. A similar effect was found when astrocytes were exposed to a higher riluzole concentration (10 microM) but with greater dilutions of the conditioned medium. This trophic activity was abolished by boiling or after treatment with trypsin. These findings strongly suggest the existence of a new trophic mechanism, through which riluzole may exert motoneuron protection.

IA-type K+ channel blockers promote survival of cortical neurons in culture: involvement of L-type Ca2+ channels.

Dendrotoxin (DTX), a well characterized IA-type potassium channel blocker, directly added to the culture medium had no effect on survival of cultured cortical neurons at 6 or 14 days in vitro. On the contrary, neurons exposed to DTX remained in better condition than untreated ones. In an attempt to demonstrate the mechanisms by which DTX may affect neuronal survival we studied its effect in co-cultures of cortical neurons and astrocytes submitted to successive medium changes. After the second change of medium, at 9 days in vitro, the neuronal number in controls decreased by 43%, while in cultures receiving astrocyte-conditioned medium the cell loss was significantly reduced (15%, p < 0.01) with respect to control conditions. When DTX was added to the culture medium neuronal loss was also significantly prevented (25% for 1 microM DTX, p < 0.01) with respect to control conditions. 4-Aminopyridine (4-AP) and 21 mM K+ also preserved neurons. The L-type calcium channel antagonist nifedipine (5 microM) abolished the protective effect of DTX and 4-AP. These results show that K+ channel blockade induces protection against damage produced by repetitive medium change and that this effect is mediated by L-type Ca2+ channels.

Acidic fibroblast growth factor enhances peroxynitrite-induced apoptosis in primary murine fibroblasts.

Oxidative stress is considered a major mediator of apoptosis in several cellular systems. Peroxynitrite is a highly toxic oxidant formed by the reaction of nitric oxide with superoxide. Primary embryonic murine fibroblasts, exposed to 1 mM peroxynitrite, resulted in delayed cell death characterized by membrane blebbing, cytoplasmic shrinkage, nuclear condensation, and DNA fragmentation that were more characteristic of apoptosis than necrosis. In addition, both morphological alterations and DNA fragmentation were inhibited by the endonuclease inhibitor aurintricarboxylic acid. Pretreatment of fibroblasts with acidic fibroblast growth factor (FGF-1) markedly enhanced peroxynitrite-induced apoptosis, an observation restricted to immediate-early transcriptional and activated tyrosine phosphorylation processes. FGF-1 pretreatment had no modulatory effect on cell death elicited by other reactive oxygen species, suggesting that enhancement of apoptosis involves a unique relationship between peroxynitrite and the growth factor. Exposure of cells to peroxynitrite resulted in immediate tyrosine nitration of several polypeptides, including major targets with estimated molecular masses of 62, 68, and 77 kDa. Pretreatment with FGF-1 did not alter targets of peroxynitrite-mediated tyrosine nitration, but rather increased the total amount of this amino acid modification. Treatment with other reactive oxygen species failed to induce tyrosine nitration. Collectively, these efforts demonstrate that FGF-1 transiently renders primary fibroblasts more sensitive to peroxynitrite-induced apoptosis. In addition, results presented here predict a pivotal role for FGF-1 and peroxynitrite-induced cytotoxicity during the resolution of inflammation and repair processes in vivo.

N-acetylsuccinimidylglutamate, a cyclic imide form of the peptide N-acetylaspartylglutamate, is present in low micromolar concentrations in murine and bovine CNS.

N-Acetylsuccinimidylglutamate [(asu)NAAG], a cyclic form of the peptide N-acetylaspartylglutamate (NAAG) in which the aspartyl residue is linked to glutamate via the alpha- and beta-carboxylates, was identified and quantified by HPLC in the murine and bovine CNS. In the rat, the highest concentrations of (asu)NAAG were detected in the spinal cord (1.83 +/- 0.15 pmol/mg of wet tissue weight) and brainstem (1.16 +/- 0.08 pmol/mg wet weight), whereas the levels were below the limit of detection in cerebellum, hippocampus, and cerebral cortex. (Asu)NAAG was also detected in significant amount in the superior colliculus and lateral geniculate nucleus (1.17 +/- 0.05 and 0.82 +/- 0.13 pmol/mg we weight, respectively). Although the tissue content of (asu)NAAG was about three orders of magnitude lower than that of NAAG, levels of both peptides were positively correlated among different CNS regions (r=0.74, p<0.003). In the rat spinal cord, (asu)NAAG levels progressively increased from week 2 to month 12 after birth. In bovine spinal cord, the contents of (asu)NAAG and NAAG were comparable in gray and white matter as well as in the dorsal and ventral horns. These results suggest that NAAG and (asu)-NAAG are closely related metabolically and raise the question of the physiological significance of such a cyclic peptide.

N-acetylaspartylglutamate acetoxymethyl triester (NAAG.AM) as a tool for loading the neuropeptides NAAG and succinimidyl-NAAG into intact cells: effect on [3H]-dopamine exocytosis.

Although N-acetylaspartylglutamate (NAAG) is one of the neuropeptides found in highest concentrations in the mammalian central nervous system, its functional role in neuronal signaling has not been definitively established. In some neuronal populations, NAAG is concentrated in nerve terminals and thus, it may play a role in the cytoplasmic events underlying neurotransmitter exocytosis. In the present study we have validated the use of the synthetic derivative NAAG-acetoxymethyl triester (NAAG.AM) as a tool to increase the intracellular levels of the peptide and assessed the ability of NAAG to regulate [3H]-dopamine ([3H]-DA) secretion in PC12 cells. Enzymatic degradation of NAAG.AM by nonspecific brain esterases resulted in the progressive formation of NAAG and succinimidyl-NAAG (Asu-NAAG). However, only 8% of NAAG.AM was converted to NAAG. Significant amounts of NAAG (1 nmol/mg protein) were demonstrable in cultures of the neuroblastoma cell line N2A following incubation with NAAG.AM for 2 h, with the concentration of (Asu)-NAAG being at least 100-fold higher. The pheochromocytoma cell line PC12 was used to assess the influence of loaded NAAG derivatives on [3H]-DA exocytosis. Incubation with 0.1-1 mM NAAG.AM did not affect the basal efflux or total content of [3H]-DA. However, it induced a dose-dependent decrease of [3H]-DA secretion in response to 56 mM KCl depolarization reaching an inhibition of 49% with 1 mM NAAG.AM. In contrast, NAAG.AM did not affect secretion induced by the calcium ionophore A23187 (100 microM). The present study validates the use of NAAG.AM as a tool to load NAAG derivatives into intact cells and provides preliminary evidence for an intracellular role of the peptide.