The metallochaperone Atox1 plays a critical role in perinatal copper homeostasis

Copper plays a fundamental role in the biochemistry of all aerobic organisms. The delivery of this metal to specific intracellular targets is mediated by metallochaperones. To elucidate the role of the metallochaperone Atox1, we analyzed mice with a disruption of the Atox1 locus. Atox1−/− mice failed to thrive immediately after birth, with 45% of pups dying before weaning. Surviving animals exhibited growth failure, skin laxity, hypopigmentation, and seizures because of perinatal copper deficiency. Maternal Atox1 deficiency markedly increased the severity of Atox1−/− phenotype, resulting in increased perinatal mortality as well as severe growth retardation and congenital malformations among surviving Atox1−/− progeny. Furthermore, Atox1-deficient cells accumulated high levels of intracellular copper, and metabolic studies indicated that this defect was because of impaired cellular copper efflux. Taken together, these data reveal a direct role for Atox1 in trafficking of intracellular copper to the secretory pathway of mammalian cells and demonstrate that this metallochaperone plays a critical role in perinatal copper homeostasis.

Lipid thioesters derived from acylated proteins accumulate in infantile neuronal ceroid lipofuscinosis: correction of the defect in lymphoblasts by recombinant palmitoyl-protein thioesterase.

Palmitoyl-protein thioesterase is a lysosomal long-chain fatty acyl hydrolase that removes fatty acyl groups from modified cysteine residues in proteins. Mutations in palmitoyl-protein thioesterase were recently found to cause the neurodegenerative disorder infantile neuronal ceroid lipofuscinosis, a disease characterized by accumulation of amorphous granular deposits in cortical neurons, leading to blindness, seizures, and brain death by the age of three. In the current study, we demonstrate that [35S]cysteine-labeled lipid thioesters accumulate in immortalized lymphoblasts of patients with infantile neuronal ceroid lipofuscinosis. The accumulation in cultured cells is reversed by the addition of recombinant palmitoyl-protein thioesterase that is competent for lysosomal uptake through the mannose-6-phosphate receptor. The [35S]cysteine-labeled lipids are substrates for palmitoyl-protein thioesterase in vitro, and their formation requires prior protein synthesis. These data support a role for palmitoyl-protein thioesterase in the lysosomal degradation of S-acylated proteins and define a major new pathway for the catabolism of acylated proteins in the lysosome.

Long-lasting reduction of inhibitory function and gamma-aminobutyric acid type A receptor subunit mRNA expression in a model of temporal lobe epilepsy.

This study evaluated hippocampal inhibitory function and the level of expression of gamma-aminobutyric acid type A (GABAA) receptor mRNA in an in vivo model of epilepsy. Chronic recurrent limbic seizures were induced in rats using injections of pilocarpine. Electrophysiological studies performed on hippocampal slices prepared from control and epileptic animals 1 to 2 months after pilocarpine injections demonstrated a significant hyperexcitability in the epileptic animals. Reduced levels of mRNA expression for the alpha 2 and alpha 5 subunits of the GABAA receptors were evident in the CA1, CA2, and CA3 regions of the hippocampus of epileptic animals. No decrease in mRNA encoding alpha 1, beta 2, or gamma 2 GABAA receptor subunits was observed. In addition, no change in the mRNA levels of alpha CaM kinase II was seen. Selective decreases in mRNA expression did not correlate with neuronal cell loss. The results indicate that selective, long-lasting reduction of GABAA subunit mRNA expression and increased excitability, possibly reflecting loss of GABAergic inhibition, occur in an in vivo model of partial complex epilepsy.

Reconstitution of the hippocampal mossy fiber and associational-commissural pathways in a novel dissociated cell culture system.

Synapses of the hippocampal mossy fiber pathway exhibit several characteristic features, including a unique form of long-term potentiation that does not require activation of the N-methyl-D-aspartate receptor by glutamate, a complex postsynaptic architecture, and sprouting in response to seizures. However, these connections have proven difficult to study in hippocampal slices because of their relative paucity (<0.4%) compared to commissural-collateral synapses. To overcome this problem, we have developed a novel dissociated cell culture system in which we have enriched mossy fiber synapses by increasing the ratio of granule-to-pyramidal cells. As in vivo, mossy fiber connections are composed of large dynorphin A-positive varicosities contacting complex spines (but without a restricted localization). The elementary synaptic connections are glutamatergic, inhibited by dynorphin A, and exhibit N-methyl-D-aspartate-independent long-term potentiation. Thus, the simplicity and experimental accessibility of this enriched in vitro mossy fiber pathway provides a new perspective for studying nonassociative plasticity in the mammalian central nervous system.

The ALIAmide palmitoylethanolamide and cannabinoids, but not anandamide, are protective in a delayed postglutamate paradigm of excitotoxic death in cerebellar granule neurons.

The amino acid L-glutamate is a neurotransmitter that mediates fast neuronal excitation in a majority of synapses in the central nervous system. Glutamate stimulates both N-methyl-D-aspartate (NMDA) and non-NMDA receptors. While activation of NMDA receptors has been implicated in a variety of neurophysiologic processes, excessive NMDA receptor stimulation (excitotoxicity) is thought to be primarily responsible for neuronal injury in a wide variety of acute neurological disorders including hypoxia-ischemia, seizures, and trauma. Very little is known about endogenous molecules and mechanisms capable of modulating excitotoxic neuronal death. Saturated N-acylethanolamides like palmitoylethanolamide accumulate in ischemic tissues and are synthesized by neurons upon excitatory amino acid receptor activation. Here we report that palmitoylethanolamide, but not the cognate N-acylamide anandamide (the ethanolamide of arachidonic acid), protects cultured mouse cerebellar granule cells against glutamate toxicity in a delayed postagonist paradigm. Palmitoylethanolamide reduced this injury in a concentration-dependent manner and was maximally effective when added 15-min postglutamate. Cannabinoids, which like palmitoylethanolamide are functionally active at the peripheral cannabinoid receptor CB2 on mast cells, also prevented neuron loss in this delayed postglutamate model. Furthermore, the neuroprotective effects of palmitoylethanolamide, as well as that of the active cannabinoids, were efficiently antagonized by the candidate central cannabinoid receptor (CB1) agonist anandamide. Analogous pharmacological behaviors have been observed for palmitoylethanolamide (ALI-Amides) in downmodulating mast cell activation. Cerebellar granule cells expressed mRNA for CB1 and CB2 by in situ hybridization, while two cannabinoid binding sites were detected in cerebellar membranes. The results suggest that (i) non-CB1 cannabinoid receptors control, upon agonist binding, the downstream consequences of an excitotoxic stimulus; (ii) palmitoylethanolamide, unlike anandamide, behaves as an endogenous agonist for CB2-like receptors on granule cells; and (iii) activation of such receptors may serve to downmodulate deleterious cellular processes following pathological events or noxious stimuli in both the nervous and immune systems.

Temporal fluctuations in coherence of brain waves.

As a measure of dynamical structure, short-term fluctuations of coherence between 0.3 and 100 Hz in the electroencephalogram (EEG) of humans were studied from recordings made by chronic subdural macroelectrodes 5-10 mm apart, on temporal, frontal, and parietal lobes, and from intracranial probes deep in the temporal lobe, including the hippocampus, during sleep, alert, and seizure states. The time series of coherence between adjacent sites calculated every second or less often varies widely in stability over time; sometimes it is stable for half a minute or more. Within 2-min samples, coherence commonly fluctuates by a factor up to 2-3, in all bands, within the time scale of seconds to tens of seconds. The power spectrum of the time series of these fluctuations is broad, extending to 0.02 Hz or slower, and is weighted toward the slower frequencies; little power is faster than 0.5 Hz. Some records show conspicuous swings with a preferred duration of 5-15s, either irregularly or quasirhythmically with a broad peak around 0.1 Hz. Periodicity is not statistically significant in most records. In our sampling, we have not found a consistent difference between lobes of the brain, subdural and depth electrodes, or sleeping and waking states. Seizures generally raise the mean coherence in all frequencies and may reduce the fluctuations by a ceiling effect. The coherence time series of different bands is positively correlated (0.45 overall); significant nonindependence extends for at least two octaves. Coherence fluctuations are quite local; the time series of adjacent electrodes is correlated with that of the nearest neighbor pairs (10 mm) to a coefficient averaging approximately 0.4, falling to approximately 0.2 for neighbors-but-one (20 mm) and to < 0.1 for neighbors-but-two (30 mm). The evidence indicates fine structure in time and space, a dynamic and local determination of this measure of cooperativity. Widely separated frequencies tending to fluctuate together exclude independent oscillators as the general or usual basis of the EEG, although a few rhythms are well known under special conditions. Broad-band events may be the more usual generators. Loci only a few millimeters apart can fluctuate widely in seconds, either in parallel or independently. Scalp EEG coherence cannot be predicted from subdural or deep recordings, or vice versa, and intracortical microelectrodes show still greater coherence fluctuation in space and time. Widely used computations of chaos and dimensionality made upon data from scalp or even subdural or depth electrodes, even when reproducible in successive samples, cannot be considered representative of the brain or the given structure or brain state but only of the scale or view (receptive field) of the electrodes used. Relevant to the evolution of more complex brains, which is an outstanding fact of animal evolution, we believe that measures of cooperativity are likely to be among the dynamic features by which major evolutionary grades of brains differ.

Spontaneous inflammatory demyelinating disease in transgenic mice showing central nervous system-specific expression of tumor necrosis factor alpha.

Cytokines are now recognized to play important roles in the physiology of the central nervous system (CNS) during health and disease. Tumor necrosis factor alpha (TNF-alpha) has been implicated in the pathogenesis of several human CNS disorders including multiple sclerosis, AIDS dementia, and cerebral malaria. We have generated transgenic mice that constitutively express a murine TNF-alpha transgene, under the control of its own promoter, specifically in their CNS and that spontaneously develop a chronic inflammatory demyelinating disease with 100% penetrance from around 3-8 weeks of age. High-level expression of the transgene was seen in neurons distributed throughout the brain. Disease is manifested by ataxia, seizures, and paresis and leads to early death. Histopathological analysis revealed infiltration of the meninges and CNS parenchyma by CD4+ and CD8+ T lymphocytes, widespread reactive astrocytosis and microgliosis, and focal demyelination. The direct action of TNF-alpha in the pathogenesis of this disease was confirmed by peripheral administration of a neutralizing anti-murine TNF-alpha antibody. This treatment completely prevented the development of neurological symptoms, T-cell infiltration into the CNS parenchyma, astrocytosis, and demyelination, and greatly reduced the severity of reactive microgliosis. These results demonstrate that overexpression of TNF-alpha in the CNS can cause abnormalities in nervous system structure and function. The disease induced in TNF-alpha transgenic mice shows clinical and histopathological features characteristic of inflammatory demyelinating CNS disorders in humans, and these mice represent a relevant in vivo model for their further study.

A nerve growth factor peptide retards seizure development and inhibits neuronal sprouting in a rat model of epilepsy.

Kindling, an animal model of epilepsy wherein seizures are induced by subcortical electrical stimulation, results in the upregulation of neurotrophin mRNA and protein in the adult rat forebrain and causes mossy fiber sprouting in the hippocampus. Intraventricular infusion of a synthetic peptide mimic of a nerve growth factor domain that interferes with the binding of neurotrophins to their receptors resulted in significant retardation of kindling and inhibition of mossy fiber sprouting. These findings suggest a critical role for neurotrophins in both kindling and kindling-induced synaptic reorganization.

Impairment of synaptic vesicle clustering and of synaptic transmission, and increased seizure propensity, in synapsin I-deficient mice.

Synapsin I has been proposed to be involved in the modulation of neurotransmitter release by controlling the availability of synaptic vesicles for exocytosis. To further understand the role of synapsin I in the function of adult nerve terminals, we studied synapsin I-deficient mice generated by homologous recombination. The organization of synaptic vesicles at presynaptic terminals of synapsin I-deficient mice was markedly altered: densely packed vesicles were only present in a narrow rim at active zones, whereas the majority of vesicles were dispersed throughout the terminal area. This was in contrast to the organized vesicle clusters present in terminals of wild-type animals. Release of glutamate from nerve endings, induced by K+,4-aminopyridine, or a Ca2+ ionophore, was markedly decreased in synapsin I mutant mice. The recovery of synaptic transmission after depletion of neurotransmitter by high-frequency stimulation was greatly delayed. Finally, synapsin I-deficient mice exhibited a strikingly increased response to electrical stimulation, as measured by electrographic and behavioral seizures. These results provide strong support for the hypothesis that synapsin I plays a key role in the regulation of nerve terminal function in mature synapses.

Stress and antidepressants differentially regulate neurotrophin 3 mRNA expression in the locus coeruleus.

The mechanisms by which stress and anti-depressants exert opposite effects on the course of clinical depression are not known. However, potential candidates might include neurotrophic factors that regulate the development, plasticity, and survival of neurons. To explore this hypothesis, we examined the effects of stress and antidepressants on neurotrophin expression in the locus coeruleus (LC), which modulates many of the behavioral and physiological responses to stress and has been implicated in mood disorders. Using in situ hybridization, we demonstrate that neurotrophin 3 (NT-3) is expressed in noradrenergic neurons of the LC. Recurrent, but not acute, immobilization stress increased NT-3 mRNA levels in the LC. In contrast, chronic treatment with antidepressants decreased NT-3 mRNA levels. The effect occurred in response to antidepressants that blocked norepinephrine uptake, whereas serotonin-specific reuptake inhibitors did not alter NT-3 levels. Electroconvulsive seizures also decreased NT-3 expression in the LC as well as the hippocampus. Ntrk3 (neurotrophic tyrosine kinase receptor type 3; formerly TrkC), the receptor for NT-3, is expressed in the LC, but its mRNA levels did not change with stress or antidepressant treatments. Because, NT-3 is known to be trophic for LC neurons, our results raise the possibility that some of the effects of stress and antidepressants on LC function and plasticity could be mediated through NT-3. Moreover, the coexpression of NT-3 and its receptor in the LC suggests the potential for autocrine mechanisms of action.

Limbic epilepsy in transgenic mice carrying a Ca2+/calmodulin-dependent kinase II alpha-subunit mutation.

Multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMK) phosphorylates proteins pivotally involved in diverse neuronal processes and thereby coordinates cellular responses to external stimuli that regulate intracellular Ca2+ [Hanson, P. I. & Schulman, H. (1992) Annu. Rev. Biochem. 61, 559-664]. Despite extensive study, the impact of this enzyme on control of the excitability of neuron populations in the mammalian nervous system in situ is unknown. To address this question, we studied transgenic mice carrying a null mutation (-/-) for the alpha subunit of CaMK. In contrast to wild-type littermates, null mutants exhibit profound hyperexcitability, evident in epileptic seizures involving limbic structures including the hippocampus. No evidence of increased excitability was detected in mice carrying null mutations of the gamma isoform of protein kinase C, underscoring the specificity of the effect of CaMK. CaMK plays a powerful and previously underappreciated role in control of neuronal excitability in the mammalian nervous system. These insights have important implications for analyses of mechanisms of epilepsy and, perhaps, learning and memory.

Emergent spindle oscillations and intermittent burst firing in a thalamic model: specific neuronal mechanisms.

The rhythmogenesis of 10-Hz sleep spindles is studied in a large-scale thalamic network model with two cell populations: the excitatory thalamocortical (TC) relay neurons and the inhibitory nucleus reticularis thalami (RE) neurons. Spindle-like bursting oscillations emerge naturally from reciprocal interactions between TC and RE neurons. We find that the network oscillations can be synchronized coherently, even though the RE-TC connections are random and sparse, and even though individual neurons fire rebound bursts intermittently in time. When the fast gamma-aminobutyrate type A synaptic inhibition is blocked, synchronous slow oscillations resembling absence seizures are observed. Near-maximal network synchrony is established with even modest convergence in the RE-to-TC projection (as few as 5-10 RE inputs per TC cell suffice). The hyperpolarization-activated cation current (Ih) is found to provide a cellular basis for the intermittency of rebound bursting that is commonly observed in TC neurons during spindles. Such synchronous oscillations with intermittency can be maintained only with a significant degree of convergence for the TC-to-RE projection.

Somatodendritic expression of an immediate early gene is regulated by synaptic activity.

Trans-synaptic activation of gene expression is linked to long-term plastic adaptations in the nervous system. To examine the molecular program induced by synaptic activity, we have employed molecular cloning techniques to identify an immediate early gene that is rapidly induced in the brain. We here report the entire nucleotide sequence of the cDNA, which encodes an open reading frame of 396 amino acids. Within the hippocampus, constitutive expression was low. Basal levels of expression in the cortex were high but can be markedly reduced by blockade of N-methyl-D-aspartate receptors. By contrast, synaptic activity induced by convulsive seizures increased mRNA levels in neurons of the cortex and hippocampus. High-frequency stimulation of the perforant path resulted in long-term potentiation and a spatially confined dramatic increase in the level of mRNA in the granule cells of the ipsilateral dentate gyrus. Transcripts were localized to the soma and to the dendrites of the granule cells. The dendritic localization of the transcripts offers the potential for local synthesis of the protein at activated postsynaptic sites and may underlie synapse-specific modifications during long-term plastic events.

Notificación de reacciones adversas a la vacuna frente al virus del papiloma humano en la Comunidad Valenciana (2007-2011)

Introducción: En 2009, 2 casos de convulsiones en adolescentes tras la administración de la vacuna tetravalente frente al virus del papiloma humano (VPH) generaron impacto mediático y afectaron negativamente la confianza del público en esta vacuna. Nuestros objetivos fueron describir las sospechas de reacciones adversas (SRA) notificadas al Centro Autonómico de Farmacovigilancia de la Comunidad Valenciana (CAFCV) tras la administración de la vacuna frente al VPH y comparar la tasa de notificación de síncope y convulsiones de esta vacuna con la de otras vacunas administradas en adolescentes. Material y métodos: Estudio descriptivo de las notificaciones de SRA relacionadas con esta vacuna recibidas por el CAFCV entre 2007 y 2011. Resultados: Las manifestaciones clínicas más comunicadas fueron mareos, cefalea y síncope. Las tasas de notificación de síncope o pérdida de conciencia y convulsiones con la vacuna frente al VPH fueron de 17 y 3,2 por 100.000 dosis administradas, respectivamente, y de 15 y 1,6 para síncope o pérdida de conciencia y convulsiones sincopales ocurridas el día de la vacunación. Las tasas de notificación de síncope o pérdida de conciencia y convulsiones fueron de 6,4 y 0,4 para otras vacunas. Conclusiones: Las tasas de notificación de síncope o pérdida de conciencia y convulsiones fueron mayores para la vacuna frente al VPH que para otras vacunas administradas en adolescentes; esto es consistente con la atención mediática originada por la vacuna y con hallazgos de estudios previos. No obstante, la información obtenida sobre las SRA a la vacuna sugiere un buen perfil de seguridad.

Quistos aracnoideus : quais as indicações cirúrgicas?

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014