RESUMEN
BACKGROUND AND PURPOSE: Duplications of lamin B1 (LMNB1) at 5q23 are implicated in adult-onset autosomal dominant leukodystrophy (ADLD) having been described in six families with diverse ethnic background but with a homogeneous phenotype. In a large Italian family, we recently identified a variant form of ADLD characterized clinically by absence of the autonomic dysfunction at onset described in ADLD and, on MRI, by milder cerebellar involvement with sparing of hemispheric white matter. Aim of this study was to investigate the genetic basis of this variant form of ADLD. METHODS: We carried out a genome-wide linkage analysis using microsatellite markers, and the genes in the candidate region were screened for point mutations. LMNB1 was also screened for deletions/duplications by real-time PCR, multiplex ligation-dependent probe amplification and Southern blot. RESULTS: We mapped the variant ADLD locus to 5q23.2-q23.3, a genomic region containing 11 genes including LMNB1. Neither gene copy-number defects nor point mutations in the LMNB1 gene were found. We also excluded point mutations in the coding exons of the other ten genes in the candidate region. However, expression of lamin B1 evaluated in lymphoblastoid cells was higher in patients than in healthy controls, and was similar to the lamin B1 expression levels found in a patient with LMNB1 duplication. CONCLUSIONS: This observation suggests that a mutation in an LMNB1 regulatory sequence underlies the variant ADLD phenotype. Thus, adult forms of ADLD linked to 5q23 appear to be more heterogeneous clinically and genetically than previously thought.
Asunto(s)
Cromosomas Humanos Par 5 , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Lamina Tipo B/genética , Leucodistrofia de Células Globoides/genética , Leucoencefalopatías/genética , Adulto , Edad de Inicio , Anciano , Variaciones en el Número de Copia de ADN , Familia , Femenino , Duplicación de Gen , Ligamiento Genético , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/metabolismo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/patología , Humanos , Italia , Lamina Tipo B/metabolismo , Leucodistrofia de Células Globoides/metabolismo , Leucodistrofia de Células Globoides/patología , Leucoencefalopatías/metabolismo , Leucoencefalopatías/patología , Masculino , Repeticiones de Microsatélite , Persona de Mediana Edad , Mutación , Fenotipo , Mutación Puntual , Eliminación de SecuenciaRESUMEN
BACKGROUND: Whole gene duplication of the lamin B1 gene (LMNB1), encoding for a protein of the nuclear lamina, causes an adult-onset autosomal dominant leukodystrophy (ADLD). Clinical features of ADLD (onset in adult life, dysautonomic symptoms, followed by pyramidal and cerebellar dysfunctions) partially resemble those of multiple sclerosis (MS), particularly the primary-progressive form. Our aim was to test whether LMNB1 gene mutations were present amongst patients with a diagnosis of MS. METHODS: One hundred eighty-two MS patients were screened for copy number variations of the LMNB1 gene using a qPCR assay. Point mutations in the LMNB1 gene were searched by denaturing high-performance liquid chromatography and direct sequencing in a subgroup of 16 patients with familial MS. RESULTS: No duplication/deletion of the lamin B1 gene was found amongst MS patients, and no point mutation was identified in the familial cases. CONCLUSION: Our work indicates that lamin B1 defects are probably not responsible for signs and symptoms resembling multiple sclerosis.
Asunto(s)
Lamina Tipo B/genética , Esclerosis Múltiple/genética , Cromatografía Líquida de Alta Presión , Familia , Femenino , Duplicación de Gen , Humanos , Masculino , Fenotipo , Mutación Puntual , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
BACKGROUND AND AIMS: Duplication of the lamin B1 gene (LMNB1) has recently been described in a rare form of autosomal dominant adult-onset leucoencephalopathy. The aim of the study was to evaluate the presence of LMNB1 gene defects in a series of eight patients with diffuse adult-onset hereditary leucoencephalopathy. METHODS: Clinical features of tested patients included a variable combination of pyramidal, cerebellar, cognitive and autonomic dysfunction. Neuroradiological data (MRI) showed symmetrical and diffuse white-matter lesions in six cases, and multifocal confluent lesions in two. LMNB1 full gene deletion/duplication and point mutations were searched using a TaqMan real-time PCR assay and direct sequencing of all coding exons. RESULTS: One patient carried a 140-190 kb duplication involving the entire LMNB1 gene, the AX748201 transcript and the 3' end of the MARCH3 gene. Clinical and neuroimaging data of this proband and an affected relative overlapped with the features already described in patients with LMNB1 duplication. Lamin B1 expression was found increased in lymphoblasts. No LMNB1 gene defect was identified in the remaining seven probands. CONCLUSIONS: LMNB1 gene duplication appears characteristic of a subset of adult-onset autosomal dominant leucoencephalopathies, sharing autonomic dysfunction at onset, diffuse T2-hyperintensity of supra- and infratentorial white matter, sparing of U-fibres and optic radiations. The variable phenotypes in the remaining cases lacking LMNB1 defects (five with autosomal dominant transmission) suggest that adult-onset leucoencephalopathies are genetically heterogeneous.
Asunto(s)
Genes Duplicados/genética , Lamina Tipo B/genética , Leucoencefalopatía Multifocal Progresiva/genética , Leucoencefalopatía Multifocal Progresiva/patología , Adulto , Cerebelo/patología , Análisis Mutacional de ADN , Exones , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Linaje , Mutación Puntual/genética , Tractos Piramidales/patología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In neurons, apoptosis can be triggered by a variety of exogenous stimuli. In spite of a significant diversity in the nature of apoptotic signals, it is possible to identify points of convergence common to neuronal apoptotic processes irrespective of the nature of the signal that has initiated apoptosis. These points of convergence can be defined as a common mechanism that is activated prior to neuronal death and, if inhibited, it can prevent apoptosis. The stress-activated protein kinases (SAPK) are activated in response to a variety of cellular stresses that lead to apoptosis. The SAPK-mediated apoptosis is also a ceramide-dependent processes. Recently, we reported that stress-induced neuronal apoptosis is prevented by the isoquinolinesulfonamides (IQS) H7, H8, and H9, which are known protein kinase inhibitors. We hypothesized that IQS will prevent ceramide-induced neuronal death. We observed in primary cultures of rat cerebellar granule neurons that C2-ceramide induced morphological signs of apoptosis (assayed by propidium iodide staining) and cell death. We treated cultures with C2-ceramide in the presence or absence of IQS and assessed cell death. The IQS provided neuroprotection, suggesting that ceramide-activated SAPK might play a role in IQS-sensitive neuronal death. Similarities between the ceramide-dependent and IQS-sensitive pathways indicate that they may utilize a common principle.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Inhibidores Enzimáticos/farmacología , Isoquinolinas/farmacología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Esfingosina/análogos & derivados , Sulfonamidas , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas , Ratas , Esfingosina/farmacologíaRESUMEN
Although zinc is essential for the activity of numerous biological systems, and zinc deficiency has been associated with various pathologies, this metal can also exert direct neurotoxic action. In primary cultures of rat cerebellar granule neurons, a brief, 15- to 30-min exposure to zinc (100-500 microM) resulted in concentration-dependent delayed neuronal death. The toxicity of zinc depended on the maturity of the neuronal cultures-it was not apparent prior to Day 5 and it reached a plateau at about 9-10 days in vitro. We assayed cell injury by measuring mitochondrial functioning (MTT assay) and cell death with the trypan blue exclusion assay. Apoptosis was assayed by the morphological appearance of cells following fluorescence staining with propidium iodide and by the in situ TUNEL technique. Mitochondrial injury was an early result of zinc treatment. Actinomycin D, an inhibitor of macromolecular synthesis, attenuated delayed cell death. The calcium channel blockers nimodipine and amlodipine reduced both mitochondrial injury and cell death; the blockade of ionotropic glutamate receptors with MK-801 or CNQX was ineffective. These results suggest that calcium channel-blocker-sensitive mitochondrial injury and DNA damage are operative in the protein-synthesis-dependent neurotoxicity of zinc. An imbalance of zinc homeostasis might play a role in the pathophysiology of apoptosis-associated neurodegenerative disorders.
Asunto(s)
Apoptosis/efectos de los fármacos , Cerebelo/citología , Neuronas/efectos de los fármacos , Neurotoxinas , Zinc/toxicidad , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Amlodipino/farmacología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Muerte Celular/efectos de los fármacos , Células Cultivadas , Cerebelo/patología , Fragmentación del ADN , Dactinomicina/farmacología , Maleato de Dizocilpina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Neuronas/citología , Neuronas/fisiología , Nimodipina/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Factores de TiempoRESUMEN
Cerebellar granule neurons undergo apoptosis when deprived of chronic depolarization; serum deprivation has not been considered as a trigger of apoptosis in this culture. Here we report that serum removal triggers cell injury, which is characterized by signs of apoptosis. Actual cell death (trypan blue permeability) occurred 24 and 48 hr after serum removal. At earlier times (6 and 8 hr after serum removal) we found significant impairment of mitochondrial functioning [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay] and an increase in the percentage of neurons showing signs of DNA fragmentation (insitu terminal deoxynucleotidyl transferase assay, fluorescent assay). Protection was obtained by inhibiting RNA synthesis with actinomycin D and by antioxidants [1mM: 1,4-diazobicyclo(2.2.2)octane, histidine, mannitol; 1% dimethyl sulfoxide; 0.01-1 microM ascorbic acid]. We also measured neuronal oxidation utilizing the oxidation-sensitive fluorescent dye 2', 7'-dichloro- fluorescin diacetate, and found a significant increase in the rate of neuronal oxidation as early as 15 min after serum deprivation. The blockade of glutamate receptors by (+)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imine (MK-801) and 6-cyano-7-nitroquinoxaline-2,3-dione also provided neuroprotection. However, oxidative stress appears to precede glutamate receptor activation: within the 8 hr period of serum deprivation, mannitol was protective when present either during only the first or last 4 hr; MK-801 was protective only when present for the entire 8 hr period or in the last, but not first 4 hr of serum deprivation. Serum deprivation of mature cerebellar granule neurons can be used to study mechanisms of oxidative stress-induced apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Cerebelo/efectos de los fármacos , Daño del ADN , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Animales , Antioxidantes/farmacología , Células Cultivadas , Cerebelo/citología , Medio de Cultivo Libre de Suero , Antagonistas de Aminoácidos Excitadores/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
The full range of physiological actions of melatonin is not completely known. In mammals, it modulates gonadal function and regulates biological rhythms. Furthermore, it has also been reported to have anxyolitic, sedative, and anticonvulsant properties, both in human and animals. Recently it has been shown that melantonin is a potent, endogenous hydroxyl radical scavenger suggesting that it might interfere with neurodegenerative processing involving free-radical formation and excitatory amino acid release. Using primary cultures of rat cerebellar neurons and in vivo models of brain injury in rats, we demonstrate that melatonin might be considered an endogenous neuroprotective factor useful for the pharmacological treatment of neurological disorders and neural degeneration produced by glutamate excitotoxicity and oxidative stress.
Asunto(s)
Encéfalo/fisiología , Melatonina/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Animales , Muerte Celular/efectos de los fármacos , Cerebelo/citología , Cerebelo/efectos de los fármacos , Cerebelo/fisiología , Hipocampo/efectos de los fármacos , Hipocampo/patología , Humanos , Ácido Kaínico/toxicidad , Neuronas/citología , Neurotoxinas/farmacología , Neurotoxinas/toxicidad , Estrés Oxidativo , RatasRESUMEN
Protein phosphorylation is kept in balance by an orchestrated action of kinases and phosphatases; when this balance is lost, neuronal apoptosis may occur. Okadaic acid (OKA), a marine toxin that inhibits specifically protein phosphatases 1 and 2A (EC 3.1.3.16), and staurosporine, an inhibitor of protein kinase C (PKC; EC 2.7.1.37), induced apoptosis in primary cultures of rat cerebellar granule neurons. We assayed apoptosis by the DNA gel electrophoresis, by the in situ TUNEL assay, and by morphological appearance following propidium iodide staining. Cell viability was assessed by the Trypan blue assay. Both OKA- and staurosporine-induced neuronal apoptosis were prevented by a macromolecular synthesis inhibitor actinomycin D and by a group of isoquinolinesulfonamide kinase inhibitors (H-7, 1-[5-isoquinolinesulfonyl]-2-methylpiperazine; H-8, N-¿2-[methylamino]ethyl¿-5-isoquinolinesulfonamide; H-9, N-(2-aminoethyl)-5-isoquinolinesulfonamide, but not by inhibitors of PKC, cyclic-GMP- and cyclic-AMP-dependent kinases, calcium/calmodulin-dependent kinases, tyrosine kinases, or by antioxidants. We postulate that a common mechanism, possibly an increased protein phosphorylation, is responsible for apoptosis triggered by an inhibition of phosphatases 1 and 2A and PKC. Elucidating the isoquinolinesulfonamide-sensitive mechanism may help us find new therapies for neurodegenerative diseases that involve apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Isoquinolinas/farmacología , Neuronas/citología , Ácido Ocadaico/farmacología , Estaurosporina/farmacología , Sulfonamidas , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Cerebelo/citología , Neuronas/efectos de los fármacos , Neuronas/enzimología , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Ratas Sprague-DawleyRESUMEN
We have previously reported that a group of isoquinolinesulfonamide kinase inhibitors (H7, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine; H8, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide; and H9, N-2-(aminoethyl)-5-isoquinolinesulfonamide) prevents apoptosis triggered in neurons by an inhibition of phosphatases 1 and 2A with okadaic acid, and by inhibition of protein kinase C with staurosporine and chelerythrine. In the present study we assessed the capability of isoquinolinesulfonamides (IQS) to prevent different types of apoptosis in primary cultures of cerebellar granule neurons. We induced apoptosis by: a) serum removal, b) potassium deficiency, c) serum removal + potassium deficiency, and d) beta-amyloid peptide (beta AP). The IQS prevented apoptosis in all the above models. Thus, it appears that the IQS-sensitive pathway is a common mechanism in different types of neuronal apoptosis, and that it might be used as a target in the development of novel neuroprotective drugs.
Asunto(s)
Apoptosis/efectos de los fármacos , Cerebelo/efectos de los fármacos , Isoquinolinas/farmacología , Neuronas/efectos de los fármacos , Piperazinas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Sulfonamidas , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Animales , Células Cultivadas , Cerebelo/citología , Gránulos Citoplasmáticos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
We recently reported that the pineal hormone melatonin protected neuronal cultures from excitotoxicity mediated via kainate-sensitive glutamate receptors and from oxidative stress-induced apoptosis. It has been shown that in rats, a systemic administration of kainate induces apoptotic cell death in various brain regions. In this study, we assayed the extent of brain injury after intraperitoneal (i.p.) administration of 10 mg/kg kainate to rats, using the quantitative TUNEL technique and Nissl staining. We examined the role of melatonin on kainate-induced brain injury by (a) injecting melatonin (4 × 2.5 mg/kg i.p.) prior to and after kainate injection and (b) injecting kainate at the time of low circulating melatonin levels (day/light), and high melatonin levels (night/dark). The extent of kainate-triggered DNA damage and the loss of Nissl staining were lower in animals treated with melatonin, or when kainate was injected at night, i.e. in the presence of high endogenous levels of melatonin. Our results suggest that both the pharmacological use of melatonin and the circadian secretion of endogenous melatonin during the night may reduce the extent of excitotoxic brain injury. Further studies are needed to fully characterize the relevance of our findings for the treatment of progressive neurodegenerative processes which involve excitotoxicity and apoptotic neuronal death.
RESUMEN
Typically, primary cultures of rat cerebellar granule neurons are grown in the presence of 25 mM KCl and are considered to mature by approximately 7 days in vitro. Potassium deficiency was created by growing the neurons from days 1 to 4 in the presence of 12.5 mM KCl (immature cultures) or by switching the mature neurons grown with 25 mM KCl to 12.5 mM KCl. In both conditions we observed neuronal death that bears the signs of apoptosis, i.e., DNA fragmentation determined qualitatively by agarose gel electrophoresis of DNA and quantitatively by in situ terminal deoxynucleotidyl transferase assay. The protein synthesis inhibitors cycloheximide and anisomycin provided neuroprotection in the mature cultures but potentiated the toxic effect of KCl deprivation in the immature neurons. The results suggest that a prudent use of protein synthesis inhibitors is critical in experiments with primary neuronal cultures.
Asunto(s)
Anisomicina/farmacología , Apoptosis , Cerebelo/citología , Cicloheximida/farmacología , Neuronas/fisiología , Potasio/fisiología , Animales , Células Cultivadas , Cerebelo/efectos de los fármacos , ADN/metabolismo , Neuronas/efectos de los fármacos , Potasio/administración & dosificación , Cloruro de Potasio/administración & dosificación , Ratas , Ratas Sprague-DawleyRESUMEN
In neurons, oxidative stress can be triggered by neurotransmitter-linked mechanisms and may lead to apoptotic cell death. A simple and reproducible model of inducing oxidative stress is needed to elucidate mechanisms which link oxidative stress and neuronal apoptosis. We report here a method of inducing apoptosis in cell cultures by loading them with a photosensitive dye, rose bengal, and exposing the cultures to light, a procedure which generates reactive singlet oxygen. We used this model in primary culture of rat cerebellar granule neurons, and in a nonneuronal human embryonic kidney 293 cell line. We have measured the following: (a) metabolic activity of the mitochondria by quantitative staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), (b) DNA fragmentation by quantitative in situ terminal deoxynucleotidyl transferase assay, and (c) cell viability by a trypan blue exclusion test. The oxidative stress caused an early impairment of mitochondrial function (MTT assay). This was followed by DNA fragmentation and ultimately by cell death. Protection was obtained with an inhibitor of macromolecular synthesis, anisomycin, and with antioxidant, vitamin E. This model can be used to study the mechanism of oxidative stress-triggered neuronal apoptosis, and it may help in discovering new targets for neuroprotective drugs.
Asunto(s)
Apoptosis , Luz , Neuronas/fisiología , Estrés Oxidativo/fisiología , Animales , Mitocondrias/fisiología , Neuronas/efectos de los fármacos , Neuronas/efectos de la radiación , Fármacos Neuroprotectores/farmacología , Ratas , Ratas Sprague-Dawley , Rosa Bengala/farmacología , Factores de Tiempo , Vitamina E/farmacologíaRESUMEN
Singlet oxygen (O2[1 delta g]) is a very reactive molecule that can be produced by living cells and may contribute to cytotoxicity. The pineal hormone melatonin has been reported to possess potent antioxidant activity, and to be capable of scavenging O2(1 delta g). We investigated whether melatonin might reduce the neurotoxic action of O2(1 delta g). The cytotoxic effect of singlet oxygen was studied in primary cultures of cerebellar granule neurons pretreated with a photosensitive dye, rose bengal, and exposed to light--a procedure that generates O2(1 delta g). We found that this procedure triggers neuronal death, which is preceded by mitochondrial impairment (assayed by the rate of the reduction of MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, into formazan), and by DNA fragmentation--a marker of apoptosis. DNA fragmentation was determined in situ by terminal deoxynucleotidyl transferase assay; cell death was assayed with 0.4% trypan blue solution--viable cells with an intact membrane are not permeable to trypan blue; dead cells are, and thus, they are stained blue. Neuroprotection was obtained with the pineal hormone melatonin. In a cell-free system, melatonin also protected the enzyme creatine kinase (EC 2.7.3.2) from the rose bengal-induced injury. The results suggest that melatonin might counteract the cytotoxic action of singlet oxygen. Further studies are needed to clarify the exact role singlet oxygen and melatonin might play in neurodegenerative diseases.
Asunto(s)
Melatonina/farmacología , Neuronas/efectos de los fármacos , Animales , Apoptosis , Células Cultivadas , Cerebelo/citología , Luz , Neuronas/fisiología , Oxígeno/toxicidad , Ratas , Ratas Sprague-Dawley , Rosa BengalaRESUMEN
Cyclic AMP is a second messenger by which different extracellular signals are transduced into biological responses. Within the cell, most of the effects of cAMP are mediated through the cAMP protein kinase which appears to be localized in specific compartments of the cell near to their substrate proteins. In the present study, we have investigated the possible association of cAMP-dependent protein kinase, its substrate proteins and RII binding proteins in stable microtubules from rat cerebral cortex. The results show that in this fraction there is a cAMP binding protein of 52-54 kDa. This cAMP receptor is in the inactive holoenzyme form, since the addition of cAMP (5 microM) induces an increase in the endogenous phosphorylation of different stable microtubules polypeptides, which is completely inhibited in the presence of a specific protein kinase inhibitor (PKI 5-24 1 microM). Interestingly, overlay binding assay reveals that beside MAP2, 32P/R II is able to bind stable microtubule proteins of M(r) 150 and 75 kDa which, according to their electrophoretic mobility, can also be endogenous substrates for the enzyme. We conclude that cAMP-dependent phosphorylation system is indeed associated with stable microtubules from rat cerebral cortex.