RESUMEN
Autism spectrum disorder (ASD) is associated with enhanced processing of amyloid-ß precursor protein (APP) by secretase-α, higher blood levels of sAPPα and intraneuronal accumulation of N-terminally truncated Aß peptides in the brain cortex - mainly in the GABAergic neurons expressing parvalbumin - and subcortical structures. Brain Aß accumulation has been also described in epilepsy-the frequent ASD co-morbidity. Furthermore, Aß peptides have been shown to induce electroconvulsive episodes. Enhanced production and altered processing of APP, as well as accumulation of Aß in the brain are also frequent consequences of traumatic brain injuries which result from self-injurious behaviors, another ASD co-morbidity. We discuss distinct consequences of accumulation of Aß in the neurons and synapses depending on the Aß species, their posttranslational modifications, concentration, level of aggregation and oligomerization, as well as brain structures, cell types and subcellular structures where it occurs. The biological effects of Aß species which are discussed in the context of the pathomechanisms of ASD, epilepsy, and self-injurious behavior include modulation of transcription-both activation and repression; induction of oxidative stress; activation and alteration of membrane receptors' signaling; formation of calcium channels causing hyper-activation of neurons; reduction of GABAergic signaling - all of which lead to disruption of functions of synapses and neuronal networks. We conclude that ASD, epilepsy, and self-injurious behaviors all contribute to the enhanced production and accumulation of Aß peptides which in turn cause and enhance dysfunctions of the neuronal networks that manifest as autism clinical symptoms, epilepsy, and self-injurious behaviors.
RESUMEN
Autism, the most frequent neurodevelopmental disorder of a very complex etiopathology, is associated with dysregulation of cellular homeostatic mechanisms, including processing of amyloid-ß precursor protein (APP). Products of APP processing - N-terminally truncated amyloid-ß peptide (N-tr-Aß) species - are accumulated in autism in neurons and glia in the cortex, cerebellum, and subcortical structures of the brain. This process in neurons is correlated with increased oxidative stress. Because abnormally high levels of N-tr-Aß are detected in only a fraction of neurons in the prefrontal cortex, we applied immunocytochemical staining and confocal microscopy in autopsy brain material from idiopathic and chromosome 15q11.2-q13 duplication (dup-15) autism to measure the load of N-tr-Aß in the cells and synapses and to identify the subpopulation of neurons affected by these pathophysiological processes. The peptides accumulated in autism are N-terminally truncated; therefore, we produced a new antibody against Aß truncated at N-terminal amino acid 11 modified to pyroglutamate to evaluate the presence and distribution of this peptide species in autism. We also quantified and characterized the oligomerization patterns of the Aß-immunoreactive peptides in autism and control frozen brain samples. We provide morphological evidence, that in idiopathic and dup-15 autism, accumulation of N-tr-Aß with and without pyroglutamate-11 modified N-terminus affects mainly the parvalbumin-expressing subpopulation of GABAergic neurons. N-tr-Aß peptides are accumulated in neurons' cytoplasm and nucleus as well as in GABAergic synapses. Aß peptides with both C-terminus 40 and 42 were detected by immunoblotting in frozen cortex samples, in the form of dimers and complexes of the molecular sizes of 18-24kD and 32-34kD. We propose that deposition of N-tr-Aß specifically affects the functions of the parvalbumin-expressing GABAergic neurons and results in a dysregulation of brain excitatory-inhibitory homeostasis in autism. This process may be the target of new therapies.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Trastorno Autístico/patología , Neuronas GABAérgicas/patología , Corteza Prefrontal/patología , Adolescente , Adulto , Trastorno Autístico/genética , Trastorno Autístico/metabolismo , Niño , Duplicación Cromosómica/genética , Cromosomas Humanos Par 15/genética , Femenino , Neuronas GABAérgicas/metabolismo , Humanos , Masculino , Parvalbúminas/metabolismo , Corteza Prefrontal/metabolismo , Ácido Pirrolidona Carboxílico/metabolismo , Adulto JovenRESUMEN
N-terminally truncated pyroglutamate amyloid-ß (Aß) peptide starting at position 3 represents a significant fraction of Aß peptides (pE3-Aß) in amyloid plaques of postmortem brains from patients with Alzheimer's disease (AD) and older persons with Down syndrome (DS). Studies in transgenic mouse models of AD also showed that pE3-Aß is a major component of plaques, and mouse monoclonal antibody to pE3-Aß appears to be a desirable therapeutic agent for AD. Since small peptides do not typically elicit a good immune response in mice, but do so favorably in rabbits, our aims were to generate and partially characterize a rabbit monoclonal antibody (RabmAb) to pE3-Aß. The generated RabmAb was found to be specific for pE3-Aß, since it showed no reactivity with Aß16, Aß40, Aß42, Aß3-11, and pE11-17 Aß peptides in an enzyme linked immunosorbent assay (ELISA). The isotype of the antibody was found to be IgG class. The antibody possesses high affinity to pE3-Aß with dissociation constant (KD) for the antibody of 1 nM. The epitope of the antibody lies within the sequence of pE3-FRHD. In dot blotting, the optimal detection of pE3-Aß was at an antibody concentration of 0.5 µg/ml. The threshold of pE3-Aß detection was 2 fmol. The antibody was sensitive enough to detect 10 pg/ml of pE3-Aß in sandwich ELISA. pE3-Aß was detected in AD and DS brain extracts in ELISA and immunoblotting. Immunohistological studies showed immunolabeling of plaques and blood vessels in brains from patients with AD, and DS showing AD pathology. Thus, the antibody can be widely applied in AD and DS research, and therapeutic applications.
Asunto(s)
Péptidos beta-Amiloides/inmunología , Anticuerpos Monoclonales , Fragmentos de Péptidos/inmunología , Adulto , Anciano , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Síndrome de Down/metabolismo , Síndrome de Down/patología , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Técnica del Anticuerpo Fluorescente , Lóbulo Frontal/metabolismo , Lóbulo Frontal/patología , Humanos , Persona de Mediana Edad , ConejosRESUMEN
Secreted soluble amyloid-ß 1-37 (Aß37) peptide is one of the prominent Aß forms next to Aß40, and is found in cerebrospinal fluid (CSF) and blood. Recent studies have shown the importance of quantitation of CSF Aß37 levels in combination with Aß38, Aß40, and Aß42 to support the diagnosis of patients with probable Alzheimer's disease (AD), and the value of antibody to Aß37 to facilitate drug discovery studies. However, the availability of reliable and specific monoclonal antibody to Aß37 is very limited. Our aims were: 1) to generate and partially characterize rabbit monoclonal antibody (RabmAb) to Aß37, and 2) to determine whether the antibody detects changes in Aß37 levels produced by a γ-secretase modulator (GSM). Our generated RabmAb to Aß37 was found to be specific to Aß37, since it did not react with Aß36, Aß38, Aß39, Aß40, and Aß42 in an ELISA or immunoblotting. The epitope of the antibody was contained in the seven C-terminal residues of Aß37. The antibody was sensitive enough to measure CSF and plasma Aß37 levels in ELISA. Immunohistological studies showed the presence of Aß37-positive deposits in the brain of AD, and Down syndrome persons diagnosed with AD. Our studies also showed that the antibody detected Aß37 increases in CSF and brains of rodents following treatment with a GSM. Thus, our antibody can be widely applied to AD research, and in a panel based approach it may have potential to support the diagnosis of probable AD, and in testing the effect of GSMs to target AD.
Asunto(s)
Péptidos beta-Amiloides/inmunología , Anticuerpos Monoclonales/inmunología , Fragmentos de Péptidos/inmunología , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Encéfalo/metabolismo , Encéfalo/patología , Síndrome de Down/metabolismo , Síndrome de Down/patología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunización , Immunoblotting , Inmunohistoquímica , Persona de Mediana Edad , Conejos , Sensibilidad y EspecificidadRESUMEN
Secreted soluble amyloid-ß (Aß)38 is the second most prominent Aß form next to Aß40, and is found in cerebrospinal fluid (CSF) and blood. Recent studies have shown the importance of quantitation of CSF Aß38 levels in combination with those of Aß40 and Aß42 to support the diagnosis of Alzheimer's disease (AD), and other neurodegenerative diseases, and to facilitate drug discovery studies. However, the availability of reliable and specific Aß38 monoclonal antibody is limited. Our first aim was to generate and partially characterize rabbit monoclonal antibody (RabmAb) to Aß38. The antibody was specific to Aß38, since it did not react with Aß37, Aß39, Aß40, or Aß42 in ELISA or immunoblotting. The antibody was sensitive enough to measure Aß38 levels in plasma. Our second aim was to quantitate Aß38 levels in plasma from older Down syndrome (DS) persons and age-matched controls. Persons with DS (35 years and older) have neuropathological changes characteristic of AD. Studies have shown that plasma Aß40 and Aß42 levels are higher in older persons with DS than in controls. However, none examined Aß38 levels in DS. Our quantitation data showed that, like Aß40 and Aß42 plasma levels, Aß38 plasma levels were higher in DS than in controls. Longitudinal studies will determine whether plasma Aß38 levels in combination with levels of Aß40 and Aß42 are useful to predict early signs of AD in DS.
Asunto(s)
Péptidos beta-Amiloides/sangre , Péptidos beta-Amiloides/inmunología , Anticuerpos Monoclonales/metabolismo , Síndrome de Down/sangre , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/inmunología , Animales , Apolipoproteínas E/genética , Encéfalo/metabolismo , Estudios de Casos y Controles , Síndrome de Down/patología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Conejos , Sensibilidad y EspecificidadRESUMEN
INTRODUCTION: Characterization of the type and topography of structural changes and their alterations throughout the lifespan of individuals with autism is essential for understanding the mechanisms contributing to the autistic phenotype. The aim of this stereological study of neurons in 16 brain structures of 14 autistic and 14 control subjects from 4 to 64 years of age was to establish the course of neuronal nuclear and cytoplasmic volume changes throughout the lifespan of individuals with autism. RESULTS: Our data indicate that a deficit of neuronal soma volume in children with autism is associated with deficits in the volume of the neuronal nucleus and cytoplasm. The significant deficits of neuronal nuclear and cytoplasmic volumes in 13 of 16 examined subcortical structures, archicortex, cerebellum, and brainstem in 4- to 8-year-old autistic children suggest a global nature of brain developmental abnormalities, but with region-specific differences in the severity of neuronal pathology. The observed increase in nuclear volumes in 8 of 16 structures in the autistic teenagers/young adults and decrease in nuclear volumes in 14 of 16 regions in the age-matched control subjects reveal opposite trajectories throughout the lifespan. The deficit in neuronal nuclear volumes, ranging from 7% to 42% in the 16 examined regions in children with autism, and in neuronal cytoplasmic volumes from 1% to 31%, as well as the broader range of interindividual differences for the nuclear than the cytoplasmic volume deficits, suggest a partial distinction between nuclear and cytoplasmic pathology. CONCLUSIONS: The most severe deficit of both neuronal nucleus and cytoplasm volume in 4-to 8-year-old autistic children appears to be a reflection of early developmental alterations that may have a major contribution to the autistic phenotype. The broad range of functions of the affected structures implies that their developmental and age-associated abnormalities contribute not only to the diagnostic features of autism but also to the broad spectrum of clinical alterations associated with autism. Lack of clinical improvement in autistic teenagers and adults indicates that the observed increase in neuron nucleus and cytoplasm volume close to control level does not normalize brain function.
Asunto(s)
Trastorno Autístico/patología , Encéfalo/crecimiento & desarrollo , Encéfalo/patología , Núcleo Celular/patología , Citoplasma/patología , Neuronas/patología , Adolescente , Adulto , Factores de Edad , Trastorno Autístico/fisiopatología , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Humanos , Masculino , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
The pathological role of autoantibodies in development of CNS disorders is a new idea with growing interest among neuroscientists. The involvement of autoimmune response in the pathogenesis of autism spectrum disorders (ASD) has been suggested by the presence of multiple brain-specific autoantibodies in children with ASD and in their mothers. The possibility of the effect of autoimmunity on neurogenesis and postnatal brain plasticity has not been determined. The presence of autoantibodies against human neuronal progenitor cells (NPCs) stimulated for neuronal differentiation in culture was tested in sera from children with autism (n=20) and age-matched controls (n=18) by immunoblotting and immunocytochemistry. Immunoreactivity against multiple NPCs proteins of molecular sizes of approximately 55 kDa, 105 kDa, 150 kDa, and 210 kDa in sera from individuals with autism had a higher incidence and was stronger than in control sera which immunoreacted mainly with a 150 kDa protein. The sera from children with autism immunoreacted the strongest with NPCs expressing neuronal markers Tuj1 and doublecortin, but not astrocyte marker GFAP. The epitopes recognized by antibodies from sera were not human-specific because they detected also NPCs in situ in murine hippocampus. The autoimmune reactions against NPCs suggest an impaired tolerance to neural antigens in autism. These autoantibodies may be symptomatic for autism and furthermore, their presence suggests that autoimmunity may affect postnatal neuronal plasticity particularly after impairment of blood-brain barrier. Future studies will determine the diagnostic value of the presence of autoantibodies in autism and the therapeutic value of prevention of autoimmunity in autism.
Asunto(s)
Trastorno Autístico/sangre , Autoanticuerpos/sangre , Proteínas del Tejido Nervioso/inmunología , Células-Madre Neurales/inmunología , Neurogénesis/inmunología , Animales , Células Cultivadas , Preescolar , Proteínas de Dominio Doblecortina , Femenino , Técnica del Anticuerpo Fluorescente , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipocampo/inmunología , Humanos , Immunoblotting , Inmunohistoquímica , Lactante , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Neuropéptidos/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
The gene encoding dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is located within the Down syndrome (DS) critical region of chromosome 21. DYRK1A interacts with a plethora of substrates in the cytosol, cytoskeleton, and nucleus. Its overexpression is a contributing factor to the developmental alterations and age-associated pathology observed in DS. We hypothesized that the intracellular distribution of DYRK1A and cell-compartment-specific functions are associated with DYRK1A posttranslational modifications. Fractionation showed that, in both human and mouse brain, almost 80% of DYRK1A was associated with the cytoskeleton, and the remaining DYRK1A was present in the cytosolic and nuclear fractions. Coimmunoprecipitation revealed that DYRK1A in the brain cytoskeleton fraction forms complexes with filamentous actin, neurofilaments, and tubulin. Two-dimensional gel analysis of the fractions revealed DYRK1A with distinct isoelectric points: 5.5-6.5 in the nucleus, 7.2-8.2 in the cytoskeleton, and 8.7 in the cytosol. Phosphate-affinity gel electrophoresis demonstrated several bands of DYRK1A with different mobility shifts for nuclear, cytoskeletal, and cytosolic DYRK1A, indicating modification by phosphorylation. Mass spectrometry analysis disclosed one phosphorylated site in the cytosolic DYRK1A and multiple phosphorylated residues in the cytoskeletal DYRK1A, including two not previously described. This study supports the hypothesis that intracellular distribution and compartment-specific functions of DYRK1A may depend on its phosphorylation pattern.
Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Lóbulo Frontal/química , Lóbulo Frontal/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Adulto , Anciano , Animales , Western Blotting , Electroforesis en Gel Bidimensional , Humanos , Inmunoprecipitación , Ratones , Persona de Mediana Edad , Fosforilación , Proteínas Serina-Treonina Quinasas/análisis , Proteínas Tirosina Quinasas/análisis , Quinasas DyrKRESUMEN
BACKGROUND: Autism is a neurodevelopmental disorder of unknown etiopathogenesis associated with structural and functional abnormalities of neurons and increased formation of reactive oxygen species. Our previous study revealed enhanced accumulation of amino-terminally truncated amyloid-ß (Aß) in brain neurons and glia in children and adults with autism. Verification of the hypothesis that intraneuronal Aß may cause oxidative stress was the aim of this study. RESULTS: The relationships between neuronal Aß and oxidative stress markers-4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA)-were examined in the frontal cortex from individuals aged 7-32 years with idiopathic autism or with chromosome 15q11.2-q13 duplications (dup(15)) with autism, and age-matched controls. Quantification of confocal microscopy images revealed significantly higher levels of neuronal N-truncated Aß and HNE and MDA in idiopathic autism and dup(15)/autism than in controls. Lipid peroxidation products were detected in all mitochondria and lipofuscin deposits, in numerous autophagic vacuoles and lysosomes, and in less than 5% of synapses. Neuronal Aß was co-localized with HNE and MDA, and increased Aß levels correlated with higher levels of HNE and MDA. CONCLUSIONS: The results suggest a self-enhancing pathological process in autism that is initiated by intraneuronal deposition of N-truncated Aß in childhood. The cascade of events includes altered APP metabolism and abnormal intracellular accumulation of N-terminally truncated Aß which is a source of reactive oxygen species, which in turn increase the formation of lipid peroxidation products. The latter enhance Aß deposition and sustain the cascade of changes contributing to metabolic and functional impairments of neurons in autism of an unknown etiology and caused by chromosome 15q11.2-q13 duplication.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Trastorno Autístico/metabolismo , Encéfalo/metabolismo , Discapacidad Intelectual/metabolismo , Neuronas/metabolismo , Adolescente , Adulto , Aldehídos/metabolismo , Trastorno Autístico/complicaciones , Niño , Aberraciones Cromosómicas , Cromosomas Humanos Par 15/metabolismo , Femenino , Humanos , Discapacidad Intelectual/complicaciones , Peroxidación de Lípido/fisiología , Lisosomas/metabolismo , Masculino , Malondialdehído/metabolismo , Mitocondrias/metabolismo , Sinapsis/metabolismo , Vacuolas/metabolismo , Adulto JovenRESUMEN
BACKGROUND: It has been shown that amyloid ß (Aß), a product of proteolytic cleavage of the amyloid ß precursor protein (APP), accumulates in neuronal cytoplasm in non-affected individuals in a cell type-specific amount. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we found that the percentage of amyloid-positive neurons increases in subjects diagnosed with idiopathic autism and subjects diagnosed with duplication 15q11.2-q13 (dup15) and autism spectrum disorder (ASD). In spite of interindividual differences within each examined group, levels of intraneuronal Aß load were significantly greater in the dup(15) autism group than in either the control or the idiopathic autism group in 11 of 12 examined regions (p<0.0001 for all comparisons; Kruskall-Wallis test). In eight regions, intraneuronal Aß load differed significantly between idiopathic autism and control groups (p<0.0001). The intraneuronal Aß was mainly N-terminally truncated. Increased intraneuronal accumulation of Aß(17-40/42) in children and adults suggests a life-long enhancement of APP processing with α-secretase in autistic subjects. Aß accumulation in neuronal endosomes, autophagic vacuoles, Lamp1-positive lysosomes and lipofuscin, as revealed by confocal microscopy, indicates that products of enhanced α-secretase processing accumulate in organelles involved in proteolysis and storage of metabolic remnants. Diffuse plaques containing Aß(1-40/42) detected in three subjects with ASD, 39 to 52 years of age, suggest that there is an age-associated risk of alterations of APP processing with an intraneuronal accumulation of a short form of Aß and an extracellular deposition of full-length Aß in nonfibrillar plaques. CONCLUSIONS/SIGNIFICANCE: The higher prevalence of excessive Aß accumulation in neurons in individuals with early onset of intractable seizures, and with a high risk of sudden unexpected death in epilepsy in autistic subjects with dup(15) compared to subjects with idiopathic ASD, supports the concept of mechanistic and functional links between autism, epilepsy and alterations of APP processing leading to neuronal and astrocytic Aß accumulation and diffuse plaque formation.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Trastorno Autístico/metabolismo , Trastornos Generalizados del Desarrollo Infantil/metabolismo , Neuronas/metabolismo , Adolescente , Adulto , Astrocitos/metabolismo , Western Blotting , Niño , Preescolar , Femenino , Humanos , Inmunohistoquímica , Masculino , Microscopía Confocal , Persona de Mediana Edad , Adulto JovenRESUMEN
Overexpression of dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (DYRK1A), encoded by a gene located in the Down syndrome (DS) critical region, is considered a major contributor to developmental abnormalities in DS. DYRK1A regulates numerous genes involved in neuronal commitment, differentiation, maturation, and apoptosis. Because alterations of neurogenesis could lead to impaired brain development and mental retardation in individuals with DS, pharmacological normalization of DYRK1A activity has been postulated as DS therapy. We tested the effect of harmine, a specific DYRK1A inhibitor, on the development of neuronal progenitor cells (NPCs) isolated from the periventricular zone of newborn mice with segmental trisomy 16 (Ts65Dn mice), a mouse model for DS that overexpresses Dyrk1A by 1.5-fold. Trisomy did not affect the ability of NPCs to expand in culture. Twenty-four hours after stimulation of migration and neuronal differentiation, NPCs showed increased expression of Dyrk1A, particularly in the trisomic cultures. After 7 days, NPCs developed into a heterogeneous population of differentiating neurons and astrocytes that expressed Dyrk1A in the nuclei. In comparison with disomic cells, NPCs with trisomy showed premature neuronal differentiation and enhanced γ-aminobutyric acid (GABA)-ergic differentiation, but astrocyte development was unchanged. Harmine prevented premature neuronal maturation of trisomic NPCs but not acceleration of GABA-ergic development. In control NPCs, harmine treatment caused altered neuronal development of NPCs, similar to that in trisomic NPCs with Dyrk1A overexpression. This study suggests that pharmacological normalization of DYRK1A activity may have a potential role in DS therapy.
Asunto(s)
Diferenciación Celular/genética , Síndrome de Down/patología , Regulación del Desarrollo de la Expresión Génica/genética , Células-Madre Neurales/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Animales Recién Nacidos , Bromodesoxiuridina/metabolismo , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Células Cultivadas , Cromosomas Humanos Par 16/genética , Modelos Animales de Enfermedad , Síndrome de Down/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Glutamato Descarboxilasa/metabolismo , Harmina/farmacología , Ratones , Ratones Transgénicos , Inhibidores de la Monoaminooxidasa/farmacología , Mosaicismo , Células-Madre Neurales/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Factores de Tiempo , Trisomía/genética , Quinasas DyrKRESUMEN
Triplication of chromosome 21 in Down syndrome (DS) results in overexpression of the minibrain kinase/dual-specificity tyrosine phosphorylated and regulated kinase 1A gene (DYRK1A). DYRK1A phosphorylates cytoplasmic tau protein and appears in intraneuronal neurofibrillary tangles (NFTs). We have previously shown significantly more DYRK1A-positive NFTs in DS brains than in sporadic Alzheimer disease (AD) brains. This study demonstrates a gene dosage-proportional increase in the level of DYRK1A in DS in the cytoplasm and the cell nucleus, and enhanced cytoplasmic and nuclear immunoreactivity of DYRK1A in DS. The results suggest that overexpressed DYRK1A may alter both phosphorylation of tau and alternative splicing factor (ASF). Two-dimensional electrophoresis revealed modification of ASF phosphorylation in DS/AD and AD in comparison to controls. Altered phosphorylation of ASF by overexpressed nuclear DYRK1A may contribute to the alternative splicing of the tau gene and an increase by 2.68 × of the 3R/4R ratio in DS/AD, and a several-fold increase in the number of 3R tau-positive NFTs in DS/AD subjects compared with that in sporadic AD subjects. These data support the hypothesis that phosphorylation of ASF by overexpressed DYRK1A may contribute to alternative splicing of exon 10, increased expression of 3R tau, and early onset of neurofibrillary degeneration in DS.
Asunto(s)
Síndrome de Down/enzimología , Regulación Enzimológica de la Expresión Génica , Degeneración Nerviosa/enzimología , Degeneración Nerviosa/patología , Ovillos Neurofibrilares/enzimología , Ovillos Neurofibrilares/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Proteínas tau/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Bovinos , Síndrome de Down/genética , Síndrome de Down/patología , Femenino , Dosificación de Gen/genética , Humanos , Masculino , Ratones , Persona de Mediana Edad , Degeneración Nerviosa/genética , Ovillos Neurofibrilares/genética , Fenotipo , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/biosíntesis , Ratas , Repeticiones de Trinucleótidos/genética , Proteínas tau/biosíntesis , Quinasas DyrKRESUMEN
Altered brain development during embryogenesis and early postnatal life has been hypothesized to be responsible for the abnormal behaviors of people with autism. The specific genetic background that alters vulnerability to some environmental insults has been suggested in the etiology of autism; however, the specific pathomechanisms have not been identified. Recently, we showed that sera from children with autism alter the maturation of human neuronal progenitor cells (NPCs) in culture. Results suggest that pre-programmed neurogenesis, i.e., neuronal proliferation, migration, differentiation, growth, and circuit organization, can be affected differently by factors present in autistic sera. In this report, we tested the effect of autistic sera on the vulnerability of NPCs to oxidative stress-a recognized risk factor of autism. We found that mild oxidative stress reduced proliferation of differentiating NPCs but not immature NPCs. This decrease of proliferation was less prominent in cultures treated with sera from children with autism than from age-matched controls. These results suggest that altered response of NPCs to oxidative stress may play a role in the etiology of autism.
Asunto(s)
Trastorno Autístico/sangre , Proliferación Celular/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Neuronas/fisiología , Estrés Oxidativo/fisiología , Suero/química , Adolescente , Análisis de Varianza , Apoptosis/efectos de los fármacos , Bromodesoxiuridina/metabolismo , Carbocianinas/metabolismo , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Niño , Preescolar , Proteínas de Dominio Doblecortina , Femenino , Compuestos Ferrosos/farmacología , Feto , Humanos , Lactante , Proteínas de Filamentos Intermediarios/metabolismo , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Nestina , Neuronas/efectos de los fármacos , Neuropéptidos/metabolismo , Fosfopiruvato Hidratasa/metabolismo , Factores de Transcripción SOXB1/metabolismo , Factores de Tiempo , Tubulina (Proteína)/metabolismoRESUMEN
Vascular smooth muscle cells are involved in deposition of amyloid in brain blood vessels. Accumulation of amyloid-beta peptide (Abeta) in cultured brain vascular smooth muscle cells that overexpress human amyloid-beta precursor protein (APP) Swedish, is strongly enhanced by exposure to iron ions. We studied cellular accumulation of Abeta and APP processing in vascular smooth muscle cells during recovery after exposure to ferrous ions using cells cultured from Tg2576 mice. The treatment with ferrous ions for 24 and 48 h significantly increased the intracellular levels of ferric, but not ferrous iron. The treatment led to cellular accumulation of C-terminal fragments of APP and to a decreased secretion of APP, Abeta1-40, and Abeta1-42, all of which were quickly normalized in iron-free culture conditions. These effects of iron were neutralized by alpha-tocopherol, suggesting the role of oxygen reactive species in altered APP processing. Formation of abundant Abeta oligomers, mainly Abeta1-40 tetramers and pentamers, were detected in iron-treated cells, particularly during subsequent culture in iron-free media for up to 72 h. The data suggest that transient increases in local availability of iron in brain blood vessel walls in vivo, e.g., after microhemorrhages, may trigger Abeta oligomerization.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/irrigación sanguínea , Hierro/toxicidad , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Estrés Oxidativo , Animales , Células Cultivadas , Inmunohistoquímica , Ratones , Ratones Transgénicos , Microscopía Confocal , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Factores de TiempoRESUMEN
The gene encoding the minibrain kinase/dual-specificity tyrosine phosphorylated and regulated kinase 1A (DYRK1A) is located in the Down syndrome (DS) critical region of chromosome 21. The third copy of DYRK1A is believed to contribute to abnormal brain development in patients with DS. In vitro studies showing that DYRK1A phosphorylates tau protein suggest that this kinase is also involved in tau protein phosphorylation in the human brain and contributes to neurofibrillary degeneration, and that this contribution might be enhanced in patients with DS. To explore this hypothesis, the brain tissue from 57 subjects including 16 control subjects, 21 patients with DS, and 20 patients with sporadic Alzheimer's disease (AD) was examined with two antibodies to the amino-terminus of DYRK1A (7F3 and G-19), as well as two polyclonal antibodies to its carboxy-terminus (X1079 and 324446). Western blots demonstrated higher levels of full-length DYRK1A in the brains of patients with DS when compared to control brains. Immunocytochemistry revealed that DYRK1A accumulates in neurofibrillary tangles (NFTs) in subjects with sporadic AD and in subjects with DS/AD. Overexpression of DYRK1A in patients with DS was associated with an increase in DYRK1A-positive NFTs in a gene dosage-dependent manner. Results support the hypothesis that overexpressed DYRK1A contributes to neurofibrillary degeneration in DS more significantly than in subjects with two copies of the DYRK1A gene and sporadic AD. Immunoreactivity with antibodies against DYRK1A not only in NFTs but also in granules in granulovacuolar degeneration and in corpora amylacea suggests that DYRK1A is involved in all three forms of degeneration and that overexpression of this kinase may contribute to the early onset of these pathologies in DS.
Asunto(s)
Síndrome de Down/metabolismo , Síndrome de Down/patología , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Neurofibrillas/metabolismo , Neurofibrillas/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Estudios de Casos y Controles , Femenino , Dosificación de Gen , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Proteínas tau/metabolismo , Quinasas DyrKRESUMEN
Stimulation of endogenous neurogenesis and transplantation of neuronal progenitors (NPs) are considered in therapy of neuronal loss associated with ageing and in neurodegenerative diseases with amyloidosis-beta, for example, Alzheimer's disease and Down syndrome. However, the influence of brain environment altered by ageing and deposits of amyloid-beta on proliferation of endogenous and transplanted NPs and their maturation into neurons is not understood. We studied the effect of ageing and development of amyloidosis-beta on proliferation of NPs (1) in the granular layer of dentate gyrus in the hippocampi of APP-transgenic mice (Tg9291) before and after development of amyloidosis-beta, that is, in mice aged 2-4 months and 9-12 months, respectively, and in age-matched controls; and (2) in culture of NPs isolated from brains of control and Tg9291 mice, aged 3 and 9 months. We found that the number of proliferating NPs was reduced in 9-12-months-old mice, in both control and Tg9291, as compared to 2-4-months-old mice. However, the 9-12-months-old Tg9291 mice with amyloid-beta deposits had significantly more proliferating NPs than the age-matched controls. NPs proliferation in culture did not depend on the age, presence of APP-transgene, and amyloidosis-beta in donors. The results indicate that the local brain environment influences proliferation of NPs, and development of amyloidosis-beta in the neurogenic regions attenuates the age-associated reduction of proliferation of NPs. Identification of the responsible mechanisms may be important for development of a successful therapy of neurodegeneration caused by amyloidosis-beta.
Asunto(s)
Envejecimiento/patología , Precursor de Proteína beta-Amiloide/genética , Amiloidosis/patología , Proliferación Celular , Hipocampo/patología , Neuronas/patología , Células Madre/patología , Envejecimiento/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/metabolismo , Amiloidosis/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Hipocampo/metabolismo , Ratones , Ratones Transgénicos , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Neuronas/metabolismo , Células Madre/metabolismoRESUMEN
Changes of brain structure and functions in people with autism may result from altered neuronal development, however, no adequate cellular or animal models are available to study neurogenesis in autism. Neuronal development can be modeled in culture of neuronal progenitor cells (NPCs) stimulated with serum to differentiate into neurons. Because sera from people with autism and age-matched controls contain different levels of numerous biologically active factors, we hypothesized that development of human NPCs induced to differentiate into neurons with sera from children with autism reflects the altered early neuronal development that leads to autism. The control and autistic sera were collected from siblings aged below 6 years that lived in the same environment. The effect of sera on differentiation of NPC neurospheres into neuronal colonies was tested in 72-h-long cultures by morphometry, immunocytochemistry and immunoblotting. We found that sera from children with autism significantly reduced NPCs' proliferation, but stimulated cell migration, development of small neurons with processes, length of processes and synaptogenesis. These results suggest that development of network of processes and synaptogenesis--the specific events in the brain during postnatal ontogenesis--are altered in autism. Further studies in this cell culture model may explain some of the cellular alterations described in autistic patients.
Asunto(s)
Trastorno Autístico/sangre , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neuronas/efectos de los fármacos , Seroglobulinas/farmacología , Células Madre/efectos de los fármacos , Bromodesoxiuridina/metabolismo , Recuento de Células/métodos , Diferenciación Celular/fisiología , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Preescolar , Electroforesis Capilar/métodos , Femenino , Feto , Humanos , Lactante , Masculino , Proteínas del Tejido Nervioso/metabolismo , Células Madre/fisiologíaRESUMEN
Amyloid beta (Abeta) immunoreactivity in neurons was examined in brains of 32 control subjects, 31 people with Down syndrome, and 36 patients with sporadic Alzheimer's disease to determine if intraneuronal Abeta immunoreactivity is an early manifestation of Alzheimer-type pathology leading to fibrillar plaque formation and/or neurofibrillary degeneration. The appearance of Abeta immunoreactivity in neurons in infants and stable neuron-type specific Abeta immunoreactivity in a majority of brain structures during late childhood, adulthood, and normal aging does not support this hypothesis. The absence or detection of only traces of reaction with antibodies against 4-13 aa and 8-17 aa of Abeta in neurons indicated that intraneuronal Abeta was mainly a product of alpha- and gamma-secretases (Abeta(17-40/42)). The presence of N-terminally truncated Abeta(17-40) and Abeta(17-42) in the control brains was confirmed by Western blotting and the identity of Abeta(17-40) was confirmed by mass spectrometry. The prevalence of products of alpha- and gamma -secretases in neurons and beta- and gamma-secretases in plaques argues against major contribution of Abeta-immunopositive material detected in neuronal soma to amyloid deposit in plaques. The strongest intraneuronal Abeta(17-42) immunoreactivity was observed in structures with low susceptibility to fibrillar Abeta deposition, neurofibrillary degeneration, and neuronal loss compared to areas more vulnerable to Alzheimer-type pathology. These observations indicate that the intraneuronal Abeta immunoreactivity detected in this study is not a predictor of brain amyloidosis or neurofibrillary degeneration. The constant level of Abeta immunoreactivity in structures free from neuronal pathology during essentially the entire life span suggests that intraneuronal amino-terminally truncated Abeta represents a product of normal neuronal metabolism.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Encéfalo/patología , Síndrome de Down/metabolismo , Líquido Intracelular/metabolismo , Ovillos Neurofibrilares/patología , Neuronas/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Estudios de Casos y Controles , Preescolar , Síndrome de Down/patología , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Valor Predictivo de las PruebasRESUMEN
Transplantation of human neuronal progenitor cells (HNPC) is being considered for neuroreplacement therapy in beta-amyloidosis associated with neuronal loss in Down's syndrome and Alzheimer's disease. However, the influence of amyloid-beta-containing brain environment on the development of HNPCs is unknown. Recently, we demonstrated that amyloid-beta peptide (Abeta) impaired differentiation of HNPCs in culture through oxidative stress. Now we studied the effect of neprilysin, an Abeta-degrading enzyme, on development of neuronal colonies from neurospheres of HNPCs in the presence of Abeta1-40. Neprilysin increased the number of neurospheres that formed colonies of neuron-like cells. This effect of neprilysin was associated with reduced amounts of the monomeric and dimeric Abeta that remained in culture supernatants as well as the Abeta uptaken by differentiating HNPCs. Phosphoramidon, a neprilysin inhibitor, attenuated these effects of neprilysin. In control cultures of HNPCs that grew without exogenous Abeta1-40, the treatment with neprilysin reduced the number of developing colonies. This effect might result from degradation by neprilysin of endogenous Abeta produced and secreted by HNPCs or other peptides that are involved in neuronal development. The results demonstrate that even a partial reduction of extracellular Abeta levels by neprilysin may facilitate development of HNPCs into neurons in an environment overloaded with Abeta. This finding suggests that neprilysin could facilitate neuroreplacement therapy with HNPCs in treatment of neurodegenerative diseases.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Neprilisina/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/toxicidad , Células Madre/efectos de los fármacos , Bromodesoxiuridina/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Interacciones Farmacológicas , Feto , Glicopéptidos/farmacología , Humanos , Inmunohistoquímica/métodos , Proteínas del Tejido Nervioso/metabolismo , Inhibidores de Proteasas/farmacología , Factores de TiempoRESUMEN
The reduced antioxidant defense in apolipoprotein E epsilon4/epsilon4 carriers may contribute to beta-amyloidosis. Previously we found that Fe(2+)-induced oxidative stress caused greater protein oxidation in epsilon4/epsilon4 than in epsilon3/epsilon3 human brain vascular smooth muscle cells. Moreover, Fe(2+) induced lysosomal accumulation of endogenous Abeta and APOE in cultured cells, and Abeta deposition in vascular tunica media in organotypic cultures of brain vessels. Here we demonstrated that Fe(2+) enhanced an uptake of exogenous Abeta 1-40 and its deposition together with APOE in lysosomes in myocytes. Abeta deposits were associated with lipid-peroxidation and protein ubiquitination, and were more abundant and stable in epsilon4/epsilon4 than in epsilon3/epsilon3 cells. In organotypic cultures of brain vessels Fe(2+) induced deposition of non-fibrillar and fibrillar Abeta 1-40 in vascular tunica media. We hypothesize that locally increased concentrations of iron induce accumulation of exogenous and endogenous Abeta in SMCs, triggering beta-amyloid angiopathy. The greater susceptibility of epsilon4 carriers to Fe(2+) ions may result in an increased risk of beta-amyloidosis.