RESUMEN
The precise polarization and orientation of developing neurons is essential for the correct wiring of the brain. In pyramidal excitatory neurons, polarization begins with the sprouting of opposite neurites, which later define directed migration and axo-dendritic domains. We here show that endogenous N-cadherin concentrates at one pole of the newborn neuron, from where the first neurite subsequently emerges. Ectopic N-cadherin is sufficient to favour the place of appearance of the first neurite. The Golgi and centrosome move towards this newly formed morphological pole in a second step, which is regulated by PI3K and the actin/microtubule cytoskeleton. Moreover, loss of function experiments in vivo showed that developing neurons with a non-functional N-cadherin misorient their cell axis. These results show that polarization of N-cadherin in the immediate post-mitotic stage is an early and crucial mechanism in neuronal polarity.
Asunto(s)
Cadherinas/metabolismo , División Celular , Polaridad Celular , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Animales , Centrosoma/metabolismo , Citoesqueleto/metabolismo , Aparato de Golgi/metabolismo , Neuritas/fisiología , Fosfatidilinositol 3-Quinasa/metabolismo , RatasRESUMEN
Clinical, pharmacological, biochemical, and genetic evidence support the notion that alteration of cholesterol homeostasis strongly predisposes to Alzheimer disease (AD). The ATP-binding cassette transporter-2 (Abca2), which plays a role in intracellular sterol trafficking, has been genetically linked to AD. It is unclear how these two processes are related. Here we demonstrate that down-regulation of Abca2 in mammalian cells leads to decreased amyloid-ß (Aß) generation. In vitro studies revealed altered γ-secretase complex formation in Abca2 knock-out cells due to the altered levels, post-translational modification, and subcellular localization of Nicastrin. Reduced Abca2 levels in mammalian cells in vitro, in Drosophila melanogaster and in mice resulted in altered γ-secretase processing of APP, and thus Aß generation, without affecting Notch cleavage.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Drosophila/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Regulación hacia Abajo/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Ratones , Proteínas del Tejido Nervioso/genética , RatasRESUMEN
Distal hereditary motor neuropathies are pure motor disorders of the peripheral nervous system resulting in severe atrophy and wasting of distal limb muscles. In two pedigrees with distal hereditary motor neuropathy type II linked to chromosome 12q24.3, we identified the same mutation (K141N) in small heat-shock 22-kDa protein 8 (encoded by HSPB8; also called HSP22). We found a second mutation (K141E) in two smaller families. Both mutations target the same amino acid, which is essential to the structural and functional integrity of the small heat-shock protein alphaA-crystallin. This positively charged residue, when mutated in other small heat-shock proteins, results in various human disorders. Coimmunoprecipitation experiments showed greater binding of both HSPB8 mutants to the interacting partner HSPB1. Expression of mutant HSPB8 in cultured cells promoted formation of intracellular aggregates. Our findings provide further evidence that mutations in heat-shock proteins have an important role in neurodegenerative disorders.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Proteínas de Choque Térmico/genética , Mutación Puntual , Proteínas Serina-Treonina Quinasas , Secuencia de Aminoácidos , Animales , Células COS , Línea Celular , Enfermedad de Charcot-Marie-Tooth/metabolismo , Femenino , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Humanos , Masculino , Chaperonas Moleculares , Datos de Secuencia Molecular , Linaje , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Frontotemporal dementia (FTD) with ubiquitin-immunoreactive neuronal inclusions (both cytoplasmic and nuclear) of unknown nature has been linked to a chromosome 17q21 region (FTDU-17) containing MAPT (microtubule-associated protein tau). FTDU-17 patients have consistently been shown to lack a tau-immunoreactive pathology, a feature characteristic of FTD with parkinsonism linked to mutations in MAPT (FTDP-17). Furthermore, in FTDU-17 patients, mutations in MAPT and genomic rearrangements in the MAPT region have been excluded by both genomic sequencing and fluorescence in situ hybridization on mechanically stretched chromosomes. Here we demonstrate that FTDU-17 is caused by mutations in the gene coding for progranulin (PGRN), a growth factor involved in multiple physiological and pathological processes including tumorigenesis. Besides the production of truncated PGRN proteins due to premature stop codons, we identified a mutation within the splice donor site of intron 0 (IVS0 + 5G > C), indicating loss of the mutant transcript by nuclear degradation. The finding was made within an extensively documented Belgian FTDU-17 founder family. Transcript and protein analyses confirmed the absence of the mutant allele and a reduction in the expression of PGRN. We also identified a mutation (c.3G > A) in the Met1 translation initiation codon, indicating loss of PGRN due to lack of translation of the mutant allele. Our data provide evidence that PGRN haploinsufficiency leads to neurodegeneration because of reduced PGRN-mediated neuronal survival. Furthermore, in a Belgian series of familial FTD patients, PGRN mutations were 3.5 times more frequent than mutations in MAPT, underscoring a principal involvement of PGRN in FTD pathogenesis.
Asunto(s)
Cromosomas Humanos Par 17/genética , Demencia/genética , Lóbulo Frontal/fisiopatología , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Mutación/genética , Lóbulo Temporal/fisiopatología , Ubiquitina/metabolismo , Bélgica , Análisis Mutacional de ADN , Demencia/fisiopatología , Lóbulo Frontal/metabolismo , Ligamiento Genético/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Mapeo Físico de Cromosoma , Progranulinas , Sitios de Empalme de ARN/genética , Lóbulo Temporal/metabolismoRESUMEN
The varied ways in which mutations in presenilins (PSEN1 and PSEN2) affect amyloid b precursor protein (APP) processing in causing early-onset familial Alzheimer disease (FAD) are complex and not yet properly understood. Nonetheless, one useful diagnostic marker is an increased ratio of Ab42 to Ab40 (Ab42/Ab40) in patients' brain and biological fluids as well as in transgenic mice and cells. We studied Ab and APP processing for a set of nine clinical PSEN mutations on a novel and highly reproducible enzyme-linked immunosorbent assay (ELISA)-based in vitro method and also sought correlation with brain Ab analyzed by image densitometry and mass spectrometry. All mutations significantly increased Ab42/Ab40 in vitro by significantly decreasing Ab40 with accumulation of APP C-terminal fragments, a sign of decreased PSEN activity. A significant increase in absolute levels of Ab42 was observed for only half of the mutations tested. We also showed that age-of-onset of PSEN1-linked FAD correlated inversely with Ab42/Ab40 (r = -0.89; P = 0.001) and absolute levels of Ab42 (r = -0.83; P = 0.006), but directly with Ab40 levels (r = 0.69; P = 0.035). These changes also partly correlated with brain Ab42 and Ab40 levels. Together, our data suggested that Ab40 might be protective by perhaps sequestering the more toxic Ab42 and facilitating its clearance. Also, the in vitro method we describe here is a valid tool for assaying the pathogenic potential of clinical PSEN mutations in a molecular diagnostic setting.
Asunto(s)
Enfermedad de Alzheimer/diagnóstico , Péptidos beta-Amiloides/metabolismo , Proteínas de la Membrana/genética , Mutación , Fragmentos de Péptidos/metabolismo , Adulto , Edad de Inicio , Enfermedad de Alzheimer/epidemiología , Enfermedad de Alzheimer/genética , Biomarcadores , Encéfalo/metabolismo , Línea Celular , Densitometría , Ensayo de Inmunoadsorción Enzimática , Humanos , Proteínas de la Membrana/química , Persona de Mediana Edad , Presenilina-1 , Presenilina-2 , Estructura Terciaria de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The most common histologic feature in patients with frontotemporal lobar degeneration (FTLD) is intracellular brain inclusions of yet uncharacterized proteins that react with antiubiquitin (Ub) antibodies, but not with tau or synuclein (FTLD-U). We identified a four-generation Belgian FTLD family in which 8 patients had dominantly inherited FTLD. In one patient, we showed frontotemporal atrophy with filamentous Ub-positive intracellular inclusions in absence of tau pathology or any alterations in the levels of soluble tau. We characterized the cellular and subcellular localization and morphology of the inclusions. Ub-positive inclusions predominantly occurred within neurons (>97%), but were also observed within oligodendroglia (approximately 2%) and microglia (<1%), but not within astroglia. Regarding the subcellular localization, the intranuclear inclusions (INI) were up to approximately four-fold more frequent than the cytoplasmic inclusions, although the latter were more specific to neurons. The INIs frequently appeared spindle-shaped and 3-dimensional confocal reconstructions identified flattened, leaf-like structures. Ultrastructurally, straight 10- to 18-nm-diameter filaments constituted the spindle-shaped inclusions that occurred in close proximity to the nuclear membrane. Staining for HSP40, p62, and valosin/p97 was observed in only a minority of the inclusions. Whereas the precise nature of the protein remains elusive, characterization of such familial FTLD-U patients would be helpful in identifying a common denominator in the pathogenesis of familial and the more prevalent sporadic FTLD-U.
Asunto(s)
Demencia/patología , Cuerpos de Inclusión , Neuronas/metabolismo , Ubiquitina/metabolismo , Anciano , Anciano de 80 o más Años , Péptidos beta-Amiloides/metabolismo , Bélgica , Forma de la Célula , Análisis Mutacional de ADN , Demencia/genética , Demencia/metabolismo , Femenino , Humanos , Cuerpos de Inclusión/química , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/ultraestructura , Masculino , Persona de Mediana Edad , Neuronas/citología , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismoRESUMEN
Several studies suggest that the generation of Aß is highly dependent on the levels of cholesterol within membranes' detergent-resistant microdomains (DRM). Indeed, the ß-amyloid precursor protein (APP) cleaving machinery, namely ß- and γ-secretases, has been shown to be present in DRM and its activity depends on membrane cholesterol levels. Counterintuitive to the localization of the cleavage machinery, the substrate, APP, localizes to membranes' detergent-soluble microdomains enriched in phospholipids (PL), indicating that Aß generation is highly dependent on the capacity of enzyme and substrate to diffuse along the lateral plane of the membrane and therefore on the internal equilibrium of the different lipids of DRM and non-DRM domains. Here, we studied to which extent changes in the content of a main non-DRM lipid might affect the proteolytic processing of APP. As phosphatidylethanolamine (PE) accounts for the majority of PL, we focused on its impact on the regulation of APP proteolysis. In mammalian cells, siRNA-mediated knock-down of PE synthesis resulted in decreased Aß owing to a dual effect: promoted α-secretase cleavage and decreased γ-secretase processing of APP. In vivo, in Drosophila melanogaster, genetic reduction in PL synthesis results in decreased γ-secretase-dependent cleavage of APP. These results suggest that modulation of the membrane-soluble domains could be a valuable alternative to reduce excessive Aß generation.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/biosíntesis , Drosophila melanogaster/metabolismo , Neuronas/metabolismo , Fosfatidiletanolaminas/metabolismo , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Membrana Celular/genética , Membrana Celular/metabolismo , Colesterol/metabolismo , Detergentes/farmacología , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Femenino , Células HEK293 , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Masculino , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Neuronas/citología , Fosfatidiletanolaminas/genética , Proteolisis , ARN Interferente Pequeño/genética , RatasRESUMEN
Transgenic mouse models of Alzheimer's disease (AD) expressing high levels of amyloid precursor protein (APP) with familial AD (FAD) mutations have proven to be extremely useful in understanding pathogenic processes of AD especially those that involve amyloidogenesis. We earlier described Austrian APP T714I pathology that leads to one of the earliest AD age-at-onsets with abundant intracellular and extracellular amyloid deposits in brain. The latter strikingly was non-fibrillar diffuse amyloid, composed of N-truncated A beta 42 in absence of A beta 40. In vitro, this mutation leads to one of the highest A beta 42/A beta 40 ratios among all FAD mutations. We generated an APP T714I transgenic mouse model that despite having 10 times lower transgene than endogenous murine APP deposited intraneuronal A beta in brain by 6 months of age. Accumulations increased with age, and this was paralleled by decreased brain sizes on volumetric MRI, compared to age-matched and similar transgene-expressing APP wild-type mice, although, with these levels of transgenic expression we did not detect neuronal loss or significant memory impairment. Immunohistochemical studies revealed that the majority of the intraneuronal A beta deposits colocalized with late endosomal markers, although some A beta inclusions were also positive for lysosomal and Golgi markers. These data support earlier observations of A beta accumulation in the endosomal-lysosomal pathway and the hypothesis that intraneuronal accumulation of A beta could be an important factor in the AD pathogenesis.
Asunto(s)
Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Encéfalo/patología , Isoleucina/genética , Tirosina/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/fisiopatología , Análisis de Varianza , Animales , Conducta Animal/fisiología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Humanos , Imagen por Resonancia Magnética , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Transgénicos , Neuronas/citología , Fragmentos de Péptidos/metabolismo , Desempeño Psicomotor/fisiologíaRESUMEN
We, amongst others, have shown that CC homozygosity at the -22C>T promoter polymorphism in presenilin 1 (PSEN1) is associated with increased risk for Alzheimer's disease (AD). Also, studies in AD brains suggested that CC homozygosity increased the risk for AD by increasing the Abeta load. We characterized the PSEN1 promoter by deletion mapping, and analysed the effect of the -22C and -22T alleles on the transcriptional activity of PSEN1 in a transient transfection system. We showed a neuron-specific 2-fold decrease in promoter activity for the -22C risk allele, which in homozygous individuals would lead to a critical decrease in PSEN1 expression. The deletion mapping suggested that the 13 bp region (-33/-20) spanning the -22C>T polymorphism harbours a binding site for a negative regulatory factor. This factor has a higher affinity for the -22C risk allele and is strongly dependent on downstream sequences for cell-type-specific expression differences. Together, these studies provide evidence that the increased risk for AD associated with PSEN1 may result from genetic variations in the regulatory region, leading to altered expression levels of PSEN1 in neurons.
Asunto(s)
Enfermedad de Alzheimer/genética , Proteínas de la Membrana/genética , Neuronas/metabolismo , Polimorfismo Genético , Regiones Promotoras Genéticas , Alelos , Animales , Secuencia de Bases , Células Cultivadas , Ensayo de Cambio de Movilidad Electroforética , Femenino , Predisposición Genética a la Enfermedad , Humanos , Datos de Secuencia Molecular , Iniciación de la Cadena Peptídica Traduccional , Presenilina-1 , Ratas , Ratas Sprague-Dawley , Secuencias Reguladoras de Ácidos Nucleicos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Mutations in the beta-amyloid (Abeta) sequence of the amyloid precursor protein gene (APP) present with variable disease phenotypes. While patients with the Dutch APP mutation (E693Q) have predominantly hemorrhagic strokes, Flemish APP (A692G) patients develop both strokes and Alzheimer's disease (AD). To determine whether these diverse clinical and pathological presentations are due to mutant Abeta or APP, we studied the effect of Flemish, Dutch, and wild-type Abeta/APP on phosphorylation of specific tau epitopes observed in AD. No effect was observed in differentiated SH-SY5Y cells either stably expressing APP or treated with synthetic Abeta(12-42). However, we did observe a paradoxical temporal difference in the neurotoxic potential of mutant and wild-type Abeta. While long 24-h incubation at physiological levels of Abeta (2 microM) showed a higher amount of apoptosis for Dutch Abeta, a short 2-h incubation showed elevated apoptosis for Flemish and wild-type Abeta. The altered aggregating properties of Abeta, with Dutch Abeta aggregating faster and Flemish Abeta slower than wild type, elucidated a discrete two-phase Abeta neurotoxicity. We propose here that, at least in vitro, Abeta might be neurotoxic in an initial phase due to its soluble oligomeric or other early toxic Abeta intermediate(s), which is perhaps distinct from the late neurotoxicity incurred by aggregated larger assemblies of Abeta.
Asunto(s)
Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/toxicidad , Encéfalo/metabolismo , Neuronas/metabolismo , Neurotoxinas/toxicidad , Fragmentos de Péptidos/toxicidad , Accidente Cerebrovascular/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Bélgica , Encéfalo/patología , Encéfalo/fisiopatología , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Células Cultivadas , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Citoesqueleto/patología , Humanos , Cinética , Mutación/genética , Países Bajos , Neuronas/efectos de los fármacos , Neuronas/patología , Fosforilación/efectos de los fármacos , Ratas , Solubilidad , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/fisiopatología , Proteínas tau/efectos de los fármacos , Proteínas tau/metabolismoRESUMEN
Nicastrin regulates gamma-secretase cleavage of the amyloid precursor protein by forming complexes with presenilins, in which most mutations causing familial early-onset Alzheimer disease (EOAD) have been found. The gene encoding nicastrin (NCSTN) maps to 1q23, a region that has been linked and associated with late-onset Alzheimer disease (LOAD) in various genome screens. In 78 familial EOAD cases, we found 14 NCSTN single-nucleotide polymorphisms (SNPs): 10 intronic SNPs, 3 silent mutations, and 1 missense mutation (N417Y). N417Y is unlikely to be pathogenic, since it did not alter amyloid beta secretion in an in vitro assay and its frequency was similar in case and control subjects. However, SNP haplotype estimation in two population-based series of Dutch patients with EOAD (n=116) and LOAD (n=240) indicated that the frequency of one SNP haplotype (HapB) was higher in the group with familial EOAD (7%), compared with the LOAD group (3%) and control group (3%). In patients with familial EOAD without the APOE epsilon4 allele, the HapB frequency further increased, to 14%, resulting in a fourfold increased risk (odds ratio = 4.1; 95% confidence interval 1.2-13.3; P=.01). These results are compatible with an important role of gamma-secretase dysfunction in the etiology of familial EOAD.
Asunto(s)
Enfermedad de Alzheimer/epidemiología , Enfermedad de Alzheimer/genética , Endopeptidasas/química , Predisposición Genética a la Enfermedad/genética , Glicoproteínas de Membrana/genética , Edad de Inicio , Anciano , Alelos , Secretasas de la Proteína Precursora del Amiloide , Apolipoproteína E4 , Apolipoproteínas E/genética , Ácido Aspártico Endopeptidasas , Estudios de Casos y Controles , Femenino , Frecuencia de los Genes/genética , Variación Genética/genética , Haplotipos/genética , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Países Bajos/epidemiología , Oportunidad Relativa , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Alzheimer's disease (AD) is characterized by deposition of beta-amyloid (Abeta) in diffuse and senile plaques, and variably in vessels. Mutations in the Abeta-encoding region of the amyloid precursor protein (APP) gene are frequently associated with very severe forms of vascular Abeta deposition, sometimes also accompanied by AD pathology. We earlier described a Flemish APP (A692G) mutation causing a form of early-onset AD with a prominent cerebral amyloid angiopathy and unusually large senile plaque cores. The pathogenic basis of Flemish AD is unknown. By image and mass spectrometric Abeta analyses, we demonstrated that in contrast to other familial AD cases with predominant brain Abeta42, Flemish AD patients predominantly deposit Abeta40. On serial histological section analysis we further showed that the neuritic senile plaques in APP692 brains were centered on vessels. Of a total of 2400 senile plaque cores studied from various brain regions from three patients, 68% enclosed a vessel, whereas the remainder were associated with vascular walls. These observations were confirmed by electron microscopy coupled with examination of serial semi-thin plastic sections, as well as three-dimensional observations by confocal microscopy. Diffuse plaques did not associate with vessels, or with neuritic or inflammatory pathology. Together with earlier in vitro data on APP692, our analyses suggest that the altered biological properties of the Flemish APP and Abeta facilitate progressive Abeta deposition in vascular walls that in addition to causing strokes, initiates formation of dense-core senile plaques in the Flemish variant of AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/patología , Placa Amiloide/patología , Enfermedad de Alzheimer/genética , Secuencia de Aminoácidos , Precursor de Proteína beta-Amiloide/genética , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Circulación Cerebrovascular , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Mutación , LinajeRESUMEN
Familial forms of frontotemporal dementia (FTD) with tauopathy are mostly caused by mutations in the gene encoding the microtubule-associated protein tau (MAPT). However, rare forms of familial tauopathy without MAPT mutations have been reported, suggesting other tauopathy-related genetic defects. Interestingly, two presenilin 1 (PS1) mutations (Leu113Pro and insArg352) recently have been associated with familial FTD albeit without neuropathological confirmation. We report here a novel PS1 mutation in a patient with Pick-type tauopathy in the absence of extracellular beta-amyloid deposits. The mutation is predicted to substitute Gly-->Val at codon position 183 (Gly183Val) and to affect the splice signal at the junction of the sixth exon and intron. Further clinical-genetic investigation showed a positive family history of FTD-like dementia and suggested that Gly183Val is associated with a phenotypically heterogeneous neurodegenerative disorder. Our results suggest PS1 as a candidate gene for Pick-type tauopathy without MAPT mutations.