RESUMO
Mice were constructed carrying prion protein (PrP) transgenes with individual regions of putative secondary structure deleted. Transgenic mice with amino-terminal regions deleted remained healthy at >400 days of age, whereas those with either of carboxy-terminal alpha-helices deleted spontaneously developed fatal CNS illnesses similar to neuronal storage diseases. Deletion of either C-terminal helix resulted in PrP accumulation within cytoplasmic inclusions in enlarged neurons. Deletion of the penultimate C-terminal helix resulted in proliferation of rough endoplasmic reticulum. Mice with the C-terminal helix deleted were affected with nerve cell loss in the hippocampus and proliferation of smooth endoplasmic reticulum. Whether children with the human counterpart of this malady will be found remains to be determined.
Assuntos
Doenças Priônicas/genética , Príons/biossíntese , Animais , Doenças Genéticas Inatas , Humanos , Corpos de Inclusão/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Doenças Priônicas/patologia , Príons/genética , Estrutura Secundária de Proteína , Deleção de Sequência , Relação Estrutura-AtividadeRESUMO
Transgenic mice were created to assess genetic linkage between Gerstmann-Sträussler-Scheinker syndrome and a leucine substitution at codon 102 of the human prion protein gene. Spontaneous neurologic disease with spongiform degeneration and gliosis similar to that in mouse scrapie developed at a mean age of 166 days in 35 mice expressing mouse prion protein with the leucine substitution. Thus, many of the clinical and pathological features of Gerstmann-Sträussler-Scheinker syndrome are reproduced in transgenic mice containing a prion protein with a single amino acid substitution, illustrating that a neurodegenerative process similar to a human disease can be genetically modeled in animals.
Assuntos
Encefalopatias/genética , Doença de Gerstmann-Straussler-Scheinker/genética , Mutação , Príons/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/patologia , Encefalopatias/microbiologia , Encefalopatias/patologia , Códon , DNA/genética , Modelos Animais de Doenças , Endopeptidase K , Doença de Gerstmann-Straussler-Scheinker/microbiologia , Doença de Gerstmann-Straussler-Scheinker/patologia , Leucina , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Linhagem , Proteínas PrPSc , Serina Endopeptidases/metabolismo , Transfecção , Vacúolos/patologia , Proteínas Virais/metabolismoRESUMO
At the endoplasmic reticulum membrane, the prion protein (PrP) can be synthesized in several topological forms. The role of these different forms was explored with transgenic mice expressing PrP mutations that alter the relative ratios of the topological forms. Expression of a particular transmembrane form (termed CtmPrP) produced neurodegenerative changes in mice similar to those of some genetic prion diseases. Brains from these mice contained CtmPrP but not PrPSc, the PrP isoform responsible for transmission of prion diseases. Furthermore, in one heritable prion disease of humans, brain tissue contained CtmPrP but not PrPSc. Thus, aberrant regulation of protein biogenesis and topology at the endoplasmic reticulum can result in neurodegeneration.
Assuntos
Retículo Endoplasmático/metabolismo , Doenças Neurodegenerativas/etiologia , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Príons/química , Príons/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Cricetinae , Endopeptidases/metabolismo , Retículo Endoplasmático/química , Doença de Gerstmann-Straussler-Scheinker/metabolismo , Humanos , Membranas Intracelulares/química , Mesocricetus , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Proteínas PrPC/biossíntese , Proteínas PrPC/genética , Proteínas PrPSc/química , Proteínas PrPSc/metabolismo , Doenças Priônicas/etiologia , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Príons/biossíntese , Príons/genética , Conformação ProteicaRESUMO
The fundamental event in prion diseases seems to be a conformational change in cellular prion protein (PrPC) whereby it is converted into the pathologic isoform PrPSc. In fatal familial insomnia (FFI), the protease-resistant fragment of PrPSc after deglycosylation has a size of 19 kilodaltons, whereas that from other inherited and sporadic prion diseases is 21 kilodaltons. Extracts from the brains of FFI patients transmitted disease to transgenic mice expressing a chimeric human-mouse PrP gene about 200 days after inoculation and induced formation of the 19-kilodalton PrPSc fragment, whereas extracts from the brains of familial and sporadic Creutzfeldt-Jakob disease patients produced the 21-kilodalton PrPSc fragment in these mice. The results presented indicate that the conformation of PrPSc functions as a template in directing the formation of nascent PrPSc and suggest a mechanism to explain strains of prions where diversity is encrypted in the conformation of PrPSc.
Assuntos
Química Encefálica , Encéfalo/patologia , Proteínas PrPSc/química , Doenças Priônicas/etiologia , Príons/química , Conformação Proteica , Animais , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/patologia , Humanos , Camundongos , Camundongos Transgênicos , Proteínas PrPSc/análise , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Doenças Priônicas/transmissão , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Frontotemporal dementia (FTD) is a clinical syndrome characterized by progressive decline in social conduct and a focal pattern of frontal and temporal lobe damage. Its biological basis is still poorly understood but the focality of the brain degeneration provides a powerful model to study the cognitive and anatomical basis of social cognition. Here, we present Dr. A, a patient with a rare hereditary bone disease (hereditary multiple exostoses) and FTD (pathologically characterized as Pick's disease), who presented with a profound behavioral disturbance characterized by acquired sociopathy. We conducted a detailed genetic, pathological, neuroimaging and cognitive study, including a battery of tests designed to investigate Dr. A's abilities to understand emotional cues and to infer mental states and intentions to others (theory of mind). Dr. A's genetic profile suggests the possibility that a mutation causing hereditary multiple exostoses, Ext2, may play a role in the pattern of neurodegeneration in frontotemporal dementia since knockout mice deficient in the Ext gene family member, Ext1, show severe CNS defects including loss of olfactory bulbs and abnormally small cerebral cortex. Dr. A showed significant impairment in emotion comprehension, second order theory of mind, attribution of intentions, and empathy despite preserved general cognitive abilities. Voxel-based morphometry on structural MRI images showed significant atrophy in the medial and right orbital frontal and anterior temporal regions with sparing of dorsolateral frontal cortex. This case demonstrates that social and emotional dysfunction in FTD can be dissociated from preserved performance on classic executive functioning tasks. The specific pattern of anatomical damage shown by VBM emphasizes the importance of the network including the superior medial frontal gyrus as well as temporal polar areas, in regulation of social cognition and theory of mind. This case provides new evidence regarding the neural basis of social cognition and suggests a possible genetic link between bone disease and FTD.
Assuntos
Exostose Múltipla Hereditária/epidemiologia , Exostose Múltipla Hereditária/genética , Demência Frontotemporal/epidemiologia , Demência Frontotemporal/genética , Predisposição Genética para Doença/genética , N-Acetilglucosaminiltransferases/genética , Idoso , Síndrome de Beckwith-Wiedemann/genética , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Osso e Ossos/fisiopatologia , Comorbidade , Análise Mutacional de DNA , Progressão da Doença , Empatia/genética , Exostose Múltipla Hereditária/fisiopatologia , Evolução Fatal , Demência Frontotemporal/fisiopatologia , Testes Genéticos , Genótipo , Humanos , Padrões de Herança/genética , Masculino , Neurônios/metabolismo , Neurônios/patologia , Linhagem , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/patologia , Córtex Pré-Frontal/fisiopatologia , Transtornos do Comportamento Social/etiologia , Transtornos do Comportamento Social/patologia , Transtornos do Comportamento Social/fisiopatologia , Lobo Temporal/metabolismo , Lobo Temporal/patologia , Lobo Temporal/fisiopatologia , Teoria da Mente/fisiologiaRESUMO
Prolonged incubation times for experimental scrapie in I/LnJ mice are dictated by a dominant gene linked to the prion protein gene (Prn-p). Transgenic mice were analyzed to discriminate between an effect of the I/LnJ Prn-pb allele and a distinct incubation time locus designated Prn-i. Paradoxically, 4 independent Prn-pb transgenic mouse lines had scrapie incubation times shorter than nontransgenic controls, instead of the anticipated prolonged incubation periods. Aberrant or overexpression of the Prn-pb transgenes may dictate abbreviated incubation times, masking genuine Prn-p/Prn-i congruence; alternatively, a discrete Prn-i gene lies adjacent to Prn-p.
Assuntos
Expressão Gênica , Scrapie/fisiopatologia , Proteínas Virais/genética , Animais , Encéfalo/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas PrPSc , Príons/genética , Príons/metabolismo , RNA Mensageiro/metabolismo , Fatores de Tempo , Proteínas Virais/metabolismoRESUMO
The pattern of scrapie prion protein (PrP(Sc)) accumulation in the brain is different for each prion strain. We tested whether the PrP(Sc) deposition pattern is influenced by the Asn-linked oligosaccharides of PrP(C) in transgenic mice. Deletion of the first oligosaccharide altered PrP(C) trafficking and prevented infection with two prion strains. Deletion of the second did not alter PrP(C) trafficking, permitted infection with one prion strain, and had a profound effect on the PrP(Sc) deposition pattern. Our data raise the possibility that glycosylation can modify the conformation of PrP(C). Glycosylation could affect the affinity of PrP(C) for a particular conformer of PrP(Sc), thereby determining the rate of nascent PrP(Sc) formation and the specific patterns of PrP(Sc) deposition.
Assuntos
Encéfalo/metabolismo , Proteínas PrPC/biossíntese , Doenças Priônicas/metabolismo , Animais , Encéfalo/patologia , Cricetinae , Mesocricetus , Camundongos , Camundongos Transgênicos , Mutagênese , Oligossacarídeos/metabolismo , Fases de Leitura Aberta , Especificidade de Órgãos , Proteínas PrPC/química , Proteínas PrPC/genética , Doenças Priônicas/patologia , Deleção de SequênciaRESUMO
We applied optimised voxel based morphometry (VBM) to brain MRIs from autopsy proven cases of tau positive frontotemporal lobar degeneration (FTLD-T, n = 6), ubiquitin and TDP-43 positive/tau negative FTLD (FTLD-U, n = 8) and cognitively normal controls (n = 61). The analysis revealed that FTLD-T and FTLD-U both show atrophy in the frontal cortex and striatum, but striatal atrophy is more severe in FTLD-T. Manual region of interest tracing of caudate and putamen volumes confirmed the VBM findings. These anatomical differences may help distinguish between FTLD spectrum pathological subtypes in vivo.
Assuntos
Demência/metabolismo , Demência/patologia , Imageamento por Ressonância Magnética , Ubiquitina/metabolismo , Proteínas tau/metabolismo , Idoso , Atrofia/patologia , Autopsia , Núcleo Caudado/anatomia & histologia , Demência/complicações , Demência/diagnóstico , Demência/etiologia , Feminino , Humanos , Masculino , Testes Neuropsicológicos , Putamen/anatomia & histologia , Índice de Gravidade de DoençaRESUMO
Given the critical role of the prion protein (PrP) in the transmission and pathogenesis of experimental scrapie, we investigated the PrP gene and its protein products in three hamster species, Chinese (CHa), Armenian (AHa), and Syrian (SHa), each of which were found to have distinctive scrapie incubation times. Passaging studies demonstrated that the host species, and not the source of scrapie prions, determined the incubation time for each species, and histochemical studies of hamsters with clinical signs of scrapie revealed characteristic patterns of neuropathology. Northern (RNA) analysis showed the size of PrP mRNA from CHa, AHa, and SHa hamsters to be 2.5, 2.4, and 2.1 kilobases, respectively. Immunoblotting demonstrated that the PrP isoforms were of similar size (33 to 35 kilodaltons); however, the monoclonal antibody 13A5 raised against SHa PrP did not react with the CHa or AHa PrP molecules. Comparison of the three predicted amino acid sequences revealed that each is distinct. Furthermore, differences within the PrP open reading frame that uniquely distinguish the three hamster species are within a hydrophilic segment of 11 amino acids that includes polymorphisms linked to scrapie incubation times in inbred mice and an inherited prion disease of humans. Single polymorphisms in this region correlate with the presence or absence of amyloid plaques for a given hamster species or mouse inbred strain. Our findings demonstrate distinctive molecular, pathological, and clinical characteristics of scrapie in three related species and are consistent with the hypothesis that molecular properties of the host PrP play a pivotal role in determining the incubation time and neuropathological features of scrapie.
Assuntos
Príons/genética , Scrapie/genética , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Western Blotting , Clonagem Molecular , Cricetinae , Cricetulus/genética , Mesocricetus/genética , Dados de Sequência Molecular , Oligonucleotídeos , Reação em Cadeia da Polimerase , Príons/metabolismo , RNA Mensageiro/genética , Mapeamento por Restrição , Scrapie/patologia , Especificidade da EspécieRESUMO
An abridged prion protein (PrP) molecule of 106 amino acids, designated PrP106, is capable of forming infectious miniprions in transgenic mice (S. Supattapone, P. Bosque, T. Muramoto, H. Wille, C. Aagaard, D. Peretz, H.-O. B. Nguyen, C. Heinrich, M. Torchia, J. Safar, F. E. Cohen, S. J. DeArmond, S. B. Prusiner, and M. Scott, Cell 96:869-878, 1999). We removed additional sequences from PrP106 and identified a 61-residue peptide, designated PrP61, that spontaneously adopted a protease-resistant conformation in neuroblastoma cells. Synthetic PrP61 bearing a carboxy-terminal lipid moiety polymerized into protease-resistant, beta-sheet-enriched amyloid fibrils at a physiological salt concentration. Transgenic mice expressing low levels of PrP61 died spontaneously with ataxia. Neuropathological examination revealed accumulation of protease-resistant PrP61 within neuronal dendrites and cell bodies, apparently causing apoptosis. PrP61 may be a useful model for deciphering the mechanism by which PrP molecules acquire protease resistance and become neurotoxic.
Assuntos
Camundongos Transgênicos , Doenças Neurodegenerativas/genética , Príons/genética , Animais , Camundongos , Doenças Neurodegenerativas/etiologia , Peptídeos/genéticaRESUMO
To better define the anatomic distinctions between Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD), we retrospectively applied voxel-based morphometry to the earliest magnetic resonance imaging scans of autopsy-proven AD (N = 11), FTLD (N = 18), and controls (N = 40). Compared with controls, AD patients showed gray matter reductions in posterior temporoparietal and occipital cortex; FTLD patients showed atrophy in medial prefrontal and medial temporal cortex, insula, hippocampus, and amygdala; and patients with both disorders showed atrophy in dorsolateral and orbital prefrontal cortex and lateral temporal cortex (P(FWE-corr) < .05). Compared with FTLD, AD patients had decreased gray matter in posterior parietal and occipital cortex, whereas FTLD patients had selective atrophy in anterior cingulate, frontal insula, subcallosal gyrus, and striatum (P < .001, uncorrected). These findings suggest that AD and FTLD are anatomically distinct, with degeneration of a posterior parietal network in AD and degeneration of a paralimbic fronto-insular-striatal network in FTLD.
Assuntos
Doença de Alzheimer/patologia , Encéfalo/patologia , Demência/patologia , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Imageamento por Ressonância Magnética , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/diagnóstico , Tonsila do Cerebelo/patologia , Atrofia , Córtex Cerebral/patologia , Corpo Estriado/patologia , Demência/diagnóstico , Diagnóstico Diferencial , Dominância Cerebral/fisiologia , Feminino , Giro do Cíngulo/patologia , Hipocampo/patologia , Humanos , Doença por Corpos de Lewy/diagnóstico , Doença por Corpos de Lewy/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
We devised a model system to study the effects of extracellular matrix proteins on the malignant phenotype of an anaplastic glioma cell line, U 343 MG-A. Well-characterized cultures derived from normal human leptomeninges were grown to confluence and maintained for 2 weeks. The leptomeningeal cells were then removed with base and detergent, leaving behind an extracellular matrix enriched in laminin, fibronectin, type I and IV collagen, and procollagen III. U 343 MG-A tumor cells planted on top of this normal extracellular matrix were profoundly growth inhibited compared with glioma cells grown on plastic alone. Glioma cells grown on the extracellular matrix developed multiple, slender processes and assumed a more differentiated astrocytic phenotype; immunostains for glial fibrillary acidic protein revealed a more extensive intracytoplasmic network of intensely staining filaments than in control glioma cells. When glioma cells grown on the extracellular matrix were analyzed by an enzyme-linked immunosorbent assay for glial fibrillary acidic protein, the amount of this intermediate filament per cell was increased 20-fold compared with glioma cells growing on plastic. The growth and differentiation of U 343 MG-A glioma cells in flasks coated with purified fibronectin or laminin was not significantly perturbed; however, glioma cell cultures grown in flasks coated with purified type I or IV collagen showed decreased cellular proliferation, stellate cell formation, and increased levels of glial fibrillary acidic protein per cell compared with glioma cells growing on plastic. Gelatin gel analysis showed that U 343 MG-A glioma cells growing on plastic secreted a 65,000-D metalloproteinase that was not secreted by glioma cells grown on the leptomeningeal extracellular matrix. We conclude that in this system, the extracellular matrix of a normal human leptomeningeal culture substantially inhibited the proliferation of and induced differentiation in an anaplastic glioma cell line. Our analysis of single components of the extracellular matrix suggests that these effects may be mediated in part by type I and IV collagen. The mechanism by which the leptomeningeal extracellular matrix inhibits glioma cell proliferation may be by diminishing tumor-associated protease secretion so that the degradation of extracellular matrix macromolecules in the tumor cell microenvironment is prevented and tumor cell migration becomes less likely.
Assuntos
Matriz Extracelular/fisiologia , Glioma/patologia , Diferenciação Celular , Divisão Celular , Linhagem Celular , Colágeno/fisiologia , Fibronectinas/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Laminina/fisiologia , Meninges/ultraestrutura , Peptídeo Hidrolases/metabolismoRESUMO
To identify molecular interaction partners of the cellular prion protein (PrP(C)), we sought to apply an in situ crosslinking method that maintains the microenvironment of PrP(C). Mild formaldehyde crosslinking of mouse neuroblastoma cells (N2a) that are susceptible to prion infection revealed the presence of PrP(C) in high molecular mass (HMM) protein complexes of 200 to 225 kDa. LC/MS/MS analysis identified three murine splice-variants of the neural cell adhesion molecule (N-CAM) in the complexes, which isolate with caveolae-like domains (CLDs). Enzymatic removal of N-linked sugar moieties did not disrupt the complexes, arguing that the interaction of PrP with N-CAM occurs through amino acid side-chains. Additionally, similar levels of PrP/N-CAM complexes were found in N2a and prion-infected N2a (ScN2a) cells. With the use of an N-CAM-specific peptide library, the PrP-binding site was determined to comprise beta-strands C and C' within the two consecutive fibronectin type III (FNIII) modules found in proximity of the membrane-attachment site of N-CAM. As revealed by in situ crosslinking of PrP deletion mutants, the PrP face of the binding site is formed by the N terminus, helix A (residues 144-154) and the adjacent loop region of PrP. N-CAM-deficient (N-CAM(-/-)) mice that were intracerebrally challenged with scrapie prions succumbed to disease with a mean incubation period of 122 (+/-4.1, SEM) days, arguing that N-CAM is not involved in PrP(Sc) replication. Our findings raise the possibility that N-CAM may join with PrP(C) in carrying out some as yet unidentified physiologic cellular function.
Assuntos
Moléculas de Adesão de Célula Nervosa/química , Moléculas de Adesão de Célula Nervosa/metabolismo , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Processamento Alternativo/genética , Amidoidrolases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cavéolas/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , Endopeptidase K/metabolismo , Formaldeído/metabolismo , Substâncias Macromoleculares , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Peso Molecular , Mutação/genética , Moléculas de Adesão de Célula Nervosa/genética , Neuroblastoma/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Fosfatidilinositol Diacilglicerol-Liase , Proteínas PrPC/genética , Proteínas PrPSc/farmacologia , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Sítios de Splice de RNA/genética , Células Tumorais Cultivadas , Fosfolipases Tipo C/metabolismoRESUMO
The molecular basis of the infectious, inherited and sporadic forms of prion diseases is best explained by a conformationally dimorphic protein that can exist in distinct normal and disease-causing isoforms. We identified a 55-residue peptide of a mutant prion protein that can be refolded into at least two distinct conformations. When inoculated intracerebrally into the appropriate transgenic mouse host, 20 of 20 mice receiving the beta-form of this peptide developed signs of central nervous system dysfunction at approximately 360 days, with neurohistologic changes that are pathognomonic of Gerstmann-Sträussler-Scheinker disease. By contrast, eight of eight mice receiving a non-beta-form of the peptide failed to develop any neuropathologic changes more than 600 days after the peptide injections. We conclude that a chemically synthesized peptide refolded into the appropriate conformation can accelerate or possibly initiate prion disease.
Assuntos
Encéfalo/patologia , Doença de Gerstmann-Straussler-Scheinker/genética , Fragmentos de Peptídeos/química , Príons/genética , Sequência de Aminoácidos , Animais , Encéfalo/efeitos dos fármacos , Doença de Gerstmann-Straussler-Scheinker/patologia , Doença de Gerstmann-Straussler-Scheinker/fisiopatologia , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/toxicidade , Príons/química , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Scrapie/patologia , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The eight amino acid sequence, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, representing the FLAG peptide, was inserted after codons 22 or 88 of the mouse (Mo) prion protein (PrP) gene. Inclusion of the FLAG sequence at these locations interfered neither with the cellular processing of PrPC nor its conversion into PrPSc. Inclusion of the FLAG epitope at residue 22 but not at residue 88 facilitated immunodetection of tagged PrP by anti-FLAG monoclonal antibodies (mAbs). Inoculation of transgenic (Tg) mice expressing N-terminally tagged MoPrP with Mo prions resulted in abbreviated incubation times, indicating that the FLAG sequence was not deleterious to prion propagation. Immunopurification of FLAG-tagged MoPrPC in the brains of Tg mice was achieved using the calcium-dependent anti-FLAG M1 mAb and non-denaturing procedures. Although the function of PrPC remains unknown, our studies demonstrate that some modifications of PrPC do not inhibit the one biological activity that can be measured, i.e., conversion into PrPSc. Tagged PrP molecules may prove useful in the development of improved assays for prions as well as structural studies of the PrP isoforms.
Assuntos
Príons/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Camundongos , Camundongos Transgênicos , Príons/química , Príons/isolamento & purificação , Células Tumorais CultivadasRESUMO
Basal ganglia, cerebral cortex and subcortical white matter were studied for glial fibrillary acidic protein (GFAP) in four cases of hepatic encephalopathy (HE) with Alzheimer II gliosis and five age-matched controls. GFAP was measured by enzyme-linked immunosorbent assay (ELISA) and reflectance densitometry of immunoperoxidase-stained western blots. Both methods revealed a 70% decrease in the average amount of GFAP in the cerebral cortex and a 60% decrease in the basal ganglia in HE cases. GFAP in white matter was not significantly changed. In contrast to GFAP, the average protein content was not significantly different for HE and controls. These findings support the view that HE is a neurological disease in which there is a selective loss of GFAP from the grey matter.
Assuntos
Química Encefálica , Proteína Glial Fibrilar Ácida/análise , Encefalopatia Hepática/metabolismo , Idoso , Gânglios da Base/análise , Córtex Cerebral/análise , Colódio , Densitometria/métodos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas Imunoenzimáticas , Pessoa de Meia-Idade , Peso MolecularRESUMO
The kinetics of PrP(Sc) and insoluble PrP accumulation in the spleens and brains of CD-1 mice were studied. The mice were inoculated intracerebrally with RML prions and euthanized at various times between inoculation and the onset of illness at approximately 130 days. Protease-resistant PrP(Sc), PrP 27-30, was first detected in brain by histoblotting 49 days after inoculation and by Western immunoblotting at 70 days. In spleen, PrP 27-30 was first detected by Western immunoblotting at 28 days after inoculation. Like PrP 27-30, substantial increases in detergent-insoluble PrP were first detected at 70 days after inoculation in brain and 28 days in spleen. In addition, a progressive increase in detergent-soluble PrP was detected beginning 70 days after inoculation. Further characterization of detergent soluble and insoluble PrP with respect to protease-sensitive PrP(Sc) and prion infectivity will be of considerable interest.
Assuntos
Encéfalo/metabolismo , Príons/metabolismo , Scrapie/metabolismo , Animais , Western Blotting , Detergentes/farmacologia , Cinética , Camundongos , Proteína PrP 27-30/metabolismo , Proteínas PrPSc/metabolismo , Solubilidade , Baço/metabolismo , Fatores de Tempo , Distribuição TecidualRESUMO
Basal ganglia, thalamus, cerebral cortex, and subcortical white matter were studied in ten cases of hepatic encephalopathy (HE), including three cases of acquired hepatocerebral degeneration (HCD), and in thirteen age-matched controls using the peroxidase-antiperoxidase immunohistochemical staining technique for glial fibrillary acidic (GFA) protein. HE cases all had pronounced Alzheimer type II astrocytosis. The perikarya and processes of Alzheimer type II glia did not stain for GFA protein. Staining of perivascular endfeet was evaluated by first selecting blood vessels throughout the gray and white matter in hematoxylin and eosin-stained slides to eliminate bias. The vessels were then identified in sections stained for GFA protein and graded as to complete circumferential, partial circumferential, or absence of staining. Both the degree and frequency of staining in the basal ganglia, thalamus, and cerebral cortex were significantly decreased in cases of HE; no statistically significant differences were found for the white matter. There were no significant differences in staining between HCD and other HE cases. These findings show that the Alzheimer II change is associated with a loss of immunohistochemically detectable GFA protein in cerebral gray matter.
Assuntos
Encefalopatia Hepática/imunologia , Proteínas do Tecido Nervoso/imunologia , Idoso , Astrócitos/imunologia , Encéfalo/imunologia , Feminino , Proteína Glial Fibrilar Ácida , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Neuroglia/imunologia , Estatística como AssuntoRESUMO
Using immunocytochemical methods, we localized several glycoproteins of the extracellular matrix to leptomeningeal cells and meningiomas in vitro. Three cell lines derived from normal human leptomeninges and seven from meningiomas were studied by indirect immunofluorescence to evaluate the cellular production of fibronectin, laminin, collagen type IV, and procollagen type III. All leptomeningeal cell lines stained intensely and uniformly for all matrix proteins; all meningioma cell cultures stained uniformly, but the intensity of staining varied considerably. After removal of the cells in culture adherent to glass with 25 mM ammonium hydroxide, indirect immunofluorescence demonstrated an exuberant residual extracellular residue enriched with fibronectin, laminin, collagen type IV, and procollagen type III. Electron microscopic examination of all leptomeningeal and meningioma cultures revealed desmosomes and dense tonofilament formation; in addition, granular, filamentous basement membrane-like material was abundant in the extracellular spaces of all cultures. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the cell layer of two leptomeningeal and four meningioma cultures showed production of interstitial collagen types I and III; diethylaminoethyl (DEAE)-cellulose chromatography of the medium demonstrated preferential production of procollagen type I. Our findings show conclusively that normal arachnoid cells in vitro synthesize several of the collagen subtypes and may be responsible for the "fibrous response" of the leptomeninges to trauma, infection, or infiltration by tumor. The similarities between leptomeningeal cells and meningiomas demonstrated by electron microscopy and by indirect immunofluorescence support the notion that meningiomas are derived from arachnoid cells. The localization of various mesenchymal glycoproteins within the intra- and extracellular spaces and the ubiquity of specialized intercellular junctions suggest that leptomeningeal cells in culture have the potential to behave like both stromal and epithelial cells.
Assuntos
Aracnoide-Máter/patologia , Neoplasias Meníngeas/patologia , Meningioma/patologia , Pia-Máter/patologia , Animais , Aracnoide-Máter/análise , Colágeno/análise , Fator VIII/análise , Proteína Glial Fibrilar Ácida/análise , Humanos , Imunoquímica , Laminina/análise , Neoplasias Meníngeas/análise , Meningioma/análise , Pia-Máter/análise , Pró-Colágeno/análise , RoedoresRESUMO
We recently found that deletion of the Asn-linked carbohydrate (CHO) at residue 197 of Syrian hamster (SHa) PrP(C) while retaining the CHO at Asn 181 has a profound effect on which population of neurons are targeted for conversion of SHaPrP(C) to SHaPrP(Sc) in transgenic (Tg) mice inoculated with scrapie prions. We hypothesized that selective targeting of neuronal populations is determined by cell-specific differences in the affinity of an infecting PrP(Sc) (prion) for PrP(C) and that the affinity might be modulated by nerve cell-specific differences in PrP(C) glycosylation. Here we tested this hypothesis by assessing whether or not each brain region in Syrian hamsters synthesizes different PrP(C) glycoforms, as inferred from 2D-gel electrophoresis. Reproducible differences in the number and isoelectric point of PrP(C) charge isomers were found as a function of brain region. The results of this study support the hypothesis that the PrP(Sc) accumulation and the vacuolation pattern phenotypes in the brain are governed by neuron-specific differences in PrP(C) glycoforms.