RESUMO
The TREM2-DAP12 receptor complex sustains microglia functions. Heterozygous hypofunctional TREM2 variants impair microglia, accelerating late-onset Alzheimer's disease. Homozygous inactivating variants of TREM2 or TYROBP-encoding DAP12 cause Nasu-Hakola disease (NHD), an early-onset dementia characterized by cerebral atrophy, myelin loss and gliosis. Mechanisms underpinning NHD are unknown. Here, single-nucleus RNA-sequencing analysis of brain specimens from DAP12-deficient NHD individuals revealed a unique microglia signature indicating heightened RUNX1, STAT3 and transforming growth factor-ß signaling pathways that mediate repair responses to injuries. This profile correlated with a wound healing signature in astrocytes and impaired myelination in oligodendrocytes, while pericyte profiles indicated vascular abnormalities. Conversely, single-nuclei signatures in mice lacking DAP12 signaling reflected very mild microglial defects that did not recapitulate NHD. We envision that DAP12 signaling in microglia attenuates wound healing pathways that, if left unchecked, interfere with microglial physiological functions, causing pathology in human. The identification of a dysregulated NHD microglia signature sparks potential therapeutic strategies aimed at resetting microglia signaling pathways.
Assuntos
Demência , Panencefalite Esclerosante Subaguda , Animais , Humanos , Camundongos , Encéfalo/metabolismo , Demência/metabolismo , Demência/patologia , Glicoproteínas de Membrana/metabolismo , Microglia/metabolismo , Receptores Imunológicos/metabolismo , Panencefalite Esclerosante Subaguda/metabolismo , Panencefalite Esclerosante Subaguda/patologiaRESUMO
Measles virus (MeV) is an enveloped RNA virus bearing two envelope glycoproteins, the hemagglutinin (H) and fusion (F) proteins. Upon receptor binding, the H protein triggers conformational changes of the F protein, causing membrane fusion and subsequent virus entry. MeV may persist in the brain, infecting neurons and causing fatal subacute sclerosing panencephalitis (SSPE). Since neurons do not express either of the MeV receptors, signaling lymphocytic activation molecule (SLAM; also called CD150) and nectin-4, how MeV propagates in neurons is unknown. Recent studies have shown that specific substitutions in the F protein found in MeV isolates from SSPE patients are critical for MeV neuropathogenicity by rendering the protein unstable and hyperfusogenic. Recombinant MeVs possessing the F proteins with such substitutions can spread in primary human neurons and in the brains of mice and hamsters and induce cell-cell fusion in cells lacking SLAM and nectin-4. Here, we show that receptor-blind mutant H proteins that have decreased binding affinities to receptors can support membrane fusion mediated by hyperfusogenic mutant F proteins, but not the wild-type F protein, in cells expressing the corresponding receptors. The results suggest that weak interactions of the H protein with certain molecules (putative neuron receptors) trigger hyperfusogenic F proteins in SSPE patients. Notably, where cell-cell contacts are ensured, the weak cis interaction of the H protein with SLAM on the same cell surface also could trigger hyperfusogenic F proteins. Some enveloped viruses may exploit such cis interactions with receptors to infect target cells, especially in cell-to-cell transmission.IMPORTANCE Measles virus (MeV) may persist in the brain, causing incurable subacute sclerosing panencephalitis (SSPE). Because neurons, the main target in SSPE, do not express receptors for wild-type (WT) MeV, how MeV propagates in the brain is a key question for the disease. Recent studies have demonstrated that specific substitutions in the MeV fusion (F) protein are critical for neuropathogenicity. Here, we show that weak cis and trans interactions of the MeV attachment protein with receptors that are not sufficient to trigger the WT MeV F protein can trigger the mutant F proteins from neuropathogenic MeV isolates. Our study not only provides an important clue to understand MeV neuropathogenicity but also reveals a novel viral strategy to expand cell tropism.
Assuntos
Moléculas de Adesão Celular/metabolismo , Hemaglutininas Virais/metabolismo , Vírus do Sarampo/metabolismo , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Panencefalite Esclerosante Subaguda/metabolismo , Proteínas Virais de Fusão/metabolismo , Animais , Moléculas de Adesão Celular/genética , Linhagem Celular , Cricetinae , Hemaglutininas Virais/genética , Humanos , Vírus do Sarampo/genética , Vírus do Sarampo/patogenicidade , Camundongos , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Panencefalite Esclerosante Subaguda/genética , Panencefalite Esclerosante Subaguda/patologia , Proteínas Virais de Fusão/genéticaRESUMO
Nasu-Hakola Disease (NHD) is a recessively inherited systemic leukodystrophy disorder characterized by a combination of frontotemporal presenile dementia and lytic bone lesions. NHD is known to be genetically related to a structural defect of TREM2 and DAP12, two genes that encode for different subunits of the membrane receptor signaling complex expressed by microglia and osteoclast cells. Because of its rarity, molecular or proteomic studies on this disorder are absent or scarce, only case reports based on neuropsychological and genetic tests being reported. In light of this, the aim of this paper is to provide evidence on the potential of a label-free proteomic platform based on the Multidimensional Protein Identification Technology (MudPIT), combined with in-house software and on-line bioinformatics tools, to characterize the protein expression trends and the most involved pathways in NHD. The application of this approach on the Lymphoblastoid cells from a family composed of individuals affected by NHD, healthy carriers and control subjects allowed for the identification of about 3000 distinct proteins within the three analyzed groups, among which proteins anomalous to each category were identified. Of note, several differentially expressed proteins were associated with neurodegenerative processes. Moreover, the protein networks highlighted some molecular pathways that may be involved in the onset or progression of this rare frontotemporal disorder. Therefore, this fully automated MudPIT platform which allowed, for the first time, the generation of the whole protein profile of Lymphoblastoid cells from Nasu-Hakola subjects, could be a valid approach for the investigation of similar neurodegenerative diseases.
Assuntos
Lipodistrofia/metabolismo , Lipodistrofia/patologia , Linfócitos/metabolismo , Linfócitos/patologia , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/patologia , Proteômica , Panencefalite Esclerosante Subaguda/metabolismo , Panencefalite Esclerosante Subaguda/patologia , Análise por Conglomerados , Análise Discriminante , Humanos , Glicoproteínas de Membrana/metabolismo , Mapas de Interação de Proteínas , Receptores Imunológicos/metabolismo , Biologia de SistemasRESUMO
Measles virus (MV) usually causes acute infection but in rare cases persists in the brain, resulting in subacute sclerosing panencephalitis (SSPE). Since human neurons, an important target affected in the disease, do not express the known MV receptors (signaling lymphocyte activation molecule [SLAM] and nectin 4), how MV infects neurons and spreads between them is unknown. Recent studies have shown that many virus strains isolated from SSPE patients possess substitutions in the extracellular domain of the fusion (F) protein which confer enhanced fusion activity. Hyperfusogenic viruses with such mutations, unlike the wild-type MV, can induce cell-cell fusion even in SLAM- and nectin 4-negative cells and spread efficiently in human primary neurons and the brains of animal models. We show here that a hyperfusogenic mutant MV, IC323-F(T461I)-EGFP (IC323 with a fusion-enhancing T461I substitution in the F protein and expressing enhanced green fluorescent protein), but not the wild-type MV, spreads in differentiated NT2 cells, a widely used human neuron model. Confocal time-lapse imaging revealed the cell-to-cell spread of IC323-F(T461I)-EGFP between NT2 neurons without syncytium formation. The production of virus particles was strongly suppressed in NT2 neurons, also supporting cell-to-cell viral transmission. The spread of IC323-F(T461I)-EGFP was inhibited by a fusion inhibitor peptide as well as by some but not all of the anti-hemagglutinin antibodies which neutralize SLAM- or nectin-4-dependent MV infection, suggesting the presence of a distinct neuronal receptor. Our results indicate that MV spreads in a cell-to-cell manner between human neurons without causing syncytium formation and that the spread is dependent on the hyperfusogenic F protein, the hemagglutinin, and the putative neuronal receptor for MV.IMPORTANCE Measles virus (MV), in rare cases, persists in the human central nervous system (CNS) and causes subacute sclerosing panencephalitis (SSPE) several years after acute infection. This neurological complication is almost always fatal, and there is currently no effective treatment for it. Mechanisms by which MV invades the CNS and causes the disease remain to be elucidated. We have previously shown that fusion-enhancing substitutions in the fusion protein of MVs isolated from SSPE patients contribute to MV spread in neurons. In this study, we demonstrate that MV bearing the hyperfusogenic mutant fusion protein spreads between human neurons in a cell-to-cell manner. Spread of the virus was inhibited by a fusion inhibitor peptide and antibodies against the MV hemagglutinin, indicating that both the hemagglutinin and hyperfusogenic fusion protein play important roles in MV spread between human neurons. The findings help us better understand the disease process of SSPE.
Assuntos
Hemaglutininas Virais/metabolismo , Vírus do Sarampo/metabolismo , Sarampo/transmissão , Neurônios/metabolismo , Panencefalite Esclerosante Subaguda/transmissão , Proteínas Virais de Fusão/metabolismo , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Chlorocebus aethiops , Hemaglutininas Virais/genética , Humanos , Sarampo/genética , Sarampo/metabolismo , Sarampo/patologia , Vírus do Sarampo/genética , Vírus do Sarampo/patogenicidade , Neurônios/patologia , Neurônios/virologia , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Panencefalite Esclerosante Subaguda/genética , Panencefalite Esclerosante Subaguda/metabolismo , Panencefalite Esclerosante Subaguda/patologia , Células Vero , Proteínas Virais de Fusão/genéticaRESUMO
Triggering receptor expressed on myeloid cells 2 (TREM2) is a single transmembrane molecule uniquely expressed in microglia. TREM2 mutations are genetically linked to Nasu-Hakola disease and associated with multiple neurodegenerative disorders, including Alzheimer's disease. TREM2 may regulate microglial inflammation and phagocytosis through coupling to the adaptor protein TYRO protein-tyrosine kinase-binding protein (TYROBP). However, there is no functional system for monitoring this protein-protein interaction. We developed a luciferase-based modality for real-time monitoring of TREM2-TYROBP coupling in live cells that utilizes split-luciferase complementation technology based on TREM2 and TYROBP fusion to the C- or N-terminal portion of the Renilla luciferase gene. Transient transfection of human embryonic kidney 293 cells with this reporter vector increased luciferase activity upon stimulation with an anti-TREM2 antibody, which induces their homodimerization. This was confirmed by ELISA-based analysis of the TREM2-TYROBP interaction. Antibody-mediated TREM2 stimulation enhanced spleen tyrosine kinase (SYK) activity and uptake of Staphylococcus aureus in microglial cell line BV-2 in a kinase-dependent manner. Interestingly, the TREM2 T66M mutation significantly enhanced luciferase activity without stimulation, indicating constitutive coupling to TYROBP. Finally, flow cytometry analyses indicated significantly lower surface expression of T66M TREM2 variant than wild type or other TREM2 variants. These results demonstrate that our TREM2 reporter vector is a novel tool for monitoring the TREM2-TYROBP interaction in real time.
Assuntos
Citometria de Fluxo/métodos , Teste de Complementação Genética/métodos , Luciferases de Renilla/metabolismo , Glicoproteínas de Membrana/metabolismo , Microglia/metabolismo , Receptores Imunológicos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Animais , Linhagem Celular , Humanos , Lipodistrofia/genética , Lipodistrofia/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Receptores Imunológicos/genética , Panencefalite Esclerosante Subaguda/genética , Panencefalite Esclerosante Subaguda/metabolismo , Quinase Syk/genética , Quinase Syk/metabolismoRESUMO
The triggering receptor expressed on myeloid cells 2 (TREM2) is an immune-modulatory receptor involved in phagocytosis and inflammation. Mutations of Q33X, Y38C and T66M cause Nasu-Hakola disease (NHD) which is characterized by early onset of dementia and bone cysts. A recent, genome-wide association study also revealed that single nucleotide polymorphism of TREM2, such as R47H, increased the risk of Alzheimer's disease (AD) similar to ApoE4. However, how these mutations affect the trafficking of TREM2, which may affect the normal functions of TREM2, was not known. In this study, we show that TREM2 with NHD mutations are impaired in the glycosylation with complex oligosaccharides in the Golgi apparatus, in the trafficking to plasma membrane and further processing by γ-secretase. Although R47H mutation in AD affected the glycosylation and normal trafficking of TREM2 less, the detailed pattern of glycosylated TREM2 differs from that of the wild type, thus suggesting that precise regulation of TREM2 glycosylation is impaired when arginine at 47 is mutated to histidine. Our results suggest that the impaired glycosylation and trafficking of TREM2 from endoplasmic reticulum/Golgi to plasma membrane by mutations may inhibit its normal functions in the plasma membrane, which may contribute to the disease.
Assuntos
Doença de Alzheimer/metabolismo , Complexo de Golgi/metabolismo , Lipodistrofia/metabolismo , Glicoproteínas de Membrana/genética , Mutação , Oligossacarídeos/metabolismo , Osteocondrodisplasias/metabolismo , Receptores Imunológicos/genética , Panencefalite Esclerosante Subaguda/metabolismo , Doença de Alzheimer/genética , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Glicosilação , Células HeLa , Humanos , Lipodistrofia/genética , Glicoproteínas de Membrana/metabolismo , Osteocondrodisplasias/genética , Polimorfismo de Nucleotídeo Único , Transporte Proteico , Receptores Imunológicos/metabolismo , Panencefalite Esclerosante Subaguda/genéticaRESUMO
The triggering receptor expressed on myeloid cells (TREM) 2 is a member of the immunoglobulin superfamily of receptors and mediates signaling in immune cells via engagement of its co-receptor DNAX-activating protein of 12 kDa (DAP12). Homozygous mutations in TREM2 or DAP12 cause Nasu-Hakola disease, which is characterized by bone abnormalities and dementia. Recently, a variant of TREM2 has also been associated with an increased risk for Alzheimer disease. The selective expression of TREM2 on immune cells and its association with different forms of dementia indicate a contribution of this receptor in common pathways of neurodegeneration.
Assuntos
Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Células Mieloides/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Receptores Imunológicos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Humanos , Lipodistrofia/genética , Lipodistrofia/imunologia , Lipodistrofia/metabolismo , Lipodistrofia/patologia , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microglia/imunologia , Microglia/metabolismo , Microglia/patologia , Mutação , Células Mieloides/imunologia , Células Mieloides/patologia , Proteínas do Tecido Nervoso/genética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/imunologia , Doenças Neurodegenerativas/patologia , Neurônios/imunologia , Neurônios/patologia , Osteocondrodisplasias/genética , Osteocondrodisplasias/imunologia , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/patologia , Receptores Imunológicos/genética , Panencefalite Esclerosante Subaguda/genética , Panencefalite Esclerosante Subaguda/imunologia , Panencefalite Esclerosante Subaguda/metabolismo , Panencefalite Esclerosante Subaguda/patologia , Receptor Gatilho 1 Expresso em Células MieloidesRESUMO
Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder, characterized by formation of multifocal bone cysts and development of leukoencephalopathy, caused by genetic mutations of either DNAX-activation protein 12 (DAP12) or triggering receptor expressed on myeloid cells 2 (TREM2). Although increasing evidence suggests a defect in microglial TREM2/DAP12 function in NHD, the molecular mechanism underlying leukoencephalopathy with relevance to microglial dysfunction remains unknown. TREM2, by transmitting signals via the immunoreceptor tyrosine-based activation motif (ITAM) of DAP12, stimulates phagocytic activity of microglia, and ITAM signaling is counterbalanced by sialic acid-binding immunoglobulin (Ig)-like lectins (Siglecs)-mediated immunoreceptor tyrosine-based inhibitory motif (ITIM) signaling. To investigate a role of CD33, a member of the Siglecs family acting as a negative regulator of microglia activation, in the pathology of NHD, we studied CD33 expression patterns in five NHD brains and 11 controls by immunohistochemistry. In NHD brains, CD33 was identified exclusively on ramified and amoeboid microglia accumulated in demyelinated white matter lesions but not expressed in astrocytes, oligodendrocytes, or neurons. However, the number of CD33-immunoreactive microglia showed great variability from case to case and from lesion to lesion without significant differences between NHD and control brains. These results do not support the view that CD33-expressing microglia play a central role in the development of leukoencephalopathy in NHD brains.
Assuntos
Lipodistrofia/metabolismo , Lipodistrofia/patologia , Microglia/metabolismo , Microglia/patologia , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/patologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/biossíntese , Panencefalite Esclerosante Subaguda/metabolismo , Panencefalite Esclerosante Subaguda/patologia , Western Blotting , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/análiseRESUMO
Subacute sclerosing panencephalitis (SSPE) is a fatal, slowly progressive brain disorder caused by a mutated measles virus. Both subacute inflammatory and neurodegenerative mechanisms appear to play significant roles in the pathogenesis. TAR DNA-binding protein 43 (TDP-43) inclusions are a common co-pathology in several neurodegenerative disorders with diverse pathogenesis. In the present study, we examined brains of 16 autopsied SSPE patients for the presence of TDP-43 pathology and possible associations with tau pathology. Immunohistochemical staining identified TDP-43 inclusions in 31% of SSPE cases. TDP-43 pathology was widely distributed in the brains, most severely in the atrophied cerebral cortex (temporal and parietal), and most frequently as tangle- and thread-like neuronal cytoplasmic inclusions. It was associated with longer disease duration (>4 years) and tau pathology (all TDP-43-positive cases had tau-positive neurofibrillary tangles). This study demonstrates for the first time an association between TDP-43 pathology and SSPE. The co-occurrence of TDP-43 and tau aggregates and correlation with the disease duration suggest that both pathological proteins are involved in the neurodegenerative process induced by viral inflammation.
Assuntos
Panencefalite Esclerosante Subaguda , Humanos , Panencefalite Esclerosante Subaguda/metabolismo , Panencefalite Esclerosante Subaguda/patologia , Vírus do Sarampo/metabolismo , Encéfalo/patologia , Emaranhados Neurofibrilares/patologia , Proteínas de Ligação a DNA/metabolismo , Inflamação/patologiaRESUMO
Measles virus (MeV), which is usually non-neurotropic, sometimes persists in the brain and causes subacute sclerosing panencephalitis (SSPE) several years after acute infection, serving as a model for persistent viral infections. The persisting MeVs have hyperfusogenic mutant fusion (F) proteins that likely enable cell-cell fusion at synapses and "en bloc transmission" between neurons. We here show that during persistence, F protein fusogenicity is generally enhanced by cumulative mutations, yet mutations paradoxically reducing the fusogenicity may be selected alongside the wild-type (non-neurotropic) MeV genome. A mutant F protein having SSPE-derived substitutions exhibits lower fusogenicity than the hyperfusogenic F protein containing some of those substitutions, but by the wild-type F protein coexpression, the fusogenicity of the former F protein is enhanced, while that of the latter is nearly abolished. These findings advance the understanding of the long-term process of MeV neuropathogenicity and provide critical insight into the genotype-phenotype relationships of en bloc transmitted viruses.
Assuntos
Panencefalite Esclerosante Subaguda , Humanos , Panencefalite Esclerosante Subaguda/genética , Panencefalite Esclerosante Subaguda/metabolismo , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Vírus do Sarampo/genética , Encéfalo/metabolismo , MutaçãoRESUMO
Microglia are unique cells in the central nervous system (CNS), being considered a sub-type of CNS macrophage. These cells monitor nearby micro-regions, having roles that far exceed immunological and scavengering functions, being fundamental for developing, protecting and maintaining the integrity of grey and white matter. Microglia might become dysfunctional, causing abnormal CNS functioning early or late in the life of patients, leading to neurologic or psychiatric disorders and premature death in some patients. Observations that the impairment of normal microglia function per se could lead to neurological or psychiatric diseases have been mainly obtained from genetic and molecular studies of Nasu-Hakola disease, caused by TYROBP or TREM2 mutations, and from studies of adult-onset leukoencephalopathy with axonal spheroids (ALSP), caused by CSF1R mutations. These classical microgliopathies are being named here Microgliopathy Type I. Recently, mutations in TREM2 have also been associated with Alzheimer Disease. However, in Alzheimer Disease TREM2 allele variants lead to an impaired, but functional TREM2 protein, so that patients do not develop Nasu-Hakola disease but are at increased risk to develop other neurodegenerative diseases. Alzheimer Disease is the prototype of the neurodegenerative disorders associated with these TREM2 variants, named here the Microgliopathies Type II. Here, we review clinical, pathological and some molecular aspects of human diseases associated with primary microglia dysfunctions and briefly comment some possible therapeutic approaches to theses microgliopathies. We hope that our review might update the interesting discussion about the impact of intrinsic microglia dysfunctions in the genesis of some pathologic processes of the CNS.
Assuntos
Doença de Alzheimer , Panencefalite Esclerosante Subaguda , Substância Branca , Adulto , Doença de Alzheimer/metabolismo , Humanos , Lipodistrofia , Microglia/metabolismo , Osteocondrodisplasias , Panencefalite Esclerosante Subaguda/genética , Panencefalite Esclerosante Subaguda/metabolismo , Panencefalite Esclerosante Subaguda/patologiaRESUMO
Subacute sclerosing panencephalitis (SSPE) is a rare progressive neurodegenerative disease caused by measles virus variants (SSPE viruses) that results in eventual death. Amino acid substitution(s) in the viral fusion (F) protein are key for viral propagation in the brain in a cell-to-cell manner, a specific trait of SSPE viruses, leading to neuropathogenicity. In this study, we passaged an SSPE virus in cultured human neuronal cells and isolated an adapted virus that propagated more efficiently in neuronal cells and exhibited increased cell-to-cell fusion. Contrary to our expectation, the virus harbored mutations in the large protein, a viral RNA-dependent RNA polymerase, and in the phosphoprotein, its co-factor, rather than in the F protein. Our results imply that upregulated RNA polymerase activity, which increases F protein expression and cell-to-cell fusion, could be a viral factor that provides a growth advantage and contributes to the adaptation of SSPE viruses to neuronal cells.
Assuntos
Doenças Neurodegenerativas , Panencefalite Esclerosante Subaguda , Humanos , Vírus do Sarampo/fisiologia , Vírus SSPE/genética , Vírus SSPE/metabolismo , Panencefalite Esclerosante Subaguda/genética , Panencefalite Esclerosante Subaguda/metabolismo , Regulação para Cima , Proteínas Virais de Fusão/genética , Proteínas do Complexo da Replicase ViralRESUMO
Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder, characterized by progressive presenile dementia and formation of multifocal bone cysts, caused by genetic mutations of DNAX-activation protein 12 (DAP12) or triggering receptor expressed on myeloid cells 2 (TREM2). TREM2 and DAP12 constitute a receptor/adapter signaling complex expressed on osteoclasts, dendritic cells (DC), macrophages and microglia. Previous studies using knockout mice and mouse brain cell cultures suggest that a loss-of-function of DAP12/TREM2 in microglia plays a central role in the neuropathological manifestation of NHD. However, there exist no immunohistochemical studies that focus attention on microglia in NHD brains. To elucidate a role of microglia in the pathogenesis of NHD, we searched NHD-specific biomarkers and characterized their expression on microglia in NHD brains. Here, we identified allograft inflammatory factor 1 (AIF1, Iba1) and sialic acid binding Ig-like lectin 1 (SIGLEC1) as putative NHD-specific biomarkers by bioinformatics analysis of microarray data of NHD DC. We studied three NHD and eight control brains by immunohistochemistry with a panel of 16 antibodies, including those against Iba1 and SIGLEC1. We verified the absence of DAP12 expression in NHD brains and the expression of DAP12 immunoreactivity on ramified microglia in control brains. Unexpectedly, TREM2 was not expressed on microglia but expressed on a small subset of intravascular monocytes/macrophages in control and NHD brains. In the cortex of NHD brains, we identified accumulation of numerous Iba1-positive microglia to an extent similar to control brains, while SIGLEC1 was undetectable on microglia in all the brains examined. These observations indicate that human microglia in brain tissues do not express TREM2 and DAP12-deficient microglia are preserved in NHD brains, suggesting that the loss of DAP2/TREM2 function in microglia might not be primarily responsible for the neuropathological phenotype of NHD.
Assuntos
Encéfalo/metabolismo , Lipodistrofia/metabolismo , Microglia/metabolismo , Osteocondrodisplasias/metabolismo , Panencefalite Esclerosante Subaguda/metabolismo , Adulto , Idoso , Proteínas de Ligação ao Cálcio , Biologia Computacional , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Lectinas/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos , Pessoa de Meia-Idade , Receptores Imunológicos/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido SiálicoRESUMO
Subacute sclerosing panencephalitis is an uncommon progressive neurological disorder caused by a persistent defective measles virus, typically affecting children. We describe a case of fulminant subacute sclerosing panencephalitis in a 25-year-old male. Brain tissue biopsy showed histologic evidence of encephalitis with eosinophilic intranuclear inclusion bodies (Cowdry Type A and B), intracytoplasmic inclusion bodies, perivascular lymphoplasmacytic infiltration and gliosis. Immunohistochemical studies were positive using an anti-measles antibody. Reverse transcriptase-PCR detected measles virus RNA and phylogenetic analysis indicated a C2 genotype. The rare adult-onset form is often atypical and difficult to diagnose and should be included in the differential diagnosis of subacute "unexplained" neurological diseases and uncommon infectious disorders.
Assuntos
Encéfalo/patologia , Filogenia , Vírus SSPE/genética , Panencefalite Esclerosante Subaguda/patologia , Panencefalite Esclerosante Subaguda/virologia , Sequência de Bases , Encéfalo/metabolismo , Genótipo , Humanos , Imuno-Histoquímica , Masculino , Dados de Sequência Molecular , RNA Viral , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Panencefalite Esclerosante Subaguda/metabolismo , Adulto JovemRESUMO
Analysis of tissue from a 34-years-old male patient from Austrian origin with a history of multiple fractures associated with painful episodes over the carpal, tarsal and at the end of the long bones respectively is presented. Radiographic images and axial 3DCT scans showed widespread defects in trabecular bone architecture and ill-defined cortices over these skeletal sites in the form of discrete cystic-like lesions. Family history indicated two sisters (one half and one full biological sisters) also with a history of fractures. Whole exome sequencing revealed two heterozygous missense mutations in TYROBP (MIM 604142; NM_003332.3) gene encoding for a cell-surface adaptor protein, which is part of a signaling complex triggering activation of immune responses. It is expressed in cells of the ectoderm cell linage such as NK and dendritic cells, macrophages, monocytes, myeloid cells, microglia cells and osteoclasts. The phenotype and genotype of the patient were consistent with the diagnosis of Nasu-Hakola disease (NHD) (OMIM 221770). Investigations at the bone material level of a transiliac bone biopsy sample from the patient using polarized light microscopy and backscatter electron imaging revealed disordered lamellar collagen fibril arrangement and extensively increased matrix mineralization. These findings are the first bone material data in a patient with NHD and point toward an osteoclast defect involvement in this genetic condition.
Assuntos
Matriz Óssea/metabolismo , Lipodistrofia/metabolismo , Lipodistrofia/fisiopatologia , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/fisiopatologia , Panencefalite Esclerosante Subaguda/metabolismo , Panencefalite Esclerosante Subaguda/fisiopatologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Biópsia , Densidade Óssea/genética , Densidade Óssea/fisiologia , Matriz Óssea/fisiopatologia , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Osteoclastos/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Sequenciamento do ExomaRESUMO
Signaling lymphocyte activating molecule (SLAM) is a receptor for measles virus which also has immunomodulatory activity. We analyzed SLAM expression in mononuclear cells (MNC) of patients with SSPE (n=7) and control subjects (n=7) from the same population. Native 10% PAGE analysis in cell and brain tissue extracts followed by Western blotting using monoclonal anti-human SLAM showed four types of bands. Differences in the type and amount of SLAM expression were observed between SSPE and control cases. Lymphocytes of SSPE patients showed two types of SLAM bands in comparison to only one in control lymphocytes. Stimulation of cells with lipopolysaccharide (80 u/ml) and concanavalin A (1 microg/ml) in vitro led to the appearance of a second isoform in both groups. Brain homogenates of SSPE patients (n=2) displayed all four types of SLAM isoforms at significantly higher levels than those of control brains (n=2). Our results show native PAGE enables the detection of all SLAM isotypes. The expression of SLAM is increased in lymphocytes, monocytes, and brain tissues of SSPE patients.
Assuntos
Antígenos CD/biossíntese , Encéfalo/metabolismo , Receptores de Superfície Celular/biossíntese , Panencefalite Esclerosante Subaguda/metabolismo , Western Blotting , Eletroforese em Gel de Poliacrilamida , Humanos , Leucócitos Mononucleares/metabolismo , Isoformas de Proteínas/biossíntese , Membro 1 da Família de Moléculas de Sinalização da Ativação LinfocitáriaRESUMO
We are presenting two cases with subacute sclerosing panencephalitis of acute and rapid form, which were initially diagnosed as acute viral encephalitis. We obtained both diffusion-weighted imaging and magnetic resonance spectroscopy, whose findings suggested the presence of acute inflammatory and metabolic changes with peculiar distribution, which were not observed on conventional MR imaging. These techniques might help to demonstrate the extent and progression of the panencephalitis.
Assuntos
Panencefalite Esclerosante Subaguda/metabolismo , Panencefalite Esclerosante Subaguda/patologia , Anticonvulsivantes/uso terapêutico , Encéfalo/patologia , Criança , Clonazepam/uso terapêutico , Imagem de Difusão por Ressonância Magnética , Eletroencefalografia , Parada Cardíaca/fisiopatologia , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Espectroscopia de Ressonância Magnética , Masculino , Membranas/patologia , Respiração Artificial , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Convulsões/etiologiaRESUMO
INTRODUCTION: Subacute sclerosing panencephalitis (SSPE) is a chronic neurodegenerative disease secondary to an infection of the central nervous system by the measles virus, with no effective treatment. The introduction of therapy with intraventricular interferon alpha (IFN-alpha) and its later association with ribavirin aroused new expectations. In experimental studies the two drugs were seen to exert a synergic effect in reducing viral replication. Therapeutic studies carried out in patients with SSPE with the two pharmaceuticals have offered contradictory findings. CASE REPORTS: We present the cases of two patients with an early-onset, fast progressing form of SSPE, who were treated with a combined regime of oral isoprenosin, intraventricular IFN-alpha and ribavirin, which was administered intravenously in one case and intraventricularly in the other. At the beginning of treatment the patients' deterioration stabilised briefly and temporarily, but then renewed its progress. CONCLUSIONS: Combined therapy with intraventricular IFN-alpha and ribavirin was not effective in our patients. The late onset and rapidly progressing symptoms of the disease may have had an effect on the poor results obtained.
Assuntos
Antivirais/uso terapêutico , Interferon-alfa/uso terapêutico , Ribavirina/uso terapêutico , Panencefalite Esclerosante Subaguda/tratamento farmacológico , Antivirais/administração & dosagem , Criança , Pré-Escolar , Quimioterapia Combinada , Evolução Fatal , Humanos , Injeções Intraventriculares , Masculino , Sarampo/complicações , Vírus do Sarampo/metabolismo , Ribavirina/administração & dosagem , Panencefalite Esclerosante Subaguda/etiologia , Panencefalite Esclerosante Subaguda/metabolismo , Panencefalite Esclerosante Subaguda/fisiopatologia , Resultado do TratamentoRESUMO
Nasu-Hakola disease (NHD) is a rare intractable autosomal recessive disorder, characterized by pathological bone fractures and progressive dementia owing to multifocal bone cysts and leukoencephalopathy, caused by various genetic mutations of either DAP12 or TREM2. Loss-of-function of TREM2-DAP12, constituting a signaling complex on osteoclasts and microglia, plays a central role in the pathogenesis of NHD. Recently, NHD has been recognized as the disease entity designated "microgliopathy". However, at present, TREM2-specific ligands in microglia and the precise molecular mechanism underlying leukoencephalopathy remain to be investigated in order to establish an effective molecular targeted therapy for NHD.
Assuntos
Encéfalo/metabolismo , Lipodistrofia/metabolismo , Osteocondrodisplasias/metabolismo , Panencefalite Esclerosante Subaguda/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Humanos , Lipodistrofia/genética , Lipodistrofia/patologia , Lipodistrofia/fisiopatologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Osteocondrodisplasias/genética , Osteocondrodisplasias/patologia , Osteocondrodisplasias/fisiopatologia , Ligação Proteica , Receptores Imunológicos/metabolismo , Transdução de Sinais , Panencefalite Esclerosante Subaguda/genética , Panencefalite Esclerosante Subaguda/patologia , Panencefalite Esclerosante Subaguda/fisiopatologiaRESUMO
A case of subacute sclerosing panencephalitis in a 27-year-old man was serially evaluated with proton magnetic resonance spectroscopy. Metabolic abnormalities included decreased N-acetylaspartate and elevated choline and myo-inositol in a lesion visible on magnetic resonance imaging and in normal-appearing white matter. Lactate appeared increased within the lesion. Metabolic impairment was persistent after intrathecal interferon-alpha treatment. Spectroscopy pointing to ongoing inflammation, gliosis, and possible membrane turnover was more sensitive than imaging in detecting widespread pathology within the white matter.