RESUMO
γ-Secretases mediate the regulated intramembrane proteolysis (RIP) of more than 150 integral membrane proteins. We developed an unbiased γ-secretase substrate identification (G-SECSI) method to study to what extent these proteins are processed in parallel. We demonstrate here parallel processing of at least 85 membrane proteins in human microglia in steady-state cell culture conditions. Pharmacological inhibition of γ-secretase caused substantial changes of human microglial transcriptomes, including the expression of genes related to the disease-associated microglia (DAM) response described in Alzheimer disease (AD). While the overall effects of γ-secretase deficiency on transcriptomic cell states remained limited in control conditions, exposure of mouse microglia to AD-inducing amyloid plaques strongly blocked their capacity to mount this putatively protective DAM cell state. We conclude that γ-secretase serves as a critical signaling hub integrating the effects of multiple extracellular stimuli into the overall transcriptome of the cell.
Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Camundongos , Animais , Humanos , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Proteoma/genética , Transdução de Sinais , Proteínas de Membrana/metabolismo , Doença de Alzheimer/genéticaRESUMO
The γ-secretase complexes are intramembrane cleaving proteases involved in the generation of the Aß peptides in Alzheimer's disease. The complex consists of four subunits, with Presenilin harboring the catalytic site. Here, we study the role of the smallest subunit, PSENEN or Presenilin enhancer 2, encoded by the gene Psenen, in vivo and in vitro. We find a profound Notch deficiency phenotype in Psenen-/- embryos confirming the essential role of PSENEN in the γ-secretase complex. We used Psenen-/- fibroblasts to explore the structure-function of PSENEN by the scanning cysteine accessibility method. Glycine 22 and proline 27, which border the membrane domains 1 and 2 of PSENEN, are involved in complex formation and stabilization of γ-secretase. The hairpin structured hydrophobic membrane domains 1 and 2 are exposed to a water-containing cavity in the complex, while transmembrane domain 3 is not water exposed. We finally demonstrate the essential role of PSENEN for the cleavage activity of the complex. PSENEN is more than a structural component of the γ-secretase complex and might contribute to the catalytic mechanism of the enzyme.
Assuntos
Secretases da Proteína Precursora do Amiloide , Animais , Feminino , Masculino , Camundongos , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Proteínas de Membrana/química , Camundongos Endogâmicos C57BL , Presenilina-1/genética , Estrutura Terciária de ProteínaRESUMO
OBJECTIVE: Mutations in presenilin genes are the major cause of Alzheimer's disease. However, little is known about their expression and function in the gut. In this study, we identify the presenilins Psen1 and Psen2 as key molecules that maintain intestinal homoeostasis. DESIGN: Human inflammatory bowel disease (IBD) and control samples were analysed for Psen1 expression. Newly generated intestinal epithelium-specific Psen1-deficient, Psen2-deficient and inducible Psen1/Psen2 double-deficient mice were used to dissect the functional role of presenilins in intestinal homoeostasis. RESULTS: Psen1 expression was regulated in experimental gut inflammation and in patients with IBD. Induced deletion of Psen1 and Psen2 in mice caused rapid weight loss and spontaneous development of intestinal inflammation. Mice exhibited epithelial barrier disruption with bacterial translocation and deregulation of key pathways for nutrient uptake. Wasting disease was independent of gut inflammation and dysbiosis, as depletion of microbiota rescued Psen-deficient animals from spontaneous colitis development but not from weight loss. On a molecular level, intestinal epithelial cells lacking Psen showed impaired Notch signalling and dysregulated epithelial differentiation. CONCLUSION: Overall, our study provides evidence that Psen1 and Psen2 are important guardians of intestinal homoeostasis and future targets for barrier-promoting therapeutic strategies in IBD.
RESUMO
Clinical development of γ-secretases, a family of intramembrane cleaving proteases, as therapeutic targets for a variety of disorders including cancer and Alzheimer's disease was aborted because of serious mechanism-based side effects in the phase III trials of unselective inhibitors. Selective inhibition of specific γ-secretase complexes, containing either PSEN1 or PSEN2 as the catalytic subunit and APH1A or APH1B as supporting subunits, does provide a feasible therapeutic window in preclinical models of these disorders. We explore here the pharmacophoric features required for PSEN1 versus PSEN2 selective inhibition. We synthesized a series of brain penetrant 2-azabicyclo[2,2,2]octane sulfonamides and identified a compound with low nanomolar potency and high selectivity (>250-fold) toward the PSEN1-APH1B subcomplex versus PSEN2 subcomplexes. We used modeling and site-directed mutagenesis to identify critical amino acids along the entry part of this inhibitor into the catalytic site of PSEN1. Specific targeting one of the different γ-secretase complexes might provide safer drugs in the future.
Assuntos
Secretases da Proteína Precursora do Amiloide , Complexos Multiproteicos , Presenilina-1 , Sulfonamidas , Humanos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Presenilina-1/antagonistas & inibidores , Presenilina-1/metabolismo , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Sulfonamidas/farmacologia , Especificidade por Substrato , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/metabolismoRESUMO
The mitochondrial intramembrane rhomboid protease PARL has been implicated in diverse functions in vitro, but its physiological role in vivo remains unclear. Here we show that Parl ablation in mouse causes a necrotizing encephalomyelopathy similar to Leigh syndrome, a mitochondrial disease characterized by disrupted energy production. Mice with conditional PARL deficiency in the nervous system, but not in muscle, develop a similar phenotype as germline Parl KOs, demonstrating the vital role of PARL in neurological homeostasis. Genetic modification of two major PARL substrates, PINK1 and PGAM5, do not modify this severe neurological phenotype. Parl-/- brain mitochondria are affected by progressive ultrastructural changes and by defects in Complex III (CIII) activity, coenzyme Q (CoQ) biosynthesis, and mitochondrial calcium metabolism. PARL is necessary for the stable expression of TTC19, which is required for CIII activity, and of COQ4, which is essential in CoQ biosynthesis. Thus, PARL plays a previously overlooked constitutive role in the maintenance of the respiratory chain in the nervous system, and its deficiency causes progressive mitochondrial dysfunction and structural abnormalities leading to neuronal necrosis and Leigh-like syndrome.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Doença de Leigh/etiologia , Metaloproteases/deficiência , Proteínas Mitocondriais/deficiência , Ubiquinona/metabolismo , Animais , Encéfalo/metabolismo , Cálcio/metabolismo , Doença de Leigh/metabolismo , Doença de Leigh/fisiopatologia , Fígado/metabolismo , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Encefalomiopatias Mitocondriais/metabolismo , Encefalomiopatias Mitocondriais/fisiopatologia , Músculo Esquelético/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
MALDI mass spectrometry imaging (MSI) enables label-free, spatially resolved analysis of a wide range of analytes in tissue sections. Quantitative analysis of MSI datasets is typically performed on single pixels or manually assigned regions of interest (ROIs). However, many sparse, small objects such as Alzheimer's disease (AD) brain deposits of amyloid peptides called plaques are neither single pixels nor ROIs. Here, we propose a new approach to facilitate the comparative computational evaluation of amyloid plaque-like objects by MSI: a fast PLAQUE PICKER tool that enables a statistical evaluation of heterogeneous amyloid peptide composition. Comparing two AD mouse models, APP NL-G-F and APP PS1, we identified distinct heterogeneous plaque populations in the NL-G-F model but only one class of plaques in the PS1 model. We propose quantitative metrics for the comparison of technical and biological MSI replicates. Furthermore, we reconstructed a high-accuracy 3D-model of amyloid plaques in a fully automated fashion, employing rigid and elastic MSI image registration using structured and plaque-unrelated reference ion images. Statistical single-plaque analysis in reconstructed 3D-MSI objects revealed the Aß1-42Arc peptide to be located either in the core of larger plaques or in small plaques without colocalization of other Aß isoforms. In 3D, a substantially larger number of small plaques were observed than that indicated by the 2D-MSI data, suggesting that quantitative analysis of molecularly diverse sparsely-distributed features may benefit from 3D-reconstruction. Data are available via ProteomeXchange with identifier PXD020824.
Assuntos
Doença de Alzheimer/complicações , Elasticidade , Imageamento Tridimensional/métodos , Imagem Molecular , Placa Amiloide/complicações , Placa Amiloide/diagnóstico por imagem , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Animais , CamundongosRESUMO
INTRODUCTION: Tauopathies are neurodegenerative diseases characterized by TAU protein-related pathology, including frontotemporal dementia and Alzheimer's disease among others. Mutant TAU animal models are available, but none of them faithfully recapitulates human pathology and are not suitable for drug screening. METHODS: To create a new in vitro tauopathy model, we generated a footprint-free triple MAPT-mutant human induced pluripotent stem cell line (N279K, P301L, and E10+16 mutations) using clustered regularly interspaced short palindromic repeats-FokI and piggyBac transposase technology. RESULTS: Mutant neurons expressed pathogenic 4R and phosphorylated TAU, endogenously triggered TAU aggregation, and had increased electrophysiological activity. TAU-mutant cells presented deficiencies in neurite outgrowth, aberrant sequence of differentiation to cortical neurons, and a significant activation of stress response pathways. RNA sequencing confirmed stress activation, demonstrated a shift toward GABAergic identity, and an upregulation of neurodegenerative pathways. DISCUSSION: In summary, we generated a novel in vitro human induced pluripotent stem cell TAU-mutant model displaying neurodegenerative disease phenotypes that could be used for disease modeling and drug screening.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Tauopatias/metabolismo , Proteínas tau/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Potenciais da Membrana/fisiologia , Mutação , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Neurogênese/fisiologia , Crescimento Neuronal/fisiologia , Neurônios/metabolismo , Neurônios/patologia , Fenótipo , Tauopatias/genética , Tauopatias/patologia , Transcriptoma , Proteínas tau/genéticaRESUMO
The mechanisms by which mutations in the presenilins (PSEN) or the amyloid precursor protein (APP) genes cause familial Alzheimer disease (FAD) are controversial. FAD mutations increase the release of amyloid ß (Aß)42 relative to Aß40 by an unknown, possibly gain-of-toxic-function, mechanism. However, many PSEN mutations paradoxically impair γ-secretase and 'loss-of-function' mechanisms have also been postulated. Here, we use kinetic studies to demonstrate that FAD mutations affect Aß generation via three different mechanisms, resulting in qualitative changes in the Aß profiles, which are not limited to Aß42. Loss of É-cleavage function is not generally observed among FAD mutants. On the other hand, γ-secretase inhibitors used in the clinic appear to block the initial É-cleavage step, but unexpectedly affect more selectively Notch than APP processing, while modulators act as activators of the carboxypeptidase-like (γ) activity. Overall, we provide a coherent explanation for the effect of different FAD mutations, demonstrating the importance of qualitative rather than quantitative changes in the Aß products, and suggest fundamental improvements for current drug development efforts.
Assuntos
Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Amiloide/metabolismo , Presenilina-1/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Receptores ErbB/metabolismo , Cinética , Receptor ErbB-4 , Receptor Notch1/metabolismoRESUMO
Amyloids are often associated with pathologic processes such as in Alzheimer's disease (AD), but can also underlie physiological processes such as pigmentation. Formation of pathological and functional amyloidogenic substrates can require precursor processing by proteases, as exemplified by the generation of Aß peptide from amyloid precursor protein (APP) by beta-site APP cleaving enzyme (BACE)1 and γ-secretase. Proteolytic processing of the pigment cell-specific Melanocyte Protein (PMEL) is also required to form functional amyloid fibrils during melanogenesis, but the enzymes involved are incompletely characterized. Here we show that the BACE1 homologue BACE2 processes PMEL to generate functional amyloids. BACE2 is highly expressed in pigment cells and Bace2(-/-) but not Bace1(-/-) mice display coat color defects, implying a specific role for BACE2 during melanogenesis. By using biochemical and morphological analyses, combined with RNA silencing, pharmacologic inhibition, and BACE2 overexpression in a human melanocytic cell line, we show that BACE2 cleaves the integral membrane form of PMEL within the juxtamembrane domain, releasing the PMEL luminal domain into endosomal precursors for the formation of amyloid fibrils and downstream melanosome morphogenesis. These studies identify an amyloidogenic substrate of BACE2, reveal an important physiological role for BACE2 in pigmentation, and highlight analogies in the generation of PMEL-derived functional amyloids and APP-derived pathological amyloids.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Amiloide/biossíntese , Ácido Aspártico Endopeptidases/metabolismo , Melanossomas/metabolismo , Antígeno gp100 de Melanoma/metabolismo , Secretases da Proteína Precursora do Amiloide/deficiência , Secretases da Proteína Precursora do Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/deficiência , Ácido Aspártico Endopeptidases/genética , Linhagem Celular , Células HeLa , Humanos , Melaninas/biossíntese , Melanócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Epitélio Pigmentado Ocular/metabolismo , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Pigmentação da Pele/genética , Pigmentação da Pele/fisiologiaRESUMO
γ-Secretase complexes are involved in the generation of amyloid-ß (Aß) in the brain. Therefore, γ-secretase has been proposed as a potential therapeutic target in Alzheimer disease (AD). Targeting γ-secretase activity in AD requires the pharmacological dissociation of the processing of physiological relevant substrates and the generation of "toxic" Aß. Previous reports suggest the differential targeting of γ-secretase complexes, based on their subunit composition, as a valid strategy. However, little is known about the biochemical properties of the different complexes, and key questions regarding their Aß product profiles should be first addressed. Here, we expressed, purified, and analyzed, under the same conditions, the endopeptidase and carboxypeptidase-like activities of the four γ-secretase complexes present in humans. We find that the nature of the catalytic subunit in the complex affects both activities. Interestingly, PSEN2 complexes discriminate between the Aß40 and Aß38 production lines, indicating that Aß generation in one or the other pathway can be dissociated. In contrast, the APH1 subunit mainly affects the carboxypeptidase-like activity, with APH1B complexes favoring the generation of longer Aß peptides. In addition, we determined that expression of a single human γ-secretase complex in cell lines retains the intrinsic attributes of the protease while present in the membrane, providing validation for the in vitro studies. In conclusion, our data show that each γ-secretase complex produces a characteristic Aß signature. The qualitative and quantitative differences between different γ-secretase complexes could be used to advance drug development in AD and other disorders.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/dietoterapia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/genética , Animais , Linhagem Celular , Endopeptidases , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Fragmentos de Peptídeos/genética , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Presenilina-2/genética , Presenilina-2/metabolismoRESUMO
miR-29 is expressed strongly in the brain and alterations in expression have been linked to several neurological disorders. To further explore the function of this miRNA in the brain, we generated miR-29a/b-1 knockout animals. Knockout mice develop a progressive disorder characterized by locomotor impairment and ataxia. The different members of the miR-29 family are strongly expressed in neurons of the olfactory bulb, the hippocampus and in the Purkinje cells of the cerebellum. Morphological analysis showed that Purkinje cells are smaller and display less dendritic arborisation compared to their wildtype littermates. In addition, a decreased number of parallel fibers form synapses on the Purkinje cells. We identified several mRNAs significantly up-regulated in the absence of the miR-29a/b-1 cluster. At the protein level, however, the voltage-gated potassium channel Kcnc3 (Kv3.3) was significantly up-regulated in the cerebella of the miR-29a/b knockout mice. Dysregulation of KCNC3 expression may contribute to the ataxic phenotype.
Assuntos
Ataxia/metabolismo , Cerebelo/metabolismo , MicroRNAs/metabolismo , Células de Purkinje/metabolismo , Canais de Potássio Shaw/metabolismo , Animais , Comportamento Animal , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade MotoraRESUMO
Microglia are central players in Alzheimer's disease pathology but analyzing microglial states in human brain samples is challenging due to genetic diversity, postmortem delay and admixture of pathologies. To circumvent these issues, here we generated 138,577 single-cell expression profiles of human stem cell-derived microglia xenotransplanted in the brain of the AppNL-G-F model of amyloid pathology and wild-type controls. Xenografted human microglia adopt a disease-associated profile similar to that seen in mouse microglia, but display a more pronounced human leukocyte antigen or HLA state, likely related to antigen presentation in response to amyloid plaques. The human microglial response also involves a pro-inflammatory cytokine/chemokine cytokine response microglia or CRM response to oligomeric Aß oligomers. Genetic deletion of TREM2 or APOE as well as APOE polymorphisms and TREM2R47H expression in the transplanted microglia modulate these responses differentially. The expression of other Alzheimer's disease risk genes is differentially regulated across the distinct cell states elicited in response to amyloid pathology. Thus, we have identified multiple transcriptomic cell states adopted by human microglia in a multipronged response to Alzheimer's disease-related pathology, which should be taken into account in translational studies.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Microglia , Transcriptoma , Animais , Humanos , Camundongos , Doença de Alzheimer/patologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Xenoenxertos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos Transgênicos , Microglia/metabolismo , Microglia/patologia , Placa Amiloide/patologia , Placa Amiloide/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismoRESUMO
Amyloid-ß pathology and neurofibrillary tangles lead to glial activation and neurodegeneration in Alzheimer's disease. In this study, we investigated the relationships between the levels of amyloid-ß oligomers, amyloid-ß plaques, glial activation and markers related to neurodegeneration in the App NL-G-F triple mutation mouse line and in a knock-in line homozygous for the common human amyloid precursor protein (App hu mouse). The relationships between neuropathological features were characterized with immunohistochemistry and imaging mass cytometry. Markers assessing human amyloid-ß proteins, microglial and astrocytic activation and neuronal and synaptic densities were used in mice between 2.5 and 12 months of age. We found that amyloid-ß oligomers were abundant in the brains of App hu mice in the absence of classical amyloid-ß plaques. These brains showed morphological changes consistent with astrocyte activation but no evidence of microglial activation or synaptic or neuronal pathology. In contrast, both high levels of amyloid-ß oligomers and numerous plaques accumulated in App NL-G-F mice in association with substantial astrocytic and microglial activation. The increase in amyloid-ß oligomers over time was more strongly correlated with astrocytic than with microglia activation. Spatial analyses suggested that activated microglia were more closely associated with amyloid-ß oligomers than with amyloid-ß plaques in App NL-G-F mice, which also showed age-dependent decreases in neuronal and synaptic density markers. A comparative study of the two models highlighted the dependence of glial and neuronal pathology on the nature and aggregation state of the amyloid-ß peptide. Astrocyte activation and neuronal pathology appeared to be more strongly associated with amyloid-ß oligomers than with amyloid-ß plaques, although amyloid-ß plaques were associated with microglia activation.
RESUMO
γ-Secretase inhibition represents a major therapeutic strategy for lowering amyloid ß (Aß) peptide production in Alzheimer's disease (AD). Progress toward clinical use of γ-secretase inhibitors has, however, been hampered due to mechanism-based adverse events, primarily related to impairment of Notch signaling. The γ-secretase inhibitor MRK-560 represents an exception as it is largely tolerable in vivo despite displaying only a small selectivity between Aß production and Notch signaling in vitro. In exploring the molecular basis for the observed tolerability, we show that MRK-560 displays a strong preference for the presenilin 1 (PS1) over PS2 subclass of γ-secretases and is tolerable in wild-type mice but causes dose-dependent Notch-related side effect in PS2-deficient mice at drug exposure levels resulting in a substantial decrease in brain Aß levels. This demonstrates that PS2 plays an important role in mediating essential Notch signaling in several peripheral organs during pharmacological inhibition of PS1 and provide preclinical in vivo proof of concept for PS2-sparing inhibition as a novel, tolerable and efficacious γ-secretase targeting strategy for AD.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Encéfalo/efeitos dos fármacos , Presenilina-2/metabolismo , Doença de Alzheimer/metabolismo , Animais , Encéfalo/metabolismo , Camundongos , Presenilina-2/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêuticoRESUMO
Neurobeachin (NBEA), a brain-enriched multidomain scaffolding protein involved in neurotransmitter release and synaptic functioning, has been identified as a candidate gene for autism spectrum disorder (ASD) in four unrelated patients haploinsufficient for NBEA. The aim of this study was to map the behavioral phenotype of Nbea(+/-) mice in order to understand its contribution to the pathogenesis of ASD. ASD-like behavioral variables of Nbea(+/-) mice were related to basal neuronal activity in different brain regions by in situ hybridizations and extracellular field recordings of synaptic plasticity in hippocampal cornu ammonis 1 (CA1) region. Levels of BDNF and phosphorylated cAMP response element-binding protein (CREB) were measured in an attempt to investigate putatively underlying changes in these neuromolecules. Nbea(+/-) mice exhibit several ASD-like features, including changes in self-grooming behavior, social behaviors, conditioned fear responses, and spatial learning and memory, which coincided with enhanced long-term potentiation (LTP) in their CA1 region. The observed alterations in learning and memory and hippocampal LTP are concomitant with decreased expression of the immediate early gene zif268 in dorsomedial striatum and hippocampal CA1 region, increased CREB phosphorylation, and increased hippocampal BDNF expression. These findings indicate that Nbea haploinsufficiency leads to various molecular and cellular changes that affect neuroplasticity and behavioral functions in mice, and could thus underlie the ASD symptomatology in NBEA deficient humans.
Assuntos
Transtorno Autístico/genética , Comportamento Animal/fisiologia , Encéfalo/fisiopatologia , Proteínas de Transporte/genética , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal/genética , Animais , Criança , Feminino , Haploinsuficiência , Humanos , Immunoblotting , Hibridização In Situ , Aprendizagem/fisiologia , Potenciação de Longa Duração/fisiologia , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Transmissão Sináptica/genéticaRESUMO
Presenilins (PSENs) form the catalytic component of the γ-secretase complex, responsible for intramembrane proteolysis of amyloid precursor protein (APP) and Notch, among many other membrane proteins. Previously, we identified a PSEN1-binding domain in APP, encompassing half of the transmembrane domain following the amyloid ß (Aß) sequence. Based on this, we designed peptides mimicking this interaction domain with the aim to selectively block APP processing and Aß generation through interfering with enzyme-substrate binding. We identified a peptide sequence that, when fused to a virally derived translocation peptide, significantly lowered Aß production (IC(50): 317 nM) in cell-free and cell-based assays using APP-carboxy terminal fragment as a direct γ-secretase substrate. Being derived from the APP sequence, this inhibitory peptide did not affect NotchΔE γ-cleavage, illustrating specificity and potential therapeutic value. In cell-based assays, the peptide strongly suppressed APP shedding, demonstrating that it exerts the inhibitory effect already upstream of γ-secretase, most likely through steric hindrance.
Assuntos
Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Membrana/metabolismo , Peptídeos/metabolismo , Presenilinas/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/química , Células HEK293 , Células HeLa , Humanos , Microscopia Confocal , Dados de Sequência Molecular , Ressonância de Plasmônio de SuperfícieRESUMO
This paper describes the rational design, synthesis, structure-activity relationship (SAR), and biological profile of presenilin-1 (PSEN-1) complex selective γ-secretase inhibitors, assessed for selectivity using a unique set of four γ-secretase subtype complexes. A set of known PSEN-1 selective γ-Secretase inhibitors (GSIs) was analyzed to understand the pharmacophoric features required for selective inhibition. Conformational modeling suggests that a characteristic 'U' shape orientation between aromatic sulfone/sulfonamide and aryl ring is crucial for PSEN-1 selectivity and potency. Using these insights, a series of brain-penetrant 2-azabicyclo[2,2,2]octane sulfonamides was devised and synthesized as a new class of PSEN-1 selective inhibitors. Compounds 13c and 13k displayed high potency towards PSEN1-APH1B complex but moderate selectivity towards PSEN2 complexes. However, compound (+)-13b displayed low nanomolar potency towards the PSEN1-APH1B complex, little (â¼4-fold) selectivity towards PSEN1-APH1A, and high selectivity (>350-fold) versus PSEN2 complexes. Excellent brain penetration, no significant CYP inhibition, or cardiotoxicity, good solubility, and permeability make (+)-13b an excellent candidate for further lead optimization.
Assuntos
Secretases da Proteína Precursora do Amiloide , Sulfonamidas , Sulfonamidas/farmacologia , Presenilina-1 , Octanos , Sulfanilamida , EncéfaloRESUMO
Neuronal cell loss is a defining feature of Alzheimer's disease (AD), but the underlying mechanisms remain unclear. We xenografted human or mouse neurons into the brain of a mouse model of AD. Only human neurons displayed tangles, Gallyas silver staining, granulovacuolar neurodegeneration (GVD), phosphorylated tau blood biomarkers, and considerable neuronal cell loss. The long noncoding RNA MEG3 was strongly up-regulated in human neurons. This neuron-specific long noncoding RNA is also up-regulated in AD patients. MEG3 expression alone was sufficient to induce necroptosis in human neurons in vitro. Down-regulation of MEG3 and inhibition of necroptosis using pharmacological or genetic manipulation of receptor-interacting protein kinase 1 (RIPK1), RIPK3, or mixed lineage kinase domain-like protein (MLKL) rescued neuronal cell loss in xenografted human neurons. This model suggests potential therapeutic approaches for AD and reveals a human-specific vulnerability to AD.
Assuntos
Doença de Alzheimer , Necroptose , Neurônios , RNA Longo não Codificante , Animais , Humanos , Camundongos , Doença de Alzheimer/patologia , Xenoenxertos , Necroptose/genética , Neurônios/patologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genéticaRESUMO
The gamma-secretase complex, consisting of presenilin, nicastrin, presenilin enhancer-2 (PEN-2), and anterior pharynx defective-1 (APH-1) cleaves type I integral membrane proteins like amyloid precursor protein and Notch in a process of regulated intramembrane proteolysis. The regulatory mechanisms governing the multistep assembly of this "proteasome of the membrane" are unknown. We characterize a new interaction partner of nicastrin, the retrieval receptor Rer1p. Rer1p binds preferentially immature nicastrin via polar residues within its transmembrane domain that are also critical for interaction with APH-1. Absence of APH-1 substantially increased binding of nicastrin to Rer1p, demonstrating the competitive nature of these interactions. Moreover, Rer1p expression levels control the formation of gamma-secretase subcomplexes and, concomitantly, total cellular gamma-secretase activity. We identify Rer1p as a novel limiting factor that negatively regulates gamma-secretase complex assembly by competing with APH-1 during active recycling between the endoplasmic reticulum (ER) and Golgi. We conclude that total cellular gamma-secretase activity is restrained by a secondary ER control system that provides a potential therapeutic value.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/análise , Secretases da Proteína Precursora do Amiloide/química , Animais , Ligação Competitiva , Regulação para Baixo , Endopeptidases , Retículo Endoplasmático/metabolismo , Glicosilação , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Peptídeo Hidrolases , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
BACKGROUND: The blood brain barrier (BBB) limits the therapeutic perspective for central nervous system (CNS) disorders. Previously we found an anti-mouse transferrin receptor (TfR) VHH (Nb62) that was able to deliver a biologically active neuropeptide into the CNS in mice. Here, we aimed to test its potential to shuttle a therapeutic relevant cargo. Since this VHH could not recognize the human TfR and hence its translational potential is limited, we also aimed to find and validate an anti-human transferrin VHH to deliver a therapeutic cargo into the CNS. METHODS: Alpaca immunizations with human TfR, and subsequent phage selection and screening for human TfR binding VHHs was performed to find a human TfR specific VHH (Nb188). Its ability to cross the BBB was determined by fusing it to neurotensin, a neuropeptide that reduces body temperature when present in the CNS but is not able to cross the BBB on its own. Next, the anti-ß-secretase 1 (BACE1) 1A11 Fab and Nb62 or Nb188 were fused to an Fc domain to generate heterodimeric antibodies (1A11AM-Nb62 and 1A11AM-Nb188). These were then administered intravenously in wild-type mice and in mice in which the murine apical domain of the TfR was replaced by the human apical domain (hAPI KI). Pharmacokinetic and pharmacodynamic (PK/PD) studies were performed to assess the concentration of the heterodimeric antibodies in the brain over time and the ability to inhibit brain-specific BACE1 by analysing the brain levels of Aß1-40. RESULTS: Selections and screening of a phage library resulted in the discovery of an anti-human TfR VHH (Nb188). Fusion of Nb188 to neurotensin induced hypothermia after intravenous injections in hAPI KI mice. In addition, systemic administration 1A11AM-Nb62 and 1A11AM-Nb188 fusions were able to reduce Aß1-40 levels in the brain whereas 1A11AM fused to an irrelevant VHH did not. A PK/PD experiment showed that this effect could last for 3 days. CONCLUSION: We have discovered an anti-human TfR specific VHH that is able to reach the CNS when administered systemically. In addition, both the currently discovered anti-human TfR VHH and the previously identified mouse-specific anti-TfR VHH, are both able to shuttle a therapeutically relevant cargo into the CNS. We suggest the mouse-specific VHH as a valuable research tool in mice and the human-specific VHH as a moiety to enhance the delivery efficiency of therapeutics into the CNS in human patients.