RESUMO
Although complex inflammatory-like alterations are observed around the amyloid plaques of Alzheimer's disease (AD), little is known about the molecular changes and cellular interactions that characterize this response. We investigate here, in an AD mouse model, the transcriptional changes occurring in tissue domains in a 100-µm diameter around amyloid plaques using spatial transcriptomics. We demonstrate early alterations in a gene co-expression network enriched for myelin and oligodendrocyte genes (OLIGs), whereas a multicellular gene co-expression network of plaque-induced genes (PIGs) involving the complement system, oxidative stress, lysosomes, and inflammation is prominent in the later phase of the disease. We confirm the majority of the observed alterations at the cellular level using in situ sequencing on mouse and human brain sections. Genome-wide spatial transcriptomics analysis provides an unprecedented approach to untangle the dysregulated cellular network in the vicinity of pathogenic hallmarks of AD and other brain diseases.
Assuntos
Doença de Alzheimer/patologia , Análise de Sequência de DNA/métodos , Transcriptoma , Doença de Alzheimer/genética , Amiloide/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas do Sistema Complemento/genética , Proteínas do Sistema Complemento/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Bainha de Mielina/genética , Bainha de Mielina/metabolismo , Estresse Oxidativo/genéticaRESUMO
Missense mutations in alanine 673 of the amyloid precursor protein (APP), which corresponds to the second alanine of the amyloid ß (Aß) sequence, have dramatic impact on the risk for Alzheimer disease; A2V is causative, and A2T is protective. Assuming a crucial role of amyloid-Aß in neurodegeneration, we hypothesized that both A2V and A2T mutations cause distinct changes in Aß properties that may at least partially explain these completely different phenotypes. Using human APP-overexpressing primary neurons, we observed significantly decreased Aß production in the A2T mutant along with an enhanced Aß generation in the A2V mutant confirming earlier data from non-neuronal cell lines. More importantly, thioflavin T fluorescence assays revealed that the mutations, while having little effect on Aß42 peptide aggregation, dramatically change the properties of the Aß40 pool with A2V accelerating and A2T delaying aggregation of the Aß peptides. In line with the kinetic data, Aß A2T demonstrated an increase in the solubility at equilibrium, an effect that was also observed in all mixtures of the A2T mutant with the wild type Aß40. We propose that in addition to the reduced ß-secretase cleavage of APP, the impaired propensity to aggregate may be part of the protective effect conferred by A2T substitution. The interpretation of the protective effect of this mutation is thus much more complicated than proposed previously.
Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/genética , Fragmentos de Peptídeos/química , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Benzotiazóis , Encéfalo/metabolismo , Técnicas de Cultura de Células/métodos , Linhagem Celular , Cricetinae , Modelos Animais de Doenças , Humanos , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese , Mutação , Neurônios/citologia , Neurônios/metabolismo , Solubilidade , Termodinâmica , Tiazóis/químicaRESUMO
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
In spite of a clear genetic link between Parkinson's disease (PD) and mutations in LRRK2, cellular localization and physiological function of LRRK2 remain debated. Here we demonstrate the immunohistochemical localization of LRRK2 in adult mouse and early postnatal mouse brain development. Antibody specificity is verified by absence of specific staining in LRRK2 knockout mouse brain. Although LRRK2 is expressed in various mouse brain regions (i.e. cortex, thalamus, hippocampus, cerebellum), strongest expression is detected in striatum, whereas LRRK2 protein expression in substantia nigra pars compacta in contrast is low. LRRK2 is highly expressed in striatal medium spiny neurons (MSN) and few cholinergic interneurons. LRRK2 expression is undetectable in other interneurons, oligodendrocytes or astrocytes of the striatum. Interestingly, LRRK2 expression is associated with striosome specific markers (i.e. MOR1, RASGRP1). Analysis of LRRK2 expression during early postnatal development and in LRRK2 knockout mice, demonstrates that LRRK2 is not required for generation or maintenance of the striosome compartment. Comparing LRRK2-WT, LRRK2-R1441G transgenic and non-transgenic mice, changes of LRRK2 expression in striosome/matrix compartments can be detected. The findings rule out a specific requirement of LRRK2 in striosome genesis but suggest a functional role for LRRK2 in striosomes.
Assuntos
Corpo Estriado/metabolismo , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/biossíntese , Animais , Western Blotting , Corpo Estriado/crescimento & desenvolvimento , Imuno-Histoquímica , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neurogênese/fisiologia , Neurônios/químicaRESUMO
The metalloproteinase and major amyloid precursor protein (APP) alpha-secretase candidate ADAM10 is responsible for the shedding of proteins important for brain development, such as cadherins, ephrins, and Notch receptors. Adam10(-/-) mice die at embryonic day 9.5, due to major defects in development of somites and vasculogenesis. To investigate the function of ADAM10 in brain, we generated Adam10 conditional knock-out (cKO) mice using a Nestin-Cre promotor, limiting ADAM10 inactivation to neural progenitor cells (NPCs) and NPC-derived neurons and glial cells. The cKO mice die perinatally with a disrupted neocortex and a severely reduced ganglionic eminence, due to precocious neuronal differentiation resulting in an early depletion of progenitor cells. Premature neuronal differentiation is associated with aberrant neuronal migration and a disorganized laminar architecture in the neocortex. Neurospheres derived from Adam10 cKO mice have a disrupted sphere organization and segregated more neurons at the expense of astrocytes. We found that Notch-1 processing was affected, leading to downregulation of several Notch-regulated genes in Adam10 cKO brains, in accordance with the central role of ADAM10 in this signaling pathway and explaining the neurogenic phenotype. Finally, we found that alpha-secretase-mediated processing of APP was largely reduced in these neurons, demonstrating that ADAM10 represents the most important APP alpha-secretase in brain. Our study reveals that ADAM10 plays a central role in the developing brain by controlling mainly Notch-dependent pathways but likely also by reducing surface shedding of other neuronal membrane proteins including APP.
Assuntos
Proteínas ADAM/fisiologia , Secretases da Proteína Precursora do Amiloide/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/enzimologia , Proteínas de Membrana/fisiologia , Proteínas ADAM/deficiência , Proteínas ADAM/genética , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/deficiência , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/biossíntese , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Córtex Cerebral/crescimento & desenvolvimento , Feminino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neurogênese/genética , Neurogênese/fisiologia , Gravidez , Receptores Notch/biossíntese , Receptores Notch/metabolismoRESUMO
BACKGROUND: Increasing evidence for a direct contribution of astrocytes to neuroinflammatory and neurodegenerative processes causing Alzheimer's disease comes from molecular and functional studies in rodent models. However, these models may not fully recapitulate human disease as human and rodent astrocytes differ considerably in morphology, functionality, and gene expression. RESULTS: To address these challenges, we established an approach to study human astrocytes within the mouse brain by transplanting human induced pluripotent stem cell (hiPSC)-derived astrocyte progenitors into neonatal brains. Xenografted hiPSC-derived astrocyte progenitors differentiated into astrocytes that integrated functionally within the mouse host brain and matured in a cell-autonomous way retaining human-specific morphologies, unique features, and physiological properties. In Alzheimer´s chimeric brains, transplanted hiPSC-derived astrocytes responded to the presence of amyloid plaques undergoing morphological changes that seemed independent of the APOE allelic background. CONCLUSIONS: In sum, we describe here a promising approach that consist of transplanting patient-derived and genetically modified astrocytes into the mouse brain to study human astrocyte pathophysiology in the context of Alzheimer´s disease.
Assuntos
Doença de Alzheimer , Células-Tronco Pluripotentes Induzidas , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Astrócitos/metabolismo , Encéfalo/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Placa Amiloide/metabolismoRESUMO
ß-Site APP-cleaving enzyme 1 (BACE1) inhibition is considered one of the most promising therapeutic strategies for Alzheimer's disease, but current BACE1 inhibitors also block BACE2. As the localization and function of BACE2 in the brain remain unknown, it is difficult to predict whether relevant side effects can be caused by off-target inhibition of BACE2 and whether it is important to generate BACE1-specific inhibitors. Here, we show that BACE2 is expressed in discrete subsets of neurons and glia throughout the adult mouse brain. We uncover four new substrates processed by BACE2 in cultured glia: vascular cell adhesion molecule 1, delta and notch-like epidermal growth factor-related receptor, fibroblast growth factor receptor 1, and plexin domain containing 2. Although these substrates were not prominently cleaved by BACE2 in healthy adult mice, proinflammatory TNF induced a drastic increase in BACE2-mediated shedding of vascular cell adhesion molecule 1 in CSF. Thus, although under steady-state conditions the effect of BACE2 cross-inhibition by BACE1-directed inhibitors is rather subtle, it is important to consider that side effects might become apparent under physiopathological conditions that induce TNF expression.
RESUMO
Synaptic dysfunction is an early pathological feature of neurodegenerative diseases associated with Tau, including Alzheimer's disease. Interfering with early synaptic dysfunction may be therapeutically beneficial to prevent cognitive decline and disease progression, but the mechanisms underlying synaptic defects associated with Tau are unclear. In disease conditions, Tau mislocalizes into pre- and postsynaptic compartments; here we show that, under pathological conditions, Tau binds to presynaptic vesicles in Alzheimer's disease patient brain. We define that the binding of Tau to synaptic vesicles is mediated by the transmembrane vesicle protein Synaptogyrin-3. In fly and mouse models of Tauopathy, reduction of Synaptogyrin-3 prevents the association of presynaptic Tau with vesicles, alleviates Tau-induced defects in vesicle mobility, and restores neurotransmitter release. This work therefore identifies Synaptogyrin-3 as the binding partner of Tau on synaptic vesicles, revealing a new presynapse-specific Tau interactor, which may contribute to early synaptic dysfunction in neurodegenerative diseases associated with Tau.
Assuntos
Doença de Alzheimer/metabolismo , Terminações Pré-Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptogirinas/metabolismo , Proteínas tau/metabolismo , Animais , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Feminino , Hipocampo/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/metabolismo , Cultura Primária de Células , Tauopatias/metabolismoRESUMO
BACKGROUND: The mechanisms behind Aß-peptide accumulation in non-familial Alzheimer's disease (AD) remain elusive. Proteins of the tetraspanin family modulate Aß production by interacting to γ-secretase. METHODS: We searched for tetraspanins with altered expression in AD brains. The function of the selected tetraspanin was studied in vitro and the physiological relevance of our findings was confirmed in vivo. RESULTS: Tetraspanin-6 (TSPAN6) is increased in AD brains and overexpression in cells exerts paradoxical effects on Amyloid Precursor Protein (APP) metabolism, increasing APP-C-terminal fragments (APP-CTF) and Aß levels at the same time. TSPAN6 affects autophagosome-lysosomal fusion slowing down the degradation of APP-CTF. TSPAN6 recruits also the cytosolic, exosome-forming adaptor syntenin which increases secretion of exosomes that contain APP-CTF. CONCLUSIONS: TSPAN6 is a key player in the bifurcation between lysosomal-dependent degradation and exosome mediated secretion of APP-CTF. This corroborates the central role of the autophagosomal/lysosomal pathway in APP metabolism and shows that TSPAN6 is a crucial player in APP-CTF turnover.
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Tetraspaninas/metabolismo , Animais , Western Blotting , Exossomos/metabolismo , Exossomos/ultraestrutura , Humanos , Imageamento Tridimensional , Imuno-Histoquímica , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Neurônios/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Human pluripotent stem cells (PSCs) provide a unique entry to study species-specific aspects of human disorders such as Alzheimer's disease (AD). However, in vitro culture of neurons deprives them of their natural environment. Here we transplanted human PSC-derived cortical neuronal precursors into the brain of a murine AD model. Human neurons differentiate and integrate into the brain, express 3R/4R Tau splice forms, show abnormal phosphorylation and conformational Tau changes, and undergo neurodegeneration. Remarkably, cell death was dissociated from tangle formation in this natural 3D model of AD. Using genome-wide expression analysis, we observed upregulation of genes involved in myelination and downregulation of genes related to memory and cognition, synaptic transmission, and neuron projection. This novel chimeric model for AD displays human-specific pathological features and allows the analysis of different genetic backgrounds and mutations during the course of the disease.
Assuntos
Doença de Alzheimer/patologia , Encéfalo , Diferenciação Celular/fisiologia , Neuritos/metabolismo , Neurônios/metabolismo , Células-Tronco Pluripotentes/citologia , Proteínas tau/metabolismo , Doença de Alzheimer/diagnóstico , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Morte Celular/fisiologia , Humanos , Camundongos , FosforilaçãoRESUMO
The orphan G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) GPR3 regulates activity of the γ-secretase complex in the absence of an effect on Notch proteolysis, providing a potential therapeutic target for Alzheimer's disease (AD). However, given the vast resources required to develop and evaluate any new therapy for AD and the multiple failures involved in translational research, demonstration of the pathophysiological relevance of research findings in multiple disease-relevant models is necessary before initiating costly drug development programs. We evaluated the physiological consequences of loss of Gpr3 in four AD transgenic mouse models, including two that contain the humanized murine Aß sequence and express similar amyloid precursor protein (APP) levels as wild-type mice, thereby reducing potential artificial phenotypes. Our findings reveal that genetic deletion of Gpr3 reduced amyloid pathology in all of the AD mouse models and alleviated cognitive deficits in APP/PS1 mice. Additional three-dimensional visualization and analysis of the amyloid plaque burden provided accurate information on the amyloid load, distribution, and volume in the structurally intact adult mouse brain. Analysis of 10 different regions in healthy human postmortem brain tissue indicated that GPR3 expression was stable during aging. However, two cohorts of human AD postmortem brain tissue samples showed a correlation between elevated GPR3 and AD progression. Collectively, these studies provide evidence that GPR3 mediates the amyloidogenic proteolysis of APP in four AD transgenic mouse models as well as the physiological processing of APP in wild-type mice, suggesting that GPR3 may be a potential therapeutic target for AD drug development.
Assuntos
Doença de Alzheimer/fisiopatologia , Doença de Alzheimer/terapia , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/fisiologia , Animais , Encéfalo/fisiologia , Deleção de Genes , Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Modelos Animais , Placa Amiloide/patologiaRESUMO
Neuregulin 1 (NRG1) and the γ-secretase subunit APH1B have been previously implicated as genetic risk factors for schizophrenia and schizophrenia relevant deficits have been observed in rodent models with loss of function mutations in either gene. Here we show that the Aph1b-γ-secretase is selectively involved in Nrg1 intracellular signalling. We found that Aph1b-deficient mice display a decrease in excitatory synaptic markers. Electrophysiological recordings show that Aph1b is required for excitatory synaptic transmission and plasticity. Furthermore, gain and loss of function and genetic rescue experiments indicate that Nrg1 intracellular signalling promotes dendritic spine formation downstream of Aph1b-γ-secretase in vitro and in vivo. In conclusion, our study sheds light on the physiological role of Aph1b-γ-secretase in brain and provides a new mechanistic perspective on the relevance of NRG1 processing in schizophrenia.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Endopeptidases/metabolismo , Regulação da Expressão Gênica , Hipocampo/embriologia , Neuregulina-1/metabolismo , Doença de Alzheimer/genética , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Eletrofisiologia , Deleção de Genes , Hipocampo/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Transgênicos , Mutação , Neurônios/metabolismo , Técnicas de Patch-Clamp , Esquizofrenia/metabolismo , Transdução de Sinais , Sinapses/metabolismoRESUMO
ß-arrestins are associated with numerous aspects of G protein-coupled receptor (GPCR) signaling and regulation and accordingly influence diverse physiological and pathophysiological processes. Here we report that ß-arrestin 2 expression is elevated in two independent cohorts of individuals with Alzheimer's disease. Overexpression of ß-arrestin 2 leads to an increase in amyloid-ß (Aß) peptide generation, whereas genetic silencing of Arrb2 (encoding ß-arrestin 2) reduces generation of Aß in cell cultures and in Arrb2(-/-) mice. Moreover, in a transgenic mouse model of Alzheimer's disease, genetic deletion of Arrb2 leads to a reduction in the production of Aß(40) and Aß(42). Two GPCRs implicated previously in Alzheimer's disease (GPR3 and the ß(2)-adrenergic receptor) mediate their effects on Aß generation through interaction with ß-arrestin 2. ß-arrestin 2 physically associates with the Aph-1a subunit of the γ-secretase complex and redistributes the complex toward detergent-resistant membranes, increasing the catalytic activity of the complex. Collectively, these studies identify ß-arrestin 2 as a new therapeutic target for reducing amyloid pathology and GPCR dysfunction in Alzheimer's disease.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/biossíntese , Arrestinas/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Animais , Arrestinas/genética , Células CHO , Linhagem Celular , Cricetinae , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , beta-Arrestina 2 , beta-ArrestinasRESUMO
Inherited familial Alzheimer's disease (AD) is characterized by small increases in the ratio of Aß42 versus Aß40 peptide which is thought to drive the amyloid plaque formation in the brain of these patients. Little is known however whether ageing, the major risk factor for sporadic AD, affects amyloid beta-peptide (Aß) generation as well. Here we demonstrate that the secretion of Aß is enhanced in an in vitro model of neuronal ageing, correlating with an increase in γ-secretase complex formation. Moreover we found that peroxynitrite (ONOO(-)), produced by the reaction of superoxide anion with nitric oxide, promoted the nitrotyrosination of presenilin 1 (PS1), the catalytic subunit of γ-secretase. This was associated with an increased association of the two PS1 fragments, PS1-CTF and PS1-NTF, which constitute the active catalytic centre. Furthermore, we found that peroxynitrite shifted the production of Aß towards Aß(42) and increased the Aß(42) /Aß(40) ratio. Our work identifies nitrosative stress as a potential mechanistic link between ageing and AD.
Assuntos
Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Senescência Celular/efeitos dos fármacos , Neurônios/metabolismo , Ácido Peroxinitroso/farmacologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Domínio Catalítico , Células Cultivadas , Humanos , Camundongos , Neurônios/citologia , Fragmentos de Peptídeos/metabolismo , Presenilina-1/química , Presenilina-1/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fatores de Risco , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
ADAM10 is involved in the proteolytic processing and shedding of proteins such as the amyloid precursor protein (APP), cadherins, and the Notch receptors, thereby initiating the regulated intramembrane proteolysis (RIP) of these proteins. Here, we demonstrate that the sheddase ADAM10 is also subject to RIP. We identify ADAM9 and -15 as the proteases responsible for releasing the ADAM10 ectodomain, and Presenilin/gamma-Secretase as the protease responsible for the release of the ADAM10 intracellular domain (ICD). This domain then translocates to the nucleus and localizes to nuclear speckles, thought to be involved in gene regulation. Thus, ADAM10 performs a dual role in cells, as a metalloprotease when it is membrane-bound, and as a potential signaling protein once cleaved by ADAM9/15 and the gamma-Secretase.
Assuntos
Proteínas ADAM/metabolismo , Proteínas ADAM/fisiologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Receptores Notch/metabolismo , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/fisiologia , Animais , Núcleo Celular/metabolismo , Camundongos , Microscopia de Fluorescência , Presenilinas/metabolismo , Estrutura Terciária de Proteína , Transdução de Sinais , Frações Subcelulares/metabolismo , Distribuição TecidualRESUMO
Mutations of the mitochondrial PTEN (phosphatase and tensin homologue)-induced kinase1 (PINK1) are important causes of recessive Parkinson disease (PD). Studies on loss of function and overexpression implicate PINK1 in apoptosis, abnormal mitochondrial morphology, impaired dopamine release and motor deficits. However, the fundamental mechanism underlying these various phenotypes remains to be clarified. Using fruit fly and mouse models we show that PINK1 deficiency or clinical mutations impact on the function of Complex I of the mitochondrial respiratory chain, resulting in mitochondrial depolarization and increased sensitivity to apoptotic stress in mammalian cells and tissues. In Drosophila neurons, PINK1 deficiency affects synaptic function, as the reserve pool of synaptic vesicles is not mobilized during rapid stimulation. The fundamental importance of PINK1 for energy maintenance under increased demand is further corroborated as this deficit can be rescued by adding ATP to the synapse. The clinical relevance of our observations is demonstrated by the fact that human wild type PINK1, but not PINK1 containing clinical mutations, can rescue Complex 1 deficiency. Our work suggests that Complex I deficiency underlies, at least partially, the pathogenesis of this hereditary form of PD. As Complex I dysfunction is also implicated in sporadic PD, a convergence of genetic and environmental causes of PD on a similar mitochondrial molecular mechanism appears to emerge.
Assuntos
Proteínas de Drosophila/genética , Complexo I de Transporte de Elétrons/metabolismo , Mutação/genética , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Sinapses/patologia , Animais , Apoptose , Proteínas de Drosophila/deficiência , Drosophila melanogaster/enzimologia , Transporte de Elétrons , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Doença de Parkinson/enzimologia , Doença de Parkinson/patologia , Proteínas Quinases/deficiência , Proteínas Serina-Treonina Quinases/deficiência , Sinapses/enzimologia , Sinapses/ultraestrutura , Transmissão Sináptica/fisiologiaRESUMO
Presenilin (PS1) and (PS2) are the centers of gamma-secretase that release Abeta from APP in Alzheimer's disease (AD). They cleave signaling proteins like Notch and downregulate beta-catenin to modulate Wnt signaling. Inactivation of PS1 or PS1 and PS2 causes a prenatally lethal 'Notch phenotype,' which has hampered investigation of PS function in adulthood seriously. We have thus turned towards PS1+/-PS2-/- mice which carry the most severe reduction of PS alleles compatible with survival, to analyze the consequences of impaired PS function especially in adulthood. In these 'partial deficient' mice, PS1 protein concentration is considerably lowered, functionally reflected by reduced gamma-secretase activity and impaired beta-catenin downregulation. Their phenotype is normal up to approximately 6 months, when the majority of the mice develop an autoimmune disease characterized by dermatitis, glomerulonephritis, keratitis and vasculitis, as seen in human systemic lupus erythematosus. Besides B-cell dominated infiltrates, we observe a hypergammaglobulinemia with immune complex deposits in several tissues, high-titer nuclear autoantibodies and an increased CD4+/CD8+ ratio. The mice further develop a benign skin hyperplasia similar to human seborrheic keratosis as opposed to malignant keratocarcinomata observed in skin-specific PS1 'full' knockouts. A partial reduction of PS function in PS1+/-PS2-/- mice causes a novel phenotype in adulthood unrelated to the developmental defects of full knockouts. As PS1+/-PS2+/- mice remain healthy, this points towards a sharply defined minimum of PS function. Skin and immune system appear to be especially sensitive targets of impaired PS function and may need careful monitoring if gamma-secretase inhibitors are envisaged for treating AD.
Assuntos
Doenças Autoimunes/genética , Ceratose Seborreica/genética , Proteínas de Membrana/genética , Doença de Alzheimer/imunologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide , Animais , Anticorpos Antinucleares/imunologia , Ácido Aspártico Endopeptidases , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Linfócitos B/imunologia , Linfócitos B/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Proteínas de Transporte/metabolismo , Endopeptidases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ceratose Seborreica/imunologia , Ceratose Seborreica/metabolismo , Ceratose Seborreica/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Presenilina-1 , Presenilina-2 , Nexinas de Proteases , Receptores de Superfície Celular , Receptores Notch , Proteínas WntRESUMO
Furin is an endoprotease of the family of mammalian proprotein convertases and is involved in the activation of a large variety of regulatory proteins by cleavage at basic motifs. A large number of substrates have been attributed to furin on the basis of in vitro and ex vivo data. However, no physiological substrates have been confirmed directly in a mammalian model system, and early embryonic lethality of a furin knock-out mouse model has precluded in vivo verification of most candidate substrates. Here, we report the generation and characterization of an interferon inducible Mx-Cre/loxP furin knock-out mouse model. Induction resulted in near-complete ablation of the floxed fur exon in liver. In sharp contrast with the general furin knock-out mouse model, no obvious adverse effects were observed in the transgenic mice after induction. Histological analysis of the liver did not reveal any overt deviations from normal morphology. Analysis of candidate substrates in liver revealed complete redundancy for the processing of the insulin receptor. Variable degrees of redundancy were observed for the processing of albumin, alpha(5) integrin, lipoprotein receptor-related protein, vitronectin and alpha(1)-microglobulin/bikunin. None of the tested substrates displayed a complete block of processing. The absence of a severe phenotype raises the possibility of using furin as a local therapeutic target in the treatment of pathologies like cancer and viral infections, although the observed redundancy may require combination therapy or the development of a more broad spectrum convertase inhibitor.
Assuntos
Furina/química , Fígado/metabolismo , Pró-Proteína Convertases/química , Albuminas/metabolismo , Alelos , alfa-Globulinas/metabolismo , Motivos de Aminoácidos , Animais , Northern Blotting , Western Blotting , Primers do DNA/química , Eletroforese em Gel de Poliacrilamida , Furina/metabolismo , Furina/fisiologia , Genótipo , Imuno-Histoquímica , Integrina alfa5/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , Neoplasias/metabolismo , Fenótipo , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Receptores de LDL/metabolismo , Proteínas Recombinantes/metabolismo , Recombinação Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por Substrato , Transgenes , Inibidor da Tripsina de Soja de Kunitz/metabolismo , Vitronectina/metabolismoRESUMO
Investigation of MR patients with 3p aberrations led to the identification of the translocation breakpoint in intron five of the neural Cell Adhesion L1-Like (CALL or CHL1) gene in a man with non-specific mental retardation and 46,Y, t(X;3)(p22.1;p26.3). The Xp breakpoint does not seem to affect a known or predicted gene. Moreover, a fusion transcript with the CALL gene could not be detected and no mutations were identified on the second allele. CALL is highly expressed in the central and peripheral nervous system, like the mouse ortholog 'close homolog to L1' (Chl1). Chl1 expression levels in the hippocampus of Chl1(+/-) mice were half of those obtained in wild-type littermates, reflecting a gene dosage effect. Timm staining and synaptophysin immunohistochemistry of the hippocampus showed focal groups of ectopic mossy fiber synapses in the lateral CA3 region, outside the trajectory of the infra-pyramidal mossy fiber bundle in Chl1(-/-) and Chl1(+/-) mice. Behavioral assessment demonstrated mild alterations in the Chl1(-/-) animals. In the probe trial of the Morris Water Maze test, Chl1(-/-) mice displayed an altered exploratory pattern. In addition, these mice were significantly more sociable and less aggressive as demonstrated in social exploration tests. The Chl1(+/-) mice showed a phenotypic spectrum ranging from wild-type to knockout behavior. We hypothesize that a 50% reduction of CALL expression in the developing brain results in cognitive deficits. This suggests that the CALL gene at 3p26.3 is a prime candidate for an autosomal form of mental retardation. So far, mutation analysis of the CALL gene in patients with non-specific MR did not reveal any disease-associated mutations.