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INTRODUCTION: In tauopathies, altered tau processing correlates with impairments in synaptic density and function. Changes in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contribute to disease-associated abnormalities in multiple neurodegenerative diseases. METHODS: To investigate the link between tau and HCN channels, we performed histological, biochemical, ultrastructural, and functional analyses of hippocampal tissues from Alzheimer's disease (AD), age-matched controls, Tau35 mice, and/or Tau35 primary hippocampal neurons. RESULTS: Expression of specific HCN channels is elevated in post mortem AD hippocampus. Tau35 mice develop progressive abnormalities including increased phosphorylated tau, enhanced HCN channel expression, decreased dendritic branching, reduced synapse density, and vesicle clustering defects. Tau35 primary neurons show increased HCN channel expression enhanced hyperpolarization-induced membrane voltage "sag" and changes in the frequency and kinetics of spontaneous excitatory postsynaptic currents. DISCUSSION: Our findings are consistent with a model in which pathological changes in tauopathies impact HCN channels to drive network-wide structural and functional synaptic deficits. HIGHLIGHTS: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are functionally linked to the development of tauopathy. Expression of specific HCN channels is elevated in the hippocampus in Alzheimer's disease and the Tau35 mouse model of tauopathy. Increased expression of HCN channels in Tau35 mice is accompanied by hyperpolarization-induced membrane voltage "sag" demonstrating a detrimental effect of tau abnormalities on HCN channel function. Tau35 expression alters synaptic organization, causing a loosened vesicle clustering phenotype in Tau35 mice.
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The autophagy-lysosomal pathway plays a critical role in the clearance of tau protein aggregates that deposit in the brain in tauopathies, and defects in this system are associated with disease pathogenesis. Here, we report that expression of Tau35, a tauopathy-associated carboxy-terminal fragment of tau, leads to lipid accumulation in cell lines and primary cortical neurons. Our findings suggest that this is likely due to a deleterious block of autophagic clearance and lysosomal degradative capacity by Tau35. Notably, upon induction of autophagy by Torin 1, Tau35 inhibited nuclear translocation of transcription factor EB (TFEB), a key regulator of lysosomal biogenesis. Both cell lines and primary cortical neurons expressing Tau35 also exhibited changes in endosomal protein expression. These findings implicate autophagic and endolysosomal dysfunction as key pathological mechanisms through which disease-associated tau fragments could lead to the development and progression of tauopathy.
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Autofagia , Endosomas , Metabolismo de los Lípidos , Lisosomas , Neuronas , Proteínas tau , Proteínas tau/metabolismo , Proteínas tau/genética , Lisosomas/metabolismo , Humanos , Neuronas/metabolismo , Animales , Endosomas/metabolismo , Tauopatías/metabolismo , Tauopatías/patología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , RatonesRESUMEN
Assembly of tau protein into paired helical filaments and straight filaments is a key feature of Alzheimer's disease. Aggregation of tau has been implicated in neurodegeneration, cellular toxicity and the propagation, which accompanies disease progression. We have reported previously that a region of tau (297-391), referred to as dGAE, assembles spontaneously in physiological conditions to form paired helical filament-like fibres in vitro in the absence of additives such as heparin. This provides a valuable tool with which to explore the effects of tau in cell culture. Here we have studied the cellular uptake of soluble oligomeric and fibrillar forms of dGAE and examined the downstream consequences of tau internalisation into differentiated SH-SY5Y neuroblastoma cells using fluorescence and electron microscopy alongside structural and biochemical analyses. The assembled dGAE shows more acute cytotoxicity than the soluble, non-aggregated form. Conversely, the soluble form is much more readily internalised and, once within the cell, is able to associate with endogenous tau resulting in increased phosphorylation and aggregation of endogenous tau, which accumulates in lysosomal/endosomal compartments. It appears that soluble oligomeric forms are able to propagate tau pathology without being acutely toxic. The model system we have developed now permits the molecular mechanisms of propagation of tau pathology to be studied in vitro in a more physiological manner with a view to development of novel therapeutic approaches.
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Neuronas/citología , Proteínas tau/química , Proteínas tau/metabolismo , Línea Celular , Proliferación Celular , Heparina/metabolismo , Humanos , Microscopía Electrónica , Microscopía Fluorescente , Neuronas/metabolismo , Dominios Proteicos , Multimerización de Proteína , Estructura Secundaria de ProteínaRESUMEN
The constituent paired helical filaments (PHFs) in neurofibrillary tangles are insoluble intracellular deposits central to the development of Alzheimer's disease (AD) and other tauopathies. Full-length tau requires the addition of anionic cofactors such as heparin to enhance assembly. We have shown that a fragment from the proteolytically stable core of the PHF, tau 297-391 known as 'dGAE', spontaneously forms cross-ß-containing PHFs and straight filaments under physiological conditions. Here, we have analysed and compared the structures of the filaments formed by dGAE in vitro with those deposited in the brains of individuals diagnosed with AD. We show that dGAE forms PHFs that share a macromolecular structure similar to those found in brain tissue. Thus, dGAEs may serve as a model system for studying core domain assembly and for screening for inhibitors of tau aggregation.
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Enfermedad de Alzheimer/metabolismo , Ovillos Neurofibrilares/ultraestructura , Proteínas tau/química , Proteínas tau/metabolismo , Encéfalo/metabolismo , Humanos , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Dominios Proteicos , Proteínas tau/ultraestructuraRESUMEN
Tau is known for its pathological role in neurodegenerative diseases, including Alzheimer's disease (AD) and other tauopathies. Tau is found in many subcellular compartments such as the cytosol and the nucleus. Although its normal role in microtubule binding is well established, its nuclear role is still unclear. Here, we reveal that tau localises to the nucleolus in undifferentiated and differentiated neuroblastoma cells (SHSY5Y), where it associates with TIP5, a key player in heterochromatin stability and ribosomal DNA (rDNA) transcriptional repression. Immunogold labelling on human brain sample confirms the physiological relevance of this finding by showing tau within the nucleolus colocalises with TIP5. Depletion of tau results in an increase in rDNA transcription with an associated decrease in heterochromatin and DNA methylation, suggesting that under normal conditions tau is involved in silencing of the rDNA. Cellular stress induced by glutamate causes nucleolar stress associated with the redistribution of nucleolar non-phosphorylated tau, in a similar manner to fibrillarin, and nuclear upsurge of phosphorylated tau (Thr231) which doesn't colocalise with fibrillarin or nucleolar tau. This suggests that stress may impact on different nuclear tau species. In addition to involvement in rDNA transcription, nucleolar non-phosphorylated tau also undergoes stress-induced redistribution similar to many nucleolar proteins.
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Nucléolo Celular/efectos de los fármacos , Nucléolo Celular/metabolismo , Agonistas de Aminoácidos Excitadores/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ácido Glutámico/farmacología , Proteínas tau/metabolismo , Encéfalo/metabolismo , Encéfalo/ultraestructura , Diferenciación Celular/fisiología , Línea Celular Tumoral , Nucléolo Celular/ultraestructura , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/ultraestructura , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Heterocromatina/fisiología , Histonas/metabolismo , Humanos , Inmunoprecipitación , Microscopía Confocal , Microscopía Electrónica , Neuroblastoma/patología , Neuroblastoma/ultraestructura , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transcripción Genética/efectos de los fármacos , Transfección , Proteínas tau/genética , Proteínas tau/ultraestructuraRESUMEN
Alzheimer's disease is a tauopathy characterized by pathological fibrillization of tau protein to form the paired helical filaments (PHFs), which constitute neurofibrillary tangles. The methylthioninium (MT) moiety reverses the proteolytic stability of the PHF core and is in clinical development for treatment of Alzheimer's disease in a stable reduced form as leuco-MT. It has been hypothesized that MT acts via oxidation of cysteine residues, which is incompatible with activity in the predominantly reducing environment of living cells. We have shown recently that the PHF-core tau unit assembles spontaneously in vitro to form PHF-like filaments. Here we describe studies using circular dichroism, SDS-PAGE, transmission electron microscopy and site-directed mutagenesis to elucidate the mechanism of action of the MT moiety. We show that MT inhibitory activity is optimal in reducing conditions, that the active moiety is the reduced leuco-MT form of the molecule and that its mechanism of action is cysteine independent.
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Cisteína/metabolismo , Azul de Metileno/análogos & derivados , Ovillos Neurofibrilares/química , Ovillos Neurofibrilares/metabolismo , Proteínas tau/química , Proteínas tau/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Heparina/química , Humanos , Azul de Metileno/química , Estructura Molecular , Ovillos Neurofibrilares/ultraestructura , Proteínas Recombinantes , Análisis EspectralRESUMEN
Alzheimer's disease is characterized by the self-assembly of tau and amyloid ß proteins into oligomers and fibrils. Tau protein assembles into paired helical filaments (PHFs) that constitute the neurofibrillary tangles observed in neuronal cell bodies in individuals with Alzheimer's disease. The mechanism of initiation of tau assembly into PHFs is not well understood. Here we report that a truncated 95-amino-acid tau fragment (corresponding to residues 297-391 of full-length tau) assembles into PHF-like fibrils in vitro without the need for other additives to initiate or template the process. Using electron microscopy, circular dichroism and X-ray fiber diffraction, we have characterized the structure of the fibrils formed from truncated tau for the first time. To explore the contribution of disulfide formation to fibril formation, we have compared the assembly of tau(297-391) under reduced and non-reducing conditions and for truncated tau carrying a C322A substitution. We show that disulfide bond formation inhibits filament assembly and that the C322A variant rapidly forms long and highly ordered PHFs.
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Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Encéfalo/metabolismo , Reactivos de Enlaces Cruzados/química , Disulfuros/química , Proteínas tau/química , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Disulfuros/metabolismo , Humanos , Ovillos Neurofibrilares , Proteínas tau/metabolismoRESUMEN
Parkinson's disease (PD) is characterized by intracellular, insoluble Lewy bodies composed of highly stable α-synuclein (α-syn) amyloid fibrils. α-synuclein is an intrinsically disordered protein that has the capacity to assemble to form ß-sheet rich fibrils. Oxidiative stress and metal rich environments have been implicated in triggering assembly. Here, we have explored the composition of Lewy bodies in post-mortem tissue using electron microscopy and immunogold labeling and revealed dityrosine crosslinks in Lewy bodies in brain tissue from PD patients. In vitro, we show that dityrosine cross-links in α-syn are formed by covalent ortho-ortho coupling of two tyrosine residues under conditions of oxidative stress by fluorescence and confirmed using mass-spectrometry. A covalently cross-linked dimer isolated by SDS-PAGE and mass analysis showed that dityrosine dimer was formed via the coupling of Y39-Y39 to give a homo dimer peptide that may play a key role in formation of oligomeric and seeds for fibril formation. Atomic force microscopy analysis reveals that the covalent dityrosine contributes to the stabilization of α-syn assemblies. Thus, the presence of oxidative stress induced dityrosine could play an important role in assembly and toxicity of α-syn in PD.