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1.
FEBS Lett ; 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31721178

RESUMO

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.

2.
J Mol Biol ; 430(21): 4119-4131, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30121297

RESUMO

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.


Assuntos
Cisteína/metabolismo , Azul de Metileno/análogos & derivados , Emaranhados Neurofibrilares/química , Emaranhados Neurofibrilares/metabolismo , Proteínas tau/química , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Heparina/química , Humanos , Azul de Metileno/química , Estrutura Molecular , Emaranhados Neurofibrilares/ultraestrutura , Proteínas Recombinantes , Análise Espectral
3.
Behav Brain Res ; 339: 153-168, 2018 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-29180135

RESUMO

Alpha-Synuclein (α-Syn) accumulation is considered a major risk factor for the development of synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies. We have generated mice overexpressing full-length human α-Syn fused to a membrane-targeting signal sequence under the control of the mouse Thy1-promotor. Three separate lines (L56, L58 and L62) with similar gene expression levels, but considerably heightened protein accumulation in L58 and L62, were established. In L62, there was widespread labelling of α-Syn immunoreactivity in brain including spinal cord, basal forebrain, cortex and striatum. Interestingly, there was no detectable α-Syn expression in dopaminergic neurones of the substantia nigra, but strong human α-Syn reactivity in glutamatergic synapses. The human α-Syn accumulated during aging and formed PK-resistant, thioflavin-binding aggregates. Mice displayed early onset bradykinesia and age progressive motor deficits. Functional alterations within the striatum were confirmed: L62 showed normal basal dopamine levels, but impaired dopamine release (upon amphetamine challenge) in the dorsal striatum measured by in vivo brain dialysis at 9 months of age. This impairment was coincident with a reduced response to amphetamine in the activity test. L62 further displayed greater sensitivity to low doses of the dopamine receptor 1 (D1) agonist SKF81297 but reacted normally to the D2 agonist quinpirole in the open field. Since accumulation of α-Syn aggregates in neurones and synapses and alterations in the dopaminergic tone are characteristics of PD, phenotypes reported for L62 present a good opportunity to further our understanding of motor dysfunction in PD and Lewy body dementia.


Assuntos
Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Doença de Parkinson/metabolismo , alfa-Sinucleína/genética , Animais , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Camundongos Transgênicos , Doença de Parkinson/genética , Fenótipo , Substância Negra/metabolismo , alfa-Sinucleína/metabolismo
4.
J Mol Biol ; 429(23): 3650-3665, 2017 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-28919235

RESUMO

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.


Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Encéfalo/metabolismo , Reagentes para Ligações Cruzadas/química , Dissulfetos/química , Proteínas tau/química , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Reagentes para Ligações Cruzadas/metabolismo , Dissulfetos/metabolismo , Humanos , Emaranhados Neurofibrilares , Proteínas tau/metabolismo
5.
Front Mol Neurosci ; 10: 447, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29375308

RESUMO

α-Synuclein (α-Syn) aggregation is a pathological feature of synucleinopathies, neurodegenerative disorders that include Parkinson's disease (PD). We have tested whether N,N,N',N'-tetramethyl-10H-phenothiazine-3,7-diaminium bis(hydromethanesulfonate) (leuco-methylthioninium bis(hydromethanesulfonate); LMTM), a tau aggregation inhibitor, affects α-Syn aggregation in vitro and in vivo. Both cellular and transgenic models in which the expression of full-length human α-Syn (h-α-Syn) fused with a signal sequence peptide to promote α-Syn aggregation were used. Aggregated α-Syn was observed following differentiation of N1E-115 neuroblastoma cells transfected with h-α-Syn. The appearance of aggregated α-Syn was inhibited by LMTM, with an EC50 of 1.1 µM, with minimal effect on h-α-Syn mRNA levels being observed. Two independent lines of mice (L58 and L62) transgenic for the same fusion protein accumulated neuronal h-α-Syn that, with aging, developed into fibrillary inclusions characterized by both resistance to proteinase K (PK)-cleavage and their ability to bind thiazin red. There was a significant decrease in α-Syn-positive neurons in multiple brain regions following oral treatment of male and female mice with LMTM administered daily for 6 weeks at 5 and 15 mg MT/kg. The early aggregates of α-Syn and the late-stage fibrillar inclusions were both susceptible to inhibition by LMTM, a treatment that also resulted in the rescue of movement and anxiety-related traits in these mice. The results suggest that LMTM may provide a potential disease modification therapy in PD and other synucleinopathies through the inhibition of α-Syn aggregation.

6.
Methods Mol Biol ; 1523: 129-140, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27975248

RESUMO

Aggregation of tau protein is a pathological hallmark of Alzheimer's disease and other neurodegenerative tauopathies. Inhibition of tau aggregation could provide a method for the treatment of these disorders. Methods to identify tau aggregation inhibitors (TAIs) in vitro are useful and here we describe assays for TAIs using purified recombinant tau protein fragments in a cell-free immunoassay format and a stably transfected cell model to create a more physiological environment.


Assuntos
Doença de Alzheimer/metabolismo , Bioensaio/métodos , Agregação Patológica de Proteínas/tratamento farmacológico , Agregação Patológica de Proteínas/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/tratamento farmacológico , Animais , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Imunoensaio , Camundongos , Fármacos Neuroprotetores/uso terapêutico , Proteínas tau/antagonistas & inibidores
7.
J Biol Chem ; 290(17): 10862-75, 2015 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-25759392

RESUMO

Alzheimer disease (AD) is a degenerative tauopathy characterized by aggregation of Tau protein through the repeat domain to form intraneuronal paired helical filaments (PHFs). We report two cell models in which we control the inherent toxicity of the core Tau fragment. These models demonstrate the properties of prion-like recruitment of full-length Tau into an aggregation pathway in which template-directed, endogenous truncation propagates aggregation through the core Tau binding domain. We use these in combination with dissolution of native PHFs to quantify the activity of Tau aggregation inhibitors (TAIs). We report the synthesis of novel stable crystalline leucomethylthioninium salts (LMTX®), which overcome the pharmacokinetic limitations of methylthioninium chloride. LMTX®, as either a dihydromesylate or a dihydrobromide salt, retains TAI activity in vitro and disrupts PHFs isolated from AD brain tissues at 0.16 µM. The Ki value for intracellular TAI activity, which we have been able to determine for the first time, is 0.12 µM. These values are close to the steady state trough brain concentration of methylthioninium ion (0.18 µM) that is required to arrest progression of AD on clinical and imaging end points and the minimum brain concentration (0.13 µM) required to reverse behavioral deficits and pathology in Tau transgenic mice.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Agregação Patológica de Proteínas/tratamento farmacológico , Agregação Patológica de Proteínas/metabolismo , Proteínas tau/química , Proteínas tau/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular , Humanos , Azul de Metileno/análogos & derivados , Azul de Metileno/síntese química , Azul de Metileno/química , Azul de Metileno/farmacologia , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Agregados Proteicos/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
8.
Behav Pharmacol ; 26(4): 353-68, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25769090

RESUMO

Given the repeated failure of amyloid-based approaches in Alzheimer's disease, there is increasing interest in tau-based therapeutics. Although methylthioninium (MT) treatment was found to be beneficial in tau transgenic models, the brain concentrations required to inhibit tau aggregation in vivo are unknown. The comparative efficacy of methylthioninium chloride (MTC) and leucomethylthioninium salts (LMTX; 5-75 mg/kg; oral administration for 3-8 weeks) was assessed in two novel transgenic tau mouse lines. Behavioural (spatial water maze, RotaRod motor performance) and histopathological (tau load per brain region) proxies were applied. Both MTC and LMTX dose-dependently rescued the learning impairment and restored behavioural flexibility in a spatial problem-solving water maze task in Line 1 (minimum effective dose: 35 mg MT/kg for MTC, 9 mg MT/kg for LMTX) and corrected motor learning in Line 66 (effective doses: 4 mg MT/kg). Simultaneously, both drugs reduced the number of tau-reactive neurons, particularly in the hippocampus and entorhinal cortex in Line 1 and in a more widespread manner in Line 66. MT levels in the brain followed a sigmoidal concentration-response relationship over a 10-fold range (0.13-1.38 µmol/l). These data establish that diaminophenothiazine compounds, like MT, can reverse both spatial and motor learning deficits and reduce the underlying tau pathology, and therefore offer the potential for treatment of tauopathies.


Assuntos
Azul de Metileno/farmacologia , Fármacos Neuroprotetores/farmacologia , Tauopatias/tratamento farmacológico , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Estudos de Coortes , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , /patologia , Aprendizagem em Labirinto/efeitos dos fármacos , Azul de Metileno/química , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/química , Oxirredução , Resolução de Problemas/efeitos dos fármacos , Distribuição Aleatória , Tauopatias/patologia , Tauopatias/fisiopatologia
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