Assays for the Screening and Characterization of Tau Aggregation Inhibitors.

Aggregation of tau protein is a pathological hallmark of Alzheimer's disease and other neurodegenerative tauopathies. Inhibition of tau aggregation may provide a method for 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 in a stably transfected cell model to create a more physiological environment.

Solid-state NMR of paired helical filaments formed by the core tau fragment tau(297-391).

Aggregation of the tau protein into fibrillar cross-ß aggregates is a hallmark of Alzheimer's diseases (AD) and many other neurodegenerative tauopathies. Recently, several core structures of patient-derived tau paired helical filaments (PHFs) have been solved revealing a structural variability that often correlates with a specific tauopathy. To further characterize the dynamics of these fibril cores, to screen for strain-specific small molecules as potential biomarkers and therapeutics, and to develop strain-specific antibodies, recombinant in-vitro models of tau filaments are needed. We recently showed that a 95-residue fragment of tau (from residue 297 to 391), termed dGAE, forms filaments in vitro in the absence of polyanionic co-factors often used for in vitro aggregation of full-length tau. Tau(297-391) was identified as the proteolytic resistant core of tau PHFs and overlaps with the structures characterized by cryo-electron microscopy in ex vivo PHFs, making it a promising model for the study of AD tau filaments in vitro. In the present study, we used solid-state NMR to characterize tau(297-391) filaments and show that such filaments assembled under non-reducing conditions are more dynamic and less ordered than those made in the presence of the reducing agent DTT. We further report the resonance assignment of tau(297-391)+DTT filaments and compare it to existing core structures of tau.

Oxidative Stress Conditions Result in Trapping of PHF-Core Tau (297-391) Intermediates.

The self-assembly of tau into paired helical filaments (PHFs) in neurofibrillary tangles (NFTs) is a significant event in Alzheimer's disease (AD) pathogenesis. Numerous post-translational modifications enhance or inhibit tau assembly into NFTs. Oxidative stress, which accompanies AD, induces multiple post-translational modifications in proteins, including the formation of dityrosine (DiY) cross-links. Previous studies have revealed that metal-catalysed oxidation (MCO) using Cu2+ and H2O2 leads to the formation of DiY cross-links in two misfolding proteins, Aß and α-synuclein, associated with AD and Parkinson's disease respectively. The effect of MCO on tau remains unknown. Here, we examined the effect of MCO and ultra-violet oxidation to study the influence of DiY cross-linking on the self-assembly of the PHF-core tau fragment. We report that DiY cross-linking facilitates tau assembly into tau oligomers that fail to bind thioflavin S, lack ß-sheet structure and prevents their elongation into filaments. At a higher concentration, Cu2+ (without H2O2) also facilitates the formation of these tau oligomers. The DiY cross-linked tau oligomers do not cause cell death. Our findings suggest that DiY cross-linking of pre-assembled tau promotes the formation of soluble tau oligomers that show no acute impact on cell viability.

Paired Helical Filament-Forming Region of Tau (297-391) Influences Endogenous Tau Protein and Accumulates in Acidic Compartments in Human Neuronal Cells.

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.

Tau (297-391) forms filaments that structurally mimic the core of paired helical filaments in Alzheimer's disease brain.

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.

Cysteine-Independent Inhibition of Alzheimer's Disease-like Paired Helical Filament Assembly by Leuco-Methylthioninium (LMT).

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.

Alpha-Synuclein transgenic mice, h-α-SynL62, display α-Syn aggregation and a dopaminergic phenotype reminiscent of Parkinson's disease.

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.

Alzheimer's Disease-like Paired Helical Filament Assembly from Truncated Tau Protein Is Independent of Disulfide Crosslinking.

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.

A Protein Aggregation Inhibitor, Leuco-Methylthioninium Bis(Hydromethanesulfonate), Decreases α-Synuclein Inclusions in a Transgenic Mouse Model of Synucleinopathy.

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.

Assays for the Screening and Characterization of Tau Aggregation Inhibitors.

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.

Cellular Models of Aggregation-dependent Template-directed Proteolysis to Characterize Tau Aggregation Inhibitors for Treatment of Alzheimer Disease.

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.

Effects of oxidized and reduced forms of methylthioninium in two transgenic mouse tauopathy models.

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.