Mitochondrial Effects of Hydromethylthionine, Rivastigmine and Memantine in Tau-Transgenic Mice.

Tau protein aggregations are important contributors to the etiology of Alzheimer's disease (AD). Hydromethylthionine (HMT) is a potent inhibitor of tau aggregation in vitro and in vivo and is being developed as a possible anti-dementia medication. HMT was also shown to affect the cholinergic system and to interact with mitochondria. Here, we used tau-transgenic (L1 and L66) and wild-type NMRI mice that were treated with HMT, rivastigmine and memantine and with combinations thereof, for 2-4 weeks. We measured HMT concentrations in both brain homogenates and isolated mitochondria and concentrations of glucose, lactate and pyruvate in brain by microdialysis. In isolated brain mitochondria, we recorded oxygen consumption of mitochondrial complexes by respirometry. While rivastigmine and memantine lowered mitochondrial respiration, HMT did not affect respiration in wild-type animals and increased respiration in tau-transgenic L1 mice. Glucose and lactate levels were not affected by HMT administration. The presence of HMT in isolated mitochondria was established. In summary, traditional anti-dementia drugs impair mitochondrial function while HMT has no adverse effects on mitochondrial respiration in tau-transgenic mice. These results support the further development of HMT as an anti-dementia drug.

Exploring the Anti-Hypoxaemia Effect of Hydromethylthionine: A Prospective Study of Phase 3 Clinical Trial Participants.

Methylthioninium chloride (MTC) is a standard treatment for methaemoglobinaemia. A preparation of reduced MTC has been reported to increase blood oxygen saturation (SpO2) and lower respiratory rates in patients with severe COVID-19. We have developed a stable form of reduced methylthionine (hydromethylthionine-mesylate, HMTM) having a benign safety profile in two Phase 3 trials in Alzheimer's disease. The aim of this prospective study was to determine the effects of oral HMTM on SpO2 and methaemoglobin (metHb) levels in a cohort of patients with mild hypoxaemia not due to COVID-19. Eighteen participants randomised to a single dose of 4, 75, 100 or 125 mg doses of HMTM had SpO2 levels below 94% at baseline. Patients were routinely monitored by pulse oximetry after 4 h, and after 2 and 6 weeks of twice daily dosing. Significant ~3% increases in SpO2 occurred within 4 h and were sustained over 2 and 6 weeks with no dose differences. There were small dose-dependent increases (0.060-0.162%) in metHb levels over 2 to 6 weeks. Minimum-energy computational chemistry revealed that HMT can bind within 2.10 Å of heme iron by donating a pair of electrons from the central nitrogen of HMT to d orbitals of heme iron, but with lower affinity than oxygen. In conclusion, HMTM can increase SpO2 without reducing metHb by acting as a strong displaceable field ligand for heme iron. We hypothesise that this facilitates a transition from the low oxygen affinity T-state of heme to the higher affinity R-state. HMTM has potential as an adjunctive treatment for hypoxaemia.

Hydromethylthionine enhancement of central cholinergic signalling is blocked by rivastigmine and memantine.

The prevention of tau protein aggregations is a therapeutic goal for the treatment of Alzheimer's disease (AD), and hydromethylthionine (HMT) (also known as leucomethylthioninium-mesylate [LMTM]), is a potent inhibitor of tau aggregation in vitro and in vivo. In two Phase 3 clinical trials in AD, HMT had greater pharmacological activity on clinical endpoints in patients not receiving approved symptomatic treatments for AD (acetylcholinesterase (AChE) inhibitors and/or memantine) despite different mechanisms of action. To investigate this drug interaction in an animal model, we used tau-transgenic L1 and wild-type NMRI mice treated with rivastigmine or memantine prior to adding HMT, and measured changes in hippocampal acetylcholine (ACh) by microdialysis. HMT given alone doubled hippocampal ACh levels in both mouse lines and increased stimulated ACh release induced by exploration of the open field or by infusion of scopolamine. Rivastigmine increased ACh release in both mouse lines, whereas memantine was more active in tau-transgenic L1 mice. Importantly, our study revealed a negative interaction between HMT and symptomatic AD drugs: the HMT effect was completely eliminated in mice that had been pre-treated with either rivastigmine or memantine. Rivastigmine was found to inhibit AChE, whereas HMT and memantine had no effects on AChE or on choline acetyltransferase (ChAT). The interactions observed in this study demonstrate that HMT enhances cholinergic activity in mouse brain by a mechanism of action unrelated to AChE inhibition. Our findings establish that the drug interaction that was first observed clinically has a neuropharmacological basis and is not restricted to animals with tau aggregation pathology. Given the importance of the cholinergic system for memory function, the potential for commonly used AD drugs to interfere with the treatment effects of disease-modifying drugs needs to be taken into account in the design of clinical trials.

Differential compartmental processing and phosphorylation of pathogenic human tau and native mouse tau in the line 66 model of frontotemporal dementia.

Synapse loss is associated with motor and cognitive decline in multiple neurodegenerative disorders, and the cellular redistribution of tau is related to synaptic impairment in tauopathies, such as Alzheimer's disease and frontotemporal dementia. Here, we examined the cellular distribution of tau protein species in human tau overexpressing line 66 mice, a transgenic mouse model akin to genetic variants of frontotemporal dementia. Line 66 mice express intracellular tau aggregates in multiple brain regions and exhibit sensorimotor and motor learning deficiencies. Using a series of anti-tau antibodies, we observed, histologically, that nonphosphorylated transgenic human tau is enriched in synapses, whereas phosphorylated tau accumulates predominantly in cell bodies and axons. Subcellular fractionation confirmed that human tau is highly enriched in insoluble cytosolic and synaptosomal fractions, whereas endogenous mouse tau is virtually absent from synapses. Cytosolic tau was resistant to solubilization with urea and Triton X-100, indicating the formation of larger tau aggregates. By contrast, synaptic tau was partially soluble after Triton X-100 treatment and most likely represents aggregates of smaller size. MS corroborated that synaptosomal tau is nonphosphorylated. Tau enriched in the synapse of line 66 mice, therefore, appears to be in an oligomeric and nonphosphorylated state, and one that could have a direct impact on cognitive function.

Current Progress and Future Directions for Tau-Based Fluid Biomarker Diagnostics in Alzheimer's Disease.

Despite continued efforts, there remain no disease-modifying drugs approved by the United States Food and Drug Administration (FDA) or European Medicines Agency (EMA) to combat the global epidemic of Alzheimer's disease. Currently approved medicines are unable to delay disease progression and are limited to symptomatic treatment. It is well established that the pathophysiology of this disease remains clinically silent for decades prior to symptomatic clinical decline. Identifying those at risk of disease progression could allow for effective treatment whilst the therapeutic window remains open for preservation of quality of life. This review aims to evaluate critically the current advances in the interpretation of tau-based biomarkers and their use to provide insights into the onset and progression of Alzheimer's disease, whilst highlighting important future directions for the field. This review emphasises the need for a more comprehensive analysis and interrogation of tau within biological fluids, to aid in obtaining a disease specific molecular signature for each stage of Alzheimer's disease. Success in achieving this could provide essential utility for presymptomatic patient selection for clinical trials, monitoring disease progression, and evaluating disease modifying therapies.

Efficacy and safety of tau-aggregation inhibitor therapy in patients with mild or moderate Alzheimer's disease: a randomised, controlled, double-blind, parallel-arm, phase 3 trial.

BACKGROUND: Leuco-methylthioninium bis(hydromethanesulfonate; LMTM), a stable reduced form of the methylthioninium moiety, acts as a selective inhibitor of tau protein aggregation both in vitro and in transgenic mouse models. Methylthioninium chloride has previously shown potential efficacy as monotherapy in patients with Alzheimer's disease. We aimed to determine whether LMTM was safe and effective in modifying disease progression in patients with mild to moderate Alzheimer's disease. METHODS: We did a 15-month, randomised, controlled double-blind, parallel-group trial at 115 academic centres and private research clinics in 16 countries in Europe, North America, Asia, and Russia with patients younger than 90 years with mild to moderate Alzheimer's disease. Patients concomitantly using other medicines for Alzheimer's disease were permitted to be included because we considered it infeasible not to allow their inclusion; however, patients using medicines carrying warnings of methaemoglobinaemia were excluded because the oxidised form of methylthioninium in high doses has been shown to induce this condition. We randomly assigned participants (3:3:4) to 75 mg LMTM twice a day, 125 mg LMTM twice a day, or control (4 mg LMTM twice a day to maintain blinding with respect to urine or faecal discolouration) administered as oral tablets. We did the randomisation with an interactive web response system using 600 blocks of length ten, and stratified patients by severity of disease, global region, whether they were concomitantly using Alzheimer's disease-labelled medications, and site PET capability. Participants, their study partners (generally carers), and all assessors were masked to treatment assignment throughout the study. The coprimary outcomes were progression on the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog) and the Alzheimer's Disease Co-operative Study-Activities of Daily Living Inventory (ADCS-ADL) scales from baseline assessed at week 65 in the modified intention-to-treat population. This trial is registered with Clinicaltrials.gov (NCT01689246) and the European Union Clinical Trials Registry (2012-002866-11). FINDINGS: Between Jan 29, 2013, and June 26, 2014, we recruited and randomly assigned 891 participants to treatment (357 to control, 268 to 75 mg LMTM twice a day, and 266 to 125 mg LMTM twice a day). The prespecified primary analyses did not show any treatment benefit at either of the doses tested for the coprimary outcomes (change in ADAS-Cog score compared with control [n=354, 6·32, 95% CI 5·31-7·34]: 75 mg LMTM twice a day [n=257] -0·02, -1·60 to 1·56, p=0·9834, 125 mg LMTM twice a day [n=250] -0·43, -2·06 to 1·20, p=0·9323; change in ADCS-ADL score compared with control [-8·22, 95% CI -9·63 to -6·82]: 75 mg LMTM twice a day -0·93, -3·12 to 1·26, p=0·8659; 125 mg LMTM twice a day -0·34, -2·61 to 1·93, p=0·9479). Gastrointestinal and urinary effects were the most common adverse events with both high doses of LMTM, and the most common causes for discontinuation. Non-clinically significant dose-dependent reductions in haemoglobin concentrations were the most common laboratory abnormality. Amyloid-related imaging abnormalities were noted in less than 1% (8/885) of participants. INTERPRETATION: The primary analysis for this study was negative, and the results do not suggest benefit of LMTM as an add-on treatment for patients with mild to moderate Alzheimer's disease. Findings from a recently completed 18-month trial of patients with mild Alzheimer's disease will be reported soon. FUNDING: TauRx Therapeutics.

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.

Complex disposition of methylthioninium redox forms determines efficacy in tau aggregation inhibitor therapy for Alzheimer's disease.

Methylthioninium (MT) is a tau aggregation inhibitor with therapeutic potential in Alzheimer's disease (AD). MT exists in equilibrium between reduced [leucomethylthioninium (LMT)] and oxidized (MT(+)) forms; as a chloride salt [methylthioninium chloride (MTC), "methylene blue"], it is stabilized in its MT(+) form. Although the results of a phase 2 study of MTC in 321 mild/moderate AD subjects identified a 138-mg MT/day dose as the minimum effective dose on cognitive and imaging end points, further clinical development of MT was delayed pending resolution of the unexpected lack of efficacy of the 228-mg MT/day dose. We hypothesized that the failure of dose response may depend on differences known at the time in dissolution in simulated gastric and intestinal fluids of the 100-mg MTC capsules used to deliver the 228-mg dose and reflect previously unsuspected differences in redox processing of MT at different levels in the gut. The synthesis of a novel chemical entity, LMTX (providing LMT in a stable anhydrous crystalline form), has enabled a systematic comparison of the pharmacokinetic properties of MTC and LMTX in preclinical and clinical studies. The quantity of MT released in water or gastric fluid within 60 minutes proved in retrospect to be an important determinant of clinical efficacy. A further factor was a dose-dependent limitation in the ability to absorb MT in the presence of food when delivered in the MT(+) form as MTC. A model is presented to account for the complexity of MT absorption, which may have relevance for other similar redox molecules.

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.

A Meta-Analysis on Presynaptic Changes in Alzheimer's Disease.

BACKGROUND: A key aspect of synaptic dysfunction in Alzheimer's disease (AD) is loss of synaptic proteins. Previous publications showed that the presynaptic machinery is more strongly affected than postsynaptic proteins. However, it has also been reported that presynaptic protein loss is highly variable and shows region- and protein-specificity. OBJECTIVE: The objective of this meta-analysis was to provide an update on the available literature and to further characterize patterns of presynaptic protein loss in AD. METHODS: Systematic literature search was conducted for studies published between 2015-2022 which quantified presynaptic proteins in postmortem tissue from AD patients and healthy controls. Three-level random effects meta-analyses of twenty-two identified studies was performed to characterize overall presynaptic protein loss and changes in specific regions, proteins, protein families, and functional categories. RESULTS: Meta-analysis confirmed overall loss of presynaptic proteins in AD patients. Subgroup analysis revealed region specificity of protein loss, with largest effects in temporal and frontal cortex. Results concerning different groups of proteins were also highly variable. Strongest and most consistently affected was the family of synaptosome associated proteins, especially SNAP25. Among the most severely affected were proteins regulating dense core vesicle exocytosis and the synaptic vesicle cycle. CONCLUSIONS: Results confirm previous literature related to presynaptic protein loss in AD patients and provide further in-depth characterization of most affected proteins and presynaptic functions.

Considerations for biomarker strategies in clinical trials investigating tau-targeting therapeutics for Alzheimer's disease.

The use of biomarker-led clinical trial designs has been transformative for investigating amyloid-targeting therapies for Alzheimer's disease (AD). The designs have ensured the correct selection of patients on these trials, supported target engagement and have been used to support claims of disease modification and clinical efficacy. Ultimately, this has recently led to approval of disease-modifying, amyloid-targeting therapies for AD; something that should be noted for clinical trials investigating tau-targeting therapies for AD. There is a clear overlap of the purpose of biomarker use at each stage of clinical development between amyloid-targeting and tau-targeting clinical trials. However, there are differences within the potential context of use and interpretation for some biomarkers in particular measurements of amyloid and utility of soluble, phosphorylated tau biomarkers. Given the complexities of tau in health and disease, it is paramount that therapies target disease-relevant tau and, in parallel, appropriate assays of target engagement are developed. Tau positron emission tomography, fluid biomarkers reflecting tau pathology and downstream measures of neurodegeneration will be important both for participant recruitment and for monitoring disease-modification in tau-targeting clinical trials. Bespoke design of biomarker strategies and interpretations for different modalities and tau-based targets should also be considered.

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.

Solubility of α-synuclein species in the L62 mouse model of synucleinopathy.

The accumulation of α-synuclein (α-Syn) into Lewy bodies is a hallmark of synucleinopathies, a group of neurological disorders that include Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Small oligomers as well as larger fibrils of α-Syn have been suggested to induce cell toxicity leading to a degenerative loss of neurones. A richer understanding of α-Syn aggregation in disease, however, requires the identification of the different α-Syn species and the characterisation of their biochemical properties. We here aimed at a more in-depth characterisation of the α-Syn transgenic mice, Line 62 (L62), and examined the deposition pattern and solubility of human and murine α-Syn in these mice using immunohistochemical and biochemical methods. Application of multiple antibodies confirmed mAb syn204 as the most discriminatory antibody for human α-Syn in L62. Syn204 revealed an intense and widespread immunohistochemical α-Syn labelling in parietal cortex and hippocampus, and to a lower level in basal forebrain and hindbrain regions. The labelled α-Syn represented somatic inclusions as well as processes and synaptic endings. Biochemical analysis revealed a Triton-resistant human α-Syn pool of large oligomers, a second pool of small oligomers that was not resistant to solubilization with urea/Triton. A third SDS-soluble pool of intermediate sized aggregates containing a mixture of both, human and mouse α-Syn was also present. These data suggest that several pools of α-Syn can exist in neurones, most likely in different cellular compartments. Information about these different pools is important for the development of novel disease modifying therapies aimed at α-Syn.

Hydromethylthionine rescues synaptic SNARE proteins in a mouse model of tauopathies: Interference by cholinesterase inhibitors.

In clinical trials for Alzheimer's disease (AD), hydromethylthionine mesylate (HMTM) showed reduced efficacy when administered as an add-on to symptomatic treatments, while it produced a significant improvement of cognitive function when taken as monotherapy. Interference of cholinesterase inhibition with HMTM was observed also in a tau transgenic mouse model, where rivastigmine reduced the pharmacological activity of HMTM at multiple brain levels including hippocampal acetylcholine release, synaptosomal glutamate release and mitochondrial activity. Here, we examined the effect of HMTM, given alone or in combination with the acetylcholinesterase inhibitor, rivastigmine, at the level of expression of selected pre-synaptic proteins (syntaxin-1; SNAP-25, VAMP-2, synaptophysin-1, synapsin-1, α-synuclein) in brain tissue harvested from tau-transgenic Line 1 (L1) and wild-type mice using immunohistochemistry. L1 mice overexpress the tau-core unit that induces tau aggregation and results in an AD-like phenotype. Synaptic proteins were lower in hippocampus and cortex but greater in basal forebrain regions in L1 compared to wild-type mice. HMTM partially normalised the expression pattern of several of these proteins in basal forebrain. This effect was diminished when HMTM was administered in combination with rivastigmine, where mean protein expression seemed supressed. This was further confirmed by group-based correlation network analyses where important levels of co-expression correlations in basal forebrain regions were lost in L1 mice and partially re-established when HMTM was given alone but not in combination with rivastigmine. These data indicate a reduction in pharmacological activity of HMTM when given as an add-on therapy, a result that is consistent with the responses observed in the clinic. Attenuation of the therapeutic effects of HMTM by cholinergic treatments may have important implications for other potential AD therapies.

Neuroprotection of Cholinergic Neurons with a Tau Aggregation Inhibitor and Rivastigmine in an Alzheimer's-like Tauopathy Mouse Model.

Basal forebrain cholinergic dysfunction, most likely linked with tau protein aggregation, is a characteristic feature of Alzheimer's disease (AD). Recent evidence suggests that tau protein is a putative target for the treatment of dementia, and the tau aggregation inhibitor, hydromethylthionine mesylate (HMTM), has emerged as a potential disease-modifying treatment. However, its efficacy was diminished in patients already receiving approved acetylcholinesterase inhibitors. In this study, we ask whether this negative interaction can also be mimicked in experimental tau models of AD and whether the underlying mechanism can be understood. From a previous age profiling study, 6-month-old line 1 (L1) tau transgenic mice were characterized by a severe reduction in several cholinergic markers. We therefore assessed whether long-term pre-exposure with the acetylcholinesterase inhibitor rivastigmine alone and in conjunction with the tau aggregation inhibitor HMTM can reverse cholinergic deficits in L1. Rivastigmine and HMTM, and combinations of the two compounds were administered orally for 11 weeks to both L1 and wild-type mice. The brains were sectioned with a focus on the basal forebrain, motor cortex and hippocampus. Immunohistochemical staining and quantification of choline acetyltransferase (ChAT), tyrosine kinase A (TrkA)-positive neurons and relative optical intensity (ROI) for vesicular acetylcholine transporter (VAChT), and acetylcholinesterase (AChE) reactivity confirmed reversal of the diminished cholinergic phenotype of interneurons (nucleus accumbens, striatum) and projection neurons (medial septum, nucleus basalis magnocellularis) by HMTM, to a greater extent than by rivastigmine alone in L1 mice. Combined administration did not yield additivity but, in most proxies, led to antagonistic effects in which rivastigmine decreased the benefits shown with HMTM alone. Local markers (VAChT and AChE) in target structures of the basal forebrain, motor cortex and hippocampal CA3 seemed to be normalized by HMTM, but not by rivastigmine or the combination of both drugs. HMTM, which was developed as a tau aggregation inhibitor, strongly decreased the tau load in L1 mice, however, not in combination with rivastigmine. Taken together, these data confirm a cholinergic phenotype in L1 tau transgenic mice that resembles the deficits observed in AD patients. This phenotype is reversible by HMTM, but at the same time appears to be subject to a homeostatic regulation induced by chronic pre-treatment with an acetylcholinesterase inhibitor, which interferes with the efficacy of HMTM. The strongest phenotypic reversal coincided with a normalization of the tau load in the cortex and hippocampus of L1, suggesting that tau accumulation underpins the loss of cholinergic markers in the basal forebrain and its projection targets.

Rescue of synaptosomal glutamate release defects in tau transgenic mice by the tau aggregation inhibitor hydromethylthionine.

Glutamatergic neurotransmission, important for learning and memory, is disrupted in different ways in patients with Alzheimer's disease (AD) and frontotemporal dementia (FTD) tauopathies. We have previously reported that two tau transgenic mouse models, L1 and L66, produce different phenotypes resembling AD and FTD, respectively. The AD-like L1 model expresses the truncated core aggregation domain of the AD paired helical filament (PHF) form of tau (tau296-390) whereas the FTD-like L66 model expresses full-length tau carrying two mutations at P301S/G335D. We have used synaptosomes isolated from these mice to investigate K+-evoked glutamate release and, if abnormal, to determine responsiveness to hydromethylthionine, a tau aggregation inhibitor previously shown to reduce tau pathology in these models. We report that the transgenes in these two mouse lines cause opposite abnormalities in glutamate release. Over-expression of the core tau unit in L1 produces a significant reduction in glutamate release and a loss of Ca2+-dependency compared with wild-type control mice. Full-length mutant tau produces an increase in glutamate release that retains normal Ca2+-dependency. Chronic pre-treatment with hydromethylthionine normalises both reduced (L1) and excessive glutamate (L66) and restores normal Ca2+-dependency in L1 mice. This implies that both patterns of impairment are the result of tau aggregation, but that the direction and Ca2+-dependency of the abnormality is determined by expression of the disease-specific transgene. Our results lead to the conclusion that the tauopathies need not be considered a single entity in terms of the downstream effects of pathological aggregation of tau protein. In this case, directionally opposite abnormalities in glutamate release resulting from different types of tau aggregation in the two mouse models can be corrected by hydromethylthionine. This may help to explain the activity of hydromethylthionine on cognitive decline and brain atrophy in both AD and behavioural-variant FTD.

LETC inhibits α-Syn aggregation and ameliorates motor deficiencies in the L62 mouse model of synucleinopathy.

Alpha-Synuclein (α-Syn) aggregation is a pathological feature of synucleinopathies, neurodegenerative disorders that include Parkinson's disease (PD). Here, we explored the efficacy of N,N,N',N'-tetraethyl-10H-phenothiazine-3,7-diamine dihydrochloride (LETC), a protein aggregation inhibitor, on α-Syn aggregation. In both cellular models and transgenic mice, α-Syn aggregation was achieved by the overexpression of full-length human α-Syn fused with a signal sequence peptide. α-Syn accumulated in transfected DH60.21 neuroblastoma cells and α-Syn aggregation was inhibited by LETC with an EC50 of 0.066 ± 0.047 µM. Full-length human α-Syn overexpressing Line 62 (L62) mice accumulated neuronal α-Syn that was associated with a decreased motor performance in the open field and automated home cage. LETC, administered orally for 6 weeks at 10 mg/kg significantly decreased α-Syn-positive neurons in multiple brain regions and this resulted in a rescue of movement deficits in the open field in these mice. LETC however, did not improve activity deficits of L62 mice in the home cage environment. The results suggest that LETC may provide a potential disease modification therapy in synucleinopathies through the inhibition of α-Syn aggregation.

Utility of Blood-Based Tau Biomarkers for Mild Cognitive Impairment and Alzheimer's Disease: Systematic Review and Meta-Analysis.

OBJECTIVES: With the development of new technologies capable of detecting low concentrations of Alzheimer's disease (AD) relevant biomarkers, the idea of a blood-based diagnosis of AD is nearing reality. This study aims to consider the evidence of total and phosphorylated tau as blood-based biomarkers for mild cognitive impairment (MCI) and AD when compared to healthy controls. METHODS: Studies published between 1 January 2012 and 1 May 2021 (Embase and MEDLINE databases) measuring plasma/serum levels of tau in AD, MCI, and control cohorts were screened for eligibility, including quality and bias assessment via a modified QUADAS. The meta-analyses comprised 48 studies assessing total tau (t-tau), tau phosphorylated at threonine 181 (p-tau181), and tau phosphorylated at threonine 217 (p-tau217), comparing the ratio of biomarker concentrations in MCI, AD, and cognitively unimpaired (CU) controls. RESULTS: Plasma/serum p-tau181 (mean effect size, 95% CI, 2.02 (1.76-2.27)) and t-tau (mean effect size, 95% CI, 1.77 (1.49-2.04)) were elevated in AD study participants compared to controls. Plasma/serum p-tau181 (mean effect size, 95% CI, 1.34 (1.20-1.49)) and t-tau (mean effect size, 95% CI, 1.47 (1.26-1.67)) were also elevated with moderate effect size in MCI study participants compared to controls. p-tau217 was also assessed, albeit in a small number of eligible studies, for AD vs. CU (mean effect size, 95% CI, 1.89 (1.86-1.92)) and for MCI vs. CU groups (mean effect size, 95% CI, 4.16 (3.61-4.71)). CONCLUSIONS: This paper highlights the growing evidence that blood-based tau biomarkers have early diagnostic utility for Alzheimer's disease. REGISTRATION: PROSPERO No. CRD42020209482.

An Additive-Free Model for Tau Self-Assembly.

Tau is a natively unfolded protein that contributes to the stability of microtubules. Under pathological conditions such as Alzheimer's disease (AD), tau protein misfolds and self-assembles to form paired helical filaments (PHFs) and straight filaments (SFs). Full-length tau protein assembles poorly and its self-assembly is enhanced with polyanions such as heparin and RNA in vitro, but a role for heparin or other polyanions in vivo remains unclear. Recently, a truncated form of tau (297-391) has been shown to self-assemble in the absence of additives which provides an alternative in vitro PHF model system. Here we describe methods to prepare in vitro PHFs and SFs from tau (297-391) named dGAE. We also discuss the range of biophysical/biochemical techniques used to monitor tau filament assembly and structure.

Crossed cerebellar diaschisis in Alzheimer's disease.

BACKGROUND: Crossed cerebellar diaschisis (CCD) is characterized by hypometabolism and hypoperfusion on molecular imaging in the cerebellum due to a supratentorial lesion on the contralateral side. CCD is a well-established phenomenon in acute or subacute conditions such as infarction but it has been less well described in chronic conditions such as neurodegenerative dementias. Here, we investigate CCD in a large sample of 830 people meeting research criteria for Alzheimer's disease (AD) using [18F]fluorodeoxyglucose-positron emission tomography (FDG-PET). MATERIALS AND METHODS: This study is based on FDG-PET data collected at baseline as part of two large-scale Phase III clinical trials of a novel tau aggregation inhibitor medication, methylthioninium in mild to moderate AD participants. Quantification of FDG-PET hypometabolism was carried out using standardized uptake value ratio (SUVR), with the pons as the comparison region. SUVR was compared in different regions of interest between the right and left hemispheres of the brain and cerebellum in people with mild AD (Mini-Mental State Examination score ≥ 20). RESULTS: Comparison of SUVR in different brain regions demonstrated significant differences in the temporal, occipital and cerebellar cortices. Right and left asymmetry was noted with lower SUVR in the left temporal and occipital regions, whereas SUVR was lower in the right side of the cerebellum. CONCLUSION: Here, we found robust evidence of CCD in a large sample of people with AD, a chronic neurodegenerative condition. The presence of this phenomenon in AD opens up a new avenue of research in AD pathogenesis and has the potential to change future diagnostic and therapeutic strategies.