Mapping the configurational landscape and aggregation phase behavior of the tau protein fragment PHF6.

The PHF6 (Val-Gln-Ile-Val-Tyr-Lys) motif, found in all isoforms of the microtubule-associated protein tau, forms an integral part of ordered cores of amyloid fibrils formed in tauopathies and is thought to play a fundamental role in tau aggregation. Because PHF6 as an isolated hexapeptide assembles into ordered fibrils on its own, it is investigated as a minimal model for insight into the initial stages of aggregation of larger tau fragments. Even for this small peptide, however, the large length and time scales associated with fibrillization pose challenges for simulation studies of its dynamic assembly, equilibrium configurational landscape, and phase behavior. Here, we develop an accurate, bottom-up coarse-grained model of PHF6 for large-scale simulations of its aggregation, which we use to uncover molecular interactions and thermodynamic driving forces governing its assembly. The model, not trained on any explicit information about fibrillar structure, predicts coexistence of formed fibrils with monomers in solution, and we calculate a putative equilibrium phase diagram in concentration-temperature space. We also characterize the configurational and free energetic landscape of PHF6 oligomers. Importantly, we demonstrate with a model of heparin that this widely studied cofactor enhances the aggregation propensity of PHF6 by ordering monomers during nucleation and remaining associated with growing fibrils, consistent with experimentally characterized heparin-tau interactions. Overall, this effort provides detailed molecular insight into PHF6 aggregation thermodynamics and pathways and, furthermore, demonstrates the potential of modern multiscale modeling techniques to produce predictive models of amyloidogenic peptides simultaneously capturing sequence-specific effects and emergent aggregate structures.

SORL1 is a receptor for tau that promotes tau seeding.

Sortilin-related receptor 1 (SORL1) is an intracellular sorting receptor genetically implicated in Alzheimer's disease (AD) that impacts amyloid precursor protein trafficking. The objective of these studies was to test the hypothesis that SORL1 binds tau, modulates its cellular trafficking and impacts the aggregation of cytoplasmic tau induced by pathological forms of tau. Using surface plasmon resonance measurements, we observed high-affinity binding of tau to SORL1 and the vacuolar protein sorting 10 domain of SORL1. Interestingly, unlike LDL receptor-related protein 1, SORL1 binds tau at both pH 7.4 and pH 5.5, revealing its ability to bind tau at endosomal pH. Immunofluorescence studies confirmed that exogenously added tau colocalized with SORL1 in H4 neuroglioma cells, while overexpression of SORL1 in LDL receptor-related protein 1-deficient Chinese hamster ovary (CHO) cells resulted in a marked increase in the internalization of tau, indicating that SORL1 can bind and mediate the internalization of monomeric forms of tau. We further demonstrated that SORL1 mediates tau seeding when tau RD P301S FRET biosensor cells expressing SORL1 were incubated with high molecular weight forms of tau isolated from the brains of patients with AD. Seeding in H4 neuroglioma cells is significantly reduced when SORL1 is knocked down with siRNA. Finally, we demonstrate that the N1358S mutant of SORL1 significantly increases tau seeding when compared to WT SORL1, identifying for the first time a potential mechanism that connects this specific SORL1 mutation to Alzheimer's disease. Together, these studies identify SORL1 as a receptor that contributes to trafficking and seeding of pathogenic tau.

The types and numbers of kinesins and dyneins transporting endocytic cargoes modulate their motility and response to tau.

Organelles and vesicular cargoes are transported by teams of kinesin and dynein motors along microtubules. We isolated endocytic organelles from cells at different stages of maturation and reconstituted their motility along microtubules in vitro. We asked how the sets of motors transporting a cargo determine its motility and response to the microtubule-associated protein tau. Here, we find that phagosomes move in both directions along microtubules, but the directional bias changes during maturation. Early phagosomes exhibit retrograde-biased transport while late phagosomes are directionally unbiased. Correspondingly, early and late phagosomes are bound by different numbers and combinations of kinesins-1, -2, -3, and dynein. Tau stabilizes microtubules and directs transport within neurons. While single-molecule studies show that tau differentially regulates the motility of kinesins and dynein in vitro, less is known about its role in modulating the trafficking of endogenous cargoes transported by their native teams of motors. Previous studies showed that tau preferentially inhibits kinesin motors, which biases late phagosome transport towards the microtubule minus-end. Here, we show that tau strongly inhibits long-range, dynein-mediated motility of early phagosomes. Tau reduces forces generated by teams of dynein motors on early phagosomes and accelerates dynein unbinding under load. Thus, cargoes differentially respond to tau, where dynein complexes on early phagosomes are more sensitive to tau inhibition than those on late phagosomes. Mathematical modeling further explains how small changes in the number of kinesins and dynein on cargoes impact the net directionality but also that cargoes with different sets of motors respond differently to tau.

A selection and optimization strategy for single-domain antibodies targeting the PHF6 linear peptide within the tau intrinsically disordered protein.

The use of variable domain of the heavy-chain of the heavy-chain-only antibodies (VHHs) as disease-modifying biomolecules in neurodegenerative disorders holds promises, including targeting of aggregation-sensitive proteins. Exploitation of their clinical values depends however on the capacity to deliver VHHs with optimal physico-chemical properties for their specific context of use. We described previously a VHH with high therapeutic potential in a family of neurodegenerative diseases called tauopathies. The activity of this promising parent VHH named Z70 relies on its binding within the central region of the tau protein. Accordingly, we carried out random mutagenesis followed by yeast two-hybrid screening to obtain optimized variants. The VHHs selected from this initial screen targeted the same epitope as VHH Z70 as shown using NMR spectroscopy and had indeed improved binding affinities according to dissociation constant values obtained by surface plasmon resonance spectroscopy. The improved affinities can be partially rationalized based on three-dimensional structures and NMR data of three complexes consisting of an optimized VHH and a peptide containing the tau epitope. Interestingly, the ability of the VHH variants to inhibit tau aggregation and seeding could not be predicted from their affinity alone. We indeed showed that the in vitro and in cellulo VHH stabilities are other limiting key factors to their efficacy. Our results demonstrate that only a complete pipeline of experiments, here described, permits a rational selection of optimized VHH variants, resulting in the selection of VHH variants with higher affinities and/or acting against tau seeding in cell models.

ER-mitochondria contacts and cholesterol metabolism are disrupted by disease-associated tau protein.

Abnormal tau protein impairs mitochondrial function, including transport, dynamics, and bioenergetics. Mitochondria interact with the endoplasmic reticulum (ER) via mitochondria-associated ER membranes (MAMs), which coordinate and modulate many cellular functions, including mitochondrial cholesterol metabolism. Here, we show that abnormal tau loosens the association between the ER and mitochondria in vivo and in vitro. Especially, ER-mitochondria interactions via vesicle-associated membrane protein-associated protein (VAPB)-protein tyrosine phosphatase-interacting protein 51 (PTPIP51) are decreased in the presence of abnormal tau. Disruption of MAMs in cells with abnormal tau alters the levels of mitochondrial cholesterol and pregnenolone, indicating that conversion of cholesterol into pregnenolone is impaired. Opposite effects are observed in the absence of tau. Besides, targeted metabolomics reveals overall alterations in cholesterol-related metabolites by tau. The inhibition of GSK3ß decreases abnormal tau hyperphosphorylation and increases VAPB-PTPIP51 interactions, restoring mitochondrial cholesterol and pregnenolone levels. This study is the first to highlight a link between tau-induced impairments in the ER-mitochondria interaction and cholesterol metabolism.

Alzheimer proteopathic tau seeds are biochemically a forme fruste of mature paired helical filaments.

Aggregation prone molecules, such as tau, form both historically well characterized fibrillar deposits (neurofibrillary tangles) and recently identified phosphate-buffered saline (PBS) extract species called proteopathic seeds. Both can cause normal endogenous tau to undergo templated misfolding. The relationship of these seeds to the fibrils that define tau-related diseases is unknown. We characterized the aqueous extractable and sarkosyl insoluble fibrillar tau species derived from human Alzheimer brain using mass spectrometry and in vitro bioassays. Post-translational modifications (PTMs) including phosphorylation, acetylation and ubiquitination are identified in both preparations. PBS extract seed competent tau can be distinguished from sarkosyl insoluble tau by the presence of overlapping, but less abundant, PTMs and an absence of some PTMs unique to the latter. The presence of ubiquitin and other PTMs on the PBS-extracted tau species correlates with the amount of tau in the seed competent size exclusion fractions, with the bioactivity and with the aggressiveness of clinical disease. These results demonstrate that the PTMs present on bioactive, seed competent PBS extract tau species are closely related to, but distinct from, the PTMs of mature paired helical filaments, consistent with the idea that they are a forme fruste of tau species that ultimately form fibrils.

Novel C. elegans models of Lewy body disease reveal pathological protein interactions and widespread miRNA dysregulation.

Lewy body diseases (LBD) comprise a group of complex neurodegenerative conditions originating from accumulation of misfolded alpha-synuclein (α-syn) in the form of Lewy bodies. LBD pathologies are characterized by α-syn deposition in association with other proteins such as Amyloid ß (Aß), Tau, and TAR-DNA-binding protein. To investigate the complex interactions of these proteins, we constructed 2 novel transgenic overexpressing (OE) C. elegans strains (α-synA53T;Taupro-agg (OE) and α-synA53T;Aß1-42;Taupro-agg (OE)) and compared them with previously established Parkinson's, Alzheimer's, and Lewy Body Dementia disease models. The LBD models presented here demonstrate impairments including uncoordinated movement, egg-laying deficits, altered serotonergic and cholinergic signaling, memory and posture deficits, as well as dopaminergic neuron damage and loss. Expression levels of total and prone to aggregation α-syn protein were increased in α-synA53T;Aß1-42 but decreased in α-synA53T;Taupro-agg animals when compared to α-synA53T animals suggesting protein interactions. These alterations were also observed at the mRNA level suggesting a pre-transcriptional mechanism. miRNA-seq revealed that cel-miR-1018 was upregulated in LBD models α-synA53T, α-synA53T;Aß1-42, and α-synA53T;Taupro-agg compared with WT. cel-miR-58c was upregulated in α-synA53T;Taupro-agg but downregulated in α-synA53T and α-synA53T;Aß1-42 compared with WT. cel-miR-41-3p and cel-miR-355-5p were significantly downregulated in 3 LBD models. Our results obtained in a model organism provide evidence of interactions between different pathological proteins and alterations in specific miRNAs that may further exacerbate or ameliorate LBD pathology.

Targeting tau only extracellularly is likely to be less efficacious than targeting it both intra- and extracellularly.

Aggregation of the tau protein is thought to be responsible for the neurodegeneration and subsequent functional impairments in diseases that are collectively named tauopathies. Alzheimer's disease is the most common tauopathy, but the group consists of over 20 different diseases, many of which have tau pathology as their primary feature. The development of tau therapies has mainly focused on preventing the formation of and/or clearing these aggregates. Of these, immunotherapies that aim to either elicit endogenous tau antibodies or deliver exogenous ones are the most common approach in clinical trials. While their mechanism of action can involve several pathways, both extra- and intracellular, pharmaceutical companies have primarily focused on antibody-mediated clearance of extracellular tau. As we have pointed out over the years, this is rather surprising because it is well known that most of pathological tau protein is found intracellularly. It has been repeatedly shown by several groups over the past decades that antibodies can enter neurons and that their cellular uptake can be enhanced by various means, particularly by altering their charge. Here, we will briefly describe the potential extra- and intracellular mechanisms involved in antibody-mediated clearance of tau pathology, discuss these in the context of recent failures of some of the tau antibody trials, and finally provide a brief overview of how the intracellular efficacy of tau antibodies can potentially be further improved by certain modifications that aim to enhance tau clearance via specific intracellular degradation pathways.

Domain-specific modulatory effects of phosphomimetic substitutions on liquid-liquid phase separation of tau protein.

Aggregation of tau is one of the major pathogenic events in Alzheimer's disease and several other neurodegenerative disorders. Recent reports demonstrated that tau can condense into liquid droplets that undergo time-dependent transition to a solid-like state, suggesting that liquid condensates may be on the pathway to pathological aggregation of tau. While hyperphosphorylation is a key feature of tau isolated from brains of patients with Alzheimer's disease and other tauopathies, the mechanistic role of phosphorylation in tau liquid-liquid phase separation (LLPS) remains largely unexplored. In an attempt to bridge this gap, here we performed systematic studies by introducing phosphomimetic substitutions of Ser/Thr residues with negatively charged Asp/Glu residues in different regions of the protein. Our data indicate that the phosphorylation patterns that increase the polarization of charge distribution in full-length tau (tau441) promote protein LLPS, whereas those that decrease charge polarization have an opposite effect. Overall, this study further supports the notion that tau LLPS is driven by attractive intermolecular electrostatic interactions between the oppositely charged domains. We also show that the phosphomimetic tau variants with low intrinsic propensity for LLPS can be efficiently recruited to droplets formed by the variants with high LLPS propensity. Furthermore, the present data demonstrate that phosphomimetic substitutions have a major effect on time-dependent material properties of tau droplets, generally slowing down their aging. The latter effect is most dramatic for the tau variant with substitutions within the repeat domain, which correlates with the decreased fibrillation rate of this variant.

Identification of small molecules and related targets that modulate tau pathology in a seeded primary neuron model.

Alzheimer's disease (AD) is characterized by the presence of tau protein inclusions and amyloid beta (Aß) plaques in the brain, with Aß peptides generated by cleavage of the amyloid precursor protein (APP) by BACE1 and γ-secretase. We previously described a primary rat neuron assay in which tau inclusions form from endogenous rat tau after seeding cells with insoluble tau isolated from the human AD brain. Here, we used this assay to screen an annotated library of ∼8700 biologically active small molecules for their ability to reduce immuno-stained neuronal tau inclusions. Compounds causing ≥30% inhibition of tau aggregates with <25% loss of DAPI-positive cell nuclei underwent further confirmation testing and assessment of neurotoxicity, and non-neurotoxic hits were subsequently analyzed for inhibitory activity in an orthogonal ELISA that quantified multimeric rat tau species. Of the 173 compounds meeting all criteria, a subset of 55 inhibitors underwent concentration-response testing and 46 elicited a concentration-dependent reduction of neuronal tau inclusions that were distinct from measures of toxicity. Among the confirmed inhibitors of tau pathology were BACE1 inhibitors, several of which, along with γ-secretase inhibitors/modulators, caused a concentration-dependent lowering of neuronal tau inclusions and a reduction of insoluble tau by immunoblotting, although they did not decrease soluble phosphorylated tau species. In conclusion, we have identified a diverse set of small molecules and related targets that reduce neuronal tau inclusions. Notably, these include BACE1 and γ-secretase inhibitors, suggesting that a cleavage product from a shared substrate, such as APP, might affect tau pathology.

MAP2 caps tau fibrils and inhibits aggregation.

Fibrils of the microtubule-associated protein tau are intimately linked to the pathology of Alzheimer's disease (AD) and related neurodegenerative disorders. A current paradigm for pathology spreading in the human brain is that short tau fibrils transfer between neurons and then recruit naive tau monomers onto their tips, perpetuating the fibrillar conformation with high fidelity and speed. Although it is known that the propagation could be modulated in a cell-specific manner and thereby contribute to phenotypic diversity, there is still limited understanding of how select molecules are involved in this process. MAP2 is a neuronal protein that shares significant sequence homology with the repeat-bearing amyloid core region of tau. There is discrepancy about MAP2's involvement in pathology and its relationship with tau fibrillization. Here, we employed the entire repeat regions of 3R and 4R MAP2, to investigate their modulatory role in tau fibrillization. We find that both proteins block the spontaneous and seeded aggregation of 4R tau, with 4R MAP2 being slightly more potent. The inhibition of tau seeding is observed in vitro, in HEK293 cells, and in AD brain extracts, underscoring its broader scope. MAP2 monomers specifically bind to the end of tau fibrils, preventing recruitment of further tau and MAP2 monomers onto the fibril tip. The findings uncover a new function for MAP2 as a tau fibril cap that could play a significant role in modulating tau propagation in disease and may hold promise as a potential intrinsic protein inhibitor.

Flanking regions, amyloid cores, and polymorphism: the potential interplay underlying structural diversity.

The ß-sheet-rich amyloid core is the defining feature of protein aggregates associated with neurodegenerative disorders. Recent investigations have revealed that there exist multiple examples of the same protein, with the same sequence, forming a variety of amyloid cores with distinct structural characteristics. These structural variants, termed as polymorphs, are hypothesized to influence the pathological profile and the progression of different neurodegenerative diseases, giving rise to unique phenotypic differences. Thus, identifying the origin and properties of these structural variants remain a focus of studies, as a preliminary step in the development of therapeutic strategies. Here, we review the potential role of the flanking regions of amyloid cores in inducing polymorphism. These regions, adjacent to the amyloid cores, show a preponderance for being structurally disordered, imbuing them with functional promiscuity. The dynamic nature of the flanking regions can then manifest in the form of conformational polymorphism of the aggregates. We take a closer look at the sequences flanking the amyloid cores, followed by a review of the polymorphic aggregates of the well-characterized proteins amyloid-ß, α-synuclein, Tau, and TDP-43. We also consider different factors that can potentially influence aggregate structure and how these regions can be viewed as novel targets for therapeutic strategies by utilizing their unique structural properties.

Microglial Drivers of Alzheimer's Disease Pathology: An Evolution of Diverse Participating States.

Microglia, the resident immune-competent cells of the brain, become dysfunctional in Alzheimer's disease (AD), and their aberrant immune responses contribute to the accumulation of pathological proteins and neuronal injury. Genetic studies implicate microglia in the development of AD, prompting interest in developing immunomodulatory therapies to prevent or ameliorate disease. However, microglia take on diverse functional states in disease, playing both protective and detrimental roles in AD, which largely overlap and may shift over the disease course, complicating the identification of effective therapeutic targets. Extensive evidence gathered using transgenic mouse models supports an active role of microglia in pathology progression, though results vary and can be contradictory between different types of models and the degree of pathology at the time of study. Here, we review microglial immune signaling and responses that contribute to the accumulation and spread of pathological proteins or directly affect neuronal health. We additionally explore the use of induced pluripotent stem cell (iPSC)-derived models to study living human microglia and how they have contributed to our knowledge of AD and may begin to fill in the gaps left by mouse models. Ultimately, mouse and iPSC-derived models have their own limitations, and a comprehensive understanding of microglial dysfunction in AD will only be established by an integrated view across models and an appreciation for their complementary viewpoints and limitations.

Involvement of Fgf2-mediated tau protein phosphorylation in cognitive deficits induced by sevoflurane in aged rats.

OBJECTIVE: Anesthetics have been linked to cognitive alterations, particularly in the elderly. The current research delineates how Fibroblast Growth Factor 2 (Fgf2) modulates tau protein phosphorylation, contributing to cognitive impairments in aged rats upon sevoflurane administration. METHODS: Rats aged 3, 12, and 18 months were subjected to a 2.5% sevoflurane exposure to form a neurotoxicity model. Cognitive performance was gauged, and the GEO database was employed to identify differentially expressed genes (DEGs) in the 18-month-old cohort post sevoflurane exposure. Bioinformatics tools, inclusive of STRING and GeneCards, facilitated detailed analysis. Experimental validations, both in vivo and in vitro, examined Fgf2's effect on tau phosphorylation. RESULTS: Sevoflurane notably altered cognitive behavior in older rats. Out of 128 DEGs discerned, Fgf2 stood out as instrumental in regulating tau protein phosphorylation. Sevoflurane exposure spiked Fgf2 expression in cortical neurons, intensifying tau phosphorylation via the PI3K/AKT/Gsk3b trajectory. Diminishing Fgf2 expression correspondingly curtailed tau phosphorylation, neurofibrillary tangles, and enhanced cognitive capacities in aged rats. CONCLUSION: Sevoflurane elicits a surge in Fgf2 expression in aging rats, directing tau protein phosphorylation through the PI3K/AKT/Gsk3b route, instigating cognitive aberrations.

Changes in lipid metabolism track with the progression of neurofibrillary pathology in tauopathies.

BACKGROUND: Accumulation of tau leads to neuroinflammation and neuronal cell death in tauopathies, including Alzheimer's disease. As the disease progresses, there is a decline in brain energy metabolism. However, the role of tau protein in regulating lipid metabolism remains less characterized and poorly understood. METHODS: We used a transgenic rat model for tauopathy to reveal metabolic alterations induced by neurofibrillary pathology. Transgenic rats express a tau fragment truncated at the N- and C-terminals. For phenotypic profiling, we performed targeted metabolomic and lipidomic analysis of brain tissue, CSF, and plasma, based on the LC-MS platform. To monitor disease progression, we employed samples from transgenic and control rats aged 4, 6, 8, 10, 12, and 14 months. To study neuron-glia interplay in lipidome changes induced by pathological tau we used well well-established multicomponent cell model system. Univariate and multivariate statistical approaches were used for data evaluation. RESULTS: We showed that tau has an important role in the deregulation of lipid metabolism. In the lipidomic study, pathological tau was associated with higher production of lipids participating in protein fibrillization, membrane reorganization, and inflammation. Interestingly, significant changes have been found in the early stages of tauopathy before the formation of high-molecular-weight tau aggregates and neurofibrillary pathology. Increased secretion of pathological tau protein in vivo and in vitro induced upregulated production of phospholipids and sphingolipids and accumulation of lipid droplets in microglia. We also found that this process depended on the amount of extracellular tau. During the later stages of tauopathy, we found a connection between the transition of tau into an insoluble fraction and changes in brain metabolism. CONCLUSION: Our results revealed that lipid metabolism is significantly affected during different stages of tau pathology. Thus, our results demonstrate that the dysregulation of lipid composition by pathological tau disrupts the microenvironment, further contributing to the propagation of pathology.

Self-Assembled BODIPY@Au Core-Shell Structures for Durable Neuroprotective Phototherapy.

BODIPY analogs are promising photosensitizers for molecular phototherapy; however, they exhibit high dark cytotoxicity and limited singlet oxygen generation capacity. In this study, we developed self-assembled core-shell nanophotosensitizers by linking a bipyridine group to BODIPY (Bpy-BODIPY) and promoting J-aggregation on gold nanourchins. This design enhances photostability and reduces the energy gap between the lowest singlet excited state and the lower triplet state, facilitating efficient singlet oxygen production. Notably, Bpy-BODIPY@Au significantly suppresses tau protein aggregation and enhances neuroprotective action, even in the presence of a phosphatase inhibitor. This work broadens the application of BODIPY chemistry to nanoagents for neuroprotective therapy.

A single-domain antibody for the detection of pathological Tau protein in the early stages of oligomerization.

BACKGROUND: Soluble oligomeric forms of Tau protein have emerged as crucial players in the propagation of Tau pathology in Alzheimer's disease (AD). Our objective is to introduce a single-domain antibody (sdAb) named 2C5 as a novel radiotracer for the efficient detection and longitudinal monitoring of oligomeric Tau species in the human brain. METHODS: The development and production of 2C5 involved llama immunization with the largest human Tau isoform oligomers of different maturation states. Subsequently, 2C5 underwent comprehensive in vitro characterization for affinity and specificity via Enzyme-Linked Immunosorbent Assay and immunohistochemistry on human brain slices. Technetium-99m was employed to radiolabel 2C5, followed by its administration to healthy mice for biodistribution analysis. RESULTS: 2C5 exhibited robust binding affinity towards Tau oligomers (Kd = 6.280 nM ± 0.557) and to Tau fibers (Kd = 5.024 nM ± 0.453), with relatively weaker binding observed for native Tau protein (Kd = 1791 nM ± 8.714) and amyloid peptide (Kd > 10,000 nM). Remarkably, this SdAb facilitated immuno-histological labeling of pathological forms of Tau in neurons and neuritic plaques, yielding a high-contrast outcome in AD patients, closely mirroring the performance of reference antibodies AT8 and T22. Furthermore, 2C5 SdAb was successfully radiolabeled with 99mTc, preserving stability for up to 6 h post-radiolabeling (radiochemical purity > 93%). However, following intravenous injection into healthy mice, the predominant uptake occurred in kidneys, amounting to 115.32 ± 3.67, 97.70 ± 43.14 and 168.20 ± 34.52% of injected dose per gram (% ID/g) at 5, 10 and 45 min respectively. Conversely, brain uptake remained minimal at all measured time points, registering at 0.17 ± 0.03, 0.12 ± 0.07 and 0.02 ± 0.01% ID/g at 5, 10 and 45 min post-injection respectively. CONCLUSION: 2C5 demonstrates excellent affinity and specificity for pathological Tau oligomers, particularly in their early stages of oligomerization. However, the current limitation of insufficient blood-brain barrier penetration necessitates further modifications before considering its application in nuclear medicine imaging for humans.

Structural determinants for activation of the Tau kinase CDK5 by the serotonin receptor 5-HT7R.

BACKGROUND: Multiple neurodegenerative diseases are induced by the formation and deposition of protein aggregates. In particular, the microtubule-associated protein Tau leads to the development of so-called tauopathies characterized by the aggregation of hyperphosphorylated Tau within neurons. We recently showed that the constitutive activity of the serotonin receptor 7 (5-HT7R) is required for Tau hyperphosphorylation and aggregation through activation of the cyclin-dependent kinase 5 (CDK5). We also demonstrated physical interaction between 5-HT7R and CDK5 at the plasma membrane suggesting that the 5-HT7R/CDK5 complex is an integral part of the signaling network involved in Tau-mediated pathology. METHODS: Using biochemical, microscopic, molecular biological, computational and AI-based approaches, we investigated structural requirements for the formation of 5-HT7R/CDK5 complex. RESULTS: We demonstrated that 5-HT7R domains responsible for coupling to Gs proteins are not involved in receptor interaction with CDK5. We also created a structural model of the 5-HT7R/CDK5 complex and refined the interaction interface. The model predicted two conserved phenylalanine residues, F278 and F281, within the third intracellular loop of 5-HT7R to be potentially important for complex formation. While site-directed mutagenesis of these residues did not influence Gs protein-mediated receptor signaling, replacement of both phenylalanines by alanine residues significantly reduced 5-HT7R/CDK5 interaction and receptor-mediated CDK5 activation, leading to reduced Tau hyperphosphorylation and aggregation. Molecular dynamics simulations of 5-HT7R/CDK5 complex for wild-type and receptor mutants confirmed binding interface stability of the initial model. CONCLUSIONS: Our results provide a structural basis for the development of novel drugs targeting the 5-HT7R/CDK5 interaction interface for the selective treatment of Tau-related disorders, including frontotemporal dementia and Alzheimer's disease.

Associations of category fluency clustering performance with in vivo brain pathology in autosomal dominant Alzheimer's disease.

OBJECTIVES: Alzheimer's disease (AD) is known to impact semantic access, which is frequently evaluated using the Category Fluency (Animals) test. Recent studies have suggested that in addition to overall category fluency scores (total number of words produced over time), poor clustering could signal AD-related cognitive difficulties. In this study, we examined the association between category fluency clustering performance (i.e., stating words sequentially that are all contained within a subcategory, such as domestic animals) and brain pathology in individuals with autosomal dominant Alzheimer's disease (ADAD). METHODS: A total of 29 non-demented carriers of the Presenilin1 E280A ADAD mutation and 32 noncarrier family members completed the category fluency test (Animals) and the Mini-Mental State Examination (MMSE). The participants also underwent positron emission tomography (PET) scans to evaluate in vivo amyloid-beta in the neocortex and tau in medial temporal lobe regions. Differences between carriers and noncarriers on cognitive tests were assessed with Mann-Whitney tests; associations between cognitive test performance and brain pathology were assessed with Spearman correlations. RESULTS: Animal fluency scores did not differ between carriers and noncarriers. Carriers, however, showed a stronger association between animal fluency clustering and in vivo AD brain pathology (neocortical amyloid and entorhinal tau) relative to noncarriers. CONCLUSION: This study indicates that using category fluency clustering, but not total score, is related to AD pathophysiology in the preclinical and early stages of the disease.

The synergistic effect of nanocurcumin and donepezil on Alzheimer's via PI3K/AKT/GSK-3ß pathway modulating.

Alzheimer's disease (AD) hallmarks include amyloid-ßeta (Aß) and tau proteins aggregates, neurite degeneration, microglial activation with cognitive impairment. Phosphatidylinositol-3-kinase/protein kinase B/Glycogen synthase kinase-3-beta (PI3K/AKT/GSK-3) pathway is essential for neuroprotection, cell survival and proliferation by blocking apoptosis. This study aimed to assess protective role of nanocurcumin (NCMN) as strong antioxidant and anti-inflammatory agent with elucidating its synergistic effects with Donepezil as acetylcholinesterase inhibitor on AD in rats via modulating PI3K/AKT/GSK-3ß pathway. The experiment was performed on 70 male Wistar albino rats divided into seven groups (control, NCMN, Donepezil, AD-model, Donepezil co-treatment, NCMN only co-treatment, and NCMN+Donepezil combined treatment). Behavioral and biochemical investigations as cholinesterase activity, oxidative stress (malondialdehyde, reduced glutathione, nitric oxide, superoxidedismutase, and catalase), tumor necrosis factor-alpha, Tau, ß-site amyloid precursor protein cleaving enzyme-1 (BACE-1), Phosphatase and tensin homolog (Pten), mitogen-activated protein kinase-1 (MAPK-1), Glycogen synthase kinase-3-beta (GSK-3ß) and toll-like receptor-4 were evaluated. Treatment with NCMN improved memory, locomotion, neuronal differentiation by activating PI3K/AKT/GSK-3ß pathway. These results were confirmed by histological studies in hippocampus.