Intracellular Tau Aggregates Research Articles

Structural Insights into the Role of the Proline Rich Region in Tau Function.

Tau is a microtubule–associated protein that plays an important role in modulating axonal microtubules in neurons. Intracellular tau aggregates are found in a broad class of disorders, including Alzheimer&#8242s disease, termed tauopathies. Tau is an intrinsically disordered protein, and its structural disorder appears to be critical to its microtubule-related functions. Tubulin binding sites are found in tau&#8242s proline-rich region (PRR), microtubule binding repeats (MTBR: R1–R4), and pseudo–repeat, R′. While many post-translational modifications have been identified on tau, phosphorylation sites, which both regulate tubulin dimer and microtubule interactions and are correlated with disease, cluster with high frequency within the PRR. Here, we use fluorescence correlation spectroscopy and structural mass spectrometry techniques to characterize the impact of phosphomimic mutations in the PRR on tubulin dimer binding and probe the structure of the PRR–tubulin dimer complex. We find that phosphomimics cumulatively diminish tubulin dimer binding and slow microtubule polymerization. Additionally, we map two ~15 residue regions of the PRR as primary tubulin dimer binding sites and propose a model in which PRR enhances lateral interactions between tubulin dimers, complementing the longitudinal interactions observed for MTBR. Together these measurements provide insight into the previously overlooked relevance of tau&#8242s PRR in functional interactions with tubulin.

Alzheimer's Disease Neuropathological Change in Aged Non-Primate Mammals.

Human brain aging is characterized by the production and deposition of β-amyloid (Aβ) in the form of senile plaques and cerebral amyloid angiopathy and the intracellular accumulation of hyper-phosphorylated tau (Hp-tau) to form neurofibrillary tangles (NFTs) and dystrophic neurites of senile plaques. The process progresses for years and eventually manifests as cognitive impairment and dementia in a subgroup of aged individuals. Aβ is produced and deposited first in the neocortex in most aged mammals, including humans; it is usually not accompanied by altered behavior and cognitive impairment. Hp-tau is less frequent than Aβ pathology, and NFTs are rare in most mammals. In contrast, NFTs are familiar from middle age onward in humans; NFTs first appear in the paleocortex and selected brain stem nuclei. NFTs precede for decades or years Aβ deposition and correlate with dementia in about 5% of individuals at the age of 65 and 25% at the age of 85. Based on these comparative data, (a) Aβ deposition is the most common Alzheimer's disease neuropathological change (ADNC) in the brain of aged mammals; (b) Hp-tau is less common, and NFTs are rare in most aged mammals; however, NFTs are the principal cytoskeletal pathology in aged humans; (c) NFT in aged humans starts in selected nuclei of the brain stem and paleocortical brain regions progressing to the most parts of the neocortex and other regions of the telencephalon; (d) human brain aging is unique among mammalian species due to the early appearance and dramatic progression of NFTs from middle age onward, matching with cognitive impairment and dementia in advanced cases; (e) neither mammalian nor human brain aging supports the concept of the amyloid cascade hypothesis.

Nasal tau immunotherapy clears intracellular tau pathology and improves cognitive functions in aged tauopathy mice.

Pathological tau aggregates cause cognitive decline in neurodegenerative tauopathies, including Alzheimer's disease (AD). These aggregates are prevalent within intracellular compartments. Current tau immunotherapies have shown limited efficacy in clearing intracellular tau aggregates and improving cognition in clinical trials. In this study, we developed toxic tau conformation-specific monoclonal antibody-2 (TTCM2), which selectively recognized pathological tau aggregates in brain tissues from patients with AD, dementia with Lewy bodies (DLB), and progressive supranuclear palsy (PSP). TTCM2 potently inhibited tau-seeding activity, an essential mechanism underlying tauopathy progression. To effectively target intracellular tau aggregates and ensure rapid delivery to the brain, TTCM2 was loaded in micelles (TTCM2-ms) and administered through the intranasal route. We found that intranasally administered TTCM2-ms efficiently entered the brain in hTau-tauopathy mice, targeting pathological tau in intracellular compartments. Moreover, a single intranasal dose of TTCM2-ms effectively cleared pathological tau, elevated synaptic proteins, and improved cognitive functions in aged tauopathy mice. Mechanistic studies revealed that TTCM2-ms cleared intracellular, synaptic, and seed-competent tau aggregates through tripartite motif-containing 21 (TRIM21), an intracellular antibody receptor and E3 ubiquitin ligase known to facilitate proteasomal degradation of cytosolic antibody-bound proteins. TRIM21 was found to be essential for TTCM2-ms-mediated clearance of tau pathology. Our study collectively provides evidence of the effectiveness of nasal tau immunotherapy in targeting and clearing intracellular tau pathology through TRIM21 and enhancing cognition in aged tauopathy mice. This study could be valuable in designing effective tau immunotherapies for AD and other tauopathies.

Intracellular tau fragment droplets serve as seeds for tau fibrils

Intracellular tau aggregation requires a local protein concentration increase, referred to as "droplets". However, the cellular mechanism for droplet formation is poorly understood. Here, we expressed OptoTau, a P301L mutant tau fused with CRY2olig, a light-sensitive protein that can form homo-oligomers. Under blue light exposure, OptoTau increased tau phosphorylation and was sequestered in aggresomes. Suppressing aggresome formation by nocodazole formed tau granular clusters in the cytoplasm. The granular clusters disappeared by discontinuing blue light exposure or 1,6-hexanediol treatment suggesting that intracellular tau droplet formation requires microtubule collapse. Expressing OptoTau-ΔN, a species of N-terminal cleaved tau observed in the Alzheimer's disease brain, formed 1,6-hexanediol and detergent-resistant tau clusters in the cytoplasm with blue light stimulation. These intracellular stable tau clusters acted as a seed for tau fibrils invitro. These results suggest that tau droplet formation and N-terminal cleavage are necessary for neurofibrillary tangles formation in neurodegenerative diseases.

Plasminogen degrades α-synuclein, Tau and TDP-43 and decreases dopaminergic neurodegeneration in mouse models of Parkinson’s disease

Parkinson's disease (PD) is the second most frequently diagnosed neurodegenerative disease, and it is characterized by the intracellular and extracellular accumulation of α-synuclein (α-syn) and Tau, which are major components of cytosolic protein inclusions called Lewy bodies, in the brain. Currently, there is a lack of effective methods that preventing PD progression. It has been suggested that the plasminogen activation system, which is a major extracellular proteolysis system, is involved in PD pathogenesis. We investigated the functional roles of plasminogen in vitro in an okadaic acid-induced Tau hyperphosphorylation NSC34 cell model, ex vivo using brains from normal controls and methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, and in vivo in a widely used MPTP-induced PD mouse model and an α-syn overexpression mouse model. The in vitro, ex vivo and in vivo results showed that the administered plasminogen crossed the blood‒brain barrier (BBB), entered cells, and migrated to the nucleus, increased plasmin activity intracellularly, bound to α-syn through lysine binding sites, significantly promoted α-syn, Tau and TDP-43 clearance intracellularly and even intranuclearly in the brain, decreased dopaminergic neurodegeneration and increased the tyrosine hydroxylase levels in the substantia nigra and striatum, and improved motor function in PD mouse models. These findings indicate that plasminogen plays a wide range of pivotal protective roles in PD and therefore may be a promising drug candidate for PD treatment.

Perineuronal net deglycosylation associates with tauopathy-induced gliosis and neurodegeneration.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by clinical symptoms of memory and cognitive deficiencies. Postmortem evaluation of AD brain tissue shows proteinopathy that closely associate with the progression of this dementing disorder, including the accumulation of extracellular beta amyloid (Aβ) and intracellular hyperphosphorylated tau (pTau) with neurofibrillary tangles (NFTs). Current therapies targeting Aβ have limited clinical efficacy and life-threatening side effects and highlight the need for alternative treatments targeting pTau and other pathophysiologic mechanisms driving AD pathogenesis. The brain's extracellular matrices (ECM), particularly perineuronal nets (PNNs), play a crucial role in brain functioning and neurocircuit stability, and reorganization of these unique PNN matrices has been associated with the progression of AD and accumulation of pTau in humans. We hypothesize that AD-associated changes in PNNs may in part be driven by the accumulation of pTau within the brain. In this work, we investigated whether the presence of pTau influenced PNN structural integrity and PNN chondroitin sulfate-glycosaminoglycan (CS-GAG) compositional changes in two transgenic mouse models expressing tauopathy-related AD pathology, PS19 (P301S) and Tau4RTg2652 mice. We show that PS19 mice exhibit an age-dependent loss of hippocampal PNN CS-GAGs, but not the underlying aggrecan core protein structures, in association with pTau accumulation, gliosis, and neurodegeneration. The loss of PNN CS-GAGs were linked to shifts in CS-GAG sulfation patterns to favor the neuroregenerative isomer, 2S6S-CS. Conversely, Tau4RTg2652 mice exhibit stable PNN structures and normal CS-GAG isomer composition despite robust pTau accumulation, suggesting a critical interaction between neuronal PNN glycan integrity and neighboring glial cell activation. Overall, our findings provide insights into the complex relationship between PNN CS-GAGs, pTau pathology, gliosis, and neurodegeneration in mouse models of tauopathy, and offer new therapeutic insights and targets for AD treatment.

Changes in the locus coeruleus during the course of Alzheimer's disease and their relationship to cortical pathology.

In Alzheimer's disease (AD), the locus coeruleus (LC) undergoes early and extensive neuronal loss, preceded by abnormal intracellular tau aggregation, decades before the onset of clinical disease. Neuromelanin-sensitive MRI has been proposed as a method to image these changes during life. Surprisingly, human post-mortem studies have not examined how changes in LC during the course of the disease relate to cerebral pathology following the loss of the LC projection to the cortex. Immunohistochemistry was used to examine markers for 4G8 (pan-Aβ) and AT8 (ptau), LC integrity (neuromelanin, dopamine β-hydroxylase [DβH], tyrosine hydroxylase [TH]) and microglia (Iba1, CD68, HLA-DR) in the LC and related temporal lobe pathology of 59 post-mortem brains grouped by disease severity determined by Braak stage (0-II, III-IV and V-VI). The inflammatory environment was assessed using multiplex assays. Changes in the LC with increasing Braak stage included increased neuronal loss (p< 0.001) and microglial Iba1 (p= 0.005) together with a reduction in neuromelanin (p< 0.001), DβH (p= 0.002) and TH (p= 0.041). Interestingly in LC, increased ptau and loss of neuromelanin were detected from Braak stage III-IV (p= 0.001). At Braak stage V/VI, the inflammatory environment was different in the LC vs TL, highlighting the anatomical heterogeneity of the inflammatory response. Here, we report the first quantification of neuromelanin during the course of AD and its relationship to AD pathology and neuroinflammation in the TL. Our findings of neuromelanin loss early in AD and before the neuroinflammatory reaction support the use of neuromelanin-MRI as a sensitive technique to identify early changes in AD.

Human Herpes Simplex Virus‐1 Encoded ICP27 Promotes the Alternative Splicing of MAPT Resulting in increased 4R Ratios within Cells of Neuronal Origin

AbstractBackgroundFamilial linked Alzheimer’s Disease (AD) accounts for a small percentage of current cases, with the remaining caused by unknown etiologies. Independent of causation, AD is defined by three common characteristics: inflammation, extracellular b‐amyloid aggregation and intracellular Tau accumulation. Human herpes simplex virus 1 (HSV‐1), a neurotropic virus found in ∼50% of the population, was recently linked to AD progression in clinical samples and animal models. HSV‐1 infections induce inflammation and APP cleavage producing Ab42, but a role for viral‐mediated Tau accumulation has not been defined. In healthy brains, alternative splicing of MAPT, the gene encoding Tau, results in equimolar ratios of 3R and 4R isoforms of Tau protein. However, in AD tissues, unequal ratios of either 3R or 4R results in Tau microtubule disassociation, hyperphosphorylation and aggregation. Host factors that regulate MAPT alternative splicing include SRPK1. Interestingly, HSV‐1 encodes a protein, ICP27, that impedes the SRPK1 functions. Thus, we hypothesized that HSV‐1 infections alter MAPT neuronal splicing resulting in altered 3R/4R Tau ratios in an ICP27 dependent fashion .MethodWe used infection with WT and mutant HSV‐1 and ectopic ICP27 expression to monitor MAPT splicing in iPSC‐derived 3D cortical organoids and 2D neuronal models.ResultsWe observed that 3D cortical organoids infected with HSV‐1 accumulate phospho‐Tau and increase MAPT 4R mRNA splicing. HSV‐1 infection of neuronal SHSY‐5Y cells, results in a significant increase in 4R isoform relative to 3R, which we did not observe when we infected these cells a virus that lacks ICP27 expression, suggesting that this viral protein is necessary for the altered splicing. Further, using lentiviruses that express either wild‐type ICP27 or a mutant ICP27 that lacks the SRPK1 interaction domain, we observed increased 4R splicing only with the wild‐type protein, revealing that ICP27 is sufficient for modulating Tau isoforms and that the SRPK1 domain is required. Finally, we found that levels of 4R splicing was increased within infected astrocytes, thus underscoring the impact HSV‐1 imparts on cells of both neuronal and non‐neuronal origin.ConclusionIn sum, our findings reveal the remaining critical third link in how neurotropic human herpesviruses can impact and influence AD progression.

Inhibition of tau phosphorylation and Aβ accumulation by S. cerevisiae-derived vacuoles in LPS-induced SH-SY5Y cells.

Neurodegenerative diseases, such as Alzheimer's disease (AD), are characterized by the accumulation of intracellular tau and amyloid beta (Aβ) proteins, which lead to neuroinflammation and neuronal apoptosis. In this study, we investigated the potential of a bioengineered vacuoles derived from Saccharomyces cerevisiae-derived vacuoles to treat neuroinflammation and protein accumulation in AD. The yeast-derived vacuole is a small organelle that achieves efficient degradation by utilizing a diverse array of hydrolytic enzymes. These hydrolytic enzymes break down and process proteins into smaller fragments. We found that vacuoles treatment significantly reduced LPS-primed cell apoptosis and diminished Aβ42 secretion in vitro, potentially through the inhibition of the NF-kB p65 signaling pathway. Additionally, vacuole pre-treatment down-regulated NF-κB translocation and reduced phosphorylated tau levels in LPS-induced SH-SY5Y cells. Our results suggest that the vacuoles have potential as a therapeutic agent for neurodegenerative diseases. The vacuole's small size and diverse hydrolytic enzymes make it a promising drug delivery system for targeting intracellular proteins. Future studies may explore the use of vacuoles in animal models of AD to determine their therapeutic potential.

Effects of Redox Homeostasis and Mitochondrial Damage on Alzheimer's Disease.

Bioenergetic mitochondrial dysfunction is a common feature of several diseases, including Alzheimer's disease (AD), where redox imbalance also plays an important role in terms of disease development. AD is an age-related disease and begins many years before the appearance of neurodegenerative symptoms. Intracellular tau aggregation, extracellular β-amyloid (Aβ) deposition in the brain, and even the APOE4 genotype contribute to the process of AD by impairing redox homeostasis and mitochondrial dysfunction. This review summarizes the evidence for the redox imbalance and mitochondrial dysfunction in AD and demonstrates the current therapeutic strategies related to mitochondrial maintenance.

ApoE4 expression disrupts tau uptake, trafficking, and clearance in astrocytes.

Tauopathies are a collection of neurodegenerative diseases characterized by the accumulation of pathogenic aggregates of the microtubule-associated protein tau. Despite the prevalence and diversity of tau astrogliopathy in tauopathies, the interactions between astrocytes and tau in the brain, and the influence of neurodegenerative genetic risk factors like the apolipoprotein E4 (apoE4) isoform, are largely unknown. Here, we leveraged primary and immortalized astrocytes expressing humanized apoE isoforms to characterize the mechanisms by which astrocytes interact with and eliminate extracellular tau, and the influence of apoE genotype on these processes. Our work indicates that astrocytes rapidly internalize, process, and release tau via an exosomal secretory mechanism under physiological conditions. However, we found that apoE4 disrupted these processes in comparison to apoE3, resulting in an astrocytic phenotype prone to intracellular tau accumulation. Furthermore, exposure to repetitive mild traumatic brain injuries exacerbated the apoE4-induced impairments in tau processing and elimination by astrocytes in apoE4 targeted-replacement mice. The diminished ability of apoE4 astrocytes to eliminate extracellular tau can lead to an accumulation of pathogenic tau, which induces mitochondrial dysfunction, as demonstrated by our studies. In total, our findings suggest that the apoE4 isoform lowers the threshold of astrocytic resilience to pathogenic tau, rendering them susceptible to bioenergetic deficits in the early stages of neurodegenerative diseases such as traumatic brain injury, potentially contributing to neurological decline.

Non-Coding RNA in Microglia Activation and Neuroinflammation in Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by complex pathophysiological features. Amyloid plaques resulting from extracellular amyloid deposition and neurofibrillary tangles formed by intracellular hyperphosphorylated tau accumulation serve as primary neuropathological criteria for AD diagnosis. The activation of microglia has been closely associated with these pathological manifestations. Non-coding RNA (ncRNA), a versatile molecule involved in various cellular functions such as genetic information storage and transport, as well as catalysis of biochemical reactions, plays a crucial role in microglial activation. This review aims to investigate the regulatory role of ncRNAs in protein expression by directly targeting genes, proteins, and interactions. Furthermore, it explores the ability of ncRNAs to modulate inflammatory pathways, influence the expression of inflammatory factors, and regulate microglia activation, all of which contribute to neuroinflammation and AD. However, there are still significant controversies surrounding microglial activation and polarization. The categorization into M1 and M2 phenotypes may oversimplify the intricate and multifaceted regulatory processes in microglial response to neuroinflammation. Limited research has been conducted on the role of ncRNAs in regulating microglial activation and inducing distinct polarization states in the context of neuroinflammation. Moreover, the regulatory mechanisms through which ncRNAs govern microglial function continue to be refined. The current understanding of ncRNA regulatory pathways involved in microglial activation remains incomplete and may be influenced by spatial, temporal, and tissue-specific factors. Therefore, further in-depth investigations are warranted. In conclusion, there are ongoing debates and uncertainties regarding the activation and polarization of microglial cells, particularly concerning the categorization into M1 and M2 phenotypes. The study of ncRNA regulation in microglial activation and polarization, as well as its mechanisms, is still in its early stages and requires further investigation. However, this review offers new insights and opportunities for therapeutic approaches in AD. The development of ncRNA-based drugs may hold promise as a new direction in AD treatment.

Abnormal phosphorylation of protein tyrosine in neurodegenerative diseases.

Neurodegenerative diseases, including Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, and multiple sclerosis, are chronic disorders of the CNS that are characterized by progressive neuronal dysfunction. These diseases have diverse clinical and pathological features and their pathogenetic mechanisms are not yet fully understood. Currently, widely accepted hypotheses include the accumulation of misfolded proteins, oxidative stress from reactive oxygen species, mitochondrial dysfunction, DNA damage, neurotrophin dysfunction, and neuroinflammatory processes. In the CNS of patients with neurodegenerative diseases, a variety of abnormally phosphorylated proteins play important roles in pathological processes such as neuroinflammation and intracellular accumulation of β-amyloid plaques and tau. In recent years, the roles of abnormal tyrosine phosphorylation of intracellular signaling molecules regulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) in neurodegenerative diseases have attracted increasing attention. Here, we summarize the roles of signaling pathways related to protein tyrosine phosphorylation in the pathogenesis of neurodegenerative diseases and the progress of therapeutic studies targeting PTKs and PTPs that provide theoretical support for future studies on therapeutic strategies for these devastating and important neurodegenerative diseases.

Progress of CNS aggregated protein degradation therapeutics from APRINOIA’s small molecule discovery platform

AbstractBackgroundThe hallmark of neurodegenerative diseases, such as Parkinson’s disease (PD) and Alzheimer’s disease (AD), is an accumulation of protein aggregates, which lead to neurotoxicity and dysfunction. Small molecule therapeutics that can remove pathological aggregates are highly desired for both diseases.MethodOur small molecule discovery platform consists of our proprietary small molecule collection, unique PET imaging biomarkers and cryoEM structures. Our proprietary collection of CNS‐focused aggregated protein binding agents, previously developed by APRINOIA to map the structure‐activity relationship of its tau PET tracer programs, is selectively coupled with agents designed to hijack cellular quality‐control systems which facilitate aggregation clearance. We have developed a screening funnel to interrogate the ability of our small molecules to affect clearance of protein aggregates.ResultTherapeutic compounds developed have been shown to bind to intracellular tau aggregates in fluorescence assays. Furthermore, we have identified compounds that show proteosome dependent elimination of aSyn aggregates in the ReNcell VM neuronal model. PK studies show that these compounds can cross the BBB. In vivo studies with rTg4510 mice show that a single dose of our degraders can affect reduction of tau protein.ConclusionAPRINOIA’s small molecule discovery platform has produced lead compounds capable of degrading protein aggregates in cellular assays and in vivo models. Further development is ongoing.

Minocycline effects on memory and learning impairment in the beta-amyloid-induced Alzheimer’s disease model in male rats using behavioral, biochemical, and histological methods

Alzheimer’s disease (AD), as an advanced neurodegenerative disease, is characterized by the everlasting impairment of memory, which is determined by hyperphosphorylation of intracellular Tau protein and accumulation of beta-amyloid (Aβ) in the extracellular space. Minocycline is an antioxidant with neuroprotective effects that can freely cross the blood-brain barrier (BBB). This study investigated the effect of minocycline on the changes in learning and memory functions, activities of blood serum antioxidant enzymes, neuronal loss, and the number of Aβ plaques after AD induced by Aβ in male rats. Healthy adult male Wistar rats (200–220g) were divided randomly into 11 groups (n = 10). The rats received minocycline (50 and 100 mg/kg/day; per os (P.O.)) before, after, and before/after AD induction for 30 days. At the end of the treatment course, behavioral performance was measured by standardized behavioral paradigms. Subsequently, brain samples and blood serum were collected for histological and biochemical analysis. The results indicated that Aβ injection impaired learning and memory performances in the Morris water maze test, reduced exploratory/locomotor activities in the open field test, and enhanced anxiety-like behavior in the elevated plus maze. The behavioral deficits were accompanied by hippocampal oxidative stress (decreased glutathione (GSH) peroxidase enzyme activity and increased malondialdehyde (MDA) levels in the brain (hippocampus) tissue), increased number of Aβ plaques, and neuronal loss in the hippocampus evidenced by Thioflavin S and H&E staining, respectively. Minocycline improved anxiety-like behavior, recovered Aβ-induced learning and memory deficits, increased GSH and decreased MDA levels, and prevented neuronal loss and the accumulation of Aβ plaques. Our results demonstrated that minocycline has neuroprotective effects and can reduce memory dysfunction, which are due to its antioxidant and anti-apoptotic effects.

Quercetagitrin Inhibits Tau Accumulation and Reverses Neuroinflammation and Cognitive Deficits in P301S-Tau Transgenic Mice.

Intracellular tau accumulation is a hallmark pathology of Alzheimer's disease (AD) and other tauopathies. Tau protein, in the hyperphosphorylated form, is the component of paired helical filaments (PHFs) and neurofibrillary tangles (NFTs) in AD. Blocking tau aggregation and/or phosphorylation is currently a promising strategy for AD treatment. Here, we elucidate that quercetagitrin, a natural compound derived from African marigold (Tagetes erecta), could inhibit tau aggregation and reduce tau phosphorylation at multiple disease-related sites in vitro. Moreover, the in vivo effect of quercetagitrin was assessed in P301S-tau transgenic via oral administration. The compound treatment restored the cognitive deficits and neuron loss in the mice. The formation of NFTs and tau phosphorylations in the hippocampus and cortex of the mice was also prevented by the compound. Moreover, quercetagitrin feeding displayed neuroinflammation protection through the inhibition of NF-κB activation in the mice. Together, our data reveal that quercetagitrin possesses the potential to further develop as a therapeutic medicine for AD and other tauopathies.

Antiepileptic Drugs Modulate Alzheimer-Related Tau Aggregation in a Neuronal Activity-Independent Manner

Introduction: A rapidly increasing number of patients with dementia present a serious social problem. Recently, the incidence of epilepsy in patients with Alzheimer’s disease (AD) is increasing, drawing attention to the pathological relationship between the two conditions. Clinical studies have suggested the protective action of antiepileptic agents on dementia; however, the underlying mechanism remains unknown. We evaluated the effects of multiple antiepileptic drugs using tau aggregation assay systems to determine the effects of antiepileptic agents on tau aggregation, a major neuropathological finding associated with AD. Methods: We evaluated the effects of seven antiepileptic agents on intracellular tau aggregation using a tau-biosensor cell-based high-throughput assay. Next, we tested these agents in a cell-free tau aggregation assay using thioflavin T (ThT). Results: The assay results revealed that phenobarbital inhibited tau aggregation, whereas sodium valproate, gabapentin, and piracetam promoted tau aggregation. In the cell-free tau aggregation assay using ThT, we confirmed that phenobarbital significantly inhibited tau aggregation. Conclusion: Antiepileptic drugs may modify the tau pathology in AD in a neural activity-independent manner. Our finding may provide an important insight into the optimization of antiepileptic drug therapy in older adults with dementia.

Cellular models for the investigation of the structural dynamics and activity of human tau protein aggregate formation.

Evidence for the significant role of tau protein in Alzheimer's disease (AD) highlights the need for deep investigation of intracellular tau aggregation pathways and formation of aberrant tau polymorphs within cellular models. Intrinsically disordered proteins with low sequence complexity like hyperphosphorylated tau protein can form condensed folded structures that contribute to neurodegeneration. Candidates for aberrant intracellular tau folding and aggregation have been identified including liquid-liquid phase separation (LLPS) and we will investigate if nanocrystal growth at ribosomes could be observed. Recent research discovered that the differences in tau folding at the core of pathological tau aggregates can differentiate AD from other tauopathies. It is hypothesized that disease specific changes in the cellular environment surrounding tau, such as patterns of hyperphosphorylation, could lead to differential core structures. Combining advanced structural determination methods including cryoEM, cryoFIB, and cryo-electron tomography, we are working on direct imaging of intracellular tau aggregates in cells as well as autopsy tissue derived from human brain. The goal is to develop new cellular models of human tau aggregation using both mammalian and insect cell lines which we employ to evaluate intracellular forces necessary to create unique pathological tau structures beginning with patterns of hyperphosphorylation. We overexpressed the six human tau isoforms in both Sf9 insect cells and SH-SY5Y mammalian cells and induced this intracellular tau to form aggregates by incubating the cells with a small molecule tau aggregation inducer Congo Red. Using cryo-focused ion beam, we can mill thin lamellae through vitrified cells used for future cryo-tomography imaging and 3D reconstruction. Using advanced structural methods, we can compare our aggregates with authentic human brain-derived AD tau aggregates as well as visualize intracellular interactions.

Characterizing tubulin interactions with the proline rich region of tau.

Tau is a microtubule-associated protein thought to function in the regulation of microtubule stability and dynamics. Normally, tau plays an important role in modulating axonal microtubules in neurons, where it is highly expressed. Intracellular tau aggregates are found in a broad class of disorders, including Alzheimer's Disease, termed tauopathies. As an intrinsically disordered protein, tau lacks stable secondary and tertiary structure, and this structural disorder is maintained even when binding to soluble tubulin and microtubules. Multiple tau-tubulin binding sites have also been identified, spanning the proline-rich region (PRR), microtubule binding repeats (MTBR: R1-R4), and pseudo-repeat, R′. Although dozens of post-translational modifications have been identified on tau, phosphorylation, and specifically hyperphosphorylation, of tau is correlated with disease and alterations to microtubule binding. Intriguingly, potential phosphorylation sites also cluster with high frequency within the PRR. Our lab has recently demonstrated that the isolated PRR both binds tubulin and has tubulin polymerization capacity. Here, we use fluorescence correlation spectroscopy (FCS) in conjunction with bulk polymerization measurements to characterize the impact of phosphomimic mutations in the PRR on tubulin binding and polymerization. We find that single phosphomimics affect tubulin binding nominally, with T231E having the most significant impact. However, as phosphorylation events rarely occur in isolation on tau, we also tested combinatorial effects. We found that multiple phosphomimics alter tubulin binding and microtubule polymerization. Furthermore, we used mass spectrometry to map the regions of the PRR involved in tubulin binding. Together these measurements provide insight into previously overlooked relevance of tau's PRR in functional interactions with tubulin.

Granulovacuolar degeneration bodies are independently induced by tau and α-synuclein pathology

BackgroundGranulovacuolar degeneration bodies (GVBs) are intracellular vesicular structures that commonly accompany pathological tau accumulations in neurons of patients with tauopathies. Recently, we developed the first model for GVBs in primary neurons, that requires exogenous tau seeds to elicit tau aggregation. This model allowed the identification of GVBs as proteolytically active lysosomes induced by tau pathology. GVBs selectively accumulate cargo in a dense core, that shows differential and inconsistent immunopositivity for (phosphorylated) tau epitopes. Despite the strong evidence connecting GVBs to tau pathology, these structures have been reported in neurons without apparent pathology in brain tissue of tauopathy patients. Additionally, GVBs and putative GVBs have also been reported in the brain of patients with non-tau proteinopathies. Here, we investigated the connection between pathological protein assemblies and GVBs in more detail.MethodsThis study combined newly developed primary neuron models for tau and α-synuclein pathology with observations in human brain tissue from tauopathy and Parkinson’s disease patients. Immunolabeling and imaging techniques were employed for extensive characterisation of pathological proteins and GVBs. Quantitative data were obtained by high-content automated microscopy as well as single-cell analysis of confocal images.ResultsEmploying a novel seed-independent neuronal tau/GVB model, we show that in the context of tauopathy, GVBs are inseparably associated with the presence of cytosolic pathological tau and that intracellular tau aggregation precedes GVB formation, strengthening the causal relationship between pathological accumulation of tau and GVBs. We also report that GVBs are inseparably associated with pathological tau at the single-cell level in the hippocampus of tauopathy patients. Paradoxically, we demonstrate the presence of GVBs in the substantia nigra of Parkinson’s disease patients and in a primary neuron model for α-synuclein pathology. GVBs in this newly developed α-synuclein/GVB model are induced in the absence of cytosolic pathological tau accumulations. GVBs in the context of tau or α-synuclein pathology showed similar immunoreactivity for different phosphorylated tau epitopes. The phosphorylated tau immunoreactivity signature of GVBs is therefore independent of the presence of cytosolic tau pathology.ConclusionOur data identify the emergence of GVBs as a more generalised response to cytosolic protein pathology.