Paired Helical Filament Tau Research Articles

Head-to-head comparison of tau PET tracers [18F]PI-2620 and [18F]RO948 in non-demented individuals with brain amyloid deposition: the TAU-PET FACEHBI cohort

BackgroundSecond-generation tau tracers for positron emission tomography (PET) show high affinity for paired helical filaments tau deposits characteristic of Alzheimer´s disease and low off-target binding. Differences in their chemical structure though may lead to variations in their regional tau uptake and off-target signal. In this work, we aimed to compare the in-vivo uptake of tau tracers [18F]PI-2620 and [18F]RO948 in the early stages of the AD continuum.MethodsData from the TAU-PET FACEHBI clinical trial (EUDRA-CT 2021–000473-83) were analyzed. All participants were non-demented and underwent tau imaging with [18F]PI-2620 and [18F]RO948 PET within 3 months, amyloid imaging with [18F]Florbetaben and brain magnetic resonance imaging. Tau PET standardized uptake values ratios (SUVR) were calculated in Braak and typical off-target regions using the inferior cerebellar cortex as a reference region.ResultsThe cohort consisted of 18 individuals with subjective cognitive decline (n = 13) and mild cognitive impairment (n = 5), with centiloid values ranging from 17 to 159. Both tau tracers showed similar tau pathology distribution but presented a distinct off-target signal pattern on visual read. SUVR measurements for [18F]PI-2620 and [18F]RO948 were highly correlated in all Braak regions (R2 range [0.65–0.80]). Regarding off-target signal, [18F]PI-2620 had higher SUVRs in vascular structures, and [18F]RO948 had higher SUVRs in the skull/meninges.ConclusionsIn a cohort of individuals at early stages of the AD continuum, tau PET tracers [18F]PI-2620 and [18F]RO948 showed similar in-vivo uptake in all Braak regions and distinct off-target signal. These preliminary results support the development of standardized quantification scales for tau deposition that are tracer-independent.Trial registrationAEMPS EudraCT 2021–000473-83. Registered 30 December 2021.

Microscopy assessment of a fluorescence [18F] flortaucipir analog (T726) shows neuropathological overlap with 3R and 4R tau lesions.

[18F] flortaucipir (FTP) binding to paired helical filament (PHF) tau in Alzheimer's disease (AD) is well accepted. Binding to 3R and 4R tau in frontotemporal lobar degeneration (FTLD) is controversial. We aimed to investigate whether an FTP fluorescent analog (T726) can help shed light on this controversy. We assessed T726 binding to amyloid beta (Aβ) and different tau isoforms in nine subjects (one control, three with Alzheimer's disease [AD], and five with FTLD) with different 3R and 4R tauopathies using fluorescence confocal microscopy. T726 did not colocalize with Aβ but showed significant co-localization with PHF tau in AD. We also observed some, albeit limited, co-localization of T726 with 3R and 4R tau lesions in FTLD. This study's findings support FTP binding to some 3R and 4R tau lesions in FTLD. Further studies are needed to understand the biology of why FTP binds some but not all FTLD tau lesions. Flortaucipir analog (T726) showed significant co-localization with paired helical filament (PHF) tau in Alzheimer's disease (AD). Colocalization between T726 with 3R and 4R tau lesions was observed in frontotemporal lobar degeneration (FTLD). Not all 4R tau lesions bind to T726 across different FTLD brain regions.

Cryo-EM structures reveal tau filaments from Down syndrome adopt Alzheimer’s disease fold

Down syndrome (DS) is a common genetic condition caused by trisomy of chromosome 21. Among their complex clinical features, including musculoskeletal, neurological, and cardiovascular disabilities, individuals with DS have an increased risk of developing progressive dementia and early-onset Alzheimer’s disease (AD). This dementia is attributed to the increased gene dosage of the amyloid-β (Aβ) precursor protein gene, the formation of self-propagating Aβ and tau prion conformers, and the deposition of neurotoxic Aβ plaques and tau neurofibrillary tangles. Tau amyloid fibrils have previously been established to adopt many distinct conformations across different neurodegenerative conditions. Here, we report the characterization of brain samples from four DS cases spanning 36–63 years of age by spectral confocal imaging with conformation-specific dyes and cryo–electron microscopy (cryo-EM) to determine structures of isolated tau fibrils. High-resolution structures revealed paired helical filament (PHF) and straight filament (SF) conformations of tau that were identical to those determined from AD cases. The PHFs and SFs are made of two C-shaped protofilaments, each containing a cross-β/β-helix motif. Similar to filaments from AD cases, most filaments from the DS cases adopted the PHF form, while a minority (approximately 20%) formed SFs. Samples from the youngest individual with no documented dementia had sparse tau deposits. To isolate tau for cryo-EM from this challenging sample we used a novel affinity-grid method involving a graphene oxide surface derivatized with anti-tau antibodies. This method improved isolation and revealed that primarily tau PHFs and a minor population of chronic traumatic encephalopathy type II–like filaments were present in this youngest case. These findings expand the similarities between AD and DS to the molecular level, providing insight into their related pathologies and the potential for targeting common tau filament folds by small-molecule therapeutics and diagnostics.

Simple modeling of familial Alzheimer’s disease using human pluripotent stem cell-derived cerebral organoid technology

BackgroundCerebral organoids (COs) are the most advanced in vitro models that resemble the human brain. The use of COs as a model for Alzheimer’s disease (AD), as well as other brain diseases, has recently gained attention. This study aimed to develop a human AD CO model using normal human pluripotent stem cells (hPSCs) that recapitulates the pathological phenotypes of AD and to determine the usefulness of this model for drug screening.MethodsWe established AD hPSC lines from normal hPSCs by introducing genes that harbor familial AD mutations, and the COs were generated using these hPSC lines. The pathological features of AD, including extensive amyloid-β (Aβ) accumulation, tauopathy, and neurodegeneration, were analyzed using enzyme-linked immunosorbent assay, Amylo-Glo staining, thioflavin-S staining, immunohistochemistry, Bielschowsky’s staining, and western blot analysis.ResultsThe AD COs exhibited extensive Aβ accumulation. The levels of paired helical filament tau and neurofibrillary tangle-like silver deposits were highly increased in the AD COs. The number of cells immunoreactive for cleaved caspase-3 was significantly increased in the AD COs. In addition, treatment of AD COs with BACE1 inhibitor IV, a β-secretase inhibitor, and compound E, a γ-secretase inhibitor, significantly attenuated the AD pathological features.ConclusionOur model effectively recapitulates AD pathology. Hence, it is a valuable platform for understanding the mechanisms underlying AD pathogenesis and can be used to test the efficacy of anti-AD drugs.

A genome‐wide association study of LATE‐NC (limbic‐predominant age‐related TDP‐43 encephalopathy neuropathologic change) reveals a distinct genetic architecture

AbstractBackgroundLimbic‐predominant age‐related TDP‐43 encephalopathy neuropathologic change (LATE‐NC) is the third leading cause of late‐life dementia. However, its pathophysiology remains largely unknown, and the relationship of LATE‐NC with Alzheimer’s disease (AD) pathology and frontotemporal lobar degeneration‐TDP (FTLD‐TDP) remains unclear. To address this knowledge gap, we performed a genome‐wide association study (GWAS) of LATE‐NC.MethodDeceased participants of European ancestry from the Religious Orders Study, the Rush Memory and Aging Project (ROSMAP, n = 1,379), and the Adult Changes in Thought study (ACT, n = 459) were included in our study. The primary phenotypes were LATE‐NC burden (a multi‐regional semi‐quantitative TDP‐43 composite, continuous) in ROSMAP, and the presence/absence of LATE‐NC (binary) in ACT, assessed with post‐mortem TDP‐43 immunohistochemistry. Quantitative amyloid‐β (Aβ) and paired helical filament tau (PHFtau) burden were also examined in ROSMAP. GWAS was performed using linear (ROSMAP) or logistic (ACT) additive models. We controlled age, sex, genotyping platforms, and the first three genotype principal components. Sample size‐weighted GWAS meta‐analysis (total n = 1,838) was performed by combining p‐values considering the direction of the association. Confounding and moderating effects of AD pathology on LATE‐NC risk variants were examined in ROSMAP.ResultWe observed two genome‐wide significant risk loci (p<5.0×10−8) in APOE (ε4) and SHANK2 (s146617441G, allele frequency 0.021). In ROSMAP, APOE ε4 was associated with higher LATE‐NC burden after controlling for quantitative Aβ and PHFtau (beta = 0.27, p = 7.3×10−6), and the APOE ε4 – LATE‐NC association was present in the subgroup without detectable Aβ (n = 170, beta = 0.50, p = 0.025). rs146617441G was not directly associated with quantitative Aβ or tau. Instead, rs146617441G×Aβ interaction term was associated with higher LATE‐NC burden (beta = 0.25, p = 0.032), suggesting the synergy between Aβ‐dependent and LATE‐NC‐specific pathways in LATE‐NC pathogenesis. Three additional loci reached a pre‐specified suggestive p‐value threshold (p<1.0×10−6; CMTM7 [rs56284874G], RNF145 [rs111653954T], and CACNA2D3 [rs71303704C]). A previously reported FTLD‐TDP risk locus in TMEM106B (rs1990622A) showed a weaker positive association with LATE‐NC (meta‐analysis p = 9.6×10−4).ConclusionOur GWAS revealed LATE‐NC‐specific risk loci (SHANK2 and other suggestive loci). The unique genetic architecture of LATE‐NC further supports the distinction between LATE‐NC and FTLD‐TDP, and the AD‐independent effect of APOE ε4 on LATE‐NC highlights the pleiotropic role of APOE in neurodegeneration.

Relationship for co‐trajectories of cognitive decline and late‐life depression with postmortem neuropathology

AbstractBackgroundThe etiopathological relationship between late‐life cognitive decline and symptoms of depression remains unclear. Existing longitudinal studies have mostly grouped participants into distinct groups based on clinical diagnoses, and very few have analyzed postmortem neuropathology. Here we identified trajectories of both cognition and depressive symptoms in an elderly cohort over up to 27 years using growth mixture modeling (GMM), identifying co‐trajectory subgroups, and characterizing them with respect to postmortem neuropathologies.MethodWe analyzed 3,010 participants enrolled without dementia (mean age = 78) from the Religious Orders Study and Rush Memory and Aging Project (ROS/MAP). All included participants were evaluated for at least three annual visits for both depressive symptoms (using a modified Center for Epidemiologic Studies Depression Scale) and 19 cognitive tasks (yielding a global cognitive function summary score). A subset of 1,732 participants had complete postmortem neuropathological assessment data available. Latent trajectory subgroups were identified independently for global cognition and depressive symptoms using GMM with non‐linear time effects. Interaction effects of cognitive and depressive trajectory subgroups on neuropathologies were assessed using linear models.ResultThree trajectory subgroups (Figure 1) were identified for both cognitive decline (minimal (n = 1,232), slow (n = 1,325) and fast (n = 302)) and depressive symptoms (sustained low (n = 1,815), moderately increasing (n = 762) and consistently elevated (n = 433)). These subgroups showed significant overlap (chi‐square p = 0.0001), and their intersections defined nine co‐trajectories. A significant interaction effect (p = 0.0062) was identified between cognitive decline and depressive symptom trajectory subgroups for brain‐wide levels of paired helical filament tau; among only individuals experiencing fast cognitive decline, sustained lower depressive symptoms were associated with higher levels of tau (Figure 2).ConclusionLongitudinal cognitive decline and depressive symptom trajectories in late‐life are not independent. Intriguingly, we observed the highest tau burden in those individuals with both sustained low depressive symptoms and fast rates of cognitive decline. Given that use of antidepressants may lower tau levels (Dafsari & Jessen, 2020), future models of neuropathology in trajectory‐based analyses will incorporate patterns of medication status.

Crosstalk between tau and alpha7 nicotinic acetylcholine receptors at the synapse

AbstractBackgroundThe failure of cholinergic transmission and the increase of amyloid‐β peptide (Aβ) and tau protein are key players in Alzheimer’s disease (AD). The interaction between α7 nicotinic acetylcholine receptors (α7‐nAChRs) and Aβ has been previously investigated, but few studies have focused on the crosstalk between α7‐nAChRs and tau. We previously reported that Aβ and tau might act independently or concomitantly to impair synaptic plasticity and memory, converging onto common targets such as amyloid precursor protein (APP). Here we investigated the role of α7‐nAChRs on tau‐mediated impairment of synaptic plasticity and memory, tau neuropathology, and their interplay with APP.MethodWe used WT (C57Bl6/J) and α7 KO at different ages. 3XTg models of AD were used as positive controls. We performed electrophysiology to investigate synaptic transmission and plasticity at the CA3‐CA1 synapses from hippocampal slices, and behavioral studies (Novel Object Recognition and Location, Fear Conditioning) to evaluate memory. We used western blot for the expression of APP, tau (Ser199, Ser396, Thr205) and GSK‐3β (Ser9) phosphorylation. Formation of tau paired helical filaments (PHFs) and neurofibrillary tangles (NFTs) were evaluated by immunohistochemistry and Bielschowsky staining. Immunoreactivity for NeuN was measured to assess neuronal loss.ResultWe found that α7 KO mice presented an AD phenotype characterized by the impairment of Long‐Term Potentiation and Paired Pulse Facilitation, and the reduction of different types of memory starting at 12 months of age. This age‐dependent phenotype was paralleled by an increase of APP expression, hyperphosphorylation of tau, and a decrease of GSK‐3β (Ser9) phosphorylation. α7 KO hippocampi presented a marked tau neuropathology with an increase of PHF‐1 immunoreactivity, the presence of PHFs and NFTs, and neuronal loss.ConclusionIn summary, the lack of α7‐nAChRs was sufficient to trigger an AD‐like phenotype characterized by a marked tau pathology. Our data also suggest that the concomitant increase of APP expression might contribute to tau neurotoxicity.

NCOG-34. BRAIN PET IMAGING OF PAIRED HELICAL FILAMENT TAU IN OLDER ADULTS WITH CANCER: PILOT STUDY TO IDENTIFY MECHANISMS OF COGNITIVE IMPAIRMENT AFTER CANCER THERAPY

Abstract BACKGROUND Cancer-related cognitive impairment (CRCI) occurs in up to 75% of patients treated with chemotherapy for systemic cancer, and older patients treated for CNS cancer receiving brain radiation therapy (RT) and chemotherapy are particularly at risk. The mechanisms of injury and CRCI remain unknown. In neurodegenerative disorders, deposition of paired helical filament tau (PHFTau) is correlated with nature and extent of cognitive symptoms. The role of PHFTau in CRCI has not been elucidated. We conducted a pilot study to determine whether CRCI is associated with PHFTau. METHODS Patients (age >60) diagnosed with mild cognitive impairment (MCI) or dementia after chemotherapy and/or brain RT underwent Tau PET imaging using [F18]Flourtaucipir. Cortical [F18]Flourtaucipir binding was quantified by standard uptake value ratio (SUVR) in 68 regions of interest (ROIs) and compared to a control group (n=22). SUVR in each ROI for each subject was considered significantly elevated when it was >2SD above the control group (p< 0.05). Neuropsychological test scores were correlated with PHFTau distribution. RESULTS Five patients with MCI/Dementia following cancer therapy participated. Two patients with prior chemo-RT for CNS cancer and prominent executive dysfunction had no significant F[18]T807 uptake. However, three patients treated with systemic chemotherapy for non-CNS cancer and neuropsychological profiles dominated by episodic memory impairment showed F[18]T807 uptake in patterns typical of AD dementia in temporal, parietal and cingulate ROI’s (n=2), or uptake in the entorhinal and inferior temporal ROIs (n=1). DISCUSSION In this pilot study, [F18]Flourtaucipir imaging of patients with cancer and MCI/Dementia showed that executive dysfunction after RT was not associated with abnormal PHFTau whereas amnestic declines after chemotherapy were associated with PHFTau in medial temporal lobes and beyond. Given the cross-sectional study, timing of tau accumulation in relation to cancer/treatment is unknown. Prospective studies are needed to understand the relevance of PHFTau in CRCI.

Structure of the nonhelical filament of the Alzheimer's disease tau core.

The microtubule-associated protein tau aggregates into neurofibrillary tangles in Alzheimer's disease (AD). The main type of aggregates, the paired helical filaments (PHF), incorporate about 20% of the full-length protein into the rigid core. Recently, cryo-electron microscopy data showed that a protease-resistant fragment of tau (residues 297-391) self-assembles in vitro in the presence of divalent cations to form twisted filaments whose molecular structure resembles that of AD PHF tau [S. Lövestam et al., Elife 11, e76494 (2022)]. To investigate whether this tau construct is uniquely predisposed to this morphology and structure, we fibrillized tau (297-391) under the reported conditions and determined its structure using solid-state NMR spectroscopy. Unexpectedly, the protein assembled predominantly into nontwisting ribbons whose rigid core spans residues 305-357. This rigid core forms a β-arch that turns at residues 322CGS324. Two protofilaments stack together via a long interface that stretches from G323 to I354. Together, these two protofilaments form a four-layered β-sheet core whose sidechains are stabilized by numerous polar and hydrophobic interactions. This structure gives insight into the fibril morphologies and molecular conformations that can be adopted by this protease-resistant core of AD tau under different pH and ionic conditions.

Selective serotonin reuptake inhibitor use, age-related neuropathology and cognition in late-life

The objective of this study was to evaluate an association of selective serotonin reuptake inhibitor (SSRI) use with late life cognitive decline and further investigate the association with brain pathology. Using the data are from two harmonized clinical-pathologic cohort studies with annual cognitive testing we found that SSRI use was associated with significantly faster global cognitive decline and this association was present in those with and without pre-existing cognitive impairment at the time of SSRI initiation. In separate analyses of persons who died during the study and underwent neuropathologic examination, SSRI use was related to higher level of paired helical filament tau tangles and faster rate of global cognitive decline. However, when SSRI use and tangles were included in the same model, the association of SSRI use with rate of global cognitive decline was reduced by more than 50% and no longer statistically significant. SSRI use was associated with higher postmortem level of tau tangles, possibly because SSRI are being used to treat neurobehavioral symptoms associated with dementia, and this relationship appears to partly account for the association of SSRI use with more rapid cognitive decline.

Decoding Angiotensin Receptors: TOMAHAQ-Based Detection and Quantification of Angiotensin Type-1 and Type-2 Receptors.

Background The renin-angiotensin system plays a crucial role in human physiology, and its main hormone, angiotensin, activates 2 G-protein-coupled receptors, the angiotensin type-1 and type-2 receptors, in almost every organ. However, controversy exists about the location, distribution, and expression levels of these receptors. Concerns have been raised over the low sensitivity, low specificity, and large variability between lots of commercially available antibodies for angiotensin type-1 and type-2 receptors, which makes it difficult to reconciliate results of different studies. Here, we describe the first non-antibody-based sensitive and specific targeted quantitative mass spectrometry assay for angiotensin receptors. Methods and Results Using a technique that allows targeted analysis of multiple peptides across multiple samples in a single mass spectrometry analysis, known as TOMAHAQ (triggered by offset, multiplexed, accurate mass, high resolution, and absolute quantification), we have identified and validated specific human tryptic peptides that permit identification and quantification of angiotensin type-1 and type-2 receptors in biological samples. Several peptide sequences are conserved in rodents, making these mass spectrometry assays amenable to both preclinical and clinical studies. We have used this method to quantify angiotensin type-1 and type-2 receptors in postmortem frontal cortex samples of older adults (n=28) with Alzheimer dementia. We correlated levels of angiotensin receptors to biomarkers classically linked to renin-angiotensin system activation, including oxidative stress, inflammation, amyloid-β load, and paired helical filament-tau tangle burden. Conclusions These robust high-throughput assays will not only catalyze novel mechanistic studies in the angiotensin research field but may also help to identify patients with an unbalanced angiotensin receptor distribution who would benefit from angiotensin receptor blocker treatment.

Region specific reduction of neurogranin immunostaining in the hippocampus of Alzheimer’s disease brains

AbstractBackgroundNeurogranin (Ng) is a post‐synaptic protein involved in formation of long‐term potentials, and thus plays an important role in learning and memory. The concentration of Ng is increased in the cerebrospinal fluid (CSF) of people with Alzheimer’s disease (AD), and CSF Ng is considered to be a biomarker for synaptic dysfunction in AD. The expression of Ng protein in the human brain and its association with AD pathology, however, are less well studied.MethodUsing immunohistochemical detection methods we investigated the protein expression of Ng, beta‐amyloid (Aβ) and hyperphosphorylated paired helical filament tau (phospho‐tau; ptau) in the post‐mortem hippocampus from 12 AD patients and 12 cognitively unaffected controls (NC). We compared the number of positively stained cells (Ng, ptau) or plaques (Aβ) in the hippocampal sub‐regions (dentate gyrus, Cornu Ammonis (CA) fields 1‐3) from the 2 groups. Donor samples with intermediate AD (i.e., Braak stage III‐IV) or NC (Braak 0‐I/II) were analyzed using Image Pro Premier automated image analysis software (version 10.01). The data were normalized to the number of cells or clusters/mm2 and analyzed using a one‐way ANOVA. Using a test of circularity (Cir = (4*A)/Pi*MaxFeret2), we further investigated morphological differences in the Ng positive neurons by measuring the Ng expression in the apical dendrite of pyramidal neurons.ResultThere was a 40% reduction of Ng immunopositive neurons in AD compared to NC in the CA3(N = 24;p = 0.0003) and in the CA1(N = 24;p = 0.014) hippocampal sub‐regions. Ng positive cells did not change significantly in AD compared to NC in the other hippocampal regions. Additionally, we showed that CA1 region, Ng was localized mainly in the neuronal cell soma of AD donors, while its expression extended into the apical dendrites of NC donors (N = 24;p = 0.006). In AD, Ng immunostaining negatively correlated with ptau immunopositive neurofibrillary tangles (r = ‐0.70;p = 0.04)ConclusionSelective loss of Ng immunostaining in certain subregions of the hippocampus suggests that synaptic damage in AD maybe a region‐specific event. The mechanism underlying such regional synaptic damage and the changes in Ng expression will be key in understanding the selective vulnerability of brain regions to AD.

Stacked binding of a PET ligand to Alzheimer’s tau paired helical filaments

Accumulation of filamentous aggregates of tau protein in the brain is a pathological hallmark of Alzheimer’s disease (AD) and many other neurodegenerative tauopathies. The filaments adopt disease-specific cross-β amyloid conformations that self-propagate and are implicated in neuronal loss. Development of molecular diagnostics and therapeutics is of critical importance. However, mechanisms of small molecule binding to the amyloid core is poorly understood. We used cryo–electron microscopy to determine a 2.7 Å structure of AD patient-derived tau paired-helical filaments bound to the PET ligand GTP-1. The compound is bound stoichiometrically at a single site along an exposed cleft of each protofilament in a stacked arrangement matching the fibril symmetry. Multiscale modeling reveals pi-pi aromatic interactions that pair favorably with the small molecule–protein contacts, supporting high specificity and affinity for the AD tau conformation. This binding mode offers critical insight into designing compounds to target different amyloid folds found across neurodegenerative diseases.

Plasma p-tau181 and p-tau217 in discriminating PART, AD and other key neuropathologies in older adults

We examined whether plasma p-tau181 and p-tau217 are specific biomarkers of pathologically confirmed Alzheimer’s disease (AD). In particular, we investigated the utility of plasma p-tau for differentiating AD from primary age-related tauopathy (PART), as well as AD with mixed pathologies. Data came from 269 older adults who participated in the Religious Orders Study or the Rush Memory and Aging Project. Blood samples were collected during annual clinical evaluations. Participants died and underwent brain autopsy. P-tau181 and p-tau217 were quantified in the plasma samples proximate to death (average interval before death: 1.4 years) using Lilly-developed MSD immunoassays. Uniform neuropathologic evaluations assessed AD, PART, and other common degenerative and cerebrovascular conditions. Plasma p-tau217 was more strongly correlated with brain β-amyloid and paired helical filament tau (PHFtau) tangles than p-tau181. Both p-tau markers were associated with greater odds of AD, but p-tau217 had higher accuracy (area under the ROC curve (AUC): 0.83) than p-tau181 (AUC: 0.76). Plasma p-tau markers were almost exclusively associated with AD pathologic indices with the exception of cerebral amyloid angiopathy. Compared to p-tau181, p-tau217 showed a higher AUC (0.82 versus 0.74) in differentiating AD from PART. For either p-tau, we did not observe a level difference between individuals with AD alone and those with mixed AD pathologies. In summary, plasma p-tau181and p-tau217 were specifically associated with AD pathological changes. Further, our data provide initial evidence that p-tau217 may be able to differentiate between AD and PART in individuals with comparable burdens of tau tangle pathology. These results demonstrate the specificity of p-tau217 for AD, supporting its use to identify patients suitable for anti-AD therapies including β-amyloid immunotherapies.

Selective In Vitro and Ex Vivo Staining of Brain Neurofibrillary Tangles and Amyloid Plaques by Novel Ethylene Ethynylene-Based Optical Sensors.

The identification of protein aggregates as biomarkers for neurodegeneration is an area of interest for disease diagnosis and treatment development. In this work, we present novel super luminescent conjugated polyelectrolyte molecules as ex vivo sensors for tau-paired helical filaments (PHFs) and amyloid-β (Aβ) plaques. We evaluated the use of two oligo-p-phenylene ethynylenes (OPEs), anionic OPE12- and cationic OPE24+, as stains for fibrillar protein pathology in brain sections of transgenic mouse (rTg4510) and rat (TgF344-AD) models of Alzheimer's disease (AD) tauopathy, and post-mortem brain sections from human frontotemporal dementia (FTD). OPE12- displayed selectivity for PHFs in fluorimetry assays and strong staining of neurofibrillary tangles (NFTs) in mouse and human brain tissue sections, while OPE24+ stained both NFTs and Aβ plaques. Both OPEs stained the brain sections with limited background or non-specific staining. This novel family of sensors outperformed the gold-standard dye Thioflavin T in sensing capacities and co-stained with conventional phosphorylated tau (AT180) and Aβ (4G8) antibodies. As the OPEs readily bind protein amyloids in vitro and ex vivo, they are selective and rapid tools for identifying proteopathic inclusions relevant to AD. Such OPEs can be useful in understanding pathogenesis and in creating in vivo diagnostically relevant detection tools for neurodegenerative diseases.

Probing the Interactions of LRP1 Ectodomain-Derived Peptides with Fibrillar Tau Protein and Its Impact on Cellular Internalization

Cellular internalization and the spreading of misfolded tau have become increasingly important for elucidating the mechanism of Tau pathology involved in Alzheimer’s disease (AD). The low-density lipoprotein-related receptor 1 (LRP1) has been implicated in the internalization of fibrillar tau. In this work, we utilized homology modeling to model the Cluster 2 domain of LRP1 and determined that a 23-amino-acid sequence is involved in binding to paired helical filaments (PHF) of Tau. Fourteen short peptide segments derived from this ectodomain region were then designed and docked with PHF Tau. Molecular dynamics studies of the optimal peptides bound to PHF Tau demonstrated that the peptides formed critical contacts through Lys and Gln residues with Tau. Based on the computational results, flow cytometry, AFM, SPR analysis and CD studies were conducted to examine binding and cellular internalization. The results showed that the peptide sequence TauRP (1–14) (DNSDEENCES) was not only associated with fibrillar Tau but was also able to mitigate its cellular internalization in LRP1-expressed HEK-293 cells. Preliminary docking studies with Aβ (1–42) revealed that the peptides also bound to Aβ (1–42). While this study focused on the CCR2 domain of LRP1 to design peptide sequences to mitigate Tau internalization, the work can be extended to other domains of the LRP1 receptor or other receptors to examine if the cellular internalization of fibrillar Tau can be deterred. These findings show that short peptides derived from the LRP1 receptor can alter the internalization of its ligands.

Cryo-EM structures of amyloid-β filaments with the Arctic mutation (E22G) from human and mouse brains

The Arctic mutation, encoding E693G in the amyloid precursor protein (APP) gene [E22G in amyloid-β (Aβ)], causes dominantly inherited Alzheimer’s disease. Here, we report the high-resolution cryo-EM structures of Aβ filaments from the frontal cortex of a previously described case (AβPParc1) with the Arctic mutation. Most filaments consist of two pairs of non-identical protofilaments that comprise residues V12–V40 (human Arctic fold A) and E11–G37 (human Arctic fold B). They have a substructure (residues F20–G37) in common with the folds of type I and type II Aβ42. When compared to the structures of wild-type Aβ42 filaments, there are subtle conformational changes in the human Arctic folds, because of the lack of a side chain at G22, which may strengthen hydrogen bonding between mutant Aβ molecules and promote filament formation. A minority of Aβ42 filaments of type II was also present, as were tau paired helical filaments. In addition, we report the cryo-EM structures of Aβ filaments with the Arctic mutation from mouse knock-in line AppNL−G−F. Most filaments are made of two identical mutant protofilaments that extend from D1 to G37 (AppNL−G−F murine Arctic fold). In a minority of filaments, two dimeric folds pack against each other in an anti-parallel fashion. The AppNL−G−F murine Arctic fold differs from the human Arctic folds, but shares some substructure.

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

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

Negative Correlation of Corpora Amylacea with pTau in the Hippocampus of People with AD

AbstractBackgroundRecent studies suggest corpora amylacea (CA) activity may be involved in modulating AD pathology. Normally, CA function in the clearance of brain waste products by amassing cellular debris and extruding it into the cerebrospinal fluid. Disruption of this process may play a role in AD pathogenesis, and its effect might be influenced by degree of tau pathology and genetic risk. In this study, we determined if the abundance of CA was correlated with paired helical filament tau pathology in the CA1 region of the hippocampus in AD postmortem tissue samples. Tau pathology was assessed using both total pTau and total NFTs. Additionally, we investigated whether that correlation was influenced by APOE genotype.MethodPostmortem brain hippocampal tissue sections from 11 brain donors with pre‐mortem cognitive impairment and neuropathologically confirmed AD were double‐stained with Periodic‐acid Schiff (PAS) stain to identify CA and AT8 antibody to identify hyperphosphorylated Tau protein (pTau). Using the Motic slide scanner, stained sections were digitized at 40x. QuPath 0.3.2 was used to extract the CA1 hippocampal region and export to ImageJ for analysis. ImageJ 1.8 color thresholding was used to quantify the number and area of CA and pTau. Neurofibrillary tangle (NFT) pathology was classified by pTau aggregations larger than 150μm2. The percent area occupied (%AO) was calculated by dividing the sum of all positively stained objects by the area of the image, then multiplying by 100. Pearson correlations between CA %AO, pTau %AO, and NFT %AO were calculated in Python 3.7.ResultWhile not statistically significant, we found negative correlations of CA %AO with pTau %AO (‐0.14), and NFT %AO (‐0.28) in the CA1 of the hippocampus. These correlations (‐0.68 and ‐0.54 respectively) were stronger in the 4 individuals who did not carry the AD risk‐increasing APOE‐4 allele.ConclusionThese results suggest that as CA decline, pTau accumulates, which is consistent with prior studies that described a decline in CA from intermediate to advanced Braak Stages (III‐VI) in the dentate gyrus. These changes are consistent with our

Neuropathology of Dementia Disorders.

This article provides an overview of the neuropathology of common age-related dementing disorders, focusing on the pathologies that underlie Alzheimer disease (AD) and related dementias, including Lewy body dementias, frontotemporal dementia, vascular dementia, limbic-predominant age-related transactive response DNA-binding protein 43 (TDP-43) encephalopathy (LATE), and mixed-etiology dementias. This article also discusses the underlying proteinopathies of neurodegenerative diseases (eg, amyloid-β, paired helical filament tau, α-synuclein, and TDP-43 pathology) and vascular pathologies, including tissue injury (eg, infarcts, hemorrhages) with or without vessel disease. New criteria for AD pathologic diagnosis highlight amyloid-β as the sine qua non of AD; they require molecular markers of amyloid and establish a minimum threshold of Braak neurofibrillary tangle stage 3. Pathologic diagnosis is separated from clinical disease (ie, pathologic diagnosis no longer requires dementia). TDP-43 pathology, a major pathology in a frontotemporal dementia subtype, was found as a central pathology in LATE, a newly named amnestic disorder. Multiple pathologies (often co-occurring with AD) contribute to dementia and add complexity to the clinical picture. Conversely, Lewy body, LATE, and vascular dementias often have accompanying AD pathology. Pathology and biomarker studies highlight subclinical pathologies in older people without cognitive impairment. This resilience to brain pathology is common and is known as cognitive reserve. The pathologies of dementia in aging are most commonly amyloid, tangles, Lewy bodies, TDP-43, hippocampal sclerosis, and vascular pathologies. These pathologies often co-occur (mixed pathologies), which may make specific clinical diagnoses difficult. In addition, dementia-related pathologies are often subclinical, suggesting varying levels of resilience in older people.