Specific detection of tau seeding activity in Alzheimer's disease using rationally designed biosensor cells.

BACKGROUND: The prion-like propagation of tau in neurodegenerative disorders implies that misfolded pathological tau can recruit the normal protein and template its aggregation. Here, we report the methods for the development of sensitive biosensor cell lines for the detection of tau seeding activity. RESULTS: We performed the rational design of novel tau probes based on the current structural knowledge of pathological tau aggregates in Alzheimer's disease. We generated Förster resonance energy transfer (FRET)-based biosensor stable cell lines and characterized their sensitivity, specificity, and overall ability to detect bioactive tau in human samples. As compared to the reference biosensor line, the optimized probe design resulted in an increased efficiency in the detection of tau seeding. The increased sensitivity allowed for the detection of lower amount of tau seeding competency in human brain samples, while preserving specificity for tau seeds found in Alzheimer's disease. CONCLUSIONS: This next generation of FRET-based biosensor cells is a novel tool to study tau seeding activity in Alzheimer's disease human samples, especially in samples with low levels of seeding activity, which may help studying early tau-related pathological events.

Tau seeding and spreading in vivo is supported by both AD-derived fibrillar and oligomeric tau.

Insoluble fibrillar tau, the primary constituent of neurofibrillary tangles, has traditionally been thought to be the biologically active, toxic form of tau mediating neurodegeneration in Alzheimer's disease. More recent studies have implicated soluble oligomeric tau species, referred to as high molecular weight (HMW), due to their properties on size-exclusion chromatography, in tau propagation across neural systems. These two forms of tau have never been directly compared. We prepared sarkosyl-insoluble and HMW tau from the frontal cortex of Alzheimer patients and compared their properties using a variety of biophysical and bioactivity assays. Sarkosyl-insoluble fibrillar tau comprises abundant paired-helical filaments (PHF) as quantified by electron microscopy (EM) and is more resistant to proteinase K, compared to HMW tau, which is mostly in an oligomeric form. Sarkosyl-insoluble and HMW tau are nearly equivalent in potency in HEK cell bioactivity assay for seeding aggregates, and their injection reveals similar local uptake into hippocampal neurons in PS19 Tau transgenic mice. However, the HMW preparation appears to be far more potent in inducing a glial response including Clec7a-positive rod microglia in the absence of neurodegeneration or synapse loss and promotes more rapid propagation of misfolded tau to distal, anatomically connected regions, such as entorhinal and perirhinal cortices. These data suggest that soluble HMW tau has similar properties to fibrillar sarkosyl-insoluble tau with regard to tau seeding potential, but may be equal or even more bioactive with respect to propagation across neural systems and activation of glial responses, both relevant to tau-related Alzheimer phenotypes.

Tau seeding and spreading in vivo is supported by both AD-derived fibrillar and oligomeric tau.

Insoluble fibrillar tau, the primary constituent of neurofibrillary tangles, has traditionally been thought to be the biologically active, toxic form of tau mediating neurodegeneration in Alzheimer's disease. More recent studies have implicated soluble oligomeric tau species, referred to as high molecular weight (HMW) due to its properties on size exclusion chromatography, in tau propagation across neural systems. These two forms of tau have never been directly compared. We prepared sarkosyl insoluble and HMW tau from the frontal cortex of Alzheimer patients and compared their properties using a variety of biophysical and bioactivity assays. Sarkosyl insoluble fibrillar tau is comprised of abundant paired helical filaments (PHF) as quantified by electron microscopy (EM), and is more resistant to proteinase K, compared to HMW tau which is mostly in an oligomeric form. Sarkosyl insoluble and HMW tau are nearly equivalent in potency in a HEK cell bioactivity assay for seeding aggregates and their injection reveals similar local uptake into hippocampal neurons in PS19 Tau transgenic mice. However, the HMW preparation appears to be far more potent in inducing a glial response including Clec7a-positive rod-microglia in the absence of neurodegeneration or synapse loss and promotes more rapid propagation of misfolded tau to distal, anatomically connected regions, such as entorhinal and perirhinal cortices. These data suggest that soluble HMW tau has similar properties to fibrillar sarkosyl insoluble tau with regard to tau seeding potential but may be equal or even more bioactive with respect to propagation across neural systems and activation of glial responses, both relevant tau-related Alzheimer phenotypes.

Tau propagation is dependent on the genetic background of mouse strains.

Progressive cognitive decline in Alzheimer's disease correlates closely with the spread of tau protein aggregation across neural networks of the cortical mantle. We tested the hypothesis that heritable factors may influence the rate of propagation of tau pathology across brain regions in a model system, taking advantage of well-defined genetically diverse background strains in mice. We virally expressed human tau locally in the hippocampus and the entorhinal cortex neurons and monitored the cell-to-cell tau protein spread by immunolabelling. Interestingly, some strains showed more tau spreading than others while tau misfolding accumulated at the same rate in all tested mouse strains. Genetic factors may contribute to tau pathology progression across brain networks, which could help refine mechanisms underlying tau cell-to-cell transfer and accumulation, and potentially provide targets for understanding patient-to-patient variability in the rate of disease progression in Alzheimer's disease.

Novel genetic variants in MAPT and alterations in tau phosphorylation in amyotrophic lateral sclerosis post-mortem motor cortex and cerebrospinal fluid.

Although the molecular mechanisms underlying amyotrophic lateral sclerosis (ALS) are not yet fully understood, several studies report alterations in tau phosphorylation in both sporadic and familial ALS. Recently, we have demonstrated that phosphorylated tau at S396 (pTau-S396) is mislocalized to synapses in ALS motor cortex (mCTX) and contributes to mitochondrial dysfunction. Here, we demonstrate that while there was no overall increase in total tau, pTau-S396, and pTau-S404 in ALS post-mortem mCTX, total tau and pTau-S396 were increased in C9ORF72-ALS. Additionally, there was a significant decrease in pTau-T181 in ALS mCTX compared controls. Furthermore, we leveraged the ALS Knowledge Portal and Project MinE data sets and identified ALS-specific genetic variants across MAPT, the gene encoding tau. Lastly, assessment of cerebrospinal fluid (CSF) samples revealed a significant increase in total tau levels in bulbar-onset ALS together with a decrease in CSF pTau-T181:tau ratio in all ALS samples, as reported previously. While increases in CSF tau levels correlated with a faster disease progression as measured by the revised ALS functional rating scale (ALSFRS-R), decreases in CSF pTau-T181:tau ratio correlated with a slower disease progression, suggesting that CSF total tau and pTau-T181 ratio may serve as biomarkers of disease in ALS. Our findings highlight the potential role of pTau-T181 in ALS, as decreases in CSF pTau-T181:tau ratio may reflect the significant decrease in pTau-T181 in post-mortem mCTX. Taken together, these results indicate that tau phosphorylation is altered in ALS post-mortem mCTX as well as in CSF and, importantly, the newly described pathogenic or likely pathogenic variants identified in MAPT in this study are adjacent to T181 and S396 phosphorylation sites further highlighting the potential role of these tau functional domains in ALS.

APOE4 derived from astrocytes leads to blood-brain barrier impairment.

Apolipoprotein E (ApoE) is a multifaceted secreted molecule synthesized in the CNS by astrocytes and microglia, and in the periphery largely by the liver. ApoE has been shown to impact the integrity of the blood-brain barrier, and, in humans, the APOE4 allele of the gene is reported to lead to a leaky blood-brain barrier. We used allele specific knock-in mice expressing each of the common (human) ApoE alleles, and longitudinal multiphoton intravital microscopy, to directly monitor the impact of various ApoE isoforms on blood-brain barrier integrity. We found that humanized APOE4, but not APOE2 or APOE3, mice show a leaky blood-brain barrier, increased MMP9, impaired tight junctions, and reduced astrocyte end-foot coverage of blood vessels. Removal of astrocyte-produced ApoE4 led to the amelioration of all phenotypes while the removal of astrocyte-produced ApoE3 had no effect on blood-brain barrier integrity. This work shows a cell specific gain of function effect of ApoE4 in the dysfunction of the BBB and implicates astrocyte production of ApoE4, possibly as a function of astrocytic end foot interactions with vessels, as a key regulator of the integrity of the blood-brain barrier.

Conformational fingerprinting of tau variants and strains by Raman spectroscopy.

Tauopathies are a group of disorders in which the deposition of abnormally folded tau protein accompanies neurodegeneration. The development of methods for detection and classification of pathological changes in protein conformation are desirable for understanding the factors that influence the structural polymorphism of aggregates in tauopathies. We have previously demonstrated the utility of Raman spectroscopy for the characterization and discrimination of different protein aggregates, including tau, based on their unique conformational signatures. Building on this, in the present study, we assess the utility of Raman spectroscopy for characterizing and distinguishing different conformers of the same protein which in the case of tau are unique tau strains generated in vitro. We now investigate the impact of aggregation environment, cofactors, post-translational modification and primary sequence on the Raman fingerprint of tau fibrils. Using quantitative conformational fingerprinting and multivariate statistical analysis, we found that the aggregation of tau in different buffer conditions resulted in the formation of distinct fibril strains. Unique spectral markers were identified for tau fibrils generated using heparin or RNA cofactors, as well as for phosphorylated tau. We also determined that the primary sequence of the tau monomer influenced the conformational signature of the resulting tau fibril, including 2N4R, 0N3R, K18 and P301S tau variants. These results highlight the conformational polymorphism of tau fibrils, which is reflected in the wide range of associated neurological disorders. Furthermore, the analyses presented in this study provide a benchmark for the Raman spectroscopic characterization of tau strains, which may shed light on how the aggregation environment, cofactors and post-translational modifications influence tau conformation in vivo in future studies.

Kinetics of tau aggregation reveals patient-specific tau characteristics among Alzheimer's cases.

The accumulation of tau aggregates throughout the human brain is the hallmark of a number of neurodegenerative conditions classified as tauopathies. Increasing evidence shows that tau aggregation occurs in a 'prion-like' manner, in which a small amount of misfolded tau protein can induce other, naïve tau proteins to aggregate. Tau aggregates have been found to differ structurally among different tauopathies. Recently, however, we have suggested that tau oligomeric species may differ biochemically among individual patients with sporadic Alzheimer disease, and have also showed that the bioactivity of the tau species, measured using a cell-based bioassay, also varied among individuals. Here, we adopted a live-cell imaging approach to the standard cell-based bioassay to explore further whether the kinetics of aggregation also differentiated these patients. We found that aggregation can be observed to follow a consistent pattern in all cases, with a lag phase, a growth phase and a plateau phase, which each provide quantitative parameters by which we characterize the aggregation kinetics. The length of the lag phase and magnitude of the plateau phase are both dependent upon the concentration of seeding-competent tau, the relative enrichment of which differs among patients. The slope of the growth phase correlates with morphological differences in the tau aggregates, which may be reflective of underlying structural differences. This kinetic assay confirms and refines the concept of heterogeneity in the characteristics of tau proteopathic seeds among individuals with Alzheimer's disease and is a method by which future studies may characterize longitudinal changes in tau aggregation and the cellular processes which may influence these changes.

Continuous Monitoring of Tau-Induced Neurotoxicity in Patient-Derived iPSC-Neurons.

Tau aggregation within neurons is a critical feature of Alzheimer's disease (AD) and related tauopathies. It is believed that soluble pathologic tau species seed the formation of tau aggregates in a prion-like manner and propagate through connected neurons during the progression of disease. Both soluble and aggregated forms of tau are thought to have neurotoxic properties. In addition, different strains of misfolded tau may cause differential neurotoxicity. In this work, we present an accelerated human neuronal model of tau-induced neurotoxicity that incorporates both soluble tau species and tau aggregation. Using patient-derived induced pluripotent stem cell (iPSC) neurons expressing a tau aggregation biosensor, we develop a cell culture system that allows continuous assessment of both induced tau aggregation and neuronal viability at single-cell resolution for periods of >1 week. We show that exogenous tau "seed" uptake, as measured by tau repeat domain (TauRD) reporter aggregation, increases the risk for subsequent neuronal death in vitro These results are the first to directly visualize neuronal TauRD aggregation and subsequent cell death in single human iPSC neurons. Specific morphologic strains or patterns of TauRD aggregation are then identified and associated with differing neurotoxicity. Furthermore, we demonstrate that familial AD iPSC neurons expressing the PSEN1 L435F mutation exhibit accelerated TauRD aggregation kinetics and a tau strain propagation bias when compared with control iPSC neurons.SIGNIFICANCE STATEMENT Neuronal intracellular aggregation of the microtubule binding protein tau occurs in Alzheimer's disease and related neurodegenerative tauopathies. Tau aggregates are believed to spread from neuron to neuron via prion-like misfolded tau seeds. Our work develops a human neuronal live-imaging system to visualize seeded tau aggregation and tau-induced neurotoxicity within single neurons. Using an aggregation-sensing tau reporter, we find that neuronal uptake and propagation of tau seeds reduces subsequent survival. In addition, human induced pluripotent stem cell (iPSC) neurons carrying an Alzheimer's disease-causing mutation in presenilin-1 undergo tau seeding more rapidly than control iPSC neurons. However, they do not show subsequent differences in neuronal survival. Finally, specific morphologies of tau aggregates are associated with increased neurotoxicity.

Persistent repression of tau in the brain using engineered zinc finger protein transcription factors.

Neuronal tau reduction confers resilience against ß-amyloid and tau-related neurotoxicity in vitro and in vivo. Here, we introduce a novel translational approach to lower expression of the tau gene MAPT at the transcriptional level using gene-silencing zinc finger protein transcription factors (ZFP-TFs). Following a single administration of adeno-associated virus (AAV), either locally into the hippocampus or intravenously to enable whole-brain transduction, we selectively reduced tau messenger RNA and protein by 50 to 80% out to 11 months, the longest time point studied. Sustained tau lowering was achieved without detectable off-target effects, overt histopathological changes, or molecular alterations. Tau reduction with AAV ZFP-TFs was able to rescue neuronal damage around amyloid plaques in a mouse model of Alzheimer's disease (APP/PS1 line). The highly specific, durable, and controlled knockdown of endogenous tau makes AAV-delivered ZFP-TFs a promising approach for the treatment of tau-related human brain diseases.

Isoform-selective decrease of glycogen synthase kinase-3-beta (GSK-3ß) reduces synaptic tau phosphorylation, transcellular spreading, and aggregation.

It has been suggested that aberrant activation of glycogen synthase kinase-3-beta (GSK-3ß) can trigger abnormal tau hyperphosphorylation and aggregation, which ultimately leads to neuronal/synaptic damage and impaired cognition in Alzheimer disease (AD). We examined if isoform-selective partial reduction of GSK-3ß can decrease pathological tau changes, including hyperphosphorylation, aggregation, and spreading, in mice with localized human wild-type tau (hTau) expression in the brain. We used adeno-associated viruses (AAVs) to express hTau locally in the entorhinal cortex of wild-type and GSK-3ß hemi-knockout (GSK-3ß-HK) mice. GSK-3ß-HK mice had significantly less accumulation of hyperphosphorylated tau in synapses and showed a significant decrease of tau protein spread between neurons. In primary neuronal cultures from GSK-3ß-HK mice, the aggregation of exogenous FTD-mutant tau was also significantly reduced. These results show that a partial decrease of GSK-3ß significantly represses tau-initiated neurodegenerative changes in the brain, and therefore is a promising therapeutic target for AD and other tauopathies.

Synaptic and metabolic gene expression alterations in neurons that are recipients of proteopathic tau seeds.

Recent studies suggest that misfolded tau molecules can be released, and taken up by adjacent neurons, propagating proteopathic seeds across neural systems. Yet critical to understanding whether tau propagation is relevant in pathophysiology of disease would be to learn if it alters neuronal properties. We utilized high resolution multi-color in situ hybridization technology, RNAScope, in a well-established tau transgenic animal, and found that a subset of neurons in the cortex do not appear to express the transgene, but do develop phospho-tau positive inclusions, consistent with having received tau seeds. Recipient neurons show decreases in their expression of synaptophysin, CAMKIIα, and mouse tau in both young and old animals. These results contrast with neurons that develop tau aggregates and also overexpress the transgene, which have few changes in expression of metabolic and synaptic markers. Taken together, these results strongly suggest that tau propagation impacts neuronal functional integrity.

Tau molecular diversity contributes to clinical heterogeneity in Alzheimer's disease.

Alzheimer's disease (AD) causes unrelenting, progressive cognitive impairments, but its course is heterogeneous, with a broad range of rates of cognitive decline1. The spread of tau aggregates (neurofibrillary tangles) across the cerebral cortex parallels symptom severity2,3. We hypothesized that the kinetics of tau spread may vary if the properties of the propagating tau proteins vary across individuals. We carried out biochemical, biophysical, MS and both cell- and animal-based-bioactivity assays to characterize tau in 32 patients with AD. We found striking patient-to-patient heterogeneity in the hyperphosphorylated species of soluble, oligomeric, seed-competent tau. Tau seeding activity correlates with the aggressiveness of the clinical disease, and some post-translational modification (PTM) sites appear to be associated with both enhanced seeding activity and worse clinical outcomes, whereas others are not. These data suggest that different individuals with 'typical' AD may have distinct biochemical features of tau. These data are consistent with the possibility that individuals with AD, much like people with cancer, may have multiple molecular drivers of an otherwise common phenotype, and emphasize the potential for personalized therapeutic approaches for slowing clinical progression of AD.

The Alzheimer Disease-Causing Presenilin-1 L435F Mutation Causes Increased Production of Soluble Aß43 Species in Patient-Derived iPSC-Neurons, Closely Mimicking Matched Patient Brain Tissue.

Familial Alzheimer disease-causing mutations in Presenilin 1 (PSEN1) are generally thought to shift the processing of APP toward longer, more amyloidogenic Aß fragments. However, certain PSEN1 mutations cause severe reduction in gamma secretase function when expressed in the homozygous state, thus challenging the amyloid hypothesis. We sought to evaluate the effects of one such mutation, PSEN1 L435F, in more physiologic conditions and genetic contexts by using human induced pluripotent stem cell (iPSC)-derived neurons from an individual with familial AD (fAD) linked to the PSEN1 L435F mutation, and compared the biochemical phenotype of the iPS-derived neurons with brain tissue obtained at autopsy from the same patient. Our results demonstrate that in the endogenous heterozygous state, the PSEN1 L435F mutation causes a large increase in soluble Aß43 but does not change the overall levels of soluble Aß40 or Aß42 when compared with control iPSC-neurons. Increased pathologically phosphorylated tau species were also observed in PSEN1-mutant iPSC-neurons. Concordant changes in Aß species were present in autopsy brain tissue from the same patient. Finally, the feasibility of using Aß43 immunohistochemistry of brain tissue to identify fAD cases was evaluated in a limited autopsy case series with the finding that strong Aß43 staining occurred only in fAD cases.

Tau impairs neural circuits, dominating amyloid-ß effects, in Alzheimer models in vivo.

The coexistence of amyloid-ß (Aß) plaques and tau neurofibrillary tangles in the neocortex is linked to neural system failure and cognitive decline in Alzheimer's disease. However, the underlying neuronal mechanisms are unknown. By employing in vivo two-photon Ca2+ imaging of layer 2/3 cortical neurons in mice expressing human Aß and tau, we reveal a dramatic tau-dependent suppression of activity and silencing of many neurons, which dominates over Aß-dependent neuronal hyperactivity. We show that neurofibrillary tangles are neither sufficient nor required for the silencing, which instead is dependent on soluble tau. Surprisingly, although rapidly effective in tau mice, suppression of tau gene expression was much less effective in rescuing neuronal impairments in mice containing both Aß and tau. Together, our results reveal how Aß and tau synergize to impair the functional integrity of neural circuits in vivo and suggest a possible cellular explanation contributing to disappointing results from anti-Aß therapeutic trials.

A flow cytometry-based in vitro assay reveals that formation of apolipoprotein E (ApoE)-amyloid beta complexes depends on ApoE isoform and cell type.

Apolipoprotein E (ApoE) is a secreted apolipoprotein with three isoforms, E2, E3, and E4, that binds to lipids and facilitates their transport in the extracellular environment of the brain and the periphery. The E4 allele is a major genetic risk factor for the sporadic form of Alzheimer's disease (AD), and studies of human brain and mouse models have revealed that E4 significantly exacerbates the deposition of amyloid beta (Aß). It has been suggested that this deposition could be attributed to the formation of soluble ApoE isoform-specific ApoE-Aß complexes. However, previous studies have reported conflicting results regarding the directionality and strength of those interactions. In this study, using a series of flow cytometry assays that maintain the physiological integrity of ApoE-Aß complexes, we systematically assessed the association of Aß with ApoE2, E3, or E4. We used ApoE secreted from HEK cells or astrocytes overexpressing ApoE fused with a GFP tag. As a source of soluble Aß peptide, we used synthetic Aß40 or Aß42 or physiological Aß secreted from CHO cell lines overexpressing WT or V717F variant amyloid precursor protein (APP). We observed significant interactions between the different ApoE isoforms and Aß, with E4 interacting with Aß more strongly than the E2 and E3 isoforms. We also found subtle differences depending on the Aß type and the ApoE-producing cell type. In conclusion, these results indicate that the strength of the ApoE-Aß association depends on the source of Aß or ApoE.

CRISPR/Cas9 Mediated Disruption of the Swedish APP Allele as a Therapeutic Approach for Early-Onset Alzheimer's Disease.

The APPswe (Swedish) mutation in the amyloid precursor protein (APP) gene causes dominantly inherited Alzheimer's disease (AD) as a result of increased ß-secretase cleavage of the amyloid-ß (Aß) precursor protein. This leads to abnormally high Aß levels, not only in brain but also in peripheral tissues of mutation carriers. Here, we selectively disrupted the human mutant APPSW allele using CRISPR. By applying CRISPR/Cas9 from Streptococcus pyogenes, we generated allele-specific deletions of either APPSW or APPWT. As measured by ELISA, conditioned media of targeted patient-derived fibroblasts displayed an approximate 60% reduction in secreted Aß. Next, coding sequences for the APPSW-specific guide RNA (gRNA) and Cas9 were packaged into separate adeno-associated viral (AAV) vectors. Site-specific indel formation was achieved both in primary neurons isolated from APPSW transgenic mouse embryos (Tg2576) and after co-injection of these vectors into hippocampus of adult mice. Taken together, we here present proof-of-concept data that CRISPR/Cas9 can selectively disrupt the APPSW allele both ex vivo and in vivo-and thereby decrease pathogenic Aß. Hence, this system may have the potential to be developed as a tool for gene therapy against AD caused by APPswe and other point mutations associated with increased Aß.

Tau reduction in the presence of amyloid-ß prevents tau pathology and neuronal death in vivo.

Several studies have now supported the use of a tau lowering agent as a possible therapy in the treatment of tauopathy disorders, including Alzheimer's disease. In human Alzheimer's disease, however, concurrent amyloid-ß deposition appears to synergize and accelerate tau pathological changes. Thus far, tau reduction strategies that have been tested in vivo have been examined in the setting of tau pathology without confounding amyloid-ß deposition. To determine whether reducing total human tau expression in a transgenic model where there is concurrent amyloid-ß plaque formation can still reduce tau pathology and protect against neuronal loss, we have taken advantage of the regulatable tau transgene in APP/PS1 × rTg4510 mice. These mice develop both neurofibrillary tangles as well as amyloid-ß plaques throughout the cortex and hippocampus. By suppressing human tau expression for 6 months in the APP/PS1 × rTg4510 mice using doxycycline, AT8 tau pathology, bioactivity, and astrogliosis were reduced, though importantly to a lesser extent than lowering tau in the rTg4510 alone mice. Based on non-denaturing gels and proteinase K digestions, the remaining tau aggregates in the presence of amyloid-ß exhibit a longer-lived aggregate conformation. Nonetheless, lowering the expression of the human tau transgene was sufficient to equally ameliorate thioflavin-S positive tangles and prevent neuronal loss equally well in both the APP/PS1 × rTg4510 mice and the rTg4510 cohort. Together, these results suggest that, although amyloid-ß stabilizes tau aggregates, lowering total tau levels is still an effective strategy for the treatment of tau pathology and neuronal loss even in the presence of amyloid-ß deposition.

Synaptic Tau Seeding Precedes Tau Pathology in Human Alzheimer's Disease Brain.

Alzheimer's disease (AD) is defined by the presence of intraneuronal neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau aggregates as well as extracellular amyloid-beta plaques. The presence and spread of tau pathology through the brain is classified by Braak stages and thought to correlate with the progression of AD. Several in vitro and in vivo studies have examined the ability of tau pathology to move from one neuron to the next, suggesting a "prion-like" spread of tau aggregates may be an underlying cause of Braak tau staging in AD. Using the HEK293 TauRD-P301S-CFP/YFP expressing biosensor cells as a highly sensitive and specific tool to identify the presence of seed competent aggregated tau in brain lysate-i.e., tau aggregates that are capable of recruiting and misfolding monomeric tau-, we detected substantial tau seeding levels in the entorhinal cortex from human cases with only very rare NFTs, suggesting that soluble tau aggregates can exist prior to the development of overt tau pathology. We next looked at tau seeding levels in human brains of varying Braak stages along six regions of the Braak Tau Pathway. Tau seeding levels were detected not only in the brain regions impacted by pathology, but also in the subsequent non-pathology containing region along the Braak pathway. These data imply that pathogenic tau aggregates precede overt tau pathology in a manner that is consistent with transneuronal spread of tau aggregates. We then detected tau seeding in frontal white matter tracts and the optic nerve, two brain regions comprised of axons that contain little to no neuronal cell bodies, implying that tau aggregates can indeed traverse along axons. Finally, we isolated cytosolic and synaptosome fractions along the Braak Tau Pathway from brains of varying Braak stages. Phosphorylated and seed competent tau was significantly enriched in the synaptic fraction of brain regions that did not have extensive cellular tau pathology, further suggesting that aggregated tau seeds move through the human brain along synaptically connected neurons. Together, these data provide further evidence that the spread of tau aggregates through the human brain along synaptically connected networks results in the pathogenesis of human Alzheimer's disease.