Propagation of Tau Misfolding from the Outside to the Inside of a Cell

Tauopathies are neurodegenerative diseases characterized by aggregation of the microtubule-associated protein Tau in neurons and glia. Although Tau is normally considered an intracellular protein, Tau aggregates are observed in the extracellular space, and Tau peptide is readily detected in the cerebrospinal fluid of patients. Tau aggregation occurs in many diseases, including Alzheimer disease and frontotemporal dementia. Tau pathology begins in discrete, disease-specific regions but eventually involves much larger areas of the brain. It is unknown how this propagation of Tau misfolding occurs. We hypothesize that extracellular Tau aggregates can transmit a misfolded state from the outside to the inside of a cell, similar to prions. Here we show that extracellular Tau aggregates, but not monomer, are taken up by cultured cells. Internalized Tau aggregates displace tubulin, co-localize with dextran, a marker of fluid-phase endocytosis, and induce fibrillization of intracellular full-length Tau. These intracellular fibrils are competent to seed fibril formation of recombinant Tau monomer in vitro. Finally, we observed that newly aggregated intracellular Tau transfers between co-cultured cells. Our data indicate that Tau aggregates can propagate a fibrillar, misfolded state from the outside to the inside of a cell. This may have important implications for understanding how protein misfolding spreads through the brains of tauopathy patients, and it is potentially relevant to myriad neurodegenerative diseases associated with protein misfolding.Tau filament deposition in Alzheimer disease (AD),² frontotemporal dementia (FTD), and other tauopathies correlates closely with cognitive dysfunction and cell death (1). Mutations in the tau gene cause autosomal dominant tauopathy, implicating Tau as the proximal cause (2–4). Specific disease phenotypes are defined by the early sites of pathology. For example, AD is characterized by memory loss that derives from involvement of hippocampal neurons, whereas FTD is characterized by personality changes that result from frontal lobe involvement (5). Pathology ultimately spreads to involve much larger regions of brain. Studies on patients with AD show a progressive, stereotyped spread of Tau deposits from the transentorhinal cortex to the hippocampus, and eventually to most cortical areas (6–8). Others have correlated the distribution of neurofibrillary tangles of Tau in AD brains with trans-synaptic distance from the affected areas (9). A similar spread affecting different subsets of neurons has been observed in other sporadic tauopathies, such as progressive supranuclear palsy (10). It is unknown why Tau misfolding progresses through the brain, whether it is a sequence of cell autonomous processes or whether a toxic factor is involved. Loss of synaptic connections and cell death may expose healthy cells to toxic factors and decrease available neurotrophins (11, 12). Another possibility is that the Tau protein itself serves as the agent of trans-cellular propagation. For example, it has been shown that extracellular Tau is toxic to cultured neuronal cells (13, 14). This is consistent with the observation that immunotherapy against Tau reduces pathology in a mouse model (15).Tau is well known as an intracellular protein that stabilizes microtubule filaments (16); however, it is readily detected in cerebrospinal fluid (17) and as extracellular aggregates, termed “ghost tangles,” in diseased brain. These are comprised predominantly of the microtubule-binding region (MTBR), the functional and pathogenic core of the Tau protein (18). We hypothesize that Tau aggregates present in the extracellular space enter naive cells and induce misfolding of intracellular Tau. We have tested this idea using cellular studies, biochemistry, and atomic force microscopy (AFM).     

Distinct Therapeutic Mechanisms of Tau Antibodies: PROMOTING MICROGLIAL CLEARANCE VERSUS BLOCKING NEURONAL UPTAKE*

Tauopathies are neurodegenerative diseases characterized by accumulation of Tau amyloids, and include Alzheimer disease and certain frontotemporal dementias. Trans-neuronal propagation of amyloid mediated by extracellular Tau may underlie disease progression. Consistent with this, active and passive vaccination studies in mouse models reduce pathology, although by unknown mechanisms. We previously reported that intracerebroventricular administration of three anti-Tau monoclonal antibodies (HJ8.5, HJ9.3, and HJ9.4) reduces pathology in a model overexpressing full-length mutant (P301S) human Tau. We now study effects of these three antibodies and a negative control antibody (HJ3.4) on Tau aggregate uptake into BV2 microglial-like cells and primary neurons. Antibody-independent Tau uptake into BV2 cells was blocked by heparin, consistent with a previously described role for heparan sulfate proteoglycans. Two therapeutic antibodies (HJ8.5 and HJ9.4) promoted uptake of full-length Tau fibrils into microglia via Fc receptors. Surprisingly, HJ9.3 promoted uptake of fibrils composed of the Tau repeat domain or Alzheimer disease-derived Tau aggregates, but failed to influence full-length recombinant Tau fibrils. Size fractionation of aggregates showed that antibodies preferentially promote uptake of larger oligomers (n ≥∼20-mer) versus smaller oligomers (n ∼10-mer) or monomer. No antibody inhibited uptake of full-length recombinant fibrils into primary neurons, but HJ9.3 blocked neuronal uptake of Tau repeat domain fibrils and Alzheimer disease-derived Tau. Antibodies thus have multiple potential mechanisms, including clearance via microglia and blockade of neuronal uptake. However these effects are epitope- and aggregate size-dependent. Establishing specific mechanisms of antibody activity in vitro may help in design and optimization of agents that are more effective in vivo.     

GPI anchoring facilitates propagation and spread of misfolded Sup35 aggregates in mammalian cells

Prion diseases differ from other amyloid‐associated protein misfolding diseases (e.g. Alzheimer's) because they are naturally transmitted between individuals and involve spread of protein aggregation between tissues. Factors underlying these features of prion diseases are poorly understood. Of all protein misfolding disorders, only prion diseases involve the misfolding of a glycosylphosphatidylinositol (GPI)‐anchored protein. To test whether GPI anchoring can modulate the propagation and spread of protein aggregates, a GPI‐anchored version of the amyloidogenic yeast protein Sup35NM (Sup35^GPI) was expressed in neuronal cells. Treatment of cells with Sup35NM fibrils induced the GPI anchor‐dependent formation of self‐propagating, detergent‐insoluble, protease‐resistant, prion‐like aggregates of Sup35^GPI. Live‐cell imaging showed intercellular spread of Sup35^GPI aggregation to involve contact between aggregate‐positive and aggregate‐negative cells and transfer of Sup35^GPI from aggregate‐positive cells. These data demonstrate GPI anchoring facilitates the propagation and spread of protein aggregation and thus may enhance the transmissibility and pathogenesis of prion diseases relative to other protein misfolding diseases.     

Prion-like propagation of cytosolic protein aggregates: Insights from cell culture models

Amyloid formation is a hallmark of several systemic and neurodegenerative diseases. Extracellular amyloid deposits or intracellular inclusions arise from the conformational transition of normally soluble proteins into highly ordered fibrillar aggregates. Amyloid fibrils are formed by nucleated polymerization, a process also shared by prions, proteinaceous infectious agents identified in mammals and fungi. Unlike so called non-infectious amyloids, the aggregation phenotype of prion proteins can be efficiently transmitted between cells and organisms. Recent discoveries in vivo now implicate that even disease-associated intracellular protein aggregates consisting of α-synuclein or Tau have the capacity to seed aggregation of homotypic native proteins and might propagate their amyloid states in a prion-like manner. Studies in tissue culture demonstrate that aggregation of diverse intracellular amyloidogenic proteins can be induced by exogenous fibrillar seeds. Still, a prerequisite for prion-like propagation is the fragmentation of proteinaceous aggregates into smaller seeds that can be transmitted to daughter cells. So far efficient propagation of the aggregation phenotype in the absence of exogenous seeds was only observed for a yeast prion domain expressed in tissue culture. Intrinsic properties of amyloidogenic protein aggregates and a suitable host environment likely determine if a protein polymer can propagate in a prion-like manner in the mammalian cytosol.Key words: prion, Sup35, huntingtin, polyglutamine, Tau, co-aggregation, amyloid, α-synuclein     

Amyloidogenesis of Tau protein

The role of microtubule‐associated protein Tau in neurodegeneration has been extensively investigated since the discovery of Tau amyloid aggregates in the brains of patients with Alzheimer's disease (AD). The process of formation of amyloid fibrils is known as amyloidogenesis and attracts much attention as a potential target in the prevention and treatment of neurodegenerative conditions linked to protein aggregation. Cerebral deposition of amyloid aggregates of Tau is observed not only in AD but also in numerous other tauopathies and prion diseases. Amyloidogenesis of intrinsically unstructured monomers of Tau can be triggered by mutations in the Tau gene, post‐translational modifications, or interactions with polyanionic molecules and aggregation‐prone proteins/peptides. The self‐assembly of amyloid fibrils of Tau shares a number of characteristic features with amyloidogenesis of other proteins involved in neurodegenerative diseases. For example, in vitro experiments have demonstrated that the nucleation phase, which is the rate‐limiting stage of Tau amyloidogenesis, is shortened in the presence of fragmented preformed Tau fibrils acting as aggregation templates (“seeds”). Accordingly, Tau aggregates released by tauopathy‐affected neurons can spread the neurodegenerative process in the brain through a prion‐like mechanism, originally described for the pathogenic form of prion protein. Moreover, Tau has been shown to form amyloid strains—structurally diverse self‐propagating aggregates of potentially various pathological effects, resembling in this respect prion strains. Here, we review the current literature on Tau aggregation and discuss mechanisms of propagation of Tau amyloid in the light of the prion‐like paradigm.     

Prion-like propagation of cytosolic protein aggregates

Amyloid formation is a hallmark of several systemic and neurodegenerative diseases. Extracellular amyloid deposits or intracellular inclusions arise from the conformational transition of normally soluble proteins into highly ordered fibrillar aggregates. Amyloid fibrils are formed by nucleated polymerization, a process also shared by prions, proteinaceous infectious agents identified in mammals and fungi. Unlike so called non-infectious amyloids, the aggregation phenotype of prion proteins can be efficiently transmitted between cells and organisms. Recent discoveries in vivo now implicate that even disease-associated intracellular protein aggregates consisting of α-synuclein or Tau have the capacity to seed aggregation of homotypic native proteins and might propagate their amyloid states in a prion-like manner. Studies in tissue culture demonstrate that aggregation of diverse intracellular amyloidogenic proteins can be induced by exogenous fibrillar seeds. Still, a prerequisite for prion-like propagation is the fragmentation of proteinaceous aggregates into smaller seeds that can be transmitted to daughter cells. So far efficient propagation of the aggregation phenotype in the absence of exogenous seeds was only observed for a yeast prion domain expressed in tissue culture. Intrinsic properties of amyloidogenic protein aggregates and a suitable host environment likely determine if a protein polymer can propagate in a prion-like manner in the mammalian cytosol.     

Remodeling of the conformational ensemble of the repeat domain of tau by an aggregation enhancer

Misfolding of the microtubule‐associated protein Tau is a hallmark of Alzheimer disease and several other neurodegenerative disorders. Because of the dynamic nature of the Tau protein, little is known about the changes in Tau structure that occur during misfolding. Here we studied the structural consequences upon binding of the repeat domain of Tau, which plays a key role in pathogenic aggregation, to an aggregation enhancer. By combining NMR experiments with molecular simulations we show that binding of the aggregation enhancer polyglutamic acid remodels the conformational ensemble of Tau. Our study thus provides insight into an early event during misfolding of Tau.     

Seeded Aggregation and Toxicity of α-Synuclein and Tau: CELLULAR MODELS OF NEURODEGENERATIVE DISEASES*

The deposition of amyloid-like filaments in the brain is the central event in the pathogenesis of neurodegenerative diseases. Here we report cellular models of intracytoplasmic inclusions of α-synuclein, generated by introducing nucleation seeds into SH-SY5Y cells with a transfection reagent. Upon introduction of preformed seeds into cells overexpressing α-synuclein, abundant, highly filamentous α-synuclein-positive inclusions, which are extensively phosphorylated and ubiquitinated and partially thioflavin-positive, were formed within the cells. SH-SY5Y cells that formed such inclusions underwent cell death, which was blocked by small molecular compounds that inhibit β-sheet formation. Similar seed-dependent aggregation was observed in cells expressing four-repeat Tau by introducing four-repeat Tau fibrils but not three-repeat Tau fibrils or α-synuclein fibrils. No aggregate formation was observed in cells overexpressing three-repeat Tau upon treatment with four-repeat Tau fibrils. Our cellular models thus provide evidence of nucleation-dependent and protein-specific polymerization of intracellular amyloid-like proteins in cultured cells.     

Tau Trimers Are the Minimal Propagation Unit Spontaneously Internalized to Seed Intracellular Aggregation

Tau amyloid assemblies propagate aggregation from the outside to the inside of a cell, which may mediate progression of the tauopathies. The critical size of Tau assemblies, or “seeds,” responsible for this activity is currently unknown, but this could be important for the design of effective therapies. We studied recombinant Tau repeat domain (RD) and Tau assemblies purified from Alzheimer disease (AD) brain composed largely of full-length Tau. Large RD fibrils were first sonicated to create a range of assembly sizes. We confirmed our ability to resolve stable assemblies ranging from n = 1 to >100 units of Tau using size exclusion chromatography, fluorescence correlation spectroscopy, cross-linking followed by Western blot, and mass spectrometry. All recombinant Tau assemblies bound heparan sulfate proteoglycans on the cell surface, which are required for Tau uptake and seeding, because they were equivalently sensitive to inhibition by heparin and chlorate. However, cells only internalized RD assemblies of n ≥ 3 units. We next analyzed Tau assemblies from AD or control brains. AD brains contained aggregated species, whereas normal brains had predominantly monomer, and no evidence of large assemblies. HEK293 cells and primary neurons spontaneously internalized Tau of n ≥ 3 units from AD brain in a heparin- and chlorate-sensitive manner. Only n ≥ 3-unit assemblies from AD brain spontaneously seeded intracellular Tau aggregation in HEK293 cells. These results indicate that a clear minimum size (n = 3) of Tau seed exists for spontaneous propagation of Tau aggregation from the outside to the inside of a cell, whereas many larger sizes of soluble aggregates trigger uptake and seeding.     

Extracellular Monomeric Tau Protein Is Sufficient to Initiate the Spread of Tau Protein Pathology

Understanding the formation and propagation of aggregates of the Alzheimer disease-associated Tau protein in vivo is vital for the development of therapeutics for this devastating disorder. Using our recently developed live-cell aggregation sensor in neuron-like cells, we demonstrate that different variants of exogenous monomeric Tau, namely full-length Tau (hTau40) and the Tau-derived construct K18 comprising the repeat domain, initially accumulate in endosomal compartments, where they form fibrillar seeds that subsequently induce the aggregation of endogenous Tau. Using superresolution imaging, we confirm that fibrils consisting of endogenous and exogenous Tau are released from cells and demonstrate their potential to spread Tau pathology. Our data indicate a greater pathological risk and potential toxicity than hitherto suspected for extracellular soluble Tau.     

血管生成素在淀粉样沉积症中发挥作用吗？

淀粉样沉积症是致命性的疾病,可以是神经退行性的,也可以是系统性的.该疾病以错误折叠蛋白质的堆积、缠绕成纤维为特征,最终导致受累组织、器官的渐进性坏死.目前,没有有效的治疗手段可以阻止该类疾病的进程.错误折叠蛋白质的累积诱导内质网应激,被认为是退行性疾病的标志.血管生成素不仅可以调节细胞生长和增殖,也在应激条件下细胞存活中发挥作用.最近,发现血管生成素介导的应激反应可以减轻蛋白聚积造成的损伤,提示该蛋白可能在退行性疾病中具有新功能.本综述概述了血管生成素在淀粉样沉积症中的研究进展,特别是描述了血管生成素失调与该类疾病的起始和进展间的关系.我们认为,深入了解血管生成素失调的分子基础有助于发展与蛋白质错误折叠和聚积相关的退行性疾病的治疗方法.     

Tau aggregates improve the Purinergic receptor P2Y12-associated podosome rearrangements in microglial cells

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is associated with protein misfolding, plaque accumulation, neuronal dysfunction, synaptic loss, and cognitive decline. The pathological cascade of AD includes the intracellular Tau hyperphosphorylation and its subsequent aggregation, extracellular Amyloid-β plaque formation and microglia-mediated neuroinflammation. The extracellular release of aggregated Tau is sensed by surveilling microglia through the involvement of various cell surface receptors. Among all, purinergic P2Y12R signaling is involved in microglial chemotaxis towards the damaged neurons. Microglial migration is highly linked with membrane-associated actin remodeling leading to the phagocytosis of extracellular Tau species. Here, we studied the formation of various actin structures such as podosome, lamellipodia and filopodia, in response to extracellular Tau monomers and aggregates. Microglial podosomes are colocalized with actin nucleator protein WASP, Arp2 and TKS5 adaptor protein during Tau-mediated migration. Moreover, the P2Y12 receptors were associated with F-actin-rich podosome structures, which signify the potential of Tau aggregates in microglial chemotaxis through the involvement of actin remodeling.     

Polyglutamine misfolding in yeast: Toxic and protective aggregation

Protein misfolding is associated with many human diseases, including neurodegenerative diseases, such as Alzheimer disease, Parkinson disease and Huntington disease. Protein misfolding often results in the formation of intracellular or extracellular inclusions or aggregates. Even though deciphering the role of these aggregates has been the object of intense research activity, their role in protein misfolding diseases is unclear. Here, I discuss the implications of studies on polyglutamine aggregation and toxicity in yeast and other model organisms. These studies provide an excellent experimental and conceptual paradigm that contributes to understanding the differences between toxic and protective trajectories of protein misfolding. Future studies like the ones discussed here have the potential to transform basic concepts of protein misfolding in human diseases and may thus help to identify new therapeutic strategies for their treatment.     

Prion-like Properties of Tau Protein: The Importance of Extracellular Tau as a Therapeutic Target

Work over the past 4 years indicates that multiple proteins associated with neurodegenerative diseases, especially Tau and α-synuclein, can propagate aggregates between cells in a prion-like manner. This means that once an aggregate is formed it can escape the cell of origin, contact a connected cell, enter the cell, and induce further aggregation via templated conformational change. The prion model predicts a key role for extracellular protein aggregates in mediating progression of disease. This suggests new therapeutic approaches based on blocking neuronal uptake of protein aggregates and promoting their clearance. This will likely include therapeutic antibodies or small molecules, both of which can be developed and optimized in vitro prior to preclinical studies.     

Fibrillar structure and charge determine the interaction of polyglutamine protein aggregates with the cell surface

The pathogenesis of most neurodegenerative diseases, including transmissible diseases like prion encephalopathy, inherited disorders like Huntington disease, and sporadic diseases like Alzheimer and Parkinson diseases, is intimately linked to the formation of fibrillar protein aggregates. It is becoming increasingly appreciated that prion-like intercellular transmission of protein aggregates can contribute to the stereotypical spread of disease pathology within the brain, but the mechanisms underlying the binding and uptake of protein aggregates by mammalian cells are largely uninvestigated. We have investigated the properties of polyglutamine (polyQ) aggregates that endow them with the ability to bind to mammalian cells in culture and the properties of the cell surface that facilitate such uptake. Binding and internalization of polyQ aggregates are common features of mammalian cells and depend upon both trypsin-sensitive and trypsin-resistant saturable sites on the cell surface, suggesting the involvement of cell surface proteins in this process. polyQ aggregate binding depends upon the presence of a fibrillar amyloid-like structure and does not depend upon electrostatic interaction of fibrils with the cell surface. Sequences in the huntingtin protein that flank the amyloid-forming polyQ tract also influence the extent to which aggregates are able to bind to cell surfaces.     

Tau Oligomers Impair Artificial Membrane Integrity and Cellular Viability

The microtubule-associated protein Tau is mainly expressed in neurons, where it binds and stabilizes microtubules. In Alzheimer disease and other tauopathies, Tau protein has a reduced affinity toward microtubules. As a consequence, Tau protein detaches from microtubules and eventually aggregates into β-sheet-containing filaments. The fibrillization of monomeric Tau to filaments is a multistep process that involves the formation of various aggregates, including spherical and protofibrillar oligomers. Previous concepts, primarily developed for Aβ and α-synuclein, propose these oligomeric intermediates as the primary cytotoxic species mediating their deleterious effects through membrane permeabilization. In the present study, we thus analyzed whether this concept can also be applied to Tau protein. To this end, viability and membrane integrity were assessed on SH-SY5Y neuroblastoma cells and artificial phospholipid vesicles, treated with Tau monomers, Tau aggregation intermediates, or Tau fibrils. Our findings suggest that oligomeric Tau aggregation intermediates are the most toxic compounds of Tau fibrillogenesis, which effectively decrease cell viability and increase phospholipid vesicle leakage. Our data integrate Tau protein into the class of amyloidogenic proteins and enforce the hypothesis of a common toxicity-mediating mechanism for amyloidogenic proteins.     

Polyglutamine misfolding in yeast: Toxic and protective aggregation

Protein misfolding is associated with many human diseases, including neurodegenerative diseases, such as Alzheimer disease, Parkinson disease and Huntington disease. Protein misfolding often results in the formation of intracellular or extracellular inclusions or aggregates. Even though deciphering the role of these aggregates has been the object of intense research activity, their role in protein misfolding diseases is unclear. Here, I discuss the implications of studies on polyglutamine aggregation and toxicity in yeast and other model organisms. These studies provide an excellent experimental and conceptual paradigm that contributes to understanding the differences between toxic and protective trajectories of protein misfolding. Future studies like the ones discussed here have the potential to transform basic concepts of protein misfolding in human diseases and may thus help to identify new therapeutic strategies for their treatment.Key words: polyglutamine proteins, neurodegeneration, aggresome, Huntington disease, yeast models     

Seeding of normal Tau by pathological Tau conformers drives pathogenesis of Alzheimer-like tangles

Neurofibrillary tangles (NFTs) in Alzheimer disease and related tauopathies are composed of insoluble hyperphosphorylated Tau protein, but the mechanisms underlying the conversion of highly soluble Tau into insoluble NFTs remain elusive. Here, we demonstrate that introduction of minute quantities of misfolded preformed Tau fibrils (Tau pffs) into Tau-expressing cells rapidly recruit large amounts of soluble Tau into filamentous inclusions resembling NFTs with unprecedented efficiency, suggesting a "seeding"-recruitment process as a highly plausible mechanism underlying NFT formation in vivo. Consistent with the emerging concept of prion-like transmissibility of disease-causing amyloidogenic proteins, we found that spontaneous uptake of Tau pffs into cells is likely mediated by endocytosis, suggesting a potential mechanism for the propagation of Tau lesions in tauopathy brains. Furthermore, sequestration of soluble Tau by pff-induced Tau aggregates attenuates microtubule overstabilization in Tau-expressing cells, supporting the hypothesis of a Tau loss-of-function toxicity in cells harboring NFTs. In summary, our study establishes a cellular system that robustly develops authentic NFT-like Tau aggregates, which provides mechanistic insights into NFT pathogenesis and a potential tool for identifying Tau-based therapeutics.     

Association of heat-shock proteins in various neurodegenerative disorders: is it a master key to open the therapeutic door?

A number of acute and chronic neurodegenerative disorders are caused due to misfolding and aggregation of many intra- and extracellular proteins. Protein misfolding and aggregation processes in cells are strongly regulated by cellular molecular chaperones known as heat-shock proteins (Hsps) that include Hsp60, Hsp70, Hsp40, and Hsp90. Recent studies have shown the evidences that Hsps are colocalized in protein aggregates in Alzheimer’s disease (AD), Parkinson’s disease (PD), Polyglutamine disease (PGD), Prion disease, and other neurodegenerative disorders. This fact indicates that Hsps might have attempted to prevent aggregate formation in cells and thus to suppress disease conditions. Experimental findings have already established in many cases that selective overexpression of Hsps like Hsp70 and Hsp40 prevented the disease progression in various animal models and cellular models. However, recently, various Hsp modulators like geldanamycin, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin, and celastrol have shown to up-regulate the expression level of Hsp70 and Hsp40, which in turn triggers the solubilization of diseased protein aggregates. Hsps are, therefore, if appropriately selected, an attractive choice for therapeutic targeting in various kinds of neurodegeneration and hence are expected to have strong potential as therapeutic agents in suppressing or curing AD, PD, PGD, and other devastative neurodegenerative disorders. In the present review, we report the experimental findings that describe the implication of Hsps in the development of neurodegeneration and explore the possibility of how Hsps can be used directly or as a target by other agents to prevent various neurodegeneration through preventing aggregation process and thus reducing the toxicity of the oligomers based on the previous reports.     

Conformation Determines the Seeding Potencies of Native and Recombinant Tau Aggregates

Intracellular Tau inclusions are a pathological hallmark of several neurodegenerative diseases, collectively known as the tauopathies. They include Alzheimer disease, tangle-only dementia, Pick disease, argyrophilic grain disease, chronic traumatic encephalopathy, progressive supranuclear palsy, and corticobasal degeneration. Tau pathology appears to spread through intercellular propagation, requiring the formation of assembled “prion-like” species. Several cell and animal models have been described that recapitulate aspects of this phenomenon. However, the molecular characteristics of seed-competent Tau remain unclear. Here, we have used a cell model to understand the relationships between Tau structure/phosphorylation and seeding by aggregated Tau species from the brains of mice transgenic for human mutant P301S Tau and full-length aggregated recombinant P301S Tau. Deletion of motifs ²⁷⁵VQIINK²⁸⁰ and ³⁰⁶VQIVYK³¹¹ abolished the seeding activity of recombinant full-length Tau, suggesting that its aggregation was necessary for seeding. We describe conformational differences between native and synthetic Tau aggregates that may account for the higher seeding activity of native assembled Tau. When added to aggregated Tau seeds from the brains of mice transgenic for P301S Tau, soluble recombinant Tau aggregated and acquired the molecular properties of aggregated Tau from transgenic mouse brain. We show that seeding is conferred by aggregated Tau that enters cells through macropinocytosis and seeds the assembly of endogenous Tau into filaments.