Recent experimental evidence suggests that transcellular propagation of fibrillar protein aggregates drives the progression of neurodegenerative diseases in a prion-like manner. This phenomenon is now well described in cell and animal models and involves the release of protein aggregates into the extracellular space. Free aggregates then enter neighboring cells to seed further fibrillization. The mechanism by which aggregated extracellular proteins such as tau and α-synuclein bind and enter cells to trigger intracellular fibril formation is unknown. Prior work indicates that prion protein aggregates bind heparan sulfate proteoglycans (HSPGs) on the cell surface to transmit pathologic processes. Here, we find that tau fibril uptake also occurs via HSPG binding. This is blocked in cultured cells and primary neurons by heparin, chlorate, heparinase, and genetic knockdown of a key HSPG synthetic enzyme, Ext1. Interference with tau binding to HSPGs prevents recombinant tau fibrils from inducing intracellular aggregation and blocks transcellular aggregate propagation. In vivo, a heparin mimetic, F6, blocks neuronal uptake of stereotactically injected tau fibrils. Finally, uptake and seeding by α-synuclein fibrils, but not huntingtin fibrils, occurs by the same mechanism as tau. This work suggests a unifying mechanism of cell uptake and propagation for tauopathy and synucleinopathy.neurodegeneration | Alzheimer's disease | prion-like mechanisms | macropinocytosis

Transcellular propagation of protein aggregates, or proteopathic seeds, may drive the progression of neurodegenerative diseases in a prion-like manner. In tauopathies such as Alzheimer's disease, this model predicts that tau seeds propagate pathology through the brain via cell-cell transfer in neural networks. The critical role of tau seeding activity is untested, however. It is unknown whether seeding anticipates and correlates with subsequent development of pathology as predicted for a causal agent. One major limitation has been the lack of a robust assay to measure proteopathic seeding activity in biological specimens. We engineered an ultrasensitive, specific, and facile FRET-based flow cytometry biosensor assay based on expression of tau or synuclein fusions to CFP and YFP, and confirmed its sensitivity and specificity to tau (∼300 fM) and synuclein (∼300 pM) fibrils. This assay readily discriminates Alzheimer's disease vs. Huntington's disease and aged control brains. We then carried out a detailed time-course study in P301S tauopathy mice, comparing seeding activity versus histological markers of tau pathology, including MC1, AT8, PG5, and Thioflavin S. We detected robust seeding activity at 1.5 mo, >1 mo before the earliest histopathological stain. Proteopathic tau seeding is thus an early and robust marker of tauopathy, suggesting a proximal role for tau seeds in neurodegeneration.amyloid | neuropathology | dementia | aging P rotein aggregation characterizes many neurodegenerative disorders, including Alzheimer's disease (AD) and the related tauopathies. These disorders feature the accumulation of fibrillar deposits of the microtubule-associated protein tau with progressive deterioration of the central nervous system. Tau pathology and its associated brain atrophy do not appear randomly throughout the brain, but rather progress along distinct neural networks (1-5). This aspect suggests a role for transcellular spread of a pathogenic agent via neural connections. Our laboratory and others have previously hypothesized that tau aggregates-or seeds-serve as this agent of spread, transmitting the aggregated state from cell to cell via prion-like mechanisms (6-15).Mounting fundamental insights support this hypothesis. Tau seeds applied to the outside of cells bind the cell surface by attaching to heparan sulfate proteoglycans, triggering uptake by macropinocytosis (13). Upon internalization, tau seeds nucleate the fibrillization of endogenous tau monomer via templated conformational change, or seeding (8, 10). Tau seeding requires a critical unit of size for activity, as only particular species propagate the aggregated state (16). In vivo studies have described tau protein spreading from local sites to distant regions, presumably via transsynaptic movement (11,12,(17)(18)(19). Finally, our laboratory and another recently demonstrated that tau propagates discrete amyloid conformations through the brains of animals that give rise to unique neuropathologies (18,20).Despite this evidence, it remains unclear wheth...

Background: Trans-cellular propagation of aggregation may be important in neurodegeneration, but mechanisms are unknown. Results: Tau fibrils are secreted into the extracellular space, where they directly trigger aggregation in recipient cells by contacting native protein. Conclusion:Trans-cellular movement of Tau fibrils seeds subsequent aggregation. Significance: Therapies that block trans-cellular movement, including antibodies, may have an important role in neurodegenerative diseases.

Accumulation of hyperphosphorylated tau directly correlates with cognitive decline in Alzheimer’s disease and other primary tauopathies. One therapeutic strategy may be to reduce total tau expression. We identified antisense oligonucleotides (ASOs) that selectively decreased human tau mRNA and protein in mice expressing mutant P301S human tau. Following reduction of human tau in this mouse model of tauopathy, fewer tau inclusions developed and pre-existing phosphorylated tau and thioflavin S pathology was reversed. The resolution of tau pathology was accompanied by the prevention of hippocampal volume loss, neuronal death, and nesting deficits. In addition, mouse survival was extended and pathological tau seeding was reversed. In non-human primates, tau ASOs distributed throughout the brain and spinal cord and reduced tau mRNA and protein in the brain, spinal cord, and cerebrospinal fluid. These data support investigation of a tau lowering therapy in human patients who have tau-positive inclusions, even after pathological tau deposition has begun.

Although tau is a cytoplasmic protein, it is also found in brain extracellular fluids, e.g., CSF. Recent findings suggest that aggregated tau can be transferred between cells and extracellular tau aggregates might mediate spread of tau pathology. Despite these data, details of whether tau is normally released into the brain interstitial fluid (ISF), its concentration in ISF in relation to CSF, and whether ISF tau is influenced by its aggregation are unknown. To address these issues, we developed a microdialysis technique to analyze monomeric ISF tau levels within the hippocampus of awake, freely moving mice. We detected tau in ISF of wild-type mice, suggesting that tau is released in the absence of neurodegeneration. ISF tau was significantly higher than CSF tau and their concentrations were not significantly correlated. Using P301S human tau transgenic mice (P301S tg mice), we found that ISF tau is fivefold higher than endogenous murine tau, consistent with its elevated levels of expression. However, following the onset of tau aggregation, monomeric ISF tau decreased markedly. Biochemical analysis demonstrated that soluble tau in brain homogenates decreased along with the deposition of insoluble tau. Tau fibrils injected into the hippocampus decreased ISF tau, suggesting that extracellular tau is in equilibrium with extracellular or intracellular tau aggregates. This technique should facilitate further studies of tau secretion, spread of tau pathology, the effects of different disease states on ISF tau, and the efficacy of experimental treatments.

Background:It is unknown what the minimum assembly of Tau is that can trigger cell uptake and seeding of intracellular aggregation. Results: Recombinant and AD-derived Tau assemblies were fractionated, and uptake and intracellular seeding activities were determined. Conclusion: Only Tau assemblies of n Ն 3 units trigger uptake and seeding. Significance: Definition of the minimal Tau propagation unit elucidates disease mechanisms for diagnosis and therapy.

Transcellular propagation of protein aggregate "seeds" has been proposed to mediate the progression of neurodegenerative diseases in tauopathies and α-synucleinopathies. We previously reported that tau and α-synuclein aggregates bind heparan sulfate proteoglycans (HSPGs) on the cell surface, promoting cellular uptake and intracellular seeding. However, the specificity and binding mode of these protein aggregates to HSPGs remain unknown. Here, we measured direct interaction with modified heparins to determine the size and sulfation requirements for tau, α-synuclein, and β-amyloid (Aβ) aggregate binding to glycosaminoglycans (GAGs). Varying the GAG length and sulfation patterns, we next conducted competition studies with heparin derivatives in cell-based assays. Tau aggregates required a precise GAG architecture with defined sulfate moieties in the - and 6--positions, whereas the binding of α-synuclein and Aβ aggregates was less stringent. To determine the genes required for aggregate uptake, we used CRISPR/Cas9 to individually knock out the major genes of the HSPG synthesis pathway in HEK293T cells. Knockouts of the extension enzymes exostosin 1 (), exostosin 2 (), and exostosin-like 3 (), as well as -sulfotransferase () or 6--sulfotransferase () significantly reduced tau uptake, consistent with our biochemical findings, and knockouts of , or but not reduced α-synuclein uptake. In summary, tau aggregates display specific interactions with HSPGs that depend on GAG length and sulfate moiety position, whereas α-synuclein and Aβ aggregates exhibit more flexible interactions with HSPGs. These principles may inform the development of mechanism-based therapies to block transcellular propagation of amyloid protein-based pathologies.

Parkinson's disease (PD) and multiple system atrophy (MSA) are distinct clinical syndromes characterized by the pathological accumulation of α-synuclein (α-syn) protein fibrils in neurons and glial cells. These disorders and other neurodegenerative diseases may progress via prion-like mechanisms. The prion model of propagation predicts the existence of “strains” that link pathological aggregate structure and neuropathology. Prion strains are aggregated conformers that stably propagate in vivo and cause disease with defined incubation times and patterns of neuropathology. Indeed, tau prions have been well defined, and research suggests that both α-syn and β-amyloid may also form strains. However, there is a lack of studies characterizing PD- versus MSA-derived α-syn strains or demonstrating stable propagation of these unique conformers between cells or animals. To fill this gap, we used an assay based on FRET that exploits a HEK293T “biosensor” cell line stably expressing α-syn (A53T)-CFP/YFP fusion proteins to detect α-syn seeds in brain extracts from PD and MSA patients. Both soluble and insoluble fractions of MSA extracts had robust seeding activity, whereas only the insoluble fractions of PD extracts displayed seeding activity. The morphology of MSA-seeded inclusions differed from PD-seeded inclusions. These differences persisted upon propagation of aggregation to second-generation biosensor cells. We conclude that PD and MSA feature α-syn conformers with very distinct biochemical properties that can be transmitted to α-syn monomers in a cell system. These findings are consistent with the idea that distinct α-syn strains underlie PD and MSA and offer possible directions for synucleinopathy diagnosis.