Brandon B. Holmes

Trans-cellular propagation of Tau aggregation by fibrillar species

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. Aggregation of the microtubule associated protein Tau is associated with several neurodegenerative disorders, including Alzheimer disease and frontotemporal dementia. In Alzheimer disease, Tau pathology spreads progressively throughout the brain, possibly along existing neural networks. However, it is still unclear how the propagation of Tau misfolding occurs. Intriguingly, in animal models, vaccine-based therapies have reduced Tau and synuclein pathology by uncertain mechanisms, given that these proteins are intracellular. We have previously speculated that trans-cellular propagation of misfolding could be mediated by a process similar to prion pathogenesis, in which fibrillar Tau aggregates spread pathology from cell to cell. However, there has been little evidence to demonstrate true trans-cellular propagation of Tau misfolding, in which Tau aggregates from one cell directly contact Tau protein in the recipient cell to trigger further aggregation. Here we have observed that intracellular Tau fibrils are directly released into the medium and then taken up by co-cultured cells. Internalized Tau aggregates induce fibrillization of intracellular Tau in these naive recipient cells via direct protein-protein contact that we demonstrate using FRET. Tau aggregation can be amplified across several generations of cells. An anti-Tau monoclonal antibody blocks Tau aggregate propagation by trapping fibrils in the extracellular space and preventing their uptake. Thus, propagation of Tau protein misfolding among cells can be mediated by release and subsequent uptake of fibrils that directly contact native protein in recipient cells. These results support the model of aggregate propagation by templated conformational change and suggest a mechanism for vaccine-based therapies in neurodegenerative diseases.

Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds

Significance Prion-like propagation of proteopathic seeds may underlie the progression of neurodegenerative diseases, including the tauopathies and synucleinopathies. Aggregate entry into the cell is a crucial step in transcellular propagation. We used chemical, enzymatic, and genetic methods to identify heparan sulfate proteoglycans as critical mediators of tau aggregate binding and uptake, and subsequent seeding of normal intracellular tau. This pathway mediates aggregate uptake in cultured cells, primary neurons, and brain. α-Synuclein fibrils use the same entry mechanism to seed intracellular aggregation, whereas huntingtin fibrils do not. This establishes the molecular basis for a key step in aggregate propagation. 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.

In Vivo Microdialysis Reveals Age-Dependent Decrease of Brain Interstitial Fluid Tau Levels in P301S Human Tau Transgenic Mice

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.

Proteopathic tau seeding predicts tauopathy in vivo

Significance Prion-like propagation of proteopathic seeds may underlie the progression of neurodegenerative diseases, including the tauopathies and synucleinopathies. We aimed to construct a versatile and simple cell assay to sensitively and specifically detect proteopathic seeding activity. Using a combination of FRET flow cytometry and a tau monoclonal FRET biosensor cell line, we report seed detection in the femtomolar range. This assay is easily applied to human brain homogenates and selectively responds to Alzheimers disease but not Huntingtons disease brains. By comparing seeding activity in a mouse model of human tauopathy, we demonstrate detection of proteopathic seeding far in advance of standard histopathological markers. Proteopathic seeding is thus an early marker of tauopathy, consistent with a causal role for tau seeds in neurodegeneration. 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. Huntingtons 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.

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.

Cellular mechanisms of protein aggregate propagation

PURPOSE OF REVIEW New research on the mechanisms of neurodegeneration highlights parallels between prion disease pathogenesis and other, more common disorders not typically thought to be infectious. This involves propagation of protein misfolding from cell to cell by templated conformational change. This review focuses on the cell biology that underlies propagation of protein aggregation between cells, including a discussion of protein biochemistry and relevant mouse models. RECENT FINDINGS Like the prion protein, several other proteins exhibit self-propagating fibrillar conformations in vitro. Multiple cellular studies have now implicated endocytic mechanisms in the uptake of aggregates into cells. Aggregates that enter cells somehow escape endocytic vesicles to contact cytosolic protein. The mechanism of release of protein monomers and aggregates from cells is not well understood. Animal models have confirmed that brain lysates and purified protein can accelerate brain pathology in a manner similar to prions. SUMMARY Aggregate flux in and out of cells likely contributes to the progression of neuropathology in neurodegenerative diseases. A better understanding of these mechanisms is emerging and can help explain local spread of protein aggregation and the role of neural networks in disease. This will also inform new therapeutic strategies aimed at blocking this process.

Tau reduction prevents neuronal loss and reverses pathological tau deposition and seeding in mice with tauopathy.

Antisense oligonucleotides that reduce tau prevent neuronal loss and reverse tau deposition and seeding in a mouse model of tauopathy. Stemming the tide of tauopathy Accumulation of the protein tau directly correlates with cognitive decline in Alzheimer’s disease and other primary tauopathies. One therapeutic option may be to reduce total tau. In a new study, DeVos et al. identified antisense oligonucleotides (ASOs) that decreased human tau in the brains of transgenic mice with tauopathy and observed the reversal of preexisting tau pathology and tau seeding activity. Further, neuronal loss was halted and mouse survival extended. In monkeys, tau ASOs reduced tau in the brain and cerebrospinal fluid. Together, these data support investigating lowering tau in human patients who have tau-positive inclusions even after pathological tau has begun to be deposited. 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. After reduction of human tau in this mouse model of tauopathy, fewer tau inclusions developed, and preexisting phosphorylated tau and Thioflavin S pathology were 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 nonhuman 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.

An N-terminal Nuclear Export Signal Regulates Trafficking and Aggregation of Huntingtin (Htt) Protein Exon 1

Background: Trafficking of huntingtin (Htt) fragments influences its toxicity. Results: A leucine-rich NES lies within the first 17 amino acids (N17) of Htt that controls subcellular localization and aggregation. Conclusion: The NES functions in cis and regulates the aggregation of Htt. Significance: This helps explain the mechanism of subcellular trafficking and aggregation of Htt fragments and may help elucidate molecular mechanisms of Htt toxicity. Huntington disease is a dominantly inherited neurodegenerative condition caused by polyglutamine expansion in the N terminus of the huntingtin protein (Htt). The first 17 amino acids (N17) of Htt play a key role in regulating its toxicity and aggregation. Both nuclear export and cytoplasm retention functions have been ascribed to N17. We have determined that N17 acts as a nuclear export sequence (NES) within Htt exon and when fused to yellow fluorescent protein. We have defined amino acids within N17 that constitute the nuclear export sequence (NES). Mutation of any of the conserved residues increases nuclear accumulation of Htt exon 1. Nuclear export of Htt is sensitive to leptomycin B and is reduced by knockdown of exportin 1. In HEK293 cells, NES mutations decrease overall Htt aggregation but increase the fraction of cells with nuclear inclusions. In primary cultured neurons, NES mutations increase nuclear accumulation and increase overall aggregation. This work defines a bona fide nuclear export sequence within N17 and links it to effects on protein aggregation. This may help explain the important role of N17 in controlling Htt toxicity.

Prions and Protein Assemblies that Convey Biological Information in Health and Disease

Prions derived from the prion protein (PrP) were first characterized as infectious agents that transmit pathology between individuals. However, the majority of cases of neurodegeneration caused by PrP prions occur sporadically. Proteins that self-assemble as cross-beta sheet amyloids are a defining pathological feature of infectious prion disorders and all major age-associated neurodegenerative diseases. In fact, multiple non-infectious proteins exhibit properties of template-driven self-assembly that are strikingly similar to PrP. Evidence suggests that like PrP, many proteins form aggregates that propagate between cells and convert cognate monomer into ordered assemblies. We now recognize that numerous proteins assemble into macromolecular complexes as part of normal physiology, some of which are self-amplifying. This review highlights similarities among infectious and non-infectious neurodegenerative diseases associated with prions, emphasizing the normal and pathogenic roles of higher-order protein assemblies. We propose that studies of the structural and cellular biology of pathological versus physiological aggregates will be mutually informative.

Macrophage migration inhibitory factor acts as a neurotrophin in the developing inner ear

This study is the first to demonstrate that macrophage migration inhibitory factor (MIF), an immune system ‘inflammatory’ cytokine that is released by the developing otocyst, plays a role in regulating early innervation of the mouse and chick inner ear. We demonstrate that MIF is a major bioactive component of the previously uncharacterized otocyst-derived factor, which directs initial neurite outgrowth from the statoacoustic ganglion (SAG) to the developing inner ear. Recombinant MIF acts as a neurotrophin in promoting both SAG directional neurite outgrowth and neuronal survival and is expressed in both the developing and mature inner ear of chick and mouse. A MIF receptor, CD74, is found on both embryonic SAG neurons and adult mouse spiral ganglion neurons. Mif knockout mice are hearing impaired and demonstrate altered innervation to the organ of Corti, as well as fewer sensory hair cells. Furthermore, mouse embryonic stem cells become neuron-like when exposed to picomolar levels of MIF, suggesting the general importance of this cytokine in neural development.