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Using Live&#45;Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal ...

Michigan State University

Aggregation and accumulation of the microtubule-associated protein tau are associated with cognitive decline and neuronal degeneration in Alzheimer's disease and other tauopathies. Thus, preventing the transition of tau from a soluble state to insoluble aggregates and/or reversing the toxicity of existing aggregates would represent a reasonable therapeutic strategy for treating these neurodegenerative diseases. Here we demonstrate that molecular chaperones of the heat shock protein 70 (Hsp70) family are potent inhibitors of tau aggregation in vitro, preventing the formation of both mature fibrils and oligomeric intermediates. Remarkably, addition of Hsp70 to a mixture of oligomeric and fibrillar tau aggregates prevents the toxic effect of these tau species on fast axonal transport, a critical process for neuronal function. When incubated with preformed tau aggregates, Hsp70 preferentially associated with oligomeric over fibrillar tau, suggesting that prefibrillar oligomeric tau aggregates play a prominent role in tau toxicity. Taken together, our data provide a novel molecular basis for the protective effect of Hsp70 in tauopathies.

Heat shock protein 70 prevents both tau aggregation and the inhibitory effects of preexisting tau aggregates on fast axonal transport.

Dementias are among the most common neurological disorders, and Alzheimer's disease (AD) is the most common cause of dementia worldwide. AD remains a looming health crisis despite great efforts to learn the mechanisms surrounding the neuron dysfunction and neurodegeneration that accompanies AD primarily in the medial temporal lobe. In addition to AD, a group of diseases known as frontotemporal dementias (FTDs) are degenerative diseases involving atrophy and degeneration in the frontal and temporal lobe regions. Importantly, AD and a number of FTDs are collectively known as tauopathies due to the abundant accumulation of pathological tau inclusions in the brain. The precise role tau plays in disease pathogenesis remains an area of strong research focus. A critical component to effectively study any human disease is the availability of models that recapitulate key features of the disease. Accordingly, a number of animal models are currently being pursued to fill the current gaps in our knowledge of the causes of dementias and to develop effective therapeutics. Recent developments in gene therapy-based approaches, particularly in recombinant adeno-associated viruses (rAAVs), have provided new tools to study AD and other related neurodegenerative disorders. Additionally, gene therapy approaches have emerged as an intriguing possibility for treating these diseases in humans. This chapter explores the current state of rAAV models of AD and other dementias, discuss recent efforts to improve these models, and describe current and future possibilities in the use of rAAVs and other viruses in treatments of disease.

Gene Therapy Models of Alzheimer's Disease and Other Dementias.

Conformational changes involving the amino terminus of the tau protein are among the earliest alterations associated with tau pathology in Alzheimer's disease and other tauopathies. This region of tau contains a phosphatase-activating domain (PAD) that is aberrantly exposed in pathological forms of the protein, an event that is associated with disruptions in anterograde fast axonal transport. We utilized four antibodies that recognize the amino terminus of tau, TNT1, TNT2 (a novel antibody), Tau12, and Tau13, to further study this important region. Using scanning alanine mutations in recombinant tau proteins, we refined the epitopes of each antibody. We examined the antibodies' relative abilities to specifically label pathological tau in non-denaturing and denaturing assays to gain insight into some of the mechanistic details of PAD exposure. We then determined the pattern of tau pathology labeled by each antibody in human hippocampal sections at various disease stages in order to characterize PAD exposure in the context of disease progression. The characteristics of reactivity for the antibodies fell into two groups. TNT1 and TNT2 recognized epitopes within amino acids 7-12 and specifically identified recombinant tau aggregates and pathological tau from Alzheimer's disease brains in a conformation-dependent manner. These antibodies labeled early pre-tangle pathology from neurons in early Braak stages and colocalized with thiazine red, a marker of fibrillar pathology, in classic neurofibrillary tangles. However, late tangles were negative for TNT1 and TNT2 indicating a loss of the epitope in later stages of tangle evolution. In contrast, Tau12 and Tau13 both identified discontinuous epitopes in the amino terminus and were unable to differentiate between normal and pathological tau in biochemical and tissue immunohistological assays. Despite the close proximity of these epitopes, the antibodies demonstrated remarkably different abilities to identify pathological changes in tau indicating that detection of conformational alterations involving PAD exposure is not achieved by all N-terminal tau antibodies and that a relatively discrete region of the N-terminus (i.e., amino acids 7-12, the TNT1 and TNT2 epitope) is central to the differences between normal and pathological tau. The appearance of PAD in early tau pathology and its disappearance in late-stage tangles suggest that toxic forms of tau are associated with the earliest forms of tau deposits. Collectively, these findings demonstrate that the TNT antibodies are useful markers for early conformational display of PAD and provide information regarding conformational changes that have potential implications in the toxic mechanisms of tau pathology.

Pathological conformations involving the amino terminus of tau occur early in Alzheimer's disease and are differentially detected by monoclonal antibodies.

In Alzheimer's disease (AD), tau undergoes numerous modifications, including increased phosphorylation at serine-422 (pS422). In the human brain, pS422 tau protein is found in prodromal AD, correlates well with cognitive decline and neuropil thread pathology, and appears associated with increased oligomer formation and exposure of the N-terminal phosphatase-activating domain (PAD). However, whether S422 phosphorylation contributes to toxic mechanisms associated with disease-related forms of tau remains unknown. Here, we report that S422-pseudophosphorylated tau (S422E) lengthens the nucleation phase of aggregation without altering the extent of aggregation or the types of aggregates formed. When compared to unmodified tau aggregates, the S422E modification significantly increased the amount of SDS-stable tau dimers, despite similar levels of immunoreactivity with an oligomer-selective antibody (TOC1) and another antibody that reports PAD exposure (TNT1). Vesicle motility assays in isolated squid axoplasm further revealed that S422E tau monomers inhibited anterograde, kinesin-1 dependent fast axonal transport (FAT). Unexpectedly, and unlike unmodified tau aggregates, which selectively inhibit anterograde FAT, aggregates composed of S422E tau were found to inhibit both anterograde and retrograde FAT. Highlighting the relevance of these findings to human disease, pS422 tau was found to colocalize with tau oligomers and with a fraction of tau showing increased PAD exposure in the human AD brain. This study identifies novel effects of pS422 on tau biochemical properties, including prolonged nucleation and enhanced dimer formation, which correlate with a distinct inhibitory effect on FAT. Taken together, these findings identify a novel mechanistic basis by which pS422 confers upon tau a toxic effect that may directly contribute to axonal dysfunction in AD and other tauopathies.

Pseudophosphorylation of tau at S422 enhances SDS-stable dimer formation and impairs both anterograde and retrograde fast axonal transport.

Misfolded tau proteins are characteristic of tauopathies, but the isoform composition of tau inclusions varies by tauopathy. Using aggregates of the longest tau isoform (containing 4 microtubule-binding repeats and 4-repeat tau), we recently described a direct mechanism of toxicity that involves exposure of the N-terminal phosphatase-activating domain (PAD) in tau, which triggers a signaling pathway that disrupts axonal transport. However, the impact of aggregation on PAD exposure for other tau isoforms was unexplored. Here, results from immunochemical assays indicate that aggregation-induced increases in PAD exposure and oligomerization are common features among all tau isoforms. The extent of PAD exposure and oligomerization was larger for tau aggregates composed of 4-repeat isoforms compared with those made of 3-repeat isoforms. Most important, aggregates of all isoforms exhibited enough PAD exposure to significantly impair axonal transport in the squid axoplasm. We also show that PAD exposure and oligomerization represent common pathological characteristics in multiple tauopathies. Collectively, these results suggest a mechanism of toxicity common to each tau isoform that likely contributes to degeneration in different tauopathies.

Analysis of isoform-specific tau aggregates suggests a common toxic mechanism involving similar pathological conformations and axonal transport inhibition.

Pathological changes to the tau protein, including conformational changes and aggregation, are major hallmarks of a group of neurodegenerative disorders known as tauopathies. Among the conformational changes are alterations involving the extreme amino terminus of the protein, known as the phosphatase-activating domain (PAD). Aberrant PAD exposure induces a signaling cascade that leads to disruption of axonal transport, a critical function for neuronal survival. Conformational display of PAD is an early marker of pathological tau in Alzheimer disease (AD), but its role in other tauopathies has yet to be firmly established. We used a relatively novel N-terminal, conformation-sensitive antibody, TNT2, to determine whether misfolding in the amino terminus (ie, PAD exposure) occurs in non-AD tauopathies. We found that TNT2 specifically labeled pathological tau in post-mortem human brain tissue from Pick disease, progressive supranuclear palsy, corticobasal degeneration, and chronic traumatic encephalopathy, but did not label nonpathological, parenchymal tau. Tau13, another N-terminal antibody, was not sensitive to pathological N-terminal conformations. Tau13 did not readily distinguish between normal (ie, parenchymal tau) and pathological tau species and showed a range of effectiveness at identifying tau pathologies in the non-AD tauopathies. These findings demonstrate that the conformational display of the PAD in tau represents a common pathological event in many tauopathies.

Exposure of the Amino Terminus of Tau Is a Pathological Event in Multiple Tauopathies.

The pathological aggregation of the tau protein is a common characteristic of many neurodegenerative diseases. There is strong interest in characterizing the potentially toxic nature of tau oligomers. These nonfibrillar, soluble multimers appear to be more toxic than neurofibrillary tangles made up of filamentous tau. However, reliable production, purification, and verification of tau oligomers can provide certain challenges. Here, we provide a series of methods that address these issues. First, recombinant tau is produced using Escherichia coli, purified through affinity, size-exclusion, and anion-exchange chromatography steps and quantified using an SDS Lowry protein quantitation assay. Aggregation of tau is induced using arachidonic acid, and oligomers are purified by centrifugation over a sucrose step gradient. Finally, we describe a sandwich enzyme-linked immunosorbent assay that utilizes the tau oligomer-specific TOC1 antibody to confirm the presence of oligomeric tau. Together, these steps provide a very simple and reliable method for producing tau oligomers that can be used in downstream applications.

Production of recombinant tau oligomers in vitro.

Tauopathies are a diverse group of diseases featuring progressive dying-back neurodegeneration of specific neuronal populations in association with accumulation of abnormal forms of the microtubule-associated protein tau. It is well-established that the clinical symptoms characteristic of tauopathies correlate with deficits in synaptic function and neuritic connectivity early in the course of disease, but mechanisms underlying these critical pathogenic events are not fully understood. Biochemical  evidence fueled the widespread notion that microtubule stabilization represents tau's primary biological role and that the marked atrophy of neurites observed in tauopathies results from loss of microtubule stability. However, this notion contrasts with the mild phenotype associated with tau deletion. Instead, an analysis of cellular hallmarks common to different tauopathies, including aberrant patterns of protein phosphorylation and early degeneration of axons, suggests that alterations in kinase-based signaling pathways and deficits in axonal transport (AT) associated with such alterations contribute to the loss of neuronal connectivity triggered by pathogenic forms of tau. Here, we review a body of literature providing evidence that axonal pathology represents an early and common pathogenic event among human tauopathies. Observations of axonal degeneration in animal models of specific tauopathies are discussed and similarities to human disease highlighted. Finally, we discuss potential mechanistic pathways other than microtubule destabilization by which disease-related forms of tau may promote axonopathy.

Axonal Degeneration in Tauopathies: Disease Relevance and Underlying Mechanisms.

More than 50 different intronic and exonic autosomal dominant mutations in the tau gene have been linked to the neurodegenerative disorder frontotemporal dementia with Parkinsonism linked to chromosome-17 (FTDP-17). Although the pathological and clinical presentation of this disorder is heterogeneous among patients, the deposition of tau as pathological aggregates is a common feature. Collectively, FTDP-17 mutations have been shown to alter tau's ability to stabilize microtubules, enhance its aggregation, alter mRNA splicing, or induce its hyperphosphorylation, among other effects. Previous in vitro studies from our lab revealed that these mutations differ markedly from each other in the longest 2N4R isoform of tau. However, it is not entirely known whether the effect of a single mutation varies when compared between different isoforms of tau. Differences in the isoelectric points of the N-terminal region of tau isoforms lead to changes in their biochemical properties, raising the possibility that isoforms could also be disproportionately affected by disease-related mechanisms such as mutations. We therefore performed a comparative study of three FTDP-17 mutations present in different regions of tau (R5L, P301L, and R406W) in the three 4R isoforms of tau. We observed significant differences in the effect these mutations exert on the total amount and kinetics of aggregation, aggregate length distributions, and microtubule stabilizing propensity of 4R tau isoforms for all three selected mutants. These results demonstrate that different combinations of FTDP-17 mutations and tau isoforms are functionally distinct and could have important implications for our understanding of disease and animal models of tauopathies.

FTDP-17 Mutations Alter the Aggregation and Microtubule Stabilization Propensity of Tau in an Isoform-Specific Fashion.

Tau is a microtubule-associated protein that is involved in both normal and pathological processes in neurons. Since the discovery and characterization of tau over 40 years ago, our understanding of tau's normal functions and toxic roles in neurodegenerative tauopathies has continued to expand. Fast axonal transport is a critical process for maintaining axons and functioning synapses, critical subcellular compartments underlying neuronal connectivity. Signs of fast axonal transport disruption are pervasive in Alzheimer's disease and other tauopathies and various mechanisms have been proposed for regulation of fast axonal transport by tau. Post-translational modifications of tau including phosphorylation at specific sites, FTDP-17 point mutations, and oligomerization, confer upon tau a toxic effect on fast axonal transport. Consistent with the well-established dependence of axons on fast axonal transport, these disease-related modifications are closely associated temporally and spatially with axonal degeneration in the early disease stages. These factors position tau as a potentially critical factor mediating the disruption of fast axonal transport that precedes synaptic dysfunction and axonal degeneration at later disease stages. In this chapter, we review the evidence that tau affects fast axonal transport and examine several potential mechanisms proposed to underlie this toxicity.

Tau and Axonal Transport Misregulation in Tauopathies.

Formation of membrane-less organelles via liquid-liquid phase separation is one way cells meet the biological requirement for spatiotemporal regulation of cellular components and reactions. Recently, tau, a protein known for its involvement in Alzheimer's disease and other tauopathies, was found to undergo liquid-liquid phase separation making it one of several proteins associated with neurodegenerative diseases to do so. Here, we demonstrate that tau forms dynamic liquid droplets in vitro at physiological protein levels upon molecular crowding in buffers that resemble physiological conditions. Tau droplet formation is significantly enhanced by disease-associated modifications, including the AT8 phospho-epitope and the P301L tau mutation linked to an inherited tauopathy. Moreover, tau droplet dynamics are significantly reduced by these modified forms of tau. Extended phase separation promoted a time-dependent adoption of toxic conformations and oligomerization, but not filamentous aggregation. P301L tau protein showed the greatest oligomer formation following extended phase separation. These findings suggest that phase separation of tau may facilitate the formation of non-filamentous pathogenic tau conformations.

Liquid-liquid phase separation induces pathogenic tau conformations in vitro.

Over four decades ago,  experiments showed that tau protein interacts with and stabilizes microtubules in a phosphorylation-dependent manner. This observation fueled the widespread hypotheses that these properties extend to living neurons and that reduced stability of microtubules represents a major disease-driving event induced by pathological forms of tau in Alzheimer's disease and other tauopathies. Accordingly, most research efforts to date have addressed this protein as a substrate, focusing on evaluating how specific mutations, phosphorylation, and other post-translational modifications impact its microtubule-binding and stabilizing properties. In contrast, fewer efforts were made to illuminate potential mechanisms linking physiological and disease-related forms of tau to the normal and pathological regulation of kinases and phosphatases. Here, we discuss published work indicating that, through interactions with various kinases and phosphatases, tau may normally act as a scaffolding protein to regulate phosphorylation-based signaling pathways. Expanding on this concept, we also review experimental evidence linking disease-related tau species to the misregulation of these pathways. Collectively, the available evidence supports the participation of tau in multiple cellular processes sustaining neuronal and glial function through various mechanisms involving the scaffolding and regulation of selected kinases and phosphatases at discrete subcellular compartments. The notion that the repertoire of tau functions includes a role as a signaling hub should widen our interpretation of experimental results and increase our understanding of tau biology in normal and disease conditions.

Tau: A Signaling Hub Protein.

Pathologic tau modifications are characteristic of Alzheimer's disease and related dementias, but mechanisms of tau toxicity continue to be debated. Inherited mutations in tau cause early onset frontotemporal lobar dementias (FTLD-tau) and are commonly used to model mechanisms of tau toxicity in tauopathies. Previous work in the isolated squid axoplasm model demonstrated that several pathogenic forms of tau inhibit axonal transport through a mechanism involving activation of protein phosphatase 1 (PP1). Here, we determined that P301L and R5L FTLD mutant tau proteins elicit a toxic effect on axonal transport as monomeric proteins. We evaluated interactions of wild-type or mutant tau with specific PP1 isoforms (α, β, and γ) to examine how the interaction contributes to this toxic effect using primary rat hippocampal neurons from both sexes. Pull-down and bioluminescence resonance energy transfer experiments revealed selective interactions of wild-type tau with PP1α and PP1γ isoforms, but not PP1β, which were significantly increased by the P301L tau mutation. The results from proximity ligation assays confirmed the interaction in primary hippocampal neurons. Moreover, expression of FTLD-linked mutant tau in these neurons enhanced levels of active PP1, also increasing the pausing frequency of fluorescently labeled vesicles in both anterograde and retrograde directions. Knockdown of PP1γ, but not PP1α, rescued the cargo-pausing effects of P301L and R5L tau, a result replicated by deleting a phosphatase-activating domain in the amino terminus of P301L tau. These findings support a model of tau toxicity involving aberrant activation of a specific PP1γ-dependent pathway that disrupts axonal transport in neurons. Tau pathology is closely associated with neurodegeneration in Alzheimer's disease and other tauopathies, but the toxic mechanisms remain a debated topic. We previously proposed that pathologic tau forms induce dysfunction and degeneration through aberrant activation of a PP1-dependent pathway that disrupts axonal transport. Here, we show that tau directly interacts with specific PP1 isoforms, increasing levels of active PP1. Pathogenic tau mutations enhance this interaction, further increasing active PP1 levels and impairing axonal transport in isolated squid axoplasm and primary hippocampal neurons. Mutant-tau-mediated impairment of axonal transport was mediated by PP1γ and a phosphatase-activating domain located at the amino terminus of tau. This work has important implications for understanding and potentially mitigating tau-mediated neurotoxicity in tauopathies.

Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism.

Aging is the primary risk factor for Alzheimer's disease (AD) and related disorders (ADRDs). Tau aggregation is a hallmark of AD and other tauopathies. Even in normal aging, tau aggregation is found in brains, but in disease states, significantly more aggregated tau is present in brain regions demonstrating synaptic degeneration and neuronal loss. It is unclear how tau aggregation and aging interact to give rise to the phenotypes observed in disease states. Most AD/ADRD animal models have focused on late stages, after significant tau aggregation has occurred. There are fewer where we can observe the early aggregation events and progression during aging. In an attempt to address this gap, we created  models expressing a GFP-tagged version of the human tau protein. Here we examined how tau-gfp behaved during aging, comparing wild-type tau (hTau40), a disease-associated mutation (P301S), and an aggregation-prone variant (3PO). We measured age-dependent changes in GFP intensity and correlated those changes to normal aging in the nematode. We found differences in tau stability and accumulation depending on the tau variant expressed. hTau40GFP and P301SGFP were localized to axons and cell bodies, while 3POGFP was more concentrated within cell bodies. Expression of 3POGFP resulted in decreased lifespan and variations in locomotor rate, consistent with a pathological effect. Finally, we found that the human tau interacted genetically with the  ortholog of human tau,  where the loss of  significantly accelerated the time to death in animals expressing 3PO.

Age-dependent accumulation of tau aggregation in .

Our understanding of the biological functions of the tau protein now includes its role as a scaffolding protein involved in signaling regulation, which also has implications for tau-mediated dysfunction and degeneration in Alzheimer's disease and other tauopathies. Recently, we found that pseudophosphorylation at sites linked to the pathology-associated AT8 phosphoepitope of tau disrupts normal fast axonal transport through a protein phosphatase 1 (PP1)-dependent pathway in squid axoplasm. Activation of the pathway and the resulting transport deficits required tau's N-terminal phosphatase-activating domain (PAD) and PP1 but the connection between tau and PP1 was not well defined. Here, we studied functional interactions between tau and PP1 isoforms and their effects on axonal transport in mammalian neurons. First, we found that wild-type tau interacted with PP1α and PP1γ primarily through its microtubule-binding repeat domain. Pseudophosphorylation of tau at S199/S202/T205 (psTau) increased PAD exposure, enhanced interactions with PP1γ, and increased active PP1γ levels in mammalian cells. Expression of psTau also significantly impaired axonal transport in primary rat hippocampal neurons. Deletion of PAD in psTau significantly reduced the interaction with PP1γ, eliminated increases of active PP1γ levels, and rescued axonal transport impairment in neurons. These data suggest that a functional consequence of phosphorylation within S199-T205 in tau, which occurs in AD and several other tauopathies, may be aberrant interaction with and activation of PP1γ and subsequent axonal transport disruption in a PAD-dependent fashion.

Article	Year
Heat shock protein 70 prevents both tau aggregation and the inhibitory effects of preexisting tau aggregates on fast axonal transport. Biochemistry\| PubMed ID: 22039833	2011
Gene Therapy Models of Alzheimer's Disease and Other Dementias. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 26611599	2016
Pathological conformations involving the amino terminus of tau occur early in Alzheimer's disease and are differentially detected by monoclonal antibodies. Neurobiology of disease\| PubMed ID: 27260838	2016
Pseudophosphorylation of tau at S422 enhances SDS-stable dimer formation and impairs both anterograde and retrograde fast axonal transport. Experimental neurology\| PubMed ID: 27373205	2016
Analysis of isoform-specific tau aggregates suggests a common toxic mechanism involving similar pathological conformations and axonal transport inhibition. Neurobiology of aging\| PubMed ID: 27574109	2016
Exposure of the Amino Terminus of Tau Is a Pathological Event in Multiple Tauopathies. The American journal of pathology\| PubMed ID: 28413156	2017
Production of recombinant tau oligomers in vitro. Methods in cell biology\| PubMed ID: 28882311	2017
Axonal Degeneration in Tauopathies: Disease Relevance and Underlying Mechanisms. Frontiers in neuroscience\| PubMed ID: 29089864	2017
FTDP-17 Mutations Alter the Aggregation and Microtubule Stabilization Propensity of Tau in an Isoform-Specific Fashion. Biochemistry\| PubMed ID: 30562452	2019
Tau and Axonal Transport Misregulation in Tauopathies. Advances in experimental medicine and biology\| PubMed ID: 32096030	2019
Liquid-liquid phase separation induces pathogenic tau conformations in vitro. Nature communications\| PubMed ID: 32499559	2020
Tau: A Signaling Hub Protein. Frontiers in molecular neuroscience\| PubMed ID: 33815057	2021
Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 34607969	2021
Age-dependent accumulation of tau aggregation in . Frontiers in aging\| PubMed ID: 36062211	2022
Phosphomimetics at Ser199/Ser202/Thr205 in Tau Impairs Axonal Transport in Rat Hippocampal Neurons. Molecular neurobiology\| PubMed ID: 36859689	2023

Benjamin Combs

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