Quantitative live cell imaging of a tauopathy model enables the identification of a polypharmacological drug candidate that restores physiological microtubule interaction.

Tauopathies such as Alzheimer's disease are characterized by aggregation and increased phosphorylation of the microtubule-associated protein tau. Tau's pathological changes are closely linked to neurodegeneration, making tau a prime candidate for intervention. We developed an approach to monitor pathological changes of aggregation-prone human tau in living neurons. We identified 2-phenyloxazole (PHOX) derivatives as putative polypharmacological small molecules that interact with tau and modulate tau kinases. We found that PHOX15 inhibits tau aggregation, restores tau's physiological microtubule interaction, and reduces tau phosphorylation at disease-relevant sites. Molecular dynamics simulations highlight cryptic channel-like pockets crossing tau protofilaments and suggest that PHOX15 binding reduces the protofilament's ability to adopt a PHF-like conformation by modifying a key glycine triad. Our data demonstrate that live-cell imaging of a tauopathy model enables screening of compounds that modulate tau-microtubule interaction and allows identification of a promising polypharmacological drug candidate that simultaneously inhibits tau aggregation and reduces tau phosphorylation.

Super-resolution imaging and quantitative analysis of microtubule arrays in model neurons show that epothilone D increases the density but decreases the length and straightness of microtubules in axon-like processes.

Microtubules are essential for the development of neurons and the regulation of their structural plasticity. Microtubules also provide the structural basis for the long-distance transport of cargo. Various factors influence the organization and dynamics of neuronal microtubules, and disturbance of microtubule regulation is thought to play a central role in neurodegenerative diseases. However, imaging and quantitative assessment of the microtubule organization in the densely packed neuronal processes is challenging. The development of super-resolution techniques combined with the use of nanobodies offers new possibilities to visualize microtubules in neurites in high resolution. In combination with recently developed computational analysis tools, this allows automated quantification of neuronal microtubule organization with high precision. Here we have implemented three-dimensional DNA-PAINT (Point Accumulation in Nanoscale Topography), a single-molecule localization microscopy (SMLM) technique, which allows us to acquire 3D arrays of the microtubule lattice in axons of model neurons (neuronally differentiated PC12 cells) and dendrites of primary neurons. For the quantitative analysis of the microtubule organization, we used the open-source software package SMLM image filament extractor (SIFNE). We found that treatment with nanomolar concentrations of the microtubule-targeting drug epothilone D (EpoD) increased microtubule density in axon-like processes of model neurons and shifted the microtubule length distribution to shorter ones, with a mean microtubule length of 2.39 µm (without EpoD) and 1.98 µm (with EpoD). We also observed a significant decrease in microtubule straightness after EpoD treatment. The changes in microtubule density were consistent with live-cell imaging measurements of ensemble microtubule dynamics using a previously established Fluorescence Decay After Photoactivation (FDAP) assay. For comparison, we determined the organization of the microtubule array in dendrites of primary hippocampal neurons. We observed that dendritic microtubules have a very similar length distribution and straightness compared to microtubules in axon-like processes of a neuronal cell line. Our data show that super-resolution imaging of microtubules followed by algorithm-based image analysis represents a powerful tool to quantitatively assess changes in microtubule organization in neuronal processes, useful to determine the effect of microtubule-modulating conditions. We also provide evidence that the approach is robust and can be applied to neuronal cell lines or primary neurons, both after incorporation of labeled tubulin and by anti-tubulin antibody staining.

Annexins A2 and A6 interact with the extreme N terminus of tau and thereby contribute to tau's axonal localization.

During neuronal development, the microtubule-associated protein tau becomes enriched in the axon, where it remains concentrated in the healthy brain. In tauopathies such as Alzheimer's disease, tau redistributes from the axon to the somatodendritic compartment. However, the cellular mechanism that regulates tau's localization remains unclear. We report here that tau interacts with the Ca2+-regulated plasma membrane-binding protein annexin A2 (AnxA2) via tau's extreme N terminus encoded by the first exon (E1). Bioinformatics analysis identified two conserved eight-amino-acids-long motifs within E1 in mammals. Using a heterologous yeast system, we found that disease-related mutations and pseudophosphorylation of Tyr-18, located within E1 but outside of the two conserved regions, do not influence tau's interaction with AnxA2. We further observed that tau interacts with the core domain of AnxA2 in a Ca2+-induced open conformation and interacts also with AnxA6. Moreover, lack of E1 moderately increased tau's association rate to microtubules, consistent with the supposition that the presence of the tau-annexin interaction reduces the availability of tau to interact with microtubules. Of note, intracellular competition through overexpression of E1-containing constructs reduced tau's axonal enrichment in primary neurons. Our results suggest that the E1-mediated tau-annexin interaction contributes to the enrichment of tau in the axon and is involved in its redistribution in pathological conditions.

Microtubule dynamics and the neurodegenerative triad of Alzheimer's disease: The hidden connection.

Alzheimer's disease (AD) is the most common neurodegenerative disorder and is, on a histopathological level, characterized by the presence of extracellular amyloid plaques composed of the protein fragment Aß, and intracellular neurofibrillary tangles, which contain the microtubule-associated protein tau in a hyperphosphorylated state. In AD defects in microtubule (MT) assembly and organization have also been reported; however, it is unclear whether MT abnormalities have a causal and early role in the disease process or represent a common end point downstream of the neurodegenerative cascade. Recent evidence indicates that microtubule-stabilizing drugs prevent axonopathy in animal models of tauopathies and reverse Aß-induced loss of synaptic connectivity in an ex vivo model of amyloidosis. This could suggest that MT dysfunction connects some of the degenerative events and provides a useful target to simultaneously prevent several neurodegenerative processes in AD. Here, we describe how changes in the structure and dynamics of MTs are involved in the different aspects of the neurodegenerative triad of AD. We discuss evidence that MTs are affected both by tau-dependent and tau-independent mechanisms but appear to be regulated in a distinct way in different neuronal compartments. We argue that modulation of MT dynamics could be of potential benefit but needs to be precisely controlled in a cell and compartment-specific manner to avoid harmful side effects. This article is part of the series "Beyond Amyloid".

Presence of a carboxy-terminal pseudorepeat and disease-like pseudohyperphosphorylation critically influence tau's interaction with microtubules in axon-like processes.

A current challenge of cell biology is to investigate molecular interactions in subcellular compartments of living cells to overcome the artificial character of in vitro studies. To dissect the interaction of the neuronal microtubule (MT)-associated protein tau with MTs in axon-like processes, we used a refined fluorescence decay after photoactivation approach and single-molecule tracking. We found that isoform variation had only a minor influence on the tau-MT interaction, whereas the presence of a C-terminal pseudorepeat region (PRR) greatly increased MT binding by a greater-than-sixfold reduction of the dissociation rate. Bioinformatic analysis revealed that the PRR contained a highly conserved motif of 18 amino acids. Disease-associated tau mutations in the PRR (K369I, G389R) did not influence apparent MT binding but increased its dynamicity. Simulation of disease-like tau hyperphosphorylation dramatically diminished the tau-MT interaction by a greater-than-fivefold decrease of the association rate with no major change in the dissociation rate. Apparent binding of tau to MTs was similar in axons and dendrites but more sensitive to increased phosphorylation in axons. Our data indicate that under the conditions of high MT density that prevail in the axon, tau's MT binding and localization are crucially affected by the presence of the PRR and tau hyperphosphorylation.

Aß-mediated spine changes in the hippocampus are microtubule-dependent and can be reversed by a subnanomolar concentration of the microtubule-stabilizing agent epothilone D.

Dendritic spines represent the major postsynaptic input of excitatory synapses. Loss of spines and changes in their morphology correlate with cognitive impairment in Alzheimer's disease (AD) and are thought to occur early during pathology. Therapeutic intervention at a preclinical stage of AD to modify spine changes might thus be warranted. To follow the development and to potentially interfere with spine changes over time, we established a long term ex vivo model from organotypic cultures of the hippocampus from APP transgenic and control mice. The cultures exhibit spine loss in principal hippocampal neurons, which closely resembles the changes occurring in vivo, and spine morphology progressively changes from mushroom-shaped to stubby. We demonstrate that spine changes are completely reversed within few days after blocking amyloid-ß (Aß) production with the gamma-secretase inhibitor DAPT. We show that the microtubule disrupting drug nocodazole leads to spine loss similar to Aß expressing cultures and suppresses DAPT-mediated spine recovery in slices from APP transgenic mice. Finally, we report that epothilone D (EpoD) at a subnanomolar concentration, which slightly stabilizes microtubules in model neurons, completely reverses Aß-induced spine loss and increases thin spine density. Taken together the data indicate that Aß causes spine changes by microtubule destabilization and that spine recovery requires microtubule polymerization. Moreover, our results suggest that a low, subtoxic concentration of EpoD is sufficient to reduce spine loss during the preclinical stage of AD.

Identification of Nucleoside Analogs as Inducers of Neuronal Differentiation in a Human Reporter Cell Line and Adult Stem Cells.

Nucleoside analogs (NSAs) were among the first chemotherapeutic agents and could also be useful for the manipulation of cell fate. To investigate the potential of NSAs for the induction of neuronal differentiation, we developed a novel phenotypic assay based on a human neuron-committed teratocarcinoma cell line (NT2) as a model for neuronal progenitors and constructed a NT2-based reporter cell line that expressed eGFP under the control of a neuron-specific promoter. We tested 38 structurally related NSAs and determined their activity to induce neuronal differentiation by immunocytochemistry of neuronal marker proteins, live cell imaging, fluorometric detection and immunoblot analysis. We identified twelve NSAs, which induced neuronal differentiation to different extents. NSAs with highest activity carried a halogen substituent at their pyrimidine nucleobase and an unmodified or 2'-O-methyl substituted 2-deoxy-ß-D-ribofuranosyl residue as glyconic moiety. Cladribine, a purine nucleoside with similar structural features and in use to treat leukemia and multiple sclerosis, induced also differentiation of adult human neural crest-derived stem cells. Our results suggest that NSAs could be useful for the manipulation of neuronal cell fate in cell replacement therapy or treatment of neurodegenerative disorders. The data on the structure and function relationship will help to design compounds with increased activity and low toxicity.

Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43.

Phosphorylation and lipidation provide posttranslational mechanisms that contribute to the distribution of cytosolic proteins in growing nerve cells. The growth-associated protein GAP43 is susceptible to both phosphorylation and S-palmitoylation and is enriched in the tips of extending neurites. However, how phosphorylation and lipidation interplay to mediate sorting of GAP43 is unclear. Using a combination of biochemical, genetic, and imaging approaches, we show that palmitoylation is required for membrane association and that phosphorylation at Ser-41 directs palmitoylated GAP43 to the plasma membrane. Plasma membrane association decreased the diffusion constant fourfold in neuritic shafts. Sorting to the neuritic tip required palmitoylation and active transport and was increased by phosphorylation-mediated plasma membrane interaction. Vesicle tracking revealed transient association of a fraction of GAP43 with exocytic vesicles and motion at a fast axonal transport rate. Simulations confirmed that a combination of diffusion, dynamic plasma membrane interaction and active transport of a small fraction of GAP43 suffices for efficient sorting to growth cones. Our data demonstrate a complex interplay between phosphorylation and lipidation in mediating the localization of GAP43 in neuronal cells. Palmitoylation tags GAP43 for global sorting by piggybacking on exocytic vesicles, whereas phosphorylation locally regulates protein mobility and plasma membrane targeting of palmitoylated GAP43.

Single-molecule tracking of tau reveals fast kiss-and-hop interaction with microtubules in living neurons.

The microtubule-associated phosphoprotein tau regulates microtubule dynamics and is involved in neurodegenerative diseases collectively called tauopathies. It is generally believed that the vast majority of tau molecules decorate axonal microtubules, thereby stabilizing them. However, it is an open question how tau can regulate microtubule dynamics without impeding microtubule-dependent transport and how tau is also available for interactions other than those with microtubules. Here we address this apparent paradox by fast single-molecule tracking of tau in living neurons and Monte Carlo simulations of tau dynamics. We find that tau dwells on a single microtubule for an unexpectedly short time of ∼40 ms before it hops to the next. This dwell time is 100-fold shorter than previously reported by ensemble measurements. Furthermore, we observed by quantitative imaging using fluorescence decay after photoactivation recordings of photoactivatable GFP-tagged tubulin that, despite this rapid dynamics, tau is capable of regulating the tubulin-microtubule balance. This indicates that tau's dwell time on microtubules is sufficiently long to influence the lifetime of a tubulin subunit in a GTP cap. Our data imply a novel kiss-and-hop mechanism by which tau promotes neuronal microtubule assembly. The rapid kiss-and-hop interaction explains why tau, although binding to microtubules, does not interfere with axonal transport.

RNA protein granules modulate tau isoform expression and induce neuronal sprouting.

The neuronal microtubule-associated protein Tau is expressed in different variants, and changes in Tau isoform composition occur during development and disease. Here, we investigate a potential role of the multivalent tau mRNA-binding proteins G3BP1 and IMP1 in regulating neuronal tau expression. We demonstrate that G3BP1 and IMP1 expression induces the formation of structures, which qualify as neuronal ribonucleoprotein (RNP) granules and concentrate multivalent proteins and mRNA. We show that RNP granule formation leads to a >30-fold increase in the ratio of high molecular weight to low molecular weight tau mRNA and an ∼12-fold increase in high molecular weight to low molecular weight Tau protein. We report that RNP granule formation is associated with increased neurite formation and enhanced process growth. G3BP1 deletion constructs that do not induce granule formation are also deficient in inducing neuronal sprouting or changing the expression pattern of tau. The data indicate that granule formation driven by multivalent proteins modulates tau isoform expression and suggest a morphoregulatory function of RNP granules during health and disease.