Axonal Microtubules Research Articles

The structure of a Tau fragment bound to tubulin prompts new hypotheses on Tau mechanism and oligomerization

Abstract Tau is a protein involved in the regulation of axonal microtubules in neurons. In pathological conditions, it forms filamentous aggregates which are molecular markers of neurodegenerative diseases known as tauopathies. Structures of Tau in fibrils or bound to the microtubule have been reported. We present here a structure of a Tau construct comprising the PHF6 motif, an oligopeptide involved in Tau aggregation, as a complex with tubulin. This Tau fragment binds as a dimer to a new site which, when transposed to the microtubule, would correspond to a pore between protofilaments. These results raise new hypotheses on Tau-induced microtubule assembly and stabilization and on Tau oligomerization.

Structural Insights into the Role of the Proline Rich Region in Tau Function.

Tau is a microtubule–associated protein that plays an important role in modulating axonal microtubules in neurons. Intracellular tau aggregates are found in a broad class of disorders, including Alzheimer&#8242s disease, termed tauopathies. Tau is an intrinsically disordered protein, and its structural disorder appears to be critical to its microtubule-related functions. Tubulin binding sites are found in tau&#8242s proline-rich region (PRR), microtubule binding repeats (MTBR: R1–R4), and pseudo–repeat, R′. While many post-translational modifications have been identified on tau, phosphorylation sites, which both regulate tubulin dimer and microtubule interactions and are correlated with disease, cluster with high frequency within the PRR. Here, we use fluorescence correlation spectroscopy and structural mass spectrometry techniques to characterize the impact of phosphomimic mutations in the PRR on tubulin dimer binding and probe the structure of the PRR–tubulin dimer complex. We find that phosphomimics cumulatively diminish tubulin dimer binding and slow microtubule polymerization. Additionally, we map two ~15 residue regions of the PRR as primary tubulin dimer binding sites and propose a model in which PRR enhances lateral interactions between tubulin dimers, complementing the longitudinal interactions observed for MTBR. Together these measurements provide insight into the previously overlooked relevance of tau&#8242s PRR in functional interactions with tubulin.

Stretch triggers microtubule stabilization and MARCKS-dependent membrane incorporation in the shaft of embryonic axons

Neurons have a unique polarized nature that must adapt to environmental changes throughout their lifespan. During embryonic development, axon elongation is led by the growth cone,1 culminating in the formation of a presynaptic terminal. After synapses are formed, axons elongate in a growth cone-independent manner to accompany body growth while maintaining their ultrastructure and function.2,3,4,5,6 To further understand mechanical strains on the axon shaft, we developed a computer-controlled stretchable microfluidic platform compatible with multi-omics and live imaging. Our data show that sensory embryonic dorsal root ganglia (DRGs) neurons have high plasticity, with axon shaft microtubules decreasing polymerization rates, aligning with the direction of tension, and undergoing stabilization. Moreover, in embryonic DRGs, stretch triggers yes-associated protein (YAP) nuclear translocation, supporting its participation in the regulatory network that enables tension-driven axon growth. Other than cytoskeleton remodeling, stretch prompted MARCKS-dependent formation of plasmalemmal precursor vesicles (PPVs), resulting in new membrane incorporation throughout the axon shaft. In contrast, adolescent DRGs showed a less robust adaptation, with axonal microtubules being less responsive to stretch. Also, while adolescent DRGs were still amenable to strain-induced PPV formation at higher stretch rates, new membrane incorporation in the axon shaft failed to occur. In summary, we developed a new resource to study the biology of axon stretch growth. By unraveling cytoskeleton adaptation and membrane remodeling in the axon shaft of stretched neurons, we are moving forward in understanding axon growth.

Differential protection by nicotinamide in a mouse model of glaucoma DBA/2J revealed by second-harmonic generation microscopy.

Glaucoma is a blinding disease where the retinal ganglion cells and their axons degenerate. Degradation of axonal microtubules is thought to play a critical role in the pathogenesis, but the mechanism is unknown. Here we investigate whether microtubule disruption in glaucoma can be alleviated by metabolic rescue. The integrity of axonal microtubules and the morphology of the retinal nerve fibers were evaluated by second-harmonic generation microscopy in a mouse model of glaucoma, DBA/2J, which received a dietary supplement of nicotinamide (NAM) for reducing metabolic stress. It was compared with control DBA/2J, which did not receive NAM, and non-glaucomatous DBA/2J-Gpnmb+. We found that the morphology of the retinal nerve fibers, but not axonal microtubules, are significantly protected by NAM. The decoupling is analogous to microtubule deficit, a glaucoma pathology in which axonal microtubules exhibit rapid degradation compared to the morphology of the retinal nerve fibers. Understanding microtubule deficit could provide insights into the divergent responses to NAM. From co-registered images of second-harmonic generation and immunofluorescence, it was determined that microtubule deficit was not due to a shortage of tubulins. Furthermore, microtubule deficit colocalized with the sectors in which the retinal ganglion cells were disconnected from the brain, suggesting that microtubule disruption is associated with axonal transport deficit in glaucoma. Together, our data suggests significant role axonal microtubules play in glaucomatous degeneration, offering a new opportunity for neuroprotection.

TRIM46 Is Required for Microtubule Fasciculation In Vivo But Not Axon Specification or Axon Initial Segment Formation.

Vertebrate nervous systems use the axon initial segment (AIS) to initiate action potentials and maintain neuronal polarity. The microtubule-associated protein tripartite motif containing 46 (TRIM46) was reported to regulate axon specification, AIS assembly, and neuronal polarity through the bundling, or fasciculation, of microtubules in the proximal axon. However, these claims are based on TRIM46 knockdown in cultured neurons. To investigate TRIM46 function in vivo, we examined male and female TRIM46 knock-out mice. Contrary to previous reports, we find that TRIM46 is dispensable for axon specification and AIS formation. TRIM46 knock-out mice are viable, have normal behavior, and have normal brain structure. Thus, TRIM46 is not required for AIS formation, axon specification, or nervous system function. However, we confirm that TRIM46 is required for microtubule fasciculation. We also show TRIM46 enrichment in the first ∼100 μm of axon occurs independently of ankyrinG (AnkG) in vivo, although AnkG is required to restrict TRIM46 only to the AIS. Our results highlight the need for further investigation of the mechanisms by which the AIS and microtubules interact to shape neuronal structure and function.

Real-time detection of Tau-381 protein using liquid crystal-based sensors for Alzheimer's disease diagnosis

Tau is a protein found in the central nervous system (CNS) and is involved in stabilizing microtubules in axons. Given the link between Tau levels in the body and Alzheimer's disease (AD), there is a demand for straightforward and precise strategies to detect Tau in body fluids. In this study, we report liquid crystal (LC)-based sensors for the real-time detection of Tau protein, a well-known AD biomarker. The sensor uses a detection method based on the orientation change of the LC because of the competitive biomolecular interaction between Tau and Tau aptamers with the cationic polymer poly-L-lysine (PLL). Tau and its aptamers form stable complexes through electrostatic interactions. Owing to the consumption of the aptamer, the positively charged PLL fails to interact with the aptamer but binds to the negatively charged 1.2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) sodium salt (DOPG). The PLL and DOPG complex alters the orientation of the LC to ensure a planar anchoring of the 4-cyano-4′-pentylbiphenyl (5CB)/aqueous interface; this anchoring intensifies with increasing Tau concentration, thus enabling the observation of a bright optical image. Our LC-based sensor demonstrated a low detection limit of 2.77 pg/mL in phosphate buffered saline (PBS) and 10.86 pg/mL and 19.31 pg/mL in human serum and plasma, respectively. Moreover, it is anticipated to be suitable for point-of-care diagnosis of AD because it does not require specialized analytical equipment and only requires microliters of sample.

KLP-7/Kinesin-13 orchestrates axon-dendrite checkpoints for polarized trafficking in neurons.

The polarized nature of neurons depends on their microtubule dynamics and orientation determined by both microtubule-stabilizing and destabilizing factors. The role of destabilizing factors in developing and maintaining neuronal polarity is unclear. We investigated the function of KLP-7, a microtubule depolymerizing motor of the Kinesin-13 family, in axon-dendrite compartmentalization using PVD neurons in Caenorhabditis elegans. Loss of KLP-7 caused a mislocalization of axonal proteins, including RAB-3, SAD-1, and their motor UNC-104, to dendrites. This is rescued by cell-autonomous expression of the KLP-7 or colchicine treatment, indicating the involvement of KLP-7-dependent microtubule depolymerization. The high mobility of KLP-7 is correlated to increased microtubule dynamics in the dendrites, which restricts the enrichment of UNC-44, an integral component of Axon Initial Segment (AIS) in these processes. Due to the loss of KLP-7, ectopic enrichment of UNC-44 in the dendrite potentially redirects axonal traffic into dendrites that include plus-end out microtubules, axonal motors, and cargoes. These observations indicate that KLP-7-mediated depolymerization defines the microtubule dynamics conducive to the specific enrichment of AIS components in dendrites. This further compartmentalizes dendritic and axonal microtubules, motors, and cargoes, thereby influencing neuronal polarity.

Autophagy initiation triggers p150Glued–AP-2β interaction on the lysosomes and facilitates their transport

The endocytic adaptor protein 2 (AP-2) complex binds dynactin as part of its noncanonical function, which is necessary for dynein-driven autophagosome transport along microtubules in neuronal axons. The absence of this AP-2-dependent transport causes neuronal morphology simplification and neurodegeneration. The mechanisms that lead to formation of the AP-2-dynactin complex have not been studied to date. However, the inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) enhances the transport of newly formed autophagosomes by influencing the biogenesis and protein interactions of Rab-interacting lysosomal protein (RILP), another dynein cargo adaptor. We tested effects of mTORC1 inhibition on interactions between the AP-2 and dynactin complexes, with a focus on their two essential subunits, AP-2β and p150Glued. We found that the mTORC1 inhibitor rapamycin enhanced p150Glued–AP-2β complex formation in both neurons and non-neuronal cells. Additional analysis revealed that the p150Glued–AP-2β interaction was indirect and required integrity of the dynactin complex. In non-neuronal cells rapamycin-driven enhancement of the p150Glued–AP-2β interaction also required the presence of cytoplasmic linker protein 170 (CLIP-170), the activation of autophagy, and an undisturbed endolysosomal system. The rapamycin-dependent p150Glued–AP-2β interaction occurred on lysosomal-associated membrane protein 1 (Lamp-1)-positive organelles but without the need for autolysosome formation. Rapamycin treatment also increased the acidification and number of acidic organelles and increased speed of the long-distance retrograde movement of Lamp-1-positive organelles. Altogether, our results indicate that autophagy regulates the p150Glued–AP-2β interaction, possibly to coordinate sufficient motor-adaptor complex availability for effective lysosome transport.

Dual Modification of Tau by Pseudophosphorylation and Glycation Does Not Enhance Amorphous Aggregation

Background/Aims: The neurofibrillary tangles consisting of Tau protein are an important pathology in Alzheimer’s disease. The paired helical filaments of Tau form most of the NFTs. These PHFs of Tau are found to carry numerous post-translational modifications, which stabilize them and aid in aggregation. The mechanistic function of Tau is to bind and stabilize the axonal microtubules. Hyperphosphorylation of Tau causes it to compromise its physiological function and accumulate in the neurons in the form of aggregates. Such residue-specific phosphorylation has been studied by employing Tau pseudophosphorylation mutants. But in addition to phosphorylation, several other modifications also aid in stabilizing the Tau PHF. Glycation is one such non-enzymatic PTM caused by sugars and their reactive intermediates. In this study, we employed the pseudophosphorylated Tau double mutants (262/404D, 262/396D, and 231/262) for studying their modification by methyl glyoxal, a reactive intermediate of glucose metabolism. Methods: We studied various biophysical properties like aggregation propensity, Advanced glycation end-product formation, and global conformation of the Tau with dual modifications. Our study includes the use of in vitro techniques e.g., ThS fluorescence assay, electron microscopy, CD spectroscopy, SDS-PAGE. Results: The overall result of the study suggest that the MG-induced Tau aggregation is influenced by the residue-specific Tau phosphorylation. Conclusion: In conclusion, the combinatorial effect of discreet PTMs on Tau function could lead to a better understanding of Tauopathy.

Nano-pulling stimulates axon regeneration in dorsal root ganglia by inducing stabilization of axonal microtubules and activation of local translation.

Axonal plasticity is strongly related to neuronal development as well as regeneration. It was recently demonstrated that active mechanical tension, intended as an extrinsic factor, is a valid contribution to the modulation of axonal plasticity. In previous publications, our team validated a the "nano-pulling" method used to apply mechanical forces to developing axons of isolated primary neurons using magnetic nanoparticles (MNP) actuated by static magnetic fields. This method was found to promote axon growth and synaptic maturation. Here, we explore the use of nano-pulling as an extrinsic factor to promote axon regeneration in a neuronal tissue explant. Whole dorsal root ganglia (DRG) were thus dissected from a mouse spinal cord, incubated with MNPs, and then stretched. We found that particles were able to penetrate the ganglion and thus become localised both in the somas and in sprouting axons. Our results highlight that nano-pulling doubles the regeneration rate, and this is accompanied by an increase in the arborizing capacity of axons, an accumulation of cellular organelles related to mass addition (endoplasmic reticulum and mitochondria) and pre-synaptic proteins with respect to spontaneous regeneration. In line with the previous results on isolated hippocampal neurons, we observed that this process is coupled to an increase in the density of stable microtubules and activation of local translation. Our data demonstrate that nano-pulling enhances axon regeneration in whole spinal ganglia exposed to MNPs and external magnetic fields. These preliminary data represent an encouraging starting point for proposing nano-pulling as a biophysical tool for the design of novel therapies based on the use of force as an extrinsic factor for promoting nerve regeneration.

A simple active fluid model unites cytokinesis, cell crawling, and axonal outgrowth.

While the structural organization and molecular biology of neurons are well characterized, the physical process of axonal elongation remains elusive. The classic view posited elongation occurs through the deposition of cytoskeletal elements in the growth cone at the tip of a stationary array of microtubules. Yet, recent studies reveal axonal microtubules and docked organelles flow forward in bulk in the elongating axons of Aplysia, chick sensory, rat hippocampal, and Drosophila neurons. Noting that the morphology, molecular components, and subcellular flow patterns of growth cones strongly resemble the leading edge of migrating cells and the polar regions of dividing cells, our working hypothesis is that axonal elongation utilizes the same physical mechanisms that drive cell crawling and cell division. As a test of that hypothesis, here we take experimental data sets of sub-cellular flow patterns in cells undergoing cytokinesis, mesenchymal migration, amoeboid migration, neuronal migration, and axonal elongation. We then apply active fluid theory to develop a biophysical model that describes the different sub-cellular flow profiles across these forms of motility and how this generates cell motility under low Reynolds numbers. The modeling suggests that mechanisms for generating motion are shared across these processes, and differences arise through modifications of sub-cellular adhesion patterns and the profiles of internal force generation. Collectively, this work suggests that ameboid and mesenchymal cell crawling may have arisen from processes that first developed to support cell division, that growth cone motility and cell crawling are closely related, and that neuronal migration and axonal elongation are fundamentally similar, differing primarily in the motion and strength of adhesion under the cell body.

Tau “islands” affects axonal transport in vitro and in vivo

AbstractBackgroundTau is a protein abundantly expressed in neurons where it modulates the stability of axonal microtubules, thus contributing to the regulation of axonal transport of several organelles. Tau aggregates in a group of neurodegenerative diseases named tauopathies, which include frontotemporal dementia (FTD) and Alzheimer’s disease. Recent in vitro work has uncovered the existence of tau “islands”, a microtubule‐bound multimeric state of tau, which is distinct from pathological aggregates. However, whether these structures exist in intact neurons remains unclear.MethodWe investigated both cultured mouse neurons and mice in vivo, using immunoistochemistry, live imaging and two photon microscopy.Resultswe found that human tau showed regions of higher density along axons, reminiscent of tau islands. FTD‐linked mutations, known to increase pathological phosphorylation and aggregation of tau, induces larger islands, an effect that is reversed by inhibition of p38 MAPK, known to phosphorylate tau at multiple sites. Functionally, axonal transport of BDNF‐containing secretory granules is affected by FTD‐linked mutant tau as assessed both in vitro and in vivo by using a new assay based on two‐photon microscopy. Interestingly, this impairment occurred very early on, before overt tau aggregation. Inhibition of p38 MAPK was able to partially rescue the defects in axonal transport both in vitro and in vivo.ConclusionOur data suggests that tau island size regulates axonal transport, an effect dependent on tau phosphorylation. Inefficient organelles transport may have severe consequences on the activity and plasticity of neuronal circuits. The evidence that reducing tau phosphorylation by inhibiting p38 MAPK potentiated axonal transport points towards inhibition of p38 MAPK as a promising therapeutic strategy in tauopathies.

A new therapeutic approach to Tauopathies: targeting presynaptic tau with antisense oligonucleotides to Synaptogyrin‐3

AbstractBackgroundThe protein Tau is central to several neurodegenerative disorders (“tauopathies”). “Tau‐tangles” (aggregations of the Tau protein) are a key hallmark of Alzheimer’s and many other diseases and Tau‐protein is mutated in several forms of dementia. In all these conditions, Tau causes complex, multifactorial defects in neurons that lead to cognitive decline. Under normal conditions, Tau is attached to axonal microtubules, but under pathogenic conditions it detaches and spreads throughout the neuron, including synapses. Work from my lab indicated that when Tau is present at pre‐synaptic terminals it binds to and clusters synaptic vesicles and identified Synaptogyrin‐3 as a physical interactor of Tau. My lab demonstrated that the partial loss of Synaptogyrin‐3 (Syngr3) restores working memory and rescues synaptic degeneration in mice that express pathogenic P301S Tau (“PS19”), a well‐accepted model for tauopathy that recapitulates features seen in patients, including synaptic loss, neuroinflammation and cognitive decline. Partial (and complete) loss of Syngr3 is benign in mice and fruit flies. Based on this, we pursued a radical alternative and innovative approach, supported by strong pre‐clinical data, to tackle Tau‐induced cognitive decline.MethodWe designed and synthetized oligonucleotides covering the whole Syngr3 transcript, and tested their activity in Neuro2A cells and primary cortical murine neurons in transfection and unassisted uptake conditions.ResultThrough this screen we found several active molecules, that were able to significantly downregulate Syngr3 transcript and protein levels. To find the best candidates, we performed dose‐response curves and time course evaluations in primary murine neurons, and we found several molecules that targeted different regions of the Syngr3 transcript with good activity, potency, and no evident toxic effects.ConclusionWe found several molecules that induce an effective and sustained reduction of Syngr3 expression. As part of the pre‐clinical evaluation, these molecules will be tested in vivo in PK/PD and acute toxicity studies in WT mice, to determine the best administration dosage and therapeutic window. Finally, we will evaluate the effect of our best candidates in a therapeutic setting, treating PS19 tau mice to assess if the acute removal of the protein can alleviate presynaptic tau toxicity and revert tau induced synaptic and cognitive defects.

Harnessing axonal transport to map reward circuitry: Differing brain-wide projections from medial prefrontal cortical domains.

Neurons project long axons that contact other distant neurons. Neurons in the medial prefrontal cortex project into the limbic system to regulate responses to reward or threat. Diminished neural activity in prefrontal cortex is associated with loss of executive function leading to drug use, yet the specific circuitry that mediate these effects is unknown. Different regions within the medial prefrontal cortex may project to differing limbic system nuclei. Here, we exploited the cell biology of intracellular membrane trafficking, fast axonal transport, to map projections from two adjacent medial prefrontal cortical regions. We used Mn(II), a calcium analog, to trace medial prefrontal cortical projections in the living animal by magnetic resonance imaging (MRI). Mn(II), a contrast agent for MRI, enters neurons through voltage-activated calcium channels and relies on kinesin-1 and amyloid-precursor protein to transport out axons to distal destinations. Aqueous MnCl2 together with fluorescent dextran (3--5nL) was stereotactically injected precisely into two adjacent regions of the medial prefrontal cortex: anterior cingulate area (ACA) or infralimbic/prelimbic (IL/PL) region. Projections were traced, first live by manganese-enhanced MRI (MEMRI) at four time points in 3D, and then after fixation by microscopy. Data-driven unbiased voxel-wise statistical maps of aligned normalized MR images after either ACA or IL/PL injections revealed statistically significant progression of Mn(II) over time into deeper brain regions: dorsal striatum, globus pallidus, amygdala, hypothalamus, substantia nigra, dorsal raphe and locus coeruleus. Quantitative comparisons of these distal accumulations at 24h revealed dramatic differences between ACA and IL/PL injection groups throughout the limbic system, and most particularly in subdomains of the hypothalamus. ACA projections targeted dorsomedial nucleus of the hypothalamus, posterior part of the periventricular region and mammillary body nuclei as well as periaqueductal gray, while IL/PL projections accumulated in anterior hypothalamic areas and lateral hypothalamic nuclei as well as amygdala. As hypothalamic subsegments relay CNS activity to the body, our results suggest new concepts about mind-body relationships and specific roles of distinct yet adjacent medial prefrontal cortical segments. Our MR imaging strategy, when applied to follow other cell biological processes in the living organism, will undoubtedly lead to an expanded perspective on how minute details of cellular processes influence whole body health and wellbeing.

Slain2 attenuates brain injury following subarachnoid hemorrhage by controlling axonal microtubule structure in mice

Neuronal injury is accountable for the poor outcome of SAH patients. In this study, oxyhemoglobin (oxyHb) was used to treat cultured primary neurons to simulate SAH, while the SAH model was established by vascular puncture in mice. First, proteomics analysis and western blot assays showed Slain2 as an increased factor in neurons exposed to oxyHb treatment, which has been reported to play an important role in axonal development by regulating microtubule stability. Upregulation of neuronal Slain2 was also detected in the murine SAH model compared with sham surgery. In addition, there was no sex difference in the protein level of Slain2 in either the sham-operated or SAH groups. Furthermore, Slain2 overexpression rescued SAH-induced sensorimotor impairments in mice, while Slain2 knockdown had the opposite effect. Finally, Slain2 overexpression rescued SAH-induced axonal injury both in vivo and in vitro, which was exacerbated by Slain2 knockdown. Thus, we demonstrate here that Slain2 acts as an endogenous protective factor of neuronal axonal microtubule structure, which plays a key role in the protection against SAH-induced neuronal axonal injury. Facilitated axonal microtubule structure by Slain2 overexpression may reduce SAH-induced axonal injury and neurobehavioral dysfunction.

A microtubule stabilizer ameliorates protein pathogenesis and neurodegeneration in mouse models of repetitive traumatic brain injury.

Tau pathogenesis is a hallmark of many neurodegenerative diseases, including Alzheimer's disease (AD). Although the events leading to initial tau misfolding and subsequent tau spreading in patient brains are largely unknown, traumatic brain injury (TBI) may be a risk factor for tau-mediated neurodegeneration. Using a repetitive TBI (rTBI) paradigm, we report that rTBI induced somatic accumulation of phosphorylated and misfolded tau, as well as neurodegeneration across multiple brain areas in 7-month-old tau transgenic PS19 mice but not wild-type (WT) mice. rTBI accelerated somatic tau pathology in younger PS19 mice and WT mice only after inoculation with tau preformed fibrils and AD brain-derived pathological tau (AD-tau), respectively, suggesting that tau seeds are needed for rTBI-induced somatic tau pathology. rTBI further disrupted axonal microtubules and induced punctate tau and TAR DNA binding protein 43 (TDP-43) pathology in the optic tracts of WT mice. These changes in the optic tract were associated with a decline of visual function. Treatment with a brain-penetrant microtubule-stabilizing molecule reduced rTBI-induced tau, TDP-43 pathogenesis, and neurodegeneration in the optic tract as well as visual dysfunction. Treatment with the microtubule stabilizer also alleviated rTBI-induced tau pathology in the cortices of AD-tau-inoculated WT mice. These results indicate that rTBI facilitates abnormal microtubule organization, pathological tau formation, and neurodegeneration and suggest microtubule stabilization as a potential therapeutic avenue for TBI-induced neurodegeneration.

Why kiss-and-hop explains that tau does not stabilize microtubules and does not interfere with axonal transport (at physiological conditions).

Tau is a microtubule-associated protein that is enriched in the axonal process of neurons. Post-translational modifications of tau have been implicated in the development of tauopathies characterized by defects in axonal transport, neuronal atrophy, and microtubule disassembly. Although tau is almost quantitatively bound to microtubules under physiological conditions, it does not significantly affect axonal transport. Furthermore, acute or chronic tau deficiency does not result in significant destabilization of neuronal microtubules, challenging the classical view that disease-related tau modifications directly cause axonal microtubule collapse. Here, we discuss how the rapid interaction kinetics of the tau-microtubule interaction, which we previously termed the kiss-and-hop interaction, explains why tau does not affect microtubule-dependent axonal transport but still allows tau to modulate microtubule polymerization. In contrast, tau modifications that slow down the kinetics of the tau-microtubule interaction and increase the residence time of tau at a microtubule interaction site can disrupt axonal transport and cause dendritic atrophy. We discuss the consequences of such a gain-of-toxicity mechanism in terms of the development of disease-modulating drugs that target the tau protein.

Axonal transport during injury on a theoretical axon.

Neurodevelopment, plasticity, and cognition are integral with functional directional transport in neuronal axons that occurs along a unique network of discontinuous polar microtubule (MT) bundles. Axonopathies are caused by brain trauma and genetic diseases that perturb or disrupt the axon MT infrastructure and, with it, the dynamic interplay of motor proteins and cargo essential for axonal maintenance and neuronal signaling. The inability to visualize and quantify normal and altered nanoscale spatio-temporal dynamic transport events prevents a full mechanistic understanding of injury, disease progression, and recovery. To address this gap, we generated DyNAMO, a Dynamic Nanoscale Axonal MT Organization model, which is a biologically realistic theoretical axon framework. We use DyNAMO to experimentally simulate multi-kinesin traffic response to focused or distributed tractable injury parameters, which are MT network perturbations affecting MT lengths and multi-MT staggering. We track kinesins with different motility and processivity, as well as their influx rates, in-transit dissociation and reassociation from inter-MT reservoirs, progression, and quantify and spatially represent motor output ratios. DyNAMO demonstrates, in detail, the complex interplay of mixed motor types, crowding, kinesin off/on dissociation and reassociation, and injury consequences of forced intermingling. Stalled forward progression with different injury states is seen as persistent dynamicity of kinesins transiting between MTs and inter-MT reservoirs. DyNAMO analysis provides novel insights and quantification of axonal injury scenarios, including local injury-affected ATP levels, as well as relates these to influences on signaling outputs, including patterns of gating, waves, and pattern switching. The DyNAMO model significantly expands the network of heuristic and mathematical analysis of neuronal functions relevant to axonopathies, diagnostics, and treatment strategies.

Tau, microtubule dynamics, and axonal transport: New paradigms for neurodegenerative disease.

The etiology of Tauopathies, a diverse class of neurodegenerative diseases associated with the Microtubule Associated Protein (MAP) Tau, is usually described by a common mechanism in which Tau dysfunction results in the loss of axonal microtubule stability. Here, we reexamine and build upon the canonical disease model to encompass other Tau functions. In addition to regulating microtubule dynamics, Tau acts as a modulator of motor proteins, a signaling hub, and a scaffolding protein. This diverse array of functions is related to the dynamic nature of Tau isoform expression, post-translational modification (PTM), and conformational flexibility. Thus, there is no single mechanism that can describe Tau dysfunction. The effects of specific pathogenic mutations or aberrant PTMs need to be examined on all of the various functions of Tau in order to understand the unique etiology of each disease state.

Understanding the effect of tau protein phosphorylation on nucleosome array compaction.

Tau is an intrinsically disordered, microtubule associated protein that is mainly expressed in neurons and responsible for stabilizing microtubules in axons. The hyperphosphorylation of tau causes it to dissociate from microtubules which promotes its aggregation into neurofibrillary tangles (NFTs). This can significantly affect brain function. Tau is also found in the nucleus and plays an important role in DNA protection, chromosome stability, and heterochromatin regulation. Recent work has found a correlation between disease-relevant mutations of tau with heterochromatin relaxation, destabilization, and aberrant gene expression. Understanding how tau interacts with nucleosome arrays would give us insight into tau's role in gene expression and regulation. Here, we investigate the question of how the concentration and phosphorylation state of tau affects interactions with nucleosome arrays and their compaction state? We explored this question by using micrococcal nuclease digestion assays to determine the extent of tau's protection on nucleosome arrays. We found that an increase in wild type tau concentration leads to a dramatic increase in array protection but phosphorylated tau shows less dramatic protection patterns. These results provide insights into how tau might be involved in DNA protection and how this process might be disrupted in disease.