Axonal Tubulin Research Articles

Overview of tau PET molecular imaging.

This article reviews tau PET imaging with an emphasis on first-generation and second-generation tau radiotracers and their application in neurodegenerative disorders, including Alzheimer's disease and non-Alzheimer's disease tauopathies. Tau is a critical protein, abundant in neurons within the central nervous system, which plays an important role in maintaining microtubules by binding to tubulin in axons. In its abnormal hyperphosphorylated form, accumulation of tau has been linked to a variety of neurodegenerative disorders, collectively referred to as tauopathies, which include Alzheimer's disease and non-Alzheimer's disease tauopathies [e.g., corticobasal degeneration (CBD), argyrophilic grain disease, progressive supranuclear palsy (PSP), and Pick's disease]. A number of first-generation and second-generation tau PET radiotracers have been developed, including the first FDA-approved agent [18F]-flortaucipir, which allow for in-vivo molecular imaging of underlying histopathology antemortem, ultimately guiding disease staging and development of disease-modifying therapeutics. Tau PET is an emerging imaging modality in the diagnosis and staging of tauopathies.

Astrocyte Structural and Molecular Response to Elevated Intraocular Pressure Occurs Rapidly and Precedes Axonal Tubulin Rearrangement within the Optic Nerve Head in a Rat Model.

Glaucomatous axon injury occurs at the level of the optic nerve head (ONH) in response to uncontrolled intraocular pressure (IOP). The temporal response of ONH astrocytes (glial cells responsible for axonal support) to elevated IOP remains unknown. Here, we evaluate the response of actin-based astrocyte extensions and integrin-based signaling within the ONH to 8 hours of IOP elevation in a rat model. IOP elevation of 60 mm Hg was achieved under isoflurane anesthesia using anterior chamber cannulation connected to a saline reservoir. ONH astrocytic extension orientation was significantly and regionally rearranged immediately after IOP elevation (inferior ONH, 43.2° ± 13.3° with respect to the anterior-posterior axis versus 84.1° ± 1.3° in controls, p<0.05), and re-orientated back to baseline orientation 1 day post IOP normalization. ONH axonal microtubule filament label intensity was significantly reduced 1 and 3 days post IOP normalization, and returned to control levels on day 5. Phosphorylated focal adhesion kinase (FAK) levels steadily decreased after IOP normalization, while levels of phosphorylated paxillin (a downstream target of FAK involved in focal adhesion dynamics) were significantly elevated 5 days post IOP normalization. The levels of phosphorylated cortactin (a downstream target of Src kinase involved in actin polymerization) were significantly elevated 1 and 3 days post IOP normalization and returned to control levels by day 5. No significant axon degeneration was noted by morphologic assessment up to 5 days post IOP normalization. Actin-based astrocyte structure and signaling within the ONH are significantly altered within hours after IOP elevation and prior to axonal cytoskeletal rearrangement, producing some responses that recover rapidly and others that persist for days despite IOP normalization.

Spatial pattern formation in microtubule post-translational modifications and the tight localization of motor-driven cargo

Microtubule (MT) "age" can be interpreted as nucleotide state, lattice defects, or post-translational modification (PTM) such as acetylation and detyrosination. In all three cases, these have been recently shown to have functionally-important effects on the dynamics of MT arrays, and can present spatial and temporal heterogeneity. While mathematical models for MT array densities are well-established, here we present equations describing MT age, defined as the mean time since the MT's building blocks (tubulin) were polymerized from their soluble dimer state. We derive the age equations using a mean first-passage time calculation and two complementary approaches: The continuum limit of spatial discretization model, and an adjoint operator approach. These equations can recapitulate the observation that the oldest (most de-tyrosinated) tubulin in axons is near the middle of axons during neuronal development in chick embryos. Furthermore, PTMs influence motor kinetics up to approximately twofold for off-rates and velocities. Our simulations demonstrate that this relatively weak dependence of motor kinetics is sufficient to target motor cargo to a specific location along the array. This localization is tightly peaked in a way that magnifies the relatively small signal of PTM spatial heterogeneity. Thus, MT age can produce long-range spatial patterning without feedbacks or diffusing signals.

Post-Translational Modification in Microtubule Arrays Exhibits Spatial Patterns that can act as a Signal to Tightly Localize Motor-Driven Cargo

Microtubule (MT) “age” has been interpreted through nucleotide state, lattice defects, and post-translational modification (PTM) such as acetylation and detyrosination. All three cases have been recently shown to have functionally-important effects on the dynamics of MT arrays, which can present spatial and temporal heterogeneity. While mathematical models for MT array densities are well-established, we present equations describing MT age, defined here as the mean time since the MT's building blocks (tubulin) were polymerized from their soluble dimer state. These equations can recapitulate the observation that the oldest (most acetylated) tubulin in axons is near the middle of axons during neuronal development. Furthermore, PTMs influence motor kinetics up to approximately 3-fold for off-rates and velocities. Our simulations demonstrate that this relatively weak dependence of motor kinetics is sufficient to target motor cargo to a specific location along the array. This localization is tightly peaked in a way that magnifies the relatively small signal of PTM spatial heterogeneity. Thus, MT age can produce long-range spatial patterning without feedbacks or diffusing signals.

Tubulin involvement in Bortezomib peripheral neurotoxicity

Axonal transport of mitochondria (Mt) controlled by specialized motor and docking proteins that distribute Mt throughout the axon where they provide energy for metabolic and synaptic activity is a vulnerable target in neuronal pathology (1). Bortezomib (BZ) is a proteasome inhibitor active in multiple myeloma (2). One of its key toxicities is painful peripheral neuropathy (BIPN), which frequently requires treatment discontinuation (3). BIPN is dose-related and predominantly sensory, resulting from axonal degeneration. Recent results indicate that BZ modifies axonal tubulin dynamic and we hypothesize that BZ alters fast axonal transport. Here we studied using time-lapse imaging the effect of different BZ concentration on axonal Mt transport in isolated dorsal root ganglion (DRG) neurons from adult male mice. We used kymograph to quantify the total number of Mt and to discriminate antero and retrogradely moving Mt from stationary Mt. Twenty-four hours of BZ treatment (0.1 to 15 µM) induced a dose-dependent reduction in Mt trafficking. Moreover, BZ had no impact on MT motion directions, but it induced a progressive reduction of both anterograde and retrograde axonal transport velocities. These events were associated with increase in tubulin polymerization and of MAP2 expression, but they occurred only after 72h of chronic BZ treatment. We have developed an in vitro model of BIPN demonstrating that transport impairment is already present before evident tubulin polymerization, suggesting that transport deficit represents an early stage of axonal dysfunction. Perpetuated transport dysfunction could impair distal organelle supply and play a critical role in advanced stages of BIPN.This work was supported by the University of Milan-Bicocca and University of Michigan research grants

Astrocyte processes label for filamentous actin and reorient early within the optic nerve head in a rat glaucoma model.

To determine if astrocyte processes label for actin and to quantify the orientation of astrocytic processes within the optic nerve head (ONH) in a rat glaucoma model. Chronic intraocular pressure (IOP) elevation was produced by episcleral hypertonic saline injection and tissues were collected after 5 weeks. For comparison, eyes with optic nerve transection were collected at 2 weeks. Fellow eyes served as controls. Axonal degeneration in retrobulbar optic nerves was graded on a scale of 1 to 5. Optic nerve head sections (n ≥ 4 eyes per group) were colabeled with phalloidin (actin marker) and antibodies to astrocytic glial fibrillary acidic protein and aquaporin 4, or axonal tubulin βIII. Confocal microscopy and FIJI software were used to quantify the orientation of actin bundles. Control ONHs showed stereotypically arranged actin bundles within astrocyte processes. Optic nerve head actin bundle orientation was nearly perpendicular to axons (82.9° ± 6.3° relative to axonal axis), unlike the retrobulbar optic nerve (45.4° ± 28.7°, P < 0.05). With IOP elevation, ONH actin bundle orientation became less perpendicular to axons, even in eyes with no perceivable axonal injury (i.e., 38.8° ± 15.1° in grade 1, P < 0.05 in comparison to control ONHs). With severe injury, ONH actin bundle orientation became more parallel to the axonal axis (24.1° ± 28.4°, P < 0.05 in comparison to control ONHs). Optic nerve head actin bundle orientation in transected optic nerves was unchanged. Actin labeling identifies fine astrocyte processes within the ONH. Optic nerve head astrocyte process reorientation occurs early in response to elevated IOP.

Stable Mossy Fiber Long-Term Potentiation Requires Calcium Influx at the Granule Cell Soma, Protein Synthesis, and Microtubule-Dependent Axonal Transport

The synapses formed by the mossy fiber (MF) axons of hippocampal dentate gyrus granule neurons onto CA3 pyramidal neurons exhibit an intriguing form of experience-dependent synaptic plasticity that is induced and expressed presynaptically. In contrast to most other CNS synapses, long-term potentiation (LTP) at the MF-CA3 synapse is readily induced even during blockade of postsynaptic glutamate receptors. Furthermore, blocking voltage-gated Ca(2+) channels prevents MF-LTP, supporting an involvement of presynaptic Ca(2+) signaling via voltage-gated Ca(2+) channels in MF-LTP induction. We examined the contribution of activity in both dentate granule cell somata and MF terminals to MF-LTP. We found that the induction of stable MF-LTP requires tetanization-induced action potentials not only at MF boutons, but also at dentate granule cell somata. Similarly, blocking Ca(2+) influx via voltage-gated Ca(2+) channels only at the granule cell soma was sufficient to disrupt MF-LTP. Finally, blocking protein synthesis or blocking fast axonal transport mechanisms via disruption of axonal tubulin filaments resulted in decremental MF-LTP. Collectively, these data suggest that-in addition to Ca(2+) influx at the MF terminals-induction of MF synaptic plasticity requires action potential-dependent Ca(2+) signaling at granule cell somata, protein synthesis, and fast axonal transport along MFs. A parsimonious interpretation of these results is that somatic activity triggers protein synthesis at the soma; newly synthesized proteins are then transported to MF terminals, where they contribute to the stabilization of MF-LTP. Finally, the present data imply that synaptic plasticity at the MF-CA3 synapse can be affected by local modulation of somatic and presynaptic Ca(2+) channel activity.

Neurofilaments Bind Tubulin and Modulate Its Polymerization

Neurofilaments assemble from three intermediate-filament proteins, contribute to the radial growth of axons, and are exceptionally stable. Microtubules are dynamic structures that assemble from tubulin dimers to support intracellular transport of molecules and organelles. We show here that neurofilaments, and other intermediate-filament proteins, contain motifs in their N-terminal domains that bind unassembled tubulin. Peptides containing such motifs inhibit the in vitro polymerization of microtubules and can be taken up by cultured cells in which they disrupt microtubules leading to altered cell shapes and an arrest of division. In transgenic mice in which neurofilaments are withheld from the axonal compartment, axonal tubulin accumulation is normal but microtubules assemble in excessive numbers. These observations suggest a model in which axonal neurofilaments modulate local microtubule assembly. This capacity also suggests novel mechanisms through which inherited or acquired disruptions in intermediate filaments might contribute to pathogenesis in multiple conditions.

Progressive Motor Neuronopathy: A Critical Role of the Tubulin Chaperone TBCE in Axonal Tubulin Routing from the Golgi Apparatus

Axonal degeneration represents one of the earliest pathological features in motor neuron diseases. We here studied the underlying molecular mechanisms in progressive motor neuronopathy (pmn) mice mutated in the tubulin-specific chaperone TBCE. We demonstrate that TBCE is a peripheral membrane-associated protein that accumulates at the Golgi apparatus. In pmn mice, TBCE is destabilized and disappears from the Golgi apparatus of motor neurons, and microtubules are lost in distal axons. The axonal microtubule loss proceeds retrogradely in parallel with the axonal dying back process. These degenerative changes are inhibited in a dose-dependent manner by transgenic TBCE complementation that restores TBCE expression at the Golgi apparatus. In cultured motor neurons, the pmn mutation, interference RNA-mediated TBCE depletion, and brefeldin A-mediated Golgi disruption all compromise axonal tubulin routing. We conclude that motor axons critically depend on axonal tubulin routing from the Golgi apparatus, a process that involves TBCE and possibly other tubulin chaperones.

Unloading kinesin transported cargoes from the tubulin track via the inflammatory c‐Jun N‐terminal kinase pathway

Axonal transport of mitochondria and synaptic vesicle precursors via kinesin motor proteins is essential to keep integrity of axons and synapses. Disturbance of axonal transport is an early sign of neuroinflammatory and neurodegenerative diseases. Treatment of cultured neurons by the inflammatory cytokine tumor necrosis factor-alpha (TNF) stimulated phosphorylation of c-Jun N-terminal kinase (JNK) in neurites. TNF treatment induced dissociation of the heavy chain kinesin family-5B (KIF5B) protein from tubulin in axons but not cell bodies as determined by lifetime-based Förster resonance energy transfer (FRET) analysis. Dissociation of KIF5B from tubulin after TNF treatment was dependent on JNK activity. Furthermore, TNF inhibited axonal transport of mitochondria and synaptophysin by reducing the mobile fraction via JNK. Thus, TNF produced by activated glial cells in inflammatory or degenerative neurological diseases acts on neurites by acting on the kinesin-tubulin complex and inhibits axonal mitochondria and synaptophysin transport via JNK.

Interactions Between Recombinant Conventional Squid Kinesin and Native Myosin-V

essential to axonal tubulin transport is lost or inhibited 2 days after axotomy. This inhibition is not due to the depletion of conventional kinesin. It could be due to the inactivation of conventional kinesin or to the inactivation or depletion of some other motor or factor essential to axonal tubulin transport. This model of protein transport in the squid giant axon may facilitate the identification of some of the factors essential to the control and maintenance of slow axonal transport. The author thanks James Galbraith and Thomas Reese for helpful discussions and comments. Literature Cited

Axonal transport of tubulin and actin.

Axonal transport is responsible for supplying the axonal processes with proteins that are synthesized in the cell body. Among the proteins that are moved by this mechanism are tubulin and actin, two major components of the cytoskeleton. Observation of the movement of metabolically labeled tubulin and actin in-vivo has demonstrated that tubulin and actin transport are reduced in various diseases and with age, but transport is increased during axonal growth and regeneration. These metabolic studies have also raised questions about the underlying mechanisms of slow axonal transport such as: what is the polymerization state of tubulin and actin during transport, what motors and tracks are responsible for their movement down the axon, and how are the transport motors coupled to tubulin and actin during transport? Since experiments using metabolically labeled tubulin and actin have not effectively addressed these questions, a variety of new in-vitro fluorescent microscopy techniques have been devised to investigate these questions. These fluorescent microscopy experiments have suggested that tubulin can be transported in the unpolymerized soluble state and that such transport of soluble tubulin relies on the presence of formed microtubule tracks. It is not yet known what motor or motors are responsible for tubulin or actin transport in axons or how such a motor(s) might be coupled to such an abundant soluble cargo.

19] Application of caged fluorescein-labeled tubulin to studies of microtubule dynamics and transport of tubulin molecules in axons

This chapter describes methods and techniques using caged fluorescein-labeled tubulin for investigations of the dynamics and transport of axonal tubulin molecules in neurons. In these studies, caged fluorescein-labeled tubulin is introduced into cultured neurons by microinjection and, after a recovery period that also allows for diffusion of the caged fluorescein tubulin throughout the cell, small segments of axons are exposed to a brief pulse of near-ultraviolet light. The caged protection groups on the fluorescein absorb these photons, and in a dark reaction, the ether bond that links the caged groups to the phenolic groups of fluorescein is cleaved, liberating highly fluorescent tubulin conjugates only in the irradiated segment. The fate of the photoactivated tubulin molecules was observed with low-light level fuorescence microscopy or with electron microscopy by staining with antibody directed against fluorescein.

Molecular determinants of paired helical filament assembly and its therapeutic implications in Alzheimer's disease

SummaryStudies of Alzheimer's disease have pointed to loss of the normal microtubule associated protein tau and accumulation of pathological paired helical filaments as strong discriminators for cognitive impairment. The most likely explanation is that the redistribution of tau protein into PHFs is associated with a failure of axonal transport in cortico-cortical association circuits through failure to maintain axonal tubulin in the polymerized state within pyramidal cells. Current understanding of and attempts to translate the key pathogenetic steps in tau pathology as they occur in the human brain are reviewed Although the phosphorylation hypothesis has dominated the application of molecular and cell biological methods, there are substantial difficulties with this account when it has been tested in clinico-pathological studies. The present review develops a more pragmatic view of PHF assembly, that it is in principle an amyloidosis, since more than 80% of the tau protein found PHFs is in the form of detergent insoluble protease-resistant polymers of molecules which have undergone substantial proteolytic processing in the course of polymerization. These molecules have also undergone a characteristic conformational shift in the course of polymerization, and acquire binding sites for planar dyes. The theoretical stance which best fits this data is that which has been proposed in the Prion diseases. A variety of factors may act to initiate a pathological conformational change which is then propagated because of the capacity of the modified form of the molecule to seed the polymerization of the normal form. Whatever the ultimate causes, the process can be shown to progress exponentially in the human brain. Pharmaceutical blockade of pathological redistribution of tau protein into PHFs would represent a desirable pharmaceutical target, since it is likely to represent a major component of the final common molecular substrate of clinical dementia of the Alzheimer-type.

Modulation of the axonal microtubule cytoskeleton by myelinating Schwann cells

The Trembler PNS myelin-deficient mutant mouse offers a unique model for the study of axon-glial interactions. Previous work in our laboratory on Trembler mouse sciatic nerve established that myelinating Schwann cells exert a profound effect on the underlying neuronal cytoskeleton. Demyelinated axon segments exhibited decreases in the rate of slow axonal transport, axonal caliber, and neurofilament phosphorylation, as well as increases in neurofilament density. The present study considers effects on the microtubule cytoskeleton. At least two aspects of the microtubule cytoskeleton in Trembler PNS axons were altered by demyelination. First, the stability of the Trembler axonal microtubule cytoskeleton is decreased, as measured by decreased levels of insoluble tubulin (Sahenk and Brady, 1987). Second, the composition and phosphorylation of axonal microtubule-associated proteins, including tau, MAP 1A, and MAP 1B, are changed in Trembler demyelinated nerves. Further, the fraction of axonal tubulin moving at slow component b rates was increased (de Waegh and Brady 1990, 1991). These results provide further evidence that cell-cell interactions between myelinating glia and their underlying axons extend beyond a structural role, actively influencing biochemical and physiological properties of the axon.

Early Effects of β,β′‐Iminodipropionitrile on Tubulin Solubility and Neurofilament Phosphorylation in the Axon

To elucidate the role of neurofilaments in microtubule stabilization in the axon, we studied the effects of beta,beta'-iminodipropionitrile (IDPN) on the solubility and transport of tubulin as well as neurofilament phosphorylation in the motor fibers of the rat sciatic nerve. IDPN is known to impair the axonal transport of neurofilaments, causing accumulation of neurofilaments in the proximal axon and segregation of neurofilaments to the peripheral axoplasm throughout the nerve. Administration of IDPN at various intervals after radioactive labeling of the spinal cord with L-[35S]methionine revealed that transport inhibition occurred all along the nerve within 1-2 days. Transport of cold-insoluble tubulin, which accounts for 50% of axonal tubulin, was also affected. A significant increase in the proportion of cold-soluble tubulin was observed, reaching a maximum at 3 days after IDPN treatment and returning to the control level in the following weeks. Preceding this change in tubulin solubility, a transient decrease in the phosphorylation level of the 200-kDa neurofilament protein was detected in the ventral root using phosphorylation-dependent antibodies. These early changes agreed in timing with the onset of segregation and transport inhibition, suggesting that interaction between neurofilaments and microtubules possibly regulated by phosphorylation plays a significant role in microtubule stabilization.

The cold stability of microtubules increases during axonal maturation

The development of cold-stable microtubules (MTs) was studied in maturing rat sciatic nerves. From 4 to 10 weeks of age, there was a large increase in the proportion of tubulin associated with stable MTs. A greater fraction of axonal tubulin than nonaxonal tubulin was associated with stable MTs. The labeled tubulin remaining behind the peak of slow axonal transport was more highly associated with stable MTs than tubulin in the peak itself. Immunoassay confirmed that the sciatic nerve contains a pool of stable MTs not identical to the peak of tubulin transport. The developmental increase in MT stability is not associated with any increase in the acetylation of tubulin or with alterations in the major MT-associated proteins. One aspect of maturation of the axonal cytoskeleton may involve deposition of tubulin into stable MTs that are either stationary or moving slowly with regard to the peak of transported tubulin.

Heterogeneity of cold-stable and cold-labile tubulin in axon- and soma-enriched portions of the adult mouse brain

Microtubules from the optic nerve (axonal tubulin) and lateral geniculate nucleus (cell tubulin) were separated by cold treatment and the cold-soluble fraction was purified in the presence of Taxol. Isoforms of cold-stable and cold-soluble tubulin were resolved by the use of high-resolution isoelectric focusing. The cold-soluble fraction of axonal tubulin has only 14 of the 20 isotypes seen in the same fraction of cell tubulin. The two cold-stable fractions have 20 isotypes but axonal tubulin has a specific pattern of isotypes 1, 2 and 5. Cold-stable fractions of both axonal and cell tubulin display the existence of an intensely stained α-isotype, isotype 7, which seems associated with the property of cold stability. Our results highly favor the hypothesis of a physiological role of the heterogeneity of tubulin in neuronal microtubules.

Axonal tubulin and microtubules: morphologic evidence for stable regions on axonal microtubules.

Biochemical studies indicate that axonal tubulin is composed of at least two distinct pools that differ in cold solubility and biochemical composition [Brady et al: J. Cell Biol. 99:1716-1724]. To determine the morphologic correlate of cold-insoluble tubulin, segments of rat optic nerves were exposed to a series of in vitro experimental conditions that affect microtubules (MTs), including cold, podophyllotoxin (PT), triflupromazine (TFP), and taxol, and then examined by electron microscopy. Longitudinal sections of control axons showed MTs oriented parallel to the long axis of the axons. Axons exposed to cold, PT, and TFP showed short segments of MTs in association with cytoskeletal disarray. Morphometric studies were used to distinguish between a simple malorientation of MTs (undulation or zigzags in their course) and the loss of labile segments of MTs, leaving the stable portions behind. The lengths of MT segments were measured in longitudinal sections, and the numbers of MTs were determined in the cross sections. All MT segment-length histograms showed a unimodal distribution. Cold and PT produced a simple shift of the control histogram to the shorter length MTs. In cross sections the numbers of MTs in cold- and PT-exposed axons were significantly decreased, indicating that the presence of short segments of MTs in the longitudinal plane of sections was due to a loss of portions of MTs. Taxol, an agent that promotes MT assembly, reversed the cold effect partially and resulted in increases in both MT segment length and number. These studies indicate that stable MT segments are portions of longer MTs containing both stable and labile regions. Furthermore, these findings are consistent with the hypothesis that cold-insoluble tubulin functions as a transportable MT-organizing complex in the axon.

Axonal transport of a subclass of tau proteins: evidence for the regional differentiation of microtubules in neurons.

Tubulin, the major constituent of microtubules, is anterogradely transported within the axon as part of slow component a (SCa; 0.2-1.0 mm/day). This raises the possibility that the microtubule-associated proteins (MAPs) may be transported at the same rate. To examine this question, the high molecular weight and tau MAPs obtained from whole brain preparations of microtubules were compared with the proteins of SCa in guinea pig retinal ganglion cell axons by using phosphocellulose chromatography and one- and two-dimensional polyacrylamide gel electrophoresis. Only two of the tau proteins were found to be cotransported with axonal tubulin, although four tau and two high molecular weight MAPs were synthesized in the retina. This result suggests either that the retinal ganglion cell synthesizes only those two tau proteins or that it synthesizes several of the MAPs, but commits to axonal transport just two of the tau proteins. In either case, these observations are consistent with the transport of intact microtubules and demonstrate that axonal microtubules represent a distinct subset of brain microtubules. Such a distinction may be related to unique properties of the axonal cytoskeleton.