Tau phosphorylation and PAD exposure in regulation of axonal growth.

Introduction: Tau is a microtubule associated phosphoprotein found principally in neurons. Prevailing dogma continues to define microtubule stabilization as the major function of tau in vivo, despite several lines of evidence suggesting this is not the case. Most importantly, tau null mice have deficits in axonal outgrowth and neuronal migration while still possessing an extensive microtubule network. Instead, mounting evidence suggests that tau may have a major function in the regulation of fast axonal transport (FAT) through activation of neuronal signaling pathways. Previous studies identified a phosphatase activating domain (PAD) at the tau N-terminal that is normally sequestered, but is constitutively exposed in tauopathies. When exposed, the PAD activates a signaling cascade involving PP1 and GSK3ß which affects cellular functions including release of cargo from kinesin. Furthermore, we discovered that PAD exposure can be regulated by a single phosphorylation at T205. Exposure of the PAD is an early event in multiple tauopathies and a major contributing factor to neurodegeneration associated with tau hyperphosphorylation. However, effects of tau PAD exposure on anterograde FAT raised the interesting possibility that this pathway may be a mechanism for physiological regulation of cargo delivery through site-specific phosphorylation of tau and transient activation of PP1 and GSK3ß. Significantly, there is already evidence of local control of PP1 and GSK3ß at sites which require cargo delivery. Methods: To investigate this hypothesis, first we evaluated cellular localization of tau PAD exposure, pT205 tau phosphorylation, and active GSK3ß in primary hippocampal neurons during development. Second, we analyzed the axonal outgrowth of tau knockout neurons following transfection with full length hTau40-WT, hTau40-ΔPAD, or hTau40-T205A. Results and Discussion: The results presented here suggest that transient activation of a PP1-GSK3ß signaling pathway through locally regulated PAD exposure is a mechanism for cargo delivery, and thereby important for neurite outgrowth of developing neurons.

Neurofilaments run sprints not marathons.

In neurons, cytoskeletal proteins are transported from where they are made - the cell body - along the axons, but it has long been disputed whether they are transported as subunits or polymers. A new analysis of neurofilament movement may help to resolve the controversy.

A kinesin medley: biochemical and functional heterogeneity.

Over the past decade, a remarkable number and diversity of molecular motors have been described in eukaryotic cells. In addition to the identification of novel forms of myosin and dynein, the kinesins have been defined as an entirely new family of molecular motors. There may be as many as 30 different genes in a single organism encoding members of the kinesin superfamily. Why is such diversity in molecular motors needed? The biochemical and functional diversity of the originally defined form of kinesin provides some insights into the roles of molecular motors in cellular dynamics.

Axonal tubulin and axonal microtubules: biochemical evidence for cold stability.

Nerve extracts containing tubulin labeled by axonal transport were analyzed by electrophoresis and differential extraction. We found that a substantial fraction of the tubulin in the axons of the retinal ganglion cell of guinea pigs is not solubilized by conventional methods for preparation of microtubules from whole brain. In two-dimensional polyacrylamide gel electrophoresis this cold-insoluble tubulin was biochemically distinct from tubulin obtained from whole brain microtubules prepared by cold cycling. Cleveland peptide maps also indicated some differences between the cold-extractable and cold-insoluble tubulins. The demonstration of cold-insoluble tubulin that is specifically axonal in origin permits consideration of the physiological role of cold-insoluble tubulin in a specific cellular structure. It appears likely that much of this material is in the form of cold-stable microtubules. We propose that the physiological role of cold-insoluble tubulin in the axon may be associated with the regulation of the axonal microtubule complexes in neurons.

Fast axonal transport of foreign synaptic vesicles in squid axoplasm.

Translocation of intracellular organelles requires interaction with the cellular cytoskeleton, but the membrane and cytoskeletal proteins involved in movement are unknown. Here we show that highly purified synaptic vesicles from electric fish added to extruded squid axoplasm can show ATP-dependent movement. The movement is indistinguishable from that of endogenous vesicles and has a slight preference for the orthograde direction. In the presence of a nonhydrolyzable ATP analog, the synaptic vesicles bind to axoplasmic fibers but do not move. Elastase treatment of vesicles inhibits both binding and movement. We conclude that a protein component on the surface of cholinergic synaptic vesicles from electric fish is conserved during evolution and so can be recognized by the organelle-translocating machinery of the squid axon, resulting in ATP-dependent movement. Synaptic vesicles apparently retain the capacity for fast axonal transport, even after they reach their intracellular destination.

Axonal transport of calmodulin: a physiologic approach to identification of long-term associations between proteins.

Calmodulin is a soluble, heat-stable protein which has been shown to modulate both membrane-bound and soluble enzymes, but relatively little has been known about the in vivo associations of calmodulin. A 17,000-dalton heat-stable protein was found to move in axonal transport in the guinea pig visual system with the proteins of slow component b (SCb; 2 mm/d) along with actin and the bulk of the soluble proteins of the axon. Co-electrophoresis of purified calmodulin and radioactively labeled SCb proteins in two dimensional polyacrylamide gel electrophoresis (PAGE) demonstrated the identity of the heat-stable SCb protein and calmodulin on the basis of pI, molecular weight, and anomalous migration in the presence of Ca2+-chelating agents. No proteins co-migrating with calmodulin in two-dimensional PAGE could be detected among the proteins of slow component a (SCa; 0.3 mm/d, microtubules and neurofilaments) or fast component (FC; 250 mm/d, membrane-associated proteins). We conclude that calmodulin is transported solely as part of the SCb complex of proteins, the axoplasmic matrix. Calmodulin moves in axonal transport independent of the movements of microtubules (SCa) and membranes (FC), which suggests that the interactions of calmodulin with these structures may represent a transient interaction between groups of proteins moving in axonal transport at different rates. Axonal transport has been shown to be an effective tool for the demonstration of long-term in vivo protein associations.

Kinesin associates with anterogradely transported membranous organelles in vivo.

Biochemical, pharmacological and immunocytochemical studies have implicated the microtubule-activated ATPase, kinesin, in the movement of membrane bounded organelles in fast axonal transport. In vitro studies suggested that kinesin moves organelles preferentially in the anterograde direction, but data about the function and precise localization of kinesin in the living axon were lacking. The current study was undertaken to establish whether kinesin associates with anterograde or retrograde moving organelles in vivo. Peripheral nerves were ligated to produce accumulations of organelles moving in defined directions. Regions proximal (anterograde) and distal (retrograde) to the ligation were analyzed for kinesin localization by immunofluorescence, and by immunogold electron microscopy using ultracryomicrotomy. Substantial amounts of kinesin were associated with anterograde moving organelles on the proximal side, while significantly less kinesin was detected distally. Statistical analyses indicated that kinesin was mostly associated with membrane-bounded organelles. These observations indicate that axonal kinesin is primarily associated with anterograde moving organelles in vivo.

GTP gamma S inhibits organelle transport along axonal microtubules.

Movements of membrane-bounded organelles through cytoplasm frequently occur along microtubules, as in the neuron-specific case of fast axonal transport. To shed light on how microtubule-based organelle motility is regulated, pharmacological probes for GTP-binding proteins, or protein kinases or phosphatases were perfused into axoplasm extruded from squid (Loligo pealei) giant axons, and effects on fast axonal transport were monitored by quantitative video-enhanced light microscopy. GTP gamma S caused concentration-dependent and time-dependent declines in organelle transport velocities. GDP beta S was a less potent inhibitor. Excess GTP, but not GDP, masked the effects of coperfused GTP gamma S. The effects of GTP gamma S on transport were not mimicked by broad spectrum inhibitors of protein kinases (K-252a) or phosphatases (microcystin LR and okadaic acid), or as shown earlier, by ATP gamma S. Therefore, suppression of organelle motility by GTP gamma S was guanine nucleotide-specific and evidently did not involve irreversible transfer of thiophosphate groups to protein. Instead, the data imply that organelle transport in the axon is modulated by cycles of GTP hydrolysis and nucleotide exchange by one or more GTP-binding proteins. Fast axonal transport was not perturbed by AlF4-, indicating that the GTP gamma S-sensitive factors do not include heterotrimeric G-proteins. Potential axoplasmic targets of GTP gamma S include dynamin and multiple small GTP-binding proteins, which were shown to be present in squid axoplasm. These collective findings suggest a novel strategy for regulating microtubule-based organelle transport and a new role for GTP-binding proteins.

Monoclonal antibodies to kinesin heavy and light chains stain vesicle-like structures, but not microtubules, in cultured cells.

Kinesin, a microtubule-activated ATPase and putative motor protein for the transport of membrane-bounded organelles along microtubules, was purified from bovine brain and used as an immunogen for the production of murine monoclonal antibodies. Hybridoma lines that secreted five distinct antikinesin IgGs were cloned. Three of the antibodies reacted on immunoblots with the 124-kD heavy chain of kinesin, while the other two antibodies recognized the 64-kD light chain. When used for immunofluorescence microscopy, the antibodies stained punctate, cytoplasmic structures in a variety of cultured mammalian cell types. Consistent with the identification of these structures as membrane-bounded organelles was the observation that cells which had been extracted with Triton X-100 before fixation contained little or no immunoreactive material. Staining of microtubules in the interphase cytoplasm or mitotic spindle was never observed, nor were associated structures, such as centrosomes and primary cilia, labeled by any of the antibodies. Nevertheless, in double-labeling experiments using antibodies to kinesin and tubulin, kinesin-containing particles were most abundant in regions where microtubules were most highly concentrated and the particles often appeared to be aligned on microtubules. These results constitute the first direct evidence for the association of kinesin with membrane-bounded organelles, and suggest a molecular mechanism for organelle motility based on transient interactions of organelle-bound kinesin with the microtubule surface.

Fast axonal transport in extruded axoplasm from squid giant axon.

Development of video-enhanced contrast-differential interference contrast for light microscopy has permitted study of both orthograde and retrograde fast axonal transport of membranous organelles in the squid giant axon. This process was found to continue normally for hours after the axoplasm was extruded from the giant axon and removed from the confines of the axonal plasma membrane. It is now possible to follow the movements of the full range of membranous organelles (30-nanometer vesicles to 5000-nanometer mitochondria) in a preparation that lacks a plasma membrane or other permeability barrier. This observation demonstrates that the plasma membrane is not required for fast axonal transport and suggests that action potentials are not involved in the regulation of fast transport. Furthermore, the absence of a permeability barrier surrounding the axoplasm makes this an important model for biochemical pharmacological, and physical manipulations of membranous organelle transport.

Fast axonal transport in squid giant axon.

Video-enhanced contrast-differential interference contrast microscopy has revealed new features of axonal transport in the giant axon of the squid, where no movement had been detected previously by conventional microscopy. The newly discovered dominant feature is vast numbers of "submicroscopic" particles, probably 30- to 50-nanometer vesicles and other tubulovesicular elements, moving parallel to linear elements, primarily in the orthograde direction but also in a retrograde direction, at a range of steady velocities up to +/- 5 micrometers per second. Medium (0.2 to 0.6 micrometer) and large (0.8 micrometer) particles move more slowly and more intermittently with a tendency at times to exhibit elastic recoil. The behavior of the smallest particles and the larger particles during actual translocation suggests that the fundamental processes in the mechanisms of organelle movement in axonal transport are not saltatory but continuous.

Immunochemical analysis of kinesin light chain function.

The kinesin heterotetramer consists of two heavy and two light chains. Kinesin light chains have been proposed to act in binding motor protein to cargo, but evidence for this has been indirect. A library of monoclonal antibodies directed against conserved epitopes throughout the kinesin light chain sequence were used to map light chain functional architecture and to assess physiological functions of these domains. Immunocytochemistry with all antibodies showed a punctate pattern that was detergent soluble. A monoclonal antibody (KLC-All) made against a highly conserved epitope in the tandem repeat domain of light chains inhibited fast axonal transport in isolated axoplasm by decreasing both the number and velocity of vesicles moving, whereas an antibody against a conserved amino terminus epitope had no effect. KLC-All was equally effective at inhibiting both anterograde and retrograde transport. Neither antibody inhibited microtubule-binding or ATPase activity in vitro. KLC-All was unique among antibodies tested in releasing kinesin from purified membrane vesicles, suggesting a mechanism of action for inhibition of axonal transport. These results provide further evidence that conventional kinesin is a motor for fast axonal transport and demonstrate that kinesin light chains play an important role in kinesin interaction with membranes.

Fast axonal transport of kinesin in the rat visual system: functionality of kinesin heavy chain isoforms.

The mechanochemical ATPase kinesin is thought to move membrane-bounded organelles along microtubules in fast axonal transport. However, fast transport includes several classes of organelles moving at rates that differ by an order of magnitude. Further, the fact that cytoplasmic forms of kinesin exist suggests that kinesins might move cytoplasmic structures such as the cytoskeleton. To define cellular roles for kinesin, the axonal transport of kinesin was characterized. Retinal proteins were pulse-labeled, and movement of radiolabeled kinesin through optic nerve and tract into the terminals was monitored by immunoprecipitation. Heavy and light chains of kinesin appeared in nerve and tract at times consistent with fast transport. Little or no kinesin moved with slow axonal transport indicating that effectively all axonal kinesin is associated with membranous organelles. Both kinesin heavy chain molecular weight variants of 130,000 and 124,000 M(r) (KHC-A and KHC-B) moved in fast anterograde transport, but KHC-A moved at 5-6 times the rate of KHC-B. KHC-A cotransported with the synaptic vesicle marker synaptophysin, while a portion of KHC-B cotransported with the mitochondrial marker hexokinase. These results suggest that KHC-A is enriched on small tubulovesicular structures like synaptic vesicles and that at least one form of KHC-B is predominantly on mitochondria. Biochemical specialization may target kinesins to appropriate organelles and facilitate differential regulation of transport.

Release of kinesin from vesicles by hsc70 and regulation of fast axonal transport.

The nature of kinesin interactions with membrane-bound organelles and mechanisms for regulation of kinesin-based motility have both been surprisingly difficult to define. Most kinesin is recovered in supernatants with standard protocols for purification of motor proteins, but kinesin recovered on membrane-bound organelles is tightly bound. Partitioning of kinesin between vesicle and cytosolic fractions is highly sensitive to buffer composition. Addition of either N-ethylmaleimide or EDTA to homogenization buffers significantly increased the fraction of kinesin bound to organelles. Given that an antibody against kinesin light chain tandem repeats also releases kinesin from vesicles, these observations indicated that specific cytoplasmic factors may regulate kinesin release from membranes. Kinesin light tandem repeats contain DnaJ-like motifs, so the effects of hsp70 chaperones were evaluated. Hsc70 released kinesin from vesicles in an MgATP-dependent and N-ethylmaleimide-sensitive manner. Recombinant kinesin light chains inhibited kinesin release by hsc70 and stimulated the hsc70 ATPase. Hsc70 actions may provide a mechanism to regulate kinesin function by releasing kinesin from cargo in specific subcellular domains, thereby effecting delivery of axonally transported materials.

Biochemical and functional diversity of microtubule motors in the nervous system.

The fact that multiple microtubule-based motors exist in brain inevitably raises questions about their function. Transcripts for at least seven kinesin superfamily genes and even more dynein heavy chain genes have been detected in brain cDNA libraries. The challenge now is to match their gene products to specific functions in cells of the nervous system. Recent studies have attempted to establish a function for each microtubule motor by using recombinant protein and immunochemical approaches.

Tau pathology-mediated presynaptic dysfunction.

Brain tauopathies are characterized by abnormal processing of tau protein. While somatodendritic tau mislocalization has attracted considerable attention in tauopathies, the role of tau pathology in axonal transport, connectivity and related dysfunctions remains obscure. We have previously shown using the squid giant synapse that presynaptic microinjection of recombinant human tau protein (htau42) results in failure of synaptic transmission. Here, we evaluated molecular mechanisms mediating this effect. Thus, the initial event, observed after htau42 presynaptic injection, was an increase in transmitter release. This event was mediated by calcium release from intracellular stores and was followed by a reduction in evoked transmitter release. The effect of htau42 on synaptic transmission was recapitulated by a peptide comprising the phosphatase-activating domain of tau, suggesting activation of phosphotransferases. Accordingly, findings indicated that htau42-mediated toxicity involves the activities of both GSK3 and Cdk5 kinases.

Changes in microtubule stability and density in myelin-deficient shiverer mouse CNS axons.

Altered axon-Schwann cell interactions in PNS myelin-deficient Trembler mice result in changed axonal transport rates, neurofilament and microtubule-associated protein phosphorylation, neurofilament density, and microtubule stability. To determine whether PNS and CNS myelination have equivalent effects on axons, neurofilaments, and microtubules in CNS, myelin-deficient shiverer axons were examined. The genetic defect in shiverer is a deletion in the myelin basic protein (MBP) gene, an essential component of CNS myelin. As a result, shiverer mice have little or no compact CNS myelin. Slow axonal transport rates in shiverer CNS axons were significantly increased, in contrast to the slowing in demyelinated PNS nerves. Even more striking were substantial changes in the composition and properties of microtubules in shiverer CNS axons. The density of axonal microtubules is increased, reflecting increased expression of tubulin in shiverer, and the stability of microtubules is drastically reduced in shiverer axons. Shiverer transgenic mice with two copies of a wild-type myelin basic protein transgene have an intermediate level of compact myelin, making it possible to determine whether the actual level of compact myelin is an important regulator of axonal microtubules. Both increased microtubule density and reduced microtubule stability were still observed in transgenic mouse nerves, indicating that signals beyond synaptogenesis and the mere presence of compact myelin are required for normal regulation of the axonal microtubule cytoskeleton.

Formation of compact myelin is required for maturation of the axonal cytoskeleton.

Although traditional roles ascribed to myelinating glial cells are structural and supportive, the importance of compact myelin for proper functioning of the nervous system can be inferred from mutations in myelin proteins and neuropathologies associated with loss of myelin. Myelinating Schwann cells are known to affect local properties of peripheral axons (de Waegh et al., 1992), but little is known about effects of oligodendrocytes on CNS axons. The shiverer mutant mouse has a deletion in the myelin basic protein gene that eliminates compact myelin in the CNS. In shiverer mice, both local axonal features like phosphorylation of cytoskeletal proteins and neuronal perikaryon functions like cytoskeletal gene expression are altered. This leads to changes in the organization and composition of the axonal cytoskeleton in shiverer unmyelinated axons relative to age-matched wild-type myelinated fibers, although connectivity and patterns of neuronal activity are comparable. Remarkably, transgenic shiverer mice with thin myelin sheaths display an intermediate phenotype indicating that CNS neurons are sensitive to myelin sheath thickness. These results indicate that formation of a normal compact myelin sheath is required for normal maturation of the neuronal cytoskeleton in large CNS neurons.

Molecular motors and cell motility in the brain.

The advent of video computer-enhanced microscopy has provided a new vision of cell migrations, growth cones, and fast axonal transport in the nervous system. In images obtained in studies of fast transport in isolated axoplasm from the squid giant axon, a virtual torrent of membrane traffic could be seen moving in both directions. Similarly, examination of growth cones and cell migrations in vitro and in vivo revealed properties of cell motility that were previously unsuspected. Evidence has accumulated that many of these activities are driven by a variety of microtubule and microfilament based motors.

Retardation in the slow axonal transport of cytoskeletal elements during maturation and aging.

Using the pulse-labeling method, the rate of the slow component (SC) of axonal transport was analyzed during maturation and aging. Ventral motor neurons and retinal ganglion cells of 3-, 6-, and 24-month-old Fischer 344 rats were radiolabeled with 35S-methionine. To measure the rates of SCa and SCb subcomponents, distributions of the total radiolabeled proteins and certain cytoskeletal proteins (actin, clathrin, tubulin, and the neurofilament proteins) were analyzed in the ventral root-sciatic nerve and optic nerve. Our results show that the rate of transport for both SCa and SCb proteins decreases with age in ventral motor axons and optic axons. For example, in ventral motor axons the rates of both SCa and SCb decreased 40% between 6 and 24 months. These results, with those of others, show that the rate of slow transport gradually decreases in the neurons of adult rats (7,11) The factors that may contribute to the slowing are discussed.