Scott T. Brady

Local modulation of neurofilament phosphorylation, axonal caliber, and slow axonal transport by myelinating Schwann cells

Studies in Trembler and control mice demonstrated that myelinating Schwann cells exert a profound influence on axons. Extensive contacts between myelin and axons have been considered structural. However, demyelination decreases neurofilament phosphorylation, slow axonal transport, and axonal diameter, as well as significantly increasing neurofilament density. In control sciatic nerves with grafted Trembler nerve segments, these changes were spatially restricted: they were confined to axon segments without normal myelination. Adjacent regions of the same axons had normal diameters, neurofilament phosphorylation, cytoskeletal organization, and axonal transport rates. Close intercellular contacts between myelinating Schwann cells and axons modulate a kinase-phosphatase system acting on neurofilaments and possibly other substrates. Myelination by Schwann cells sculpts the axon-altering functional architecture, electrical properties, and neuronal morphologies.

Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS

Many mutations confer one or more toxic function(s) on copper/zinc superoxide dismutase 1 (SOD1) that impair motor neuron viability and cause familial amyotrophic lateral sclerosis (FALS). Using a conformation-specific antibody that detects misfolded SOD1 (C4F6), we found that oxidized wild-type SOD1 and mutant SOD1 share a conformational epitope that is not present in normal wild-type SOD1. In a subset of human sporadic ALS (SALS) cases, motor neurons in the lumbosacral spinal cord were markedly C4F6 immunoreactive, indicating that an aberrant wild-type SOD1 species was present. Recombinant, oxidized wild-type SOD1 and wild-type SOD1 immunopurified from SALS tissues inhibited kinesin-based fast axonal transport in a manner similar to that of FALS-linked mutant SOD1. Our findings suggest that wild-type SOD1 can be pathogenic in SALS and identify an SOD1-dependent pathogenic mechanism common to FALS and SALS.

Submolecular domains of bovine brain kinesin identified by electron microscopy and monoclonal antibody decoration

Kinesin is a microtubule-activated ATPase thought to transport membrane-bounded organelles along MTs. To illuminate the structural basis for this function, EM was used to locate submolecular domains on bovine brain kinesin. Rotary shadowed kinesin appeared rod-shaped and approximately 80 nm long. One end of each molecule contained a pair of approximately 10 x 9 nm globular domains, while the opposite end was fan-shaped. Monoclonal antibodies against the approximately 124 kd heavy chains of kinesin decorated the globular structures, while those specific for the approximately 64 kd light chains labeled the fan-shaped end. Quick-freeze, deep-etch EM was used to analyze MTs polymerized from tubulin and cross-linked to latex microspheres by kinesin. Microspheres frequently attached to MTs by arm-like structures, 25-30 nm long. The MT attachment sites often appeared as one or two approximately 10 nm globular bulges. Morphologically similar cross-links were observed by quick-freeze, deep-etch EM between organelles and MTs in the neuronal cytoskeleton in vivo. These collective observations suggest that bovine brain kinesin binds to MTs by globular domains that contain the heavy chains, and that the attachment sites for organelles are at the opposite, fan-shaped end of kinesin, where the light chains are located.

Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

Membrane‐bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin‐based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule‐stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

Axonal Transport Defects in Neurodegenerative Diseases

Adult-onset neurodegenerative diseases (AONDs) comprise a heterogeneous group of neurological disorders characterized by a progressive, age-dependent decline in neuronal function and loss of selected neuronal populations. Alterations in synaptic function and axonal connectivity represent early and critical pathogenic events in AONDs, but molecular mechanisms underlying these defects remain elusive. The large size and complex subcellular architecture of neurons render them uniquely vulnerable to alterations in axonal transport (AT). Accordingly, deficits in AT have been documented in most AONDs, suggesting a common defect acquired through different pathogenic pathways. These observations suggest that many AONDs can be categorized as dysferopathies, diseases in which alterations in AT represent a critical component in pathogenesis. Topics here address various molecular mechanisms underlying alterations in AT in several AONDs. Illumination of such mechanisms provides a framework for the development of novel therapeutic strategies aimed to prevent axonal and synaptic dysfunction in several major AONDs.

Direct interaction of microtubule- and actin-based transport motors

The microtubule network is thought to be used for long-range transport of cellular components in animal cells whereas the actinnetwork is proposed to be used for short-range transport, although the mechanism(s) by which this transport is coordinated is poorly understood. For example, in sea urchins long-range Ca2+-regulated transport of exocytotic vesicles requires a microtubule-based motor, whereas an actin-based motor is used for short-range transport. In neurons, microtubule-based kinesin motor proteins are used for long-range vesicular transport but microtubules do not extend into the neuronal termini, where actin filaments form the cytoskeletal framework, and kinesins are rapidly degraded upon their arrival in neuronal termini, indicating that vesicles may have to be transferred from microtubules to actin tracks to reach their final destination. Here we show that an actin-based vesicle-transport motor, MyoVA (ref. 6), can interact directly with a microtubule-based transport motor, KhcU. As would be expected if these complexes were functional, they also contain kinesin light chains and the localization of MyoVA and KhcU overlaps in the cell. These results indicate that cellular transport is, in part, coordinated through the direct interaction of different motor molecules.

Neuropathogenic Forms of Huntingtin and Androgen Receptor Inhibit Fast Axonal Transport

Huntingtons and Kennedys disease are autosomal dominant neurodegenerative diseases caused by pathogenic expansion of polyglutamine tracts. Expansion of glutamine repeats must in some way confer a gain of pathological function that disrupts an essential cellular process and leads to loss of affected neurons. Association of huntingtin with vesicular structures raised the possibility that axonal transport might be altered. Here we show that polypeptides containing expanded polyglutamine tracts, but not normal N-terminal huntingtin or androgen receptor, directly inhibit both fast axonal transport in isolated axoplasm and elongation of neuritic processes in intact cells. Effects were greater with truncated polypeptides and occurred without detectable morphological aggregates.

Reelin-mediated Signaling Locally Regulates Protein Kinase B/Akt and Glycogen Synthase Kinase 3β

Reelin is a large secreted protein that controls cortical layering by signaling through the very low density lipoprotein receptor and apolipoprotein E receptor 2, thereby inducing tyrosine phosphorylation of the adaptor protein Disabled-1 (Dab1) and suppressing tau phosphorylation in vivo. Here we show that binding of Reelin to these receptors stimulates phosphatidylinositol 3-kinase, resulting in activation of protein kinase B and inhibition of glycogen synthase kinase 3β. We present genetic evidence that this cascade is dependent on apolipoprotein E receptor 2, very low density lipoprotein receptor, and Dab1. Reelin-signaling components are enriched in axonal growth cones, where tyrosine phosphorylation of Dab1 is increased in response to Reelin. These findings suggest that Reelin-mediated phosphatidylinositol 3-kinase signaling in neuronal growth cones contributes to final neuron positioning in the mammalian brain by local modulation of protein kinase B and glycogen synthase kinase 3β kinase activities.

Alzheimer's Presenilin 1 Mutations Impair Kinesin-Based Axonal Transport

Several lines of evidence indicate that alterations in axonal transport play a critical role in Alzheimers disease (AD) neuropathology, but the molecular mechanisms that control this process are not understood fully. Recent work indicates that presenilin 1 (PS1) interacts with glycogen synthase kinase 3β (GSK3β). In vivo, GSK3β phosphorylates kinesin light chains (KLC) and causes the release of kinesin-I from membrane-bound organelles (MBOs), leading to a reduction in kinesin-I driven motility (Morfini et al., 2002b). To characterize a potential role for PS1 in the regulation of kinesin-based axonal transport, we used PS1-/- and PS1 knock-inM146V (KIM146V) mice and cultured cells. We show that relative levels of GSK3β activity were increased in cells either in the presence of mutant PS1 or in the absence of PS1 (PS1-/-). Concomitant with increased GSK3β activity, relative levels of KLC phosphorylation were increased, and the amount of kinesin-I bound to MBOs was reduced. Consistent with a deficit in kinesin-I-mediated fast axonal transport, densities of synaptophysin- and syntaxin-I-containing vesicles and mitochondria were reduced in neuritic processes of KIM146V hippocampal neurons. Similarly, we found reduced levels of PS1, amyloid precursor protein, and synaptophysin in sciatic nerves of KIM146V mice. Thus PS1 appears to modulate GSK3β activity and the release of kinesin-I from MBOs at sites of vesicle delivery and membrane insertion. These findings suggest that mutations in PS1 may compromise neuronal function by affecting GSK-3 activity and kinesin-I-based motility.

A novel CDK5‐dependent pathway for regulating GSK3 activity and kinesin‐driven motility in neurons

Neuronal transmission of information requires polarized distribution of membrane proteins within axonal compartments. Membrane proteins are synthesized and packaged in membrane‐bounded organelles (MBOs) in neuronal cell bodies and later transported to axons by microtubule‐dependent motor proteins. Molecular mechanisms underlying targeted delivery of MBOs to discrete axonal subdomains (i.e. nodes of Ranvier or presynaptic terminals) are poorly understood, but regulatory pathways for microtubule motors may be an essential step. In this work, pharmacological, biochemical and in vivo experiments define a novel regulatory pathway for kinesin‐driven motility in axons. This pathway involves enzymatic activities of cyclin‐dependent kinase 5 (CDK5), protein phosphatase 1 (PP1) and glycogen synthase kinase‐3 (GSK3). Inhibition of CDK5 activity in axons leads to activation of GSK3 by PP1, phosphorylation of kinesin light chains by GSK3 and detachment of kinesin from transported cargoes. We propose that regulating the activity and localization of components in this pathway allows nerve cells to target organelle delivery to specific subcellular compartments. Implications of these findings for pathogenesis of neurodegenerative diseases such as Alzheimers disease are discussed.