Peter W. Baas

Role of cytoplasmic dynein in the axonal transport of microtubules and neurofilaments

Recent studies have shown that the transport of microtubules (MTs) and neurofilaments (NFs) within the axon is rapid, infrequent, asynchronous, and bidirectional. Here, we used RNA interference to investigate the role of cytoplasmic dynein in powering these transport events. To reveal transport of MTs and NFs, we expressed EGFP-tagged tubulin or NF proteins in cultured rat sympathetic neurons and performed live-cell imaging of the fluorescent cytoskeletal elements in photobleached regions of the axon. The occurrence of anterograde MT and retrograde NF movements was significantly diminished in neurons that had been depleted of dynein heavy chain, whereas the occurrence of retrograde MT and anterograde NF movements was unaffected. These results support a cargo model for NF transport and a sliding filament model for MT transport.

Mechanical breaking of microtubules in axons during dynamic stretch injury underlies delayed elasticity, microtubule disassembly, and axon degeneration

Little is known about which components of the axonal cytoskeleton might break during rapid mechanical deformation, such as occurs in traumatic brain injury. Here, we micropatterned neuronal cell cultures on silicone membranes to induce dynamic stretch exclusively of axon fascicles. After stretch, undulating distortions formed along the axons that gradually relaxed back to a straight orientation, demonstrating a delayed elastic response. Subsequently, swellings developed, leading to degeneration of almost all axons by 24 h. Stabilizing the microtubules with taxol maintained the undulating geometry after injury but greatly reduced axon degeneration. Conversely, destabilizing microtubules with nocodazole prevented undulations but greatly increased the rate of axon loss. Ultrastructural analyses of axons postinjury revealed immediate breakage and buckling of microtubules in axon undulations and progressive loss of microtubules. Collectively, these data suggest that dynamic stretch of axons induces direct mechanical failure at specific points along microtubules. This microtubule disorganization impedes normal relaxation of the axons, resulting in undulations. However, this physical damage also triggers progressive disassembly of the microtubules around the breakage points. While the disintegration of microtubules allows delayed recovery of the “normal” straight axon morphology, it comes at a great cost by interrupting axonal transport, leading to axonal swelling and degeneration.—Tang‐Schomer, M. D., Patel, A. R., Baas, P. W., Smith, D. H. Mechanical breaking of microtubules in axons during dynamic stretch injury underlies delayed elasticity, microtubule disassembly, and axon degeneration. FASEB J. 24, 1401–1410 (2010). www.fasebj.org

Tau-dependent microtubule disassembly initiated by prefibrillar β-amyloid

Alzheimers Disease (AD) is defined histopathologically by extracellular β-amyloid (Aβ) fibrils plus intraneuronal tau filaments. Studies of transgenic mice and cultured cells indicate that AD is caused by a pathological cascade in which Aβ lies upstream of tau, but the steps that connect Aβ to tau have remained undefined. We demonstrate that tau confers acute hypersensitivity of microtubules to prefibrillar, extracellular Aβ in nonneuronal cells that express transfected tau and in cultured neurons that express endogenous tau. Prefibrillar Aβ42 was active at submicromolar concentrations, several-fold below those required for equivalent effects of prefibrillar Aβ40, and microtubules were insensitive to fibrillar Aβ. The active region of tau was localized to an N-terminal domain that does not bind microtubules and is not part of the region of tau that assembles into filaments. These results suggest that a seminal cell biological event in AD pathogenesis is acute, tau-dependent loss of microtubule integrity caused by exposure of neurons to readily diffusible Aβ.

Microtubules and Neuronal Polarity: Lessons from Mitosis

I would like to thank Wenqian Yu for assistance in the preparation of the figures; David Sharp, Jon Scholey, Erik Dent, and Mark Black for critically reading the manuscript; and all of the past and present members of my laboratory (especially Fridoon Ahmad, Lotfi Ferhat, David Sharp, and Wenqian Yu) for their important contributions to the work discussed in this article. I would also like to thank Charles Stevens for his support and encouragement. My laboratory is funded by grants from the National Institutes of Health and the National Science Foundation.

The microtubule-severing proteins spastin and katanin participate differently in the formation of axonal branches.

Neurons express two different microtubule-severing proteins, namely P60-katanin and spastin. Here, we performed studies on cultured neurons to ascertain whether these two proteins participate differently in axonal branch formation. P60-katanin is more highly expressed in the neuron, but spastin is more concentrated at sites of branch formation. Overexpression of spastin dramatically enhances the formation of branches, whereas overexpression of P60-katanin does not. The excess spastin results in large numbers of short microtubules, whereas the excess P60-katanin results in short microtubules intermingled with longer microtubules. We hypothesized that these different microtubule-severing patterns may be due to the presence of molecules such as tau on the microtubules that more strongly shield them from being severed by P60-katanin than by spastin. Consistent with this hypothesis, we found that axons depleted of tau show a greater propensity to branch, and that this is true whether or not the axons are also depleted of spastin. We propose that there are two modes by which microtubule severing is orchestrated during axonal branch formation, one based on the local concentration of spastin at branch sites and the other based on local detachment from microtubules of molecules such as tau that regulate the severing properties of P60-katanin.

Changes in microtubule number and length during axon differentiation

Hippocampal neurons in culture initially extend several minor processes that are approximately 20 microns in length. The first minor process to grow approximately 10 microns longer than the others will continue to grow rapidly and become the axon (Goslin and Banker, 1989). We sought to define changes in the microtubule (MT) array that occur during axon differentiation. In theory, axon differentiation could involve an increase in MT number, MT length, or some combination of both. To address this issue, we first serially reconstructed the entire MT array of a minor process from a cell whose axon had not yet differentiated. This minor process contained 182 MTs that ranged in length from 0.14 to 20.09 microns. The average MT length was 3.87 +/- 3.83 microns, and the total MT length was 704 microns. We then reconstructed the MT arrays of a minor process and the 56 microns axon from a cell that had undergone axon differentiation. The minor process contained 157 MTs that ranged in length from 0.24 to 17.95 microns. The average MT length was 3.91 +/- 4.84 microns, and the total MT length was 600 microns. The axon contained 1430 MTs that ranged in length from 0.05 to 40.14 microns. The average MT length was 4.02 +/- 5.28 microns, and the total MT length was 5750 microns. These data indicate that a shift occurs toward shorter as well as longer MTs, but that virtually no change in average MT length occurs during axon differentiation. Thus, elongation of existing MTs cannot account for the major expansion of the MT array that occurs as a minor process becomes an axon. In contrast, the number of MTs increases by approximately 10-fold as a minor process differentiates and grows into an axon of the length we analyzed. Based on these data, we conclude that the MT array of a minor process is substantially expanded as it differentiates into an axon, and that the principal mechanism by which this expansion occurs is the copious addition of new MTs.

Motor proteins regulate force interactions between microtubules and microfilaments in the axon

It has long been known that microtubule depletion causes axons to retract in a microfilament-dependent manner, although it was not known whether these effects are the result of motor-generated forces on these cytoskeletal elements. Here we show that inhibition of the motor activity of cytoplasmic dynein causes the axon to retract in the presence of microtubules. This response is obliterated if microfilaments are depleted or if myosin motors are inhibited. We conclude that axonal retraction results from myosin-mediated forces on the microfilament array, and that these forces are counterbalanced or attenuated by dynein-mediated forces between the microfilament and microtubule arrays.

The basis of polarity in neurons

It has been recognized since the very early studies on the cytology of vertebrate nervous systems that neurons produce two fundamentally different types of neurite, the axon and the dendrite. Contemporary studies using electron microscopy have defined in detail the many structural differences between axons and dendrites. Perhaps the best known of these differences concerns ribosomes and Golgi elements, which are present in dendrites, but are absent from axons. In this article, we present a possible explanation for this compartmentalization which is based on current understanding of organelle transport in cells and our recent demonstration of a fundamental difference in the organization of the microtubule-based transport systems that convey organelles into the axon or into the dendrite.

Tau Protects Microtubules in the Axon from Severing by Katanin

Microtubules in the axon are more resistant to severing by katanin than microtubules elsewhere in the neuron. We have hypothesized that this is because of the presence of tau on axonal microtubules. When katanin is overexpressed in fibroblasts, the microtubules are severed into short pieces, but this phenomenon is suppressed by the coexpression of tau. Protection against severing is also afforded by microtubule-associated protein 2 (MAP2), which has a tau-like microtubule-binding domain, but not by MAP1b, which has a different microtubule-binding domain. The microtubule-binding domain of tau is required for the protection, but within itself, provides less protection than the entire molecule. When tau (but not MAP2 or MAP1b) is experimentally depleted from neurons, the microtubules in the axon lose their characteristic resistance to katanin. These results, which validate our hypothesis, also suggest a potential explanation for why axonal microtubules deteriorate in neuropathies involving the dissociation of tau from the microtubules.

Hooks and comets: The story of microtubule polarity orientation in the neuron

It is widely believed that signature patterns of microtubule polarity orientation within axons and dendrites underlie compositional and morphological differences that distinguish these neuronal processes from one another. Axons of vertebrate neurons display uniformly plus‐end‐distal microtubules, whereas their dendrites display non‐uniformly oriented microtubules. Recent studies on insect neurons suggest that it is the minus‐end‐distal microtubules that are the critical feature of the dendritic microtubule array, whether or not they are accompanied by plus‐end‐distal microtubules. Discussed in this article are the history of these findings, their implications for the regulation of neuronal polarity across the animal kingdom, and potential mechanisms by which neurons establish the distinct microtubule polarity patterns that define axons and dendrites.