Maturation of the peripheral nervous system requires specification of axonal diameter, which, in turn, has a significant influence on nerve conduction velocity. Radial axonal growth initiates with myelination, and is dependent upon the C-terminus region of neurofilament medium (NF-M). Molecular phylogenetic analysis in mammals suggested that expanded NF-M C-termini correlated with larger diameter axons. We utilized gene targeting and computational modeling to test this new hypothesis. Increasing the length of NF-M C-terminus in mice increased diameter of motor axons without altering neurofilament subunit stoichiometry. Computational modeling predicted that an expanded NF-M C-terminus extended farther from the neurofilament core independent of lysine-serine-proline (KSP) phosphorylation. However, expansion of NF-M C-terminus did not affect the distance between adjacent neurofilaments. Increased axonal diameter did not increase conduction velocity, possibly due to a failure to increase myelin thickness by the same proportion. Failure of myelin to compensate for larger axonal diameters suggested a lack of plasticity during the processes of myelination and radial axonal growth.

Traumatic brain injury (TBI) results in disrupted brain function following impact from an external force and is a risk factor for sporadic Alzheimer's disease (AD). Although neurologic symptoms triggered by mild traumatic brain injuries (mTBI), the most common form of TBI, typically resolve rapidly, even an isolated mTBI event can increase the risk to develop AD. Aberrant accumulation of amyloid b peptide (Ab), a cleaved fragment of amyloid precursor protein (APP), is a key pathologic outcome designating the progression of AD following mTBI and has also been linked to impaired axonal transport. However, relationships among mTBI, amyloidogenesis, and axonal transport remain unclear, in part because of the dearth of human models to study the neuronal response following mTBI. Here, we implemented a custom-microfabricated device to deform neurons derived from humaninduced pluripotent stem cells, derived from a cognitively unimpaired male individual, to mimic the mild stretch experienced by neurons during mTBI. Although no cell lethality or cytoskeletal disruptions were observed, mild stretch was sufficient to stimulate rapid amyloidogenic processing of APP. This processing led to abrupt cessation of APP axonal transport and progressive formation of aberrant axonal accumulations that contained APP, its processing machinery, and amyloidogenic fragments. Consistent with this sequence of events, stretch-induced defects were abrogated by reducing amyloidogenesis either pharmacologically or genetically. In sum, we have uncovered a novel and manipulable stretch-induced amyloidogenic pathway directly responsible for APP axonal transport dysregulation. Our findings may help to understand and ultimately mitigate the risk of developing AD following mTBI.

Paclitaxel is an effective chemotherapeutic that, despite its common use, frequently causes debilitating peripheral sensory neuropathy. Paclitaxel binds to and stabilizes microtubules, and through unknown mechanisms, causes abnormal microtubule aggregation. Given that microtubules contribute to the mechanical properties of cells, we tested the hypothesis that paclitaxel treatment would alter the stiffness of sensory nerves. Rat sural nerves were excised and soaked in Ringer's solution with or without paclitaxel. Nerves were secured between a force transducer and actuator, and linearly strained. Stress-strain curves were generated, from which elastic moduli were calculated. Paclitaxel treated nerves exhibited significantly higher moduli in both linear and transition regions of the curve. A composite-tissue model was then generated to estimate the stiffness increase in the cellular fraction of the nerve following paclitaxel treatment. This model was supported experimentally by data on mechanical properties of sural nerves stripped of their epineurium, and area fractions of the cellular and connective tissue components of the rat sural nerve, calculated from immunohistochemical images. Model results revealed that the cellular components of the nerve must stiffen 12x to 115x, depending on the initial axonal modulus assumed, in order to achieve the observed tissue level mechanical changes. Consistent with such an increase, electron microscopy showed increased microtubule aggregation and cytoskeletal packing, suggestive of a more cross-linked cytoskeleton. Overall, our data suggests that paclitaxel treatment induces increased microtubule bundling in axons, which leads to alterations in tissue-level mechanical properties.

Paclitaxel is an effective chemotherapeutic agent that, despite its common use, often causes peripheral sensory neuropathy. In neurons, paclitaxel binds to and stabilizes microtubules, and through unknown mechanisms, bundles microtubules and disrupts their organization. Because microtubules serve as tracks on which a variety of axonal cargoes are transported, a leading hypothesis for the etiology of paclitaxel-induced neuropathy is that these changes to microtubule organization impair axonal transport. In addition to supporting transport, microtubules also serve a structural role, accommodating axonal extension occurring during axonal growth or joint movement. In light of this dual role for microtubules, we tested the hypothesis that axonal stretch amplified the effects of paclitaxel on axonal transport. Embryonic rat dorsal root ganglia were cultured on stretchable silicone substrates, and parameters describing the axonal transport of three distinct cargoes--mitochondria, synaptophysin, and actin--were measured with and without paclitaxel treatment and axonal strain. Paclitaxel treatment, particularly in combination with stretch, led to severe perturbations in several transport parameters, including the number, velocity, and travel distance of cargoes in the axon. Our results suggest that mechanical loading of neurons can exacerbate transport deficits associated with paclitaxel treatment, raising the interesting possibility that paclitaxel influences neuronal function in a multi-factorial manner.