KLC4 shapes axon arbors during development and mediates adult behavior

Development of elaborate and polarized neuronal morphology requires precisely regulated transport of cellular cargos by motor proteins such as kinesin-1. Kinesin-1 has numerous cellular cargos which must be delivered to unique neuronal compartments. The process by which this motor selectively transports and delivers cargo to regulate neuronal morphogenesis is poorly understood. Our work implicates one kinesin light chain subunit, KLC4, as an essential regulator of axon branching and arborization pattern of sensory neurons during development. Using several live imaging approaches in klc4 mutant zebrafish, we show that KLC4 is required for stabilization of nascent axon branches and for proper microtubule (MT) dynamics. Furthermore, KLC4 is required for the contact repulsion necessary for tiling of peripheral axon arbors: in klc4 mutants, peripheral axons showed abnormal fasciculation, a behavior characteristic of central axons, suggesting that KLC4 patterns axonal compartments and helps define axon identity. Finally, we find that klc4 mutant adults show anxiety-like behavior in a novel tank test, implicating klc4 as a novel gene involved in stress response circuits.

Neurotrophin gene therapy may be able to treat individuals with noise-induced hearing loss or neural presbyacusis

Neurotrophin (NT) cochlear gene therapy might perhaps give a single treatment that might greatly enhance neuronal survival, resulting in CI patients, provided the many challenges described above can be adequately addressed and safety concerns allayed by more animal model investigations. This is particularly crucial for juvenile CI patients, who have to rely on electrical hearing for the remainder of their lives, and whose outcomes are quite different. In addition, NT gene therapy may have the potential to treat patients with noise-induced hearing loss or neural presbyacusis (e.g., age-related cochlear synaptopathy), where primary neuronal loss is a key cause of hearing loss. Animal research into noise-induced hearing loss has shown that even exposures that generate only reversible threshold alterations and no hair cell loss can lead to permanent loss of SGN synapses on hair cells, resulting in functional impairments and ultimately SGN degeneration. Cochlear synapses frequently precede both hair cell loss and threshold increases in human ears, according to current studies. Cochlear synaptopathy is characterized by ears with intact hair cell populations and normal audiograms as "hidden" hearing loss. Many frequent perceptual abnormalities, including speech-in-noise difficulties, tinnitus, and hyperacusis, are likely produced by suppressing affected neurons, which radically alters information processing. Thus, in the future, NT gene therapy may be successful in inducing SGN peripheral axon resprouting and synaptic regeneration into residual (or even regenerated) hair cell populations. We have demonstrated compelling evidence that, in this investigation, BDNF gene therapy can boost SGN survival and enhance peripheral axon maintenance or rerouting. NT-3 has been found in adult animals exposed to acoustic damage to induce synaptic regeneration of these fibers, reconnecting them to hair cells and their ribbon synapses, and restoring hearing function. Combining BDNF and NT-3 gene therapy may be the most effective way to maintain/restore a more normal cochlear neuronal substrate.

A Re-evaluation of Whether Non-monosynaptic Homonymous H Reflex Facilitation Tests Propriospinal Circuits

The C3–C4 propriospinal system is an important pathway mediating movement in cats; it contributes to movements in primates (including humans), and may have a role in recovery after lesion. Validated clinical tests of this system would find many applications, therefore we sought to test whether non-monosynaptic homonymous facilitation of the forearm flexor H reflex is mediated solely via a C3–C4 propriospinal pathway. In one anesthetized macaque monkey, median nerve stimulation elicited an H reflex in the flexor carpi radialis (FCR). Median nerve conditioning stimuli at sub-threshold intensities facilitated the H reflex, for inter-stimulus intervals up to 30 ms. Successive spinal surgical hemisections were then made. C2 lesion left the homonymous facilitation intact, suggesting mediation by spinal, not supraspinal pathways. Facilitation also remained after a second lesion at C5, indicating a major role for segmental (C7–C8) rather than propriospinal (C3–C4) interneurons. In separate experiments in five healthy human subjects, a threshold tracking approach assessed changes in peripheral axon excitability after conditioning stimulation. This was found to be enhanced up to 20 ms after the conditioning stimulus, and could partly, although not completely, underlie the H reflex facilitation seen. We conclude that homonymous facilitation of the H reflex in FCR can be produced by segmental spinal mechanisms, as well as by a supranormal period of nerve excitability. Unfortunately, this straightforward test cannot therefore be used for selective assessment of propriospinal circuits.

Structure and function of muscle

The motor unit—the final common pathway for all voluntary muscle activity—is composed of an anterior horn cell, its peripheral axon, the axon terminal branches, the associated neuromuscular junctions, and the muscle fibres innervated. The muscle cells are multinucleate units with unique structures adapted for response to metabolic, nervous, and autocrine signals. Meanwhile, there are also different types of motor units: type 1—rich in mitochondria and specialized for oxidative metabolism of fat; type 2—larger fibres with abundant glycogen that generate energy by glycosis and are critical for short-lived muscle contraction. Knowledge of the underlying molecular cell biology, neurophysiology, and biochemical energetics of muscle provides a useful basis for understanding the symptoms, signs, and pathogenesis of clinical disorders affecting the muscles. Mutations in sarcolemmal proteins, such as dystrophin, cause diseases with widespread effects on skeletal muscle function, the heart, and survival.

Effects of Repeated 20-Hz Electrical Stimulation on Functional Recovery Following Peripheral Nerve Injury

One hour of 20-Hz continuous electrical stimulation (ES) applied at the time of injury promotes the regeneration of axons in cut peripheral nerves. A more robust enhancement of peripheral axon regeneration is achieved by 2 weeks of daily treadmill exercise. We investigated whether repeated applications of brief ES (mES) would be more effective in promoting regeneration than a single application. Sciatic nerves of C57B6 mice were cut and repaired by end-to-end anastomosis. At that time and every third day for 2 weeks, the repaired nerve was stimulated for 1 hour at 20 Hz. In controls, injured mice were either untreated or treated with ES only once. Direct muscle responses recorded from reinnervated muscles in awake animals were observed earlier both in mice treated with ES and mES than untreated controls. Their amplitudes increased progressively over the post transection study period, but the rate of this progression was increased significantly only in animals treated once with ES. Monosynaptic H reflexes recovered to pretransection levels in both untreated and singly treated mice but in the animals treated repeatedly, they were maintained at more than twice that of the same reflexes recorded prior to injury. In anatomical analyses, both excitatory and inhibitory synaptic contacts with the cell bodies of injured motoneurons, including those expressing the vesicular glutamate transporter 1 (VGLUT1), were sustained in mice treated repeatedly but not in singly treated or untreated mice. Repeated ES does not enhance the rate of restoration of functional muscle reinnervation and results in the retention of exaggerated reflexes.

Tissue Engineered Axon Tracts Serve as Living Scaffolds to Accelerate Axonal Regeneration and Functional Recovery Following Peripheral Nerve Injury in Rats

Although regeneration of damaged axons in peripheral nerves has long been observed, the mechanisms facilitating this growth are not well characterized. Recently, we demonstrated that host axon regeneration could be greatly enhanced by transplanting engineered living axon tracts to guide outgrowth. Here, we used a model of rat sciatic nerve transection to explore potential mechanisms of this facilitated regeneration and its efficacy in comparison with nerve guidance tubes (NGTs) and autografts. Tissue engineered nerve grafts (TENGs) were developed via “stretch-growth” in mechanobioreactors and consisted of centimeter-scale aligned axonal tracts. Either TENGs, NGTs or autografts (reversed nerve) were then transplanted to bridge a 1 cm segmental gap in the sciatic nerve with the mechanisms of axonal regrowth assessed at 2 weeks and the extent of functional recovery assessed at 16 weeks. We observed numerous host axons growing directly along and intertwining with pre-formed axonal tracts in TENGs. This behavior appears to mimic the action of “pioneer” axons in developmental pathfinding by providing living cues for directed and accelerated outgrowth. Indeed, we found that the rates of axon regeneration were 3-4 fold faster than NGTs and equivalent to autografts. It was also observed that infiltration of host Schwann cells – traditional drivers of peripheral axon regeneration – was both accelerated and progressed directly along TENG axonal tracts. These TENG repairs resulted in levels of functional recovery equivalent to autografts, with each being several fold superior to NGT repairs. This new mechanism – which we term “axon-facilitated axon-regeneration” – may be further exploited to enhance axonal regeneration and functional recovery following neurotrauma.