UV irradiation accelerates amyloid precursor protein (APP) processing and disrupts APP axonal transport.

Overexpression and/or abnormal cleavage of amyloid precursor protein (APP) are linked to Alzheimer's disease (AD) development and progression. However, the molecular mechanisms regulating cellular levels of APP or its processing, and the physiological and pathological consequences of altered processing are not well understood. Here, using mouse and human cells, we found that neuronal damage induced by UV irradiation leads to specific APP, APLP1, and APLP2 decline by accelerating their secretase-dependent processing. Pharmacological inhibition of endosomal/lysosomal activity partially protects UV-induced APP processing implying contribution of the endosomal and/or lysosomal compartments in this process. We found that a biological consequence of UV-induced γ-secretase processing of APP is impairment of APP axonal transport. To probe the functional consequences of impaired APP axonal transport, we isolated and analyzed presumptive APP-containing axonal transport vesicles from mouse cortical synaptosomes using electron microscopy, biochemical, and mass spectrometry analyses. We identified a population of morphologically heterogeneous organelles that contains APP, the secretase machinery, molecular motors, and previously proposed and new residents of APP vesicles. These possible cargoes are enriched in proteins whose dysfunction could contribute to neuronal malfunction and diseases of the nervous system including AD. Together, these results suggest that damage-induced APP processing might impair APP axonal transport, which could result in failure of synaptic maintenance and neuronal dysfunction.

Activity-dependent synaptic capture of transiting peptidergic vesicles.

Synapses require resources synthesized in the neuronal soma, but there are no known mechanisms to overcome delays associated with the synthesis and axonal transport of new proteins generated in response to activity, or to direct resources specifically to active synapses. Here, in vivo imaging of the Drosophila melanogaster neuromuscular junction reveals a cell-biological strategy that addresses these constraints. Peptidergic vesicles continually transit through resting terminals, but retrograde peptidergic vesicle flux is accessed following activity to rapidly boost neuropeptide content in synaptic boutons. The presence of excess transiting vesicles implies that synaptic neuropeptide stores are limited by the capture of peptidergic vesicles at the terminal, rather than by synthesis in the soma or delivery via the axon. Furthermore, activity-dependent capture from a pool of transiting vesicles provides a nerve terminal-based mechanism for directing distally and slowly generated resources quickly to active synapses. Finally, retrograde transport in the nerve terminal is regulated by activity.

Radiation-induced impairment of optic nerve axonal transport in tree shrews and rats monitored by longitudinal manganese-enhanced MRI.

PURPOSE: Radiation-induced optic neuropathy (RION) is a serious complication that occurs after radiation therapy of tumors in the vicinity of the optic nerve, yet its mechanism and imaging features are poorly understood. In this study, we employed manganese-enhanced MRI (MEMRI) to assess optic nerve axonal transport in tree shrews and rats after irradiation. MATERIALS AND METHODS: A comparison of normal visual projections in tree shrews and rats was conducted by intravitreal MnCl2 injection followed by MRI. Adult male tree shrews and rats received a total dose of 20 Gy delivered in two fractions (10 Gy per fraction) within 5 days. Longitudinal MEMRI was conducted 5, 10, 20 and 30 weeks after radiation. At the end of observation, motor proteins involved in axonal transport were detected by western blotting, and the axon cytoskeleton was assessed by immunofluorescence. RESULTS: The eyeballs, lens sizes, vitreous volumes, optic nerves and superior colliculi of tree shrews were significantly larger than those of rats on MEMRI (P < 0.05). The Mn2+-enhancement of the optic nerve showed no significant changes at 5 and 10 weeks (P > 0.05) but decreased gradually from 20 to 30 weeks postirradiation (P < 0.05). The enhancement of the superior colliculus gradually decreased from 5 weeks to 30 weeks, and the decrease was most significant at 30 weeks (P < 0.05). The levels of the motor proteins cytoplasmic dynein-1, kinesin-1 and kinesin-2 in the experimental group were significantly decreased (P < 0.05). The immunofluorescence results showed that the α-tubulin, ß-tubulin and SMI 31 levels in the experimental groups and control groups were not significantly different (P > 0.05). CONCLUSION: Tree shrews show great advantages in visual neuroscience research involving MEMRI. The main cause of the decline in axonal transport in RION is an insufficient level of motor protein rather than damage to the axonal cytoskeletal structure. Longitudinal MEMRI can be used to detect changes in axonal transport function and to observe the relatively intact axon structure from the early to late stages after radiation administration.

Age-related changes in axonal transport.

In rats the rate of axonal transport (AT) or radiolabeled material decreased in the ventral roots of the spinal cord and the vagal and hypoglossal nerves with aging. A maximum AT deceleration in old age was observed in the vagus. The uncoupling of oxidative phosphorylation, inhibition of glycolysis and hypoxia induced a greater AT deceleration in old rats as compared to adults. Small doses of sodium fluoride accelerated AT, and this correlated with a rise in cAMP levels in ventral roots. High doses of sodium fluoride decelerated AT more markedly in old rats. It was shown that anabolic hormones (sex steroids and thyroxine) accelerated AT in both adult and old rats, whereas insulin induced a rise in AT rate in only adults. The catabolic steroid, hydrocortisone decelerated AT. In old rats castration diminished AT, while thyroidectomy had no effect. It was also shown that hydrocortisone and testosterone were transported along axons, reached fibers of the skeletal muscles, and hyperpolarized the plasma membrane. In old age the latent period was extended. Following 73 to 74 days of irradiation, AT slowed down in all the nerves studied in both adult and old rats. Following irradiation hormonal effects on AT changed, for example, the stimulatory effect of estradiol became weak, especially in old rats. Changes in AT could be an important mechanism of disordering the growth of neurons and innervated cells in old age.

830 nm laser irradiation induces varicosity formation, reduces mitochondrial membrane potential and blocks fast axonal flow in small and medium diameter rat dorsal root ganglion neurons: implications for the analgesic effects of 830 nm laser.

We report the formation of 830 nm (cw) laser-induced, reversible axonal varicosities, using immunostaining with beta-tubulin, in small and medium diameter, TRPV-1 positive, cultured rat DRG neurons. Laser also induced a progressive and statistically significant decrease (p<0.005) in MMP in mitochondria in and between static axonal varicosities. In cell bodies of the neuron, the decrease in MMP was also statistically significant (p<0.05), but the decrease occurred more slowly. Importantly we also report for the first time that 830 nm (cw) laser blocked fast axonal flow, imaged in real time using confocal laser microscopy and JC-1 as mitotracker. Control neurons in parallel cultures remained unaffected with no varicosity formation and no change in MMP. Mitochondrial movement was continuous and measured along the axons at a rate of 0.8 microm/s (range 0.5-2 microm/s), consistent with fast axonal flow. Photoacceptors in the mitochondrial membrane absorb laser and mediate the transduction of laser energy into electrochemical changes, initiating a secondary cascade of intracellular events. In neurons, this results in a decrease in MMP with a concurrent decrease in available ATP required for nerve function, including maintenance of microtubules and molecular motors, dyneins and kinesins, responsible for fast axonal flow. Laser-induced neural blockade is a consequence of such changes and provide a mechanism for a neural basis of laser-induced pain relief. The repeated application of laser in a clinical setting modulates nociception and reduces pain. The application of laser therapy for chronic pain may provide a non-drug alternative for the management of chronic pain.

Effect of endoscopic white light on the developing visual pathway: a histologic, histochemical, and behavioral study.

OBJECTIVE: We examined the potential teratogenic effect of endoscopic white light on the developing visual pathways. STUDY DESIGN: The right eye of chicken embryos (n = 22) was exposed to maximal endoscopic light intensity on day 10 of development. At day 17 of development the histologic characteristics of the light-exposed retinas were compared with those of the control embryos (n = 4). Normal functioning of the light-exposed eye was assessed by intravitreal injection of wheat germ agglutinin-horseradish peroxidase and observation of its axonal transport pattern to the diencephalic and mesencephalic visual centers. Axonal transport patterns were compared with those found in previous studies of normal embryos. Behavioral feeding patterns were compared between two groups of newly hatched chickens, one exposed to endoscopic light after hatching (n = 13) and the other, an unexposed control group (n = 12). RESULTS: No evidence of retinal damage, altered axonal transport or altered feeding patterns could be found between control and experimental animals. CONCLUSION: Endoscopic white light does not appear to be harmful to the developing retina and visual pathway.

Differential suppression of axoplasmic transport: effects of light irradiation to the growth cone of cultured dorsal root ganglion neurons.

1. Growth cones of cultured dorsal root ganglion neurons from mice were irradiated using a mercury lamp. 2. The flux of particles of fast retrograde axoplasmic transport decreased promptly after light irradiation without a change in velocity. 3. That of anterograde transport decreased as well, but with a significant latency. The decrease in anterograde flux was attributed to decreased velocity of particles. 4. Video-enhanced contrast microscopy of growth cones revealed transient swelling of growth cones and transient stagnation of particles in growth cones. 5. The longer the neurite, the larger the latency of the change of the anterograde transport; peripheral information was calculated to be conveyed to the cell body at a speed of 6 microns/min. 6. The mechanism of this information conveyance and the export of materials from the cell body are discussed.

Effect of light on oxygen-induced retinopathy in the rat model. Light and OIR in the rat.

The purpose of this study was to establish whether exposure to intense lighting favors the development or aggravates experimental oxygen-induced retinopathy in the newborn rat. Five groups of Wistar rats were studied. The control group was maintained for the first 14 days of life under conditions of cyclical (12L:12D) lighting at 12 Lx in room air. Two other groups were subjected, for the same amount of time, to semi-darkness (2 Lx; 12L: 12D), one with room air and the other with supplemental 80% oxygen. The final two groups were exposed to the same room air and hyperoxic treatments under intense lighting conditions (600 Lx; 12L:12D). After the treatment period, four rats were randomly chosen from each group, sacrificed and their retinas examined under electron microscope. Marked structural changes were seen only in the photoreceptor outer segments of those rats exposed to intense light. In eighty-five of the remaining rats retinal vascular morphology was examined in retinal flat mounts after intracardiac injection of India ink. Retinopathy was observed in rats treated with hyperoxia but no significant differences could be attributed to the light conditions under which the retinopathic rats had been maintained. In the rest of the rats, axonal transport along the optical pathways was evaluated after intravitreal injection of (3H) taurine. In the two groups exposed to hyperoxia, axonal transport was altered, but less markedly in those exposed to intense lighting than in those exposed to semi-darkness. Intense illumination under conditions of normoxia favors axonal transport. Exposure to intense lighting does not seem to aggravate oxygen induced retinopathy in the rat though it does produce structural lesions of the photoreceptors.

Dynein is the motor for retrograde axonal transport of organelles.

Vesicular organelles in axons of nerve cells are transported along microtubules either toward their plus ends (fast anterograde transport) or toward their minus ends (retrograde transport). Two microtubule-based motors were previously identified by examining plastic beads induced to move along microtubules by cytosol fractions from the squid giant axon: (i) an anterograde motor, kinesin, and (ii) a retrograde motor, which is characterized here. The retrograde motor, a cytosolic protein previously termed HMW1, was purified from optic lobes and extruded axoplasm by nucleotide-dependent microtubule affinity and release; microtubule gliding was used as the assay of motor activity. The following properties of the retrograde motor suggest that it is cytoplasmic dynein: (i) sedimentation at 20-22 S with a heavy chain of Mr greater than 200,000 that coelectrophoreses with the alpha and beta subunits of axonemal dynein, (ii) cleavage by UV irradiation in the presence of ATP and vanadate, and (iii) a molecular structure resembling two-headed dynein from axonemes. Furthermore, bead movement toward the minus end of microtubules was blocked when axoplasmic supernatants were treated with UV/vanadate. Treatment of axoplasmic supernatant with UV/vanadate also blocks the retrograde movement of purified organelles in vitro without changing the number of anterograde moving organelles, indicating that dynein interacts specifically with a subgroup of organelles programmed to move toward the cell body. However, purified optic lobe dynein, like purified kinesin, does not by itself promote the movement of purified organelles along microtubules, suggesting that additional axoplasmic factors are necessary for retrograde as well as anterograde transport.

Retrograde but not anterograde bead movement in intact axons requires dynein.

Dynein and kinesin have been implicated as the molecular motors that are responsible for the fast transport of axonal membranous organelles and vesicles. Experiments performed in vitro with partially reconstituted preparations have led to the hypothesis that kinesin moves organelles in the anterograde direction and dynein moves them in the retrograde direction. However, the molecular basis of transport directionality remains unclear. In the experiments described here, carboxylated fluorescent beads were injected into living Mauthner axons of lamprey and the beads were observed to move in both the anterograde and retrograde directions. The bead movement in both directions required intact microtubules, occurred at velocities approaching organelle fast transport in vivo, and was inhibited by vanadate at concentrations that inhibit organelle fast transport. When living axons were injected with micromolar concentrations of vanadate and irradiated at 365 nm prior to bead injections, a treatment that results in the V1 photolysis of dynein, the retrograde movement of the beads was specifically abolished. Neither the ultraviolet irradiation alone nor the vanadate alone produced the retrograde-specific inhibition. These results support the hypothesis that dynein is required for retrograde, but not anterograde, transport in vivo.