Assessment of Mitochondrial Trafficking as a Surrogate for Fast Axonal Transport in Human Induced Pluripotent Stem Cell-Derived Spinal Motor Neurons.

Axonal transport is essential for the development, function, and survival of the nervous system. In an energy-demanding process, motor proteins act in concert with microtubules to deliver cargoes, such as organelles, from one end of the axon to the other. Perturbations in axonal transport are a prominent phenotype of many neurodegenerative diseases, including amyotrophic lateral sclerosis. Here, we describe a simple method to fluorescently label mitochondrial cargo, a surrogate for fast axonal transport, in human induced pluripotent stem cell-derived motor neurons. This method enables the sparse labeling of axons to track directionality of movement and can be adapted to assess not only the cell autonomous effects of a genetic mutation on axonal transport but also the cell non-autonomous effects, through the use of conditioned medium and/or co-culture systems.

A possible mechanism for neurofilament slowing down in myelinated axon: Phosphorylation-induced variation of NF kinetics.

Neurofilaments(NFs) are the most abundant intermediate filaments that make up the inner volume of axon, with possible phosphorylation on their side arms, and their slow axonal transport by molecular motors along microtubule tracks in a "stop-and-go" manner with rapid, intermittent and bidirectional motion. The kinetics of NFs and morphology of axon are dramatically different between myelinate internode and unmyelinated node of Ranvier. The NFs in the node transport as 7.6 times faster as in the internode, and the distribution of NFs population in the internode is 7.6 folds as much as in the node of Ranvier. We hypothesize that the phosphorylation of NFs could reduce the on-track rate and slow down their transport velocity in the internode. By modifying the '6-state' model with (a) an extra phosphorylation kinetics to each six state and (b) construction a new '8-state' model in which NFs at off-track can be phosphorylated and have smaller on-track rate, our model and simulation demonstrate that the phosphorylation-induced decrease of on-track rate could slow down the NFs average velocity and increase the axonal caliber. The degree of phosphorylation may indicate the extent of velocity reduction. The Continuity equation used in our paper predicts that the ratio of NFs population is inverse proportional to the ratios of average velocity of NFs between node of Ranvier and internode. We speculate that the myelination of axon could increase the level of phosphorylation of NF side arms, and decrease the possibility of NFs to get on-track of microtubules, therefore slow down their transport velocity. In summary, our work provides a potential mechanism for understanding the phosphorylation kinetics of NFs in regulating their transport and morphology of axon in myelinated axons, and the different kinetics of NFs between node and internode.

Computational analysis of axonal transport: a novel assessment of neurotoxicity, neuronal development and functions.

Axonal transport plays a crucial role in neuronal morphogenesis, survival and function. Despite its importance, however, the molecular mechanisms of axonal transport remain mostly unknown because a simple and quantitative assay system for monitoring this cellular process has been lacking. In order to better characterize the mechanisms involved in axonal transport, we formulate a novel computer-assisted monitoring system of axonal transport. Potential uses of this system and implications for future studies will be discussed.

Electrophysiological and histologic assessment of retinal ganglion cell fate in a mouse model for OPA1-associated autosomal dominant optic atrophy.

PURPOSE: The main disease features of autosomal dominant optic atrophy (ADOA) are a bilateral reduction of visual acuity, cecocentral scotoma, and frequently tritanopia, which have been ascribed to a progressive loss of retinal ganglion cells (RGCs) and subsequent degeneration of the optic nerve. The main disease-causing gene is OPA1. Here, we examine a mouse carrying a pathogenic mutation in Opa1 by electrophysiological measurements and assess the fate of RGCs. METHODS: Two-year-old animals underwent a full examination by electroretinography (ERG) and visually evoked potential (VEP) measurements to assess the function of the outer and inner retina and the optic nerve. Retrograde Fluorogold labeling was performed to determine the number of surviving RGCs and to assess axonal transport by neurofilament counterstaining. Phagocytosis-dependent labeled microglial cells were identified by an Iba-1 staining. RESULTS: ERG responses were normal in aged Opa1 mice. VEP measurements revealed significantly reduced amplitudes but no change in the latencies in contrast to extended latencies found in glaucoma. Retrograde labeling of RGCs showed a significant reduction in the number of RGCs in Opa1 mice. Long-term experiments revealed the presence of microglial cells with ingested fluorescent dye. CONCLUSIONS: This is the first electrophysiological demonstration of a visual function deficit in aged Opa1 mice. VEP measurements and retrograde labeling experiments show that the number of RGCs is reduced whereas the remaining RGCs and axons function normally. Taken together, these findings support an ascending progress of degeneration from the soma toward the axon.

Chapter 8 - Nanoparticles: Transport across the olfactory epithelium and application to the assessment of brain function in health and disease.

The exciting advances within nanotechnology are beginning to be harnessed by the medical field. Nanoparticles have been used for drug delivery into the brain and have been explored for imaging, sensing, and analytical purposes. The science of nanoparticles encompasses a vast array of biological, chemical, physical, and engineering research, different aspects of which are specifically addressed in each of the chapters of this volume. Nanomaterials such as nanospheres, nanotubes, nanowires, fullerene derivatives (buckyballs), and quantum dots (Qdots) are at the forefront of scientific attention, as they provide new consumer products and advance the scientific development of novel analytical tools in medicine and in the physical sciences. This chapter will briefly survey some aspects of nanoparticle biology focusing on the following: (1) the role of olfactory nanoparticle transport into the central nervous system (CNS), both as a potential route for effective drug delivery and as a route for the passage of noxious substances into the brain proper; (2) nanoparticles as sensors of cell function and toxicity; and (3) some adverse effects of nanoparticles on the dysregulation of brain redox status.

Hippocampal neurons transplanted into ischemically lesioned hippocampus: anatomical assessment of survival, maturation and integration.

Cerebral ischemia can be caused by many diverse conditions such as cardiac arrest and severe hypotension and is often the cause of secondary brain damage following head injury or infantile birth trauma. The inadequate cerebral blood flow can result in permanent loss of essential brain circuitries and neurological deficits. The CA1 region of the hippocampal formation is the region of the brain that is most often lesioned following transient forebrain ischemia and is associated with impairments of learning and memory. Furthermore, the loss of such a large target area can lead to detrimental post-trauma synaptic reorganization. Since methods are not currently available for the prevention of neuronal loss following cerebral ischemia, a number of anatomical methodologies were utilized to investigate whether transplanted neurons had the potential to afford some measure of repair. The hippocampal CA1 region of the rat brain was lesioned by transient forebrain ischemia and subsequently repopulated with suspensions of fetal hippocampal tissue. The ability of the transplanted neurons to remain viable when placed into a degenerating environment was confirmed by the histological demonstration of 3H-thymidine labelled neurons in the lesioned region. Histological and immunohistochemical techniques showed that the transplanted neurons developed cytological features that were indistinguishable from their normal CA1 counterparts, often showed a remarkable degree of organization, and expressed some of the same neuron specific proteins; specifically calbindin-D28K and parvalbumin. Acetylcholinesterase histochemistry and retrograde axonal transport of Fluorogold demonstrated that some afferent and efferent fibre projections to and from the septal nucleus could be reinstated. The data have shown that the transplanted neurons can demonstrate many of the anatomical properties that are characteristic of the adult cells they have replaced and therefore have great potential for the reconstruction of severe focal lesions due to ischemia.