83 research outputs found
Axonal Transport: The Delivery System Keeping Nerve Cells Alive
Cells are a little bit like cities, as they have all sorts of different cargoes that need to be constantly transported to particular destinations. This delivery process is especially important for nerve cells, because they have a long, thin tube-like structure called an axon. Nerve cells need to deliver a wide range of proteins and specialized structures up and down axons if they are to remain alive and healthy. Neurons do this using a delivery system called axonal transport. In this article, we will explore what axonal transport is and how it works. We will also discover what cargoes the nerve cells need to spread along their axons, and why this is so important for keeping nerves alive. Finally, we will look at what happens when axonal transport goes wrong and see how this may cause diseases of the nervous system
Motor Neuron Gene Therapy: Lessons from Spinal Muscular Atrophy for Amyotrophic Lateral Sclerosis
Spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS) are severe nervous system diseases characterized by the degeneration of lower motor neurons. They share a number of additional pathological, cellular, and genetic parallels suggesting that mechanistic and clinical insights into one disorder may have value for the other. While there are currently no clinical ALS gene therapies, the splice-switching antisense oligonucleotide, nusinersen, was recently approved for SMA. This milestone was achieved through extensive pre-clinical research and patient trials, which together have spawned fundamental insights into motor neuron gene therapy. We have thus tried to distil key information garnered from SMA research, in the hope that it may stimulate a more directed approach to ALS gene therapy. Not only must the type of therapeutic (e.g., antisense oligonucleotide vs. viral vector) be sensibly selected, but considerable thought must be applied to the where, which, what, and when in order to enhance treatment benefit: to where (cell types and tissues) must the drug be delivered and how can this be best achieved? Which perturbed pathways must be corrected and can they be concurrently targeted? What dosing regime and concentration should be used? When should medication be administered? These questions are intuitive, but central to identifying and optimizing a successful gene therapy. Providing definitive solutions to these quandaries will be difficult, but clear thinking about therapeutic testing is necessary if we are to have the best chance of developing viable ALS gene therapies and improving upon early generation SMA treatments
Antisense oligonucleotides and other genetic therapies made simple
Many genetic neurological diseases result from the dysfunction of single proteins. Genetic therapies aim to modify these disease-associated proteins by targeting the RNA and DNA precursors. This review provides a brief overview of the main types of genetic therapies, with a focus on antisense oligonucleotides (ASOs) and RNA interference (RNAi). We use examples of new genetic therapies for spinal muscular atrophy, Duchenne muscular dystrophy and familial amyloid polyneuropathy to highlight the different mechanisms of action of ASOs and RNAi
Plexin-semaphorin signalling modifies neuromuscular defects in a Drosophila model of peripheral neuropathy
Dominant mutations in GARS, encoding the ubiquitous enzyme glycyl-tRNA synthetase (GlyRS), cause peripheral nerve degeneration and Charcot-Marie-Tooth disease type 2D (CMT2D). This genetic disorder exemplifies a recurring paradigm in neurodegeneration, in which mutations in essential genes cause selective degeneration of the nervous system. Recent evidence suggests that the mechanism underlying CMT2D involves extracellular neomorphic binding of mutant GlyRS to neuronally-expressed proteins. Consistent with this, our previous studies indicate a non-cell autonomous mechanism, whereby mutant GlyRS is secreted and interacts with the neuromuscular junction (NMJ). In this Drosophila model for CMT2D, we have previously shown that mutant gars expression decreases viability and larval motor function, and causes a concurrent build-up of mutant GlyRS at the larval neuromuscular presynapse. Here, we report additional phenotypes that closely mimic the axonal branching defects of Drosophila plexin transmembrane receptor mutants, implying interference of plexin signaling in gars mutants. Individual dosage reduction of two Drosophila Plexins, plexin A (plexA) and B (plexB) enhances and represses the viability and larval motor defects caused by mutant GlyRS, respectively. However, we find plexB levels, but not plexA levels, modify mutant GlyRS association with the presynaptic membrane. Furthermore, increasing availability of the plexB ligand, Semaphorin-2a (Sema2a), alleviates the pathology and the build-up of mutant GlyRS, suggesting competition for plexB binding may be occurring between these two ligands. This toxic gain-of-function and subversion of neurodevelopmental processes indicate that signaling pathways governing axonal guidance could be integral to neuropathology and may underlie the non-cell autonomous CMT2D mechanism
A video protocol for rapid dissection of mouse dorsal root ganglia from defined spinal levels
OBJECTIVE: Dorsal root ganglia (DRG) are heterogeneous assemblies of assorted sensory neuron cell bodies found in bilateral pairs at every level of the spinal column. Pseudounipolar afferent neurons convert external stimuli from the environment into electrical signals that are retrogradely transmitted to the spinal cord dorsal horn. To do this, they extend single axons from their DRG-resident somas that then bifurcate and project both centrally and distally. DRG can be dissected from mice at embryonic stages and any age post-natally, and have been extensively used to study sensory neuron development and function, response to injury, and pathological processes in acquired and genetic diseases. We have previously published a step-by-step dissection method for the rapid isolation of post-natal mouse DRG. Here, the objective is to extend the protocol by providing training videos that showcase the dissection in fine detail and permit the extraction of ganglia from defined spinal levels. RESULTS: By following this method, the reader will be able to swiftly and accurately isolate specific lumbar, thoracic, and cervical DRG from mice. Dissected ganglia can then be used for RNA/protein analyses, subjected to immunohistochemical examination, and cultured as explants or dissociated primary neurons, for in-depth investigations of sensory neuron biology
Methodological advances in imaging intravital axonal transport
Axonal transport is the active process whereby neurons transport cargoes such as organelles and proteins anterogradely from the cell body to the axon terminal and retrogradely in the opposite direction. Bi-directional transport in axons is absolutely essential for the functioning and survival of neurons and appears to be negatively impacted by both aging and diseases of the nervous system, such as Alzheimer's disease and amyotrophic lateral sclerosis. The movement of individual cargoes along axons has been studied in vitro in live neurons and tissue explants for a number of years; however, it is currently unclear as to whether these systems faithfully and consistently replicate the in vivo situation. A number of intravital techniques originally developed for studying diverse biological events have recently been adapted to monitor axonal transport in real-time in a range of live organisms and are providing novel insight into this dynamic process. Here, we highlight these methodological advances in intravital imaging of axonal transport, outlining key strengths and limitations while discussing findings, possible improvements, and outstanding questions
Axonal transport and neurological disease
Axonal transport is the process whereby motor proteins actively navigate microtubules to deliver diverse cargoes, such as organelles, from one end of the axon to the other, and is widely regarded as essential for nerve development, function and survival. Mutations in genes encoding key components of the transport machinery, including motor proteins, motor adaptors and microtubules, have been discovered to cause neurological disease. Moreover, disruptions in axonal cargo trafficking have been extensively reported across a wide range of nervous system disorders. However, whether these impairments have a major causative role in, are contributing to or are simply a consequence of neuronal degeneration remains unclear. Therefore, the fundamental relevance of defective trafficking along axons to nerve dysfunction and pathology is often debated. In this article, we review the latest evidence emerging from human and in vivo studies on whether perturbations in axonal transport are indeed integral to the pathogenesis of neurological disease
Neuropilin 1 sequestration by neuropathogenic mutant glycyl-tRNA synthetase is permissive to vascular homeostasis
The mechanism by which dominantly inherited mutations in the housekeeping gene GARS, which
encodes glycyl-tRNA synthetase (GlyRS), mediate selective peripheral nerve toxicity resulting in
Charcot-Marie-Tooth disease type 2D (CMT2D) is still largely unresolved. The transmembrane receptor
protein neuropilin 1 (Nrp1) was recently identifed as an aberrant extracellular binding partner of
mutant GlyRS. Formation of the Nrp1/mutant GlyRS complex antagonises Nrp1 interaction with
one of its main natural ligands, vascular endothelial growth factor-A (VEGF-A), contributing to
neurodegeneration. However, reduced extracellular binding of VEGF-A to Nrp1 is known to disrupt
post-natal blood vessel development and growth. We therefore analysed the vascular system at early
and late symptomatic time points in CMT2D mouse muscles, retina, and sciatic nerve, as well as in
embryonic hindbrain. Mutant tissues show no diference in blood vessel diameter, density/growth, and
branching from embryonic development to three months, spanning the duration over which numerous
sensory and neuromuscular phenotypes manifest. Our fndings indicate that mutant GlyRS-mediated
disruption of Nrp1/VEGF-A signalling is permissive to maturation and maintenance of the vasculature in
CMT2D mice
Aligned electrospun fibers for neural patterning
OBJECTIVES: To test a 3D approach for neural network
formation, alignment, and patterning that is reproducible
and sufficiently stable to allow for easy
manipulation.
RESULTS: A novel cell culture system was designed by
engineering a method for the directional growth of
neurons. This uses NG108-15 neuroblastoma x glioma
hybrid cells cultured on suspended and aligned
electrospun fibers. These fiber networks improved
cellular directionality, with alignment angle standard
deviations significantly lower on fibers than on regular
culture surfaces. Morphological studies found nuclear
aspect ratios and cell projection lengths to be
unchanged, indicating that cells maintained neural
morphology while growing on fibers and forming a 3D
network. Furthermore, fibronectin-coated fibers
enhanced neurite extensions for all investigated time
points. Differentiated neurons exhibited significant
increases in average neurite lengths 96 h post plating,
and formed neurite extensions parallel to suspended
fibers, as visualized through scanning electron
microscopy.
CONCLUSIONS: The developed model has the potential
to serve as the basis for advanced 3D studies,
providing an original approach to neural network
patterning and setting the groundwork for further
investigations into functionality
In vivo imaging of axonal transport in murine motor and sensory neurons
BACKGROUND: Axonal transport is essential for neuronal function and survival. Defects in axonal transport have been identified as an early pathological feature in several disorders of the nervous system. The visualisation and quantitative analysis of axonal transport in vivo in rodent models of neurological disease is therefore crucial to improve our understanding of disease pathogenesis and for the identification of novel therapeutics. NEW METHOD: Here, we describe a method for the in vivo imaging of axonal transport of signalling endosomes in the sciatic nerve of live, anaesthetised mice. RESULTS: This method allows the multiparametric, quantitative analysis of in vivo axonal transport in motor and sensory neurons of adult mice in control conditions and during disease progression. COMPARISON WITH EXISTING METHODS: Previous in vivo imaging of the axonal transport of signalling endosomes has been limited to studies in nerve explant preparations or non-invasive approaches using magnetic resonance imaging; techniques that are hampered by major drawbacks such as tissue damage and low temporal and spatial resolution. This new method allows live imaging of the axonal transport of single endosomes in the sciatic nerve in situ and a more sensitive analysis of axonal transport kinetics than previous approaches. CONCLUSIONS: The method described in this paper allows an in-depth analysis of the characteristics of axonal transport in both motor and sensory neurons in vivo. It enables the detailed study of alterations in axonal transport in rodent models of neurological diseases and can be used to identify novel pharmacological modifiers of axonal transport
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