Axonal Transport In Motor Neurons Research Articles

Axonal transport of botulinum toxin A from periphery to CNS in sensory and motor nerves

Background: Botulinum toxin A (BTX-A) has been approved for treatment of movement disorders and migraine. The widely assumed peripheral mechanism of action has been questioned by recent studies which demonstrated an axonal transport in the facial nerve and within central nerves. Findings in our laboratory suggested a central antinociceptive activity following axonal transport in the sciatic nerve. Methods: To characterize the axonal transport of BTX-A, the toxin’s enzymatic activity in the CNS was assessed using immunofluorescent detection of its cleaved substrate synaptosomal-associated protein 25(SNAP-25) following injections into the rat whisker pad and hind-paw, intramuscular injection into the gastrocnemius and intraneural injections into the sciatic nerve. Intraneural and intraganglionic colchicine was employed to block axonal transport. To investigate the significance ofaxonal transport for antinociceptive activity of BTX-A, we assessed the effect of peripheral and intraganglionic injections of low dose BTX-A in orofacial formalin-induced pain in rats. Results: Following whisker pad BTX-A injection, cleaved SNAP-25 was observed in the dorsal horn of the medulla. Cleaved SNAP-25 following subcutaneous, intramuscular and intraneural toxin injection in rat hind limbs was observed in corresponding segments of ipsilateral dorsal and ventral horn. Central SNAP-25 cleavage following BTX-A injection into the sciatic nerve was prevented by colchicine. In the ventral horn, BTX-A protease was localized within cholinergic neurons. Facial and intraganglionic injections of BTX-A prevented orofacial pain dependent on axonal transport in the trigeminal sensory nerve. Conclusions: Our results suggest that the axonal transport of BTX-A in different sensory and motor nerves commonly occurs after peripheral administration. Axonal transport in sensory neurons followed by central enzymatic activity is involved in botulinum toxin’s antinociceptive effects. The possible functional role of axonal transport in motor neurons remains to be examined. Acknowledgements: The antibody to cleaved SNAP-25 was a kind gift from Ornella Rossetto (University of Padua, Italy). The study was funded by the Croatian Ministry of Science, Education and Sport and theDeutscher Akademischer Austausch Dienst.

Tubulin-binding cofactor B is a direct interaction partner of the dynactin subunit p150Glued

The dynactin p150(Glued) subunit, encoded by the gene DCTN1, is part of the dynein-dynactin motor protein complex responsible for retrograde axonal transport in motor neurons. The p150 subunit is a candidate gene for neurodegenerative diseases, in particular motor neuron and extrapyramidal diseases. Tubulin-binding cofactors are believed to be involved in tubulin biogenesis and degradation and therefore to contribute to microtubule functional diversity and regulation. A yeast-two-hybrid screen for putative interacting proteins of dynactin p150(Glued) has revealed tubulin-folding cofactor B (TBCB). We analyzed the interaction of these proteins and investigated the impact of this complex on the microtubule network in cell lines and primary hippocampal neurons in vitro. We especially concentrated on neuronal morphology and synaptogenesis. Overexpression of both proteins or depletion of TBCB alone does not alter the microtubule network and/or neuronal morphology. The demonstration of the interaction of the transport molecule dynactin and the tubulin-regulating factor TBCB is thought to have an impact on several cellular mechanisms. TBCB expression levels have been found to have only a subtle influence on the microtubule network and neuronal morphology. However, overexpression of TBCB leads to the decreased localization of p150 to the microtubule network that might result in a functional modulation of this protein complex.

Glial-Derived Prodegenerative Signaling in the Drosophila Neuromuscular System

We provide evidence for a prodegenerative, glial-derived signaling framework in the Drosophila neuromuscular system that includes caspase and mitochondria-dependent signaling. We demonstrate that Drosophila TNF-α (eiger) is expressed in a subset of peripheral glia, and the TNF-α receptor (TNFR), Wengen, is expressed in motoneurons. NMJ degeneration caused by disruption of the spectrin/ankyrin skeleton is suppressed by an eiger mutation or by eiger knockdown within a subset of peripheral glia. Loss of wengen in motoneurons causes a similar suppression providing evidence for glial-derived prodegenerative TNF-α signaling. Neither JNK nor NFκβ is required for prodegenerative signaling. However, we provide evidence for the involvement of both an initiator and effector caspase, Dronc and Dcp-1, and mitochondrial-dependent signaling. Mutations that deplete the axon and nerve terminal of mitochondria suppress degeneration as do mutations in Drosophila Bcl-2 (debcl), a mitochondria-associated protein, and Apaf-1 (dark), which links mitochondrial signaling with caspase activity in other systems.

Impaired Axonal Transport in Motor Neurons Correlates with Clinical Prion Disease

Prion diseases are fatal neurodegenerative disorders causing motor dysfunctions, dementia and neuropathological changes such as spongiosis, astroglyosis and neuronal loss. The chain of events leading to the clinical disease and the role of distinct brain areas are still poorly understood. The role of nervous system integrity and axonal properties in prion pathology are still elusive. There is no evidence of both the functional axonal impairments in vivo and their connection with prion disease. We studied the functional axonal impairments in motor neurons at the onset of clinical prion disease using the combination of tracing as a functional assay for axonal transport with immunohistochemistry experiments. Well-established and novel confocal and ultramicroscopy techniques were used to image and quantify labeled neurons. Despite profound differences in the incubation times, 30% to 45% of neurons in the red nucleus of different mouse lines showed axonal transport impairments at the disease onset bilaterally after intracerebral prion inoculation and unilaterally—after inoculation into the right sciatic nerve. Up to 94% of motor cortex neurons also demonstrated transport defects upon analysis by alternative imaging methods. Our data connect axonal transport impairments with disease symptoms for different prion strains and inoculation routes and establish further insight on the development of prion pathology in vivo. The alterations in localization of the proteins involved in the retrograde axonal transport allow us to propose a mechanism of transport disruption, which involves Rab7-mediated cargo attachment to the dynein-dynactin pathway. These findings suggest novel targets for therapeutic and diagnostic approaches in the early stages of prion disease.

KINESIN BYPASSING BLOCKAGES ON MICROTUBULE RAILS

Kinesins are motor proteins which convert the chemical energy of ATP into mechanical energy to move along proteinaceous microtubule rails and to transport different cargoes to defined intracellular destinations. It is well documented that following the track of a single protofilament is the thermodynamically most effective mechanism of kinesin movement along microtubules. However, the question arises what happens when a kinesin molecule encounters a hindrance along the protofilament. The present study describes a simple, cell-free approach which enables to study the effects of structural blockages on kinesin-based transport. This experimental approach uses dimeric conventional kinesin moving nanometre-sized gold beads along immobilized microtubules whose surface has been irreversibly decorated by blocking proteins. We demonstrated that the continuous bead transport temporarily stopped at sites of blockages, but usually continued after a certain resting time. Our results suggest that single dimeric kinesin molecules are able to change to another protofilament if the next tubulin dimer where the second head should bind is blocked. A bypassing mechanism is discussed which is considered to be one fundamental prerequisite to realize a kinesin-mediated cargo-transport along microtubules over long distances, required for e.g., the fast axonal transport in motor neurons.

Endocytosis and retrograde axonal traffic in motor neurons.

Spinal cord motor neurons control voluntary movement by relaying messages that arrive from upper brain centres to the innervated muscles. Despite the importance of motor neurons in human health and disease, the precise control of their membrane dynamics and its effect on motor neuron homoeostasis and survival are poorly understood. In particular, the molecular basis of the co-ordination of specific endocytic events with the axonal retrograde transport pathway is largely unknown. To study these important vesicular trafficking events, we pioneered the use of atoxic fragments of tetanus and botulinum neurotoxins to follow endocytosis and retrograde axonal transport in motor neurons. These neurotoxins bind specifically to pre-synaptic nerve terminals, where they are internalized. Whereas botulinum neurotoxins remain at the neuromuscular junction, tetanus toxin is retrogradely transported along the axon to the cell body, where it is released into the intersynaptic space and is internalized by adjacent inhibitory interneurons. The high neurospecificity and the differential intracellular sorting make tetanus and botulinum neurotoxins ideal tools to study neuronal physiology. In the present review, we discuss recent developments in our understanding of the internalization and trafficking of these molecules in spinal cord motor neurons. Furthermore, we describe the development of a reliable transfection method for motor neurons based on microinjection, which will be extremely useful for dissecting further the molecular basis of membrane dynamics and axonal transport in these cells.

Mutant dynactin in motor neuron disease.

Impaired axonal transport in motor neurons has been proposed as a mechanism for neuronal degeneration in motor neuron disease. Here we show linkage of a lower motor neuron disease to a region of 4 Mb at chromosome 2p13. Mutation analysis of a gene in this interval that encodes the largest subunit of the axonal transport protein dynactin showed a single base-pair change resulting in an amino-acid substitution that is predicted to distort the folding of dynactin's microtubule-binding domain. Binding assays show decreased binding of the mutant protein to microtubules. Our results show that dysfunction of dynactin-mediated transport can lead to human motor neuron disease.

Disruption of Dynein/Dynactin Inhibits Axonal Transport in Motor Neurons Causing Late-Onset Progressive Degeneration

To test the hypothesis that inhibition of axonal transport is sufficient to cause motor neuron degeneration such as that observed in amyotrophic lateral sclerosis (ALS), we engineered a targeted disruption of the dynein-dynactin complex in postnatal motor neurons of transgenic mice. Dynamitin overexpression was found to disassemble dynactin, a required activator of cytoplasmic dynein, resulting in an inhibition of retrograde axonal transport. Mice overexpressing dynamitin demonstrate a late-onset progressive motor neuron degenerative disease characterized by decreased strength and endurance, motor neuron degeneration and loss, and denervation of muscle. Previous transgenic mouse models of ALS have shown abnormalities in microtubule-based axonal transport. In this report, we describe a mouse model that confirms the critical role of disrupted axonal transport in the pathogenesis of motor neuron degenerative disease.