Dysregulation of axonal transport and motorneuron diseases

MNDs (motorneuron diseases) are neurodegenerative disorders in which motorneurons located in the motor cortex, in the brainstem and in the spinal cord are affected. These diseases in their inherited or sporadic forms are mainly characterized by motor dysfunctions, occasionally associated with cognitive and behavioural alterations. Although these diseases show high variability in onset, progression and clinical symptoms, they share common pathological features, and motorneuronal loss invariably leads to muscle weakness and atrophy. One of the most relevant aspect of these disorders is the occurrence of defects in axonal transport, which have been postulated to be either a direct cause, or a consequence, of motorneuron degeneration. In fact, due to their peculiar morphology and high energetic metabolism, motorneurons deeply rely on efficient axonal transport processes. Dysfunction of axonal transport is known to adversely affect motorneuronal metabolism, inducing progressive degeneration and cell death. In this regard, the understanding of the fine mechanisms at the basis of the axonal transport process and of their possible alterations may help shed light on MND pathological processes. In the present review, we will summarize what is currently known about the alterations of axonal transport found to be either causative or a consequence of MNDs.     

Central generation of swimming activity in the hydrozoan jellyfish Aequorea aequorea

Swimming in Aequorea is controlled by a network of electrically coupled neurons (swim motorneurons) located in the inner nerve ring. The network is made up of the largest neurons in the ring, up to 22 microns in diameter. Intracellular recordings from swim motorneurons reveal slow membrane potential oscillations and a superimposed barrage of synaptic "noise." The synaptic noise, but not the slow oscillations, is eliminated in seawater containing an elevated Mg++ concentration. The swim motorneurons produce a rapid burst of two to eight action potentials preceding each contraction of the subumbrella. Spontaneous bursting persists in high-Mg++ seawater. Injected ramp currents indicated a "bursty" character of the swim motorneurons as suprathreshold depolarizations produced repetitive bursting with an increasing burst frequency with increased depolarization. Hyperpolarizing currents locally blocked spiking in swim motorneurons. Intercellular coupling was demonstrated with Lucifer Yellow injection and dual electrode recordings. In dye fills, only the large neurons of the inner nerve ring were dye-coupled. Two pieces of evidence suggest that swim motorneurons activate the overlying epithelial cells via chemical synapses. First, direct synaptic connections have been noted in ultrastructural examination of the inner nerve ring region. Second, dual recordings from a swim motorneuron and an epithelial cell reveal a 1:1 correspondence between neuron spikes and epithelial synaptic potentials. The synaptic potentials occur with a latency as short as 3 ms which is constant in any one recording session. The results suggest that the swim motorneuron network of Aequorea not only performs a motorneuron function, but also serves as the pattern generator for swimming activity.     

Pathfinding by cranial nerve VII (facial) motorneurons in the chick hindbrain.

Cranial nerve VII (facial) motorneurons begin extending axons through rhombomeres 4 and 5 (R4 and R5) in the chick hindbrain on the second day of incubation. Without crossing the midline, facial motorneuron axons extend laterally from a ventromedial cell body location. All facial motorneuron axons leave the hindbrain through a discrete exit site in R4. To examine the importance of the exit site in R4 on motorneuron pathfinding, we ablated R4 before motorneuron axonogenesis. We find that mechanisms intrinsic to R5 direct the initial lateral orientation of R5 motorneuron axons. Upon reaching a particular lateral position, all R5 motorneuron axons must turn. In normal embryos the axons all turn rostrally to reach the nerve exit in R4. In embryos with R4 ablated, sometimes the axons turn rostrally and sometimes they turn caudally. A model combining permissive fields and chemotropic cues is presented to account for our observations.     

The effect of partial denervation of tibialis anterior (TA) muscle on the number and sizes of motorneurons in TA motornucleus of normal and dystrophic (C57BL dy2j/dy2j) mice.

The tibialis anterior (TA) muscle in one leg of normal (C57BL) and dystrophic (dy2j) mice was partially denervated by resection of a part of the lateral popliteal nerve. Two months later the muscle was injected with horseradish peroxidase to permit visualization of the motorneurons that survived. Partial denervation in both C57 and dy2j mice resulted in reduction of the number of motorneurons that supplied the muscle to approximately one-half the normal complement. The surviving motorneurons were found to be significantly larger (about 25%) than their contralateral counterparts. This condition persisted up to 18 months and is not considered to be a transient response to the trauma associated with the partial denervation. When the size of the target tissue was also reduced by extirpation of one-half of TA together with partial denervation, motorneuron size was not found to increase. It is suggested that the increase in size is a response to the metabolic demands placed upon the motorneuron by an increase in the size of the motor unit.     

Actions of cholinergic drugs in the nematode Ascaris suum. Complex pharmacology of muscle and motorneurons

The cholinergic agonists acetylcholine (ACh), nicotine, and pilocarpine produced depolarizations and contractions of muscle of the nematode Ascaris suum. Dose-dependent depolarization and contraction by ACh were suppressed by about two orders of magnitude by 100 microM d- tubocurarine (dTC), a nicotinic antagonist, but only about fivefold by 100 microM N-methyl-scopolamine (NMS), a muscarinic antagonist. NMS itself depolarized both normal and synaptically isolated muscle cells. The muscle depolarizing action of pilocarpine was not consistently antagonized by either NMS or dTC. ACh receptors were detected on motorneuron classes DE1, DE2, DI, and VI as ACh-induced reductions in input resistance. These input resistance changes were reversed by washing in drug-free saline or by application of dTC. NMS applied alone lowered input resistance in DE1, but not in DE2, DI, or VI motorneurons. In contrast to the effect of ACh, the action of NMS in DE1 was not reversed by dTC, suggesting that NMS-sensitive sites may not respond to ACh. Excitatory synaptic responses in muscle evoked by depolarizing current injections into DE1 and DE2 motorneurons were antagonized by dTC; however, NMS antagonized the synaptic output of only the DE1 and DE3 classes of motorneurons, an effect that was more likely to have been produced by motorneuron conduction failure than by pharmacological blockade of receptor. The concentration of NMS required to produce these changes in muscle polarization and contraction, ACh antagonism, input resistance reduction, and synaptic antagonism was 100 microM, or more than five orders of magnitude higher than the binding affinity for [3H]NMS in larval Ascaris homogenates and adult Caenorhabditis elegans (Segerberg, M. A. 1989. Ph.D. thesis. University of Wisconsin-Madison, Madison, WI). These results describe a nicotinic- like pharmacology, but muscle and motorneurons also have unusual responses to muscarinic agents.     

The Unc-8 and Sup-40 Genes Regulate Ion Channel Function in Caenorhabditis Elegans Motorneurons

Two Caenorhabditis elegans genes, unc-8 and sup-40, have been newly identified, by genetic criteria, as regulating ion channel function in motorneurons. Two dominant unc-8 alleles cause motorneuron swelling similar to that of other neuronal types in dominant mutants of the deg-1 gene family, which is homologous to a mammalian gene family encoding amiloride-sensitive sodium channel subunits. As for previously identified deg-1 family members, unc-8 dominant mutations are recessively suppressed by mutations in the mec-6 gene, which probably encodes a second type of channel component. An unusual dominant mutation, sup-41 (lb125), also co-suppresses unc-8 and deg-1, suggesting the existence of yet another common component of ion channels containing unc-8 or deg-1 subunits. Dominant, transacting, intragenic suppressor mutations have been isolated for both unc-8 and deg-1, consistent with the idea that, like their mammalian homologues, the two gene products function as multimers. The sup-40 (lb130) mutation dominantly suppresses unc-8 motorneuron swelling and produces a novel swelling phenotype in hypodermal nuclei. sup-40 may encode an ion channel component or regulator that can correct the osmotic defect caused by abnormal unc-8 channels.     

Dual effects of proctolin on the rhythmic burst activity of the cardiac ganglion

The neuropeptide proctolin has distinguishable excitatory effects upon premotor cells and motorneurons of Homarus cardiac ganglion. Proctolin's excitation of the small, premotor, posterior cells is rapid in onset (5–10 s) and readily reversible (< 3 min). Prolonged bursts in small cells often produce a “doublet” ganglionic burst mode via interactions with large motorneuron burst-generating driver potentials. In contrast to small cell response, proctolin's direct excitatory effects upon motorneuron are slow in onset (60–90 s to peak) and long-lasting (10–20 min). The latter include: (a) a concentration-dependent (10^?9–10^?7M) depolarization of the somatic membrane potential; (b) increases in burst frequency and (c) enhancement of the rate of depolarization of the interburst pacemaker potential. Experiments on isolated large cells indicate: (a) the slow depolarization is produced by a decrease in the resting G_K and (b) proctolin can produce or enhance motorneuron autorhythmicity. A two-tiered non-hierarchical network model is proposed. The differential pharmacodynamics exhibited by the two cell types accounts for the sequential modes of ganglionic burst activity produced by proctolin.     

Self-organizing effects of spontaneous neural activity on the development of spinal locomotor circuits in vertebrates

Presented in this paper is a neural network model that can be used to investigate the possible self-organizing mechanisms occurring during the early ontogeny of spinal neural circuits in the vertebrate motor system. The neural circuit is composed of multiple types of neurons which correspond to motorneurons, Renshaw cells and a hypothetical class of interneurons. While the connectivity of this circuit is genetically predetermined, the efficacies of these connections – the synaptic s trengths – evolve in accordance with activity-dependent mechanisms which are initiated by the intrinsic, autonomous activity present in the developing spinal cord. Using Oja's rule, a modified Hebbian learning scheme for adjusting the values of the connections, the network stably self-organizes developing, in the process, reciprocally activated motorneuron pools analogous to those which exist in vivo. Received: 30 December 1996 / Accepted in revised form: 20 June 1997     

Scanning electron microscopic investigation of mouse cerebellar macrophages in vitro

Dispersed monolayer cultures of post-natal mouse cerebella were studied using scanning electron microscopy. Special attention was given to the morphology of the brain macrophages and their interaction with neuronal cells. The brain macrophages resemble other macrophages and mononuclear phagocytes in vitro, reported in the literature. Their ability to induce neuronal growth cones to avoid contact, results in areas devoid of neuronal processes (halos) in this culture system; this seems of considerable interest in connection with their possible developmental role in post-natal reshaping of the brain. Scanning electron microscope observations indicate that these halos are not the result of phagolysis of already established neuronal networks, but can only be induced during neuronal growth and process formation. Established, intact neurons are apparently not altered or harmed by contact with brain macrophages.     

Synaptogenesis in the giant-fibre system of Drosophila: interaction of the giant fibre and its major motorneuronal target

The tergotrochanteral (jump) motorneuron is a major synaptic target of the Giant Fibre in Drosophila. These two neurons are major components of the fly's Giant-Fibre escape system. Our previous work has described the development of the Giant Fibre in early metamorphosis and the involvement of the shaking-B locus in the formation of its electrical synapses. In the present study, we have investigated the development of the tergotrochanteral motorneuron and its electrical synapses by transforming Drosophila with a Gal4 fusion construct containing sequences largely upstream of, but including, the shaking-B(lethal) promoter. This construct drives reporter gene expression in the tergotrochanteral motorneuron and some other neurons. Expression of green fluorescent protein in the motorneuron allows visualization of its cell body and its subsequent intracellular staining with Lucifer Yellow. These preparations provide high-resolution data on motorneuron morphogenesis during the first half of pupal development. Dye-coupling reveals onset of gap-junction formation between the tergotrochanteral motorneuron and other neurons of the Giant-Fibre System. The medial dendrite of the tergotrochanteral motorneuron becomes dye-coupled to the peripheral synapsing interneurons between 28 and 32 hours after puparium formation. Dye-coupling between tergotrochanteral motorneuron and Giant Fibre is first seen at 42 hours after puparium formation. All dye coupling is abolished in a shaking-B(neural) mutant. To investigate any interactions between the Giant Fibre and the tergotroachanteral motorneuron, we arrested the growth of the motorneuron's medial neurite by targeted expression of a constitutively active form of Dcdc42. This results in the Giant Fibre remaining stranded at the midline, unable to make its characteristic bend. We conclude that Giant Fibre morphogenesis normally relies on fasciculation with its major motorneuronal target.     

Serotonin and Go modulate functional states of neurons and muscles controlling C. elegans egg-laying behavior

From nematodes to humans, animals employ neuromodulators like serotonin to regulate behavioral patterns and states. In the nematode C. elegans, serotonin has been shown to act in a modulatory fashion to increase the rate and alter the temporal pattern of egg laying. Though many candidate effectors and regulators of serotonin have been identified in genetic studies, their effects on specific neurons and muscles in the egg-laying circuitry have been difficult to determine. Using the genetically encoded Ca(2+) indicator cameleon, we found that serotonin acts directly on the vulval muscles to increase the frequency of Ca(2+) transients. In contrast, we found that the spontaneous activity of the egg-laying motorneurons was silenced by serotonin. Mutations in G protein alpha subunit genes altered the responses of both vulval muscles and egg-laying neurons to serotonin; specifically, mutations in the G(q)alpha homolog egl-30 blocked serotonin stimulation of vulval muscle Ca(2+) transients, while mutations in the G(o)alpha homolog goa-1 prevented the silencing of motorneuron activity by serotonin. These data indicate that serotonin stimulates egg laying by directly modulating the functional state of the vulval muscles. In addition, serotonin inhibits the activity of the motorneurons that release it, providing a feedback regulatory effect that may contribute to serotonin adaptation.     

A role of small heat shock protein B8 (HspB8) in the autophagic removal of misfolded proteins responsible for neurodegenerative diseases

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of upper and lower motorneurons. As with other age-dependent neurodegenerative disorders, ALS is linked to the presence of misfolded proteins that may perturb several intracellular mechanisms and trigger neurotoxicity. Misfolded proteins aggregate intracellularly generating insoluble inclusions that are a key neuropathological hallmark of ALS. Proteins involved in the intracellular degradative systems, signalling pathways and the human TAR DNA-binding protein TDP-43 are major components of these inclusions. While their role and cytotoxicity are still largely debated, aggregates represent a powerful marker to follow protein misfolding in the neurodegenerative processes. Using in vitro and in vivo models of mutant SOD1 associated familial ALS (fALS), we and other groups demonstrated that protein misfolding perturbs one of the major intracellular degradative pathways, the ubiquitin proteasome system, giving rise to a vicious cycle that leads to the further deposit of insoluble proteins and finally to the formation of inclusions. The aberrant response to mutated SOD1 thus leads to the activation of the cascade of events ultimately responsible for cell death. Hence, our idea is that, by assisting protein folding, we might reduce protein aggregation, restore a fully functional proteasome activity and/or other cascades of events triggered by the mutant proteins responsible for motorneuron death in ALS. This could be obtained by stimulating mutant protein turnover, using an alternative degradative pathway that could clear mutant SOD1, namely autophagy.     

Clasp-mediated microtubule bundling regulates persistent motility and contact repulsion in Drosophila macrophages in vivo

Drosophila melanogaster macrophages are highly migratory cells that lend themselves beautifully to high resolution in vivo imaging experiments. By expressing fluorescent probes to reveal actin and microtubules, we can observe the dynamic interplay of these two cytoskeletal networks as macrophages migrate and interact with one another within a living organism. We show that before an episode of persistent motility, whether responding to developmental guidance or wound cues, macrophages assemble a polarized array of microtubules that bundle into a compass-like arm that appears to anticipate the direction of migration. Whenever cells collide with one another, their microtubule arms transiently align just before cell–cell repulsion, and we show that forcing depolymerization of microtubules by expression of Spastin leads to their defective polarity and failure to contact inhibit from one another. The same is true in orbit/clasp mutants, indicating a pivotal role for this microtubule-binding protein in the assembly and/or functioning of the microtubule arm during polarized migration and contact repulsion.     

A model of a human neuromuscular system for small isometric tensions

A model is constructed of the motor units in the human first dorsal interosseus (FDI) muscle. Each motorneuron is simulated using a pseudo-steady-state model that omits the membrane capacity and the events underlying the action potential. Properties of individual twitches in the corresponding muscle units are based on the data of Milner-Brown et al. for the FDI, while the transduction between steady firing rate and percentage of maximum tension in a muscle unit is based on the work of Rack and Westbury on the cat soleus muscle. Since we are concerned only with small isometric tensions, we ignore effects due to muscle spindles and to recurrent inhibition. The model allows one to to determine, by simulation, the tension-time functions produced by different programs of input to an entire pool of 120 motorneurons. Thus, for example, in order to produce tension rising linearly with time, it suffices to deliver to each neuron in the pool a non-linearly rising conductance; the conductance can be the same for all neurons in the pool, but can NOT be scaled in proportion to the surface area of the respective neurons. The input may be delivered to any part of the neuron's dendritic tree, as long as the electrotonic distribution of input is the same for all the neurons. For a linearly rising force produced in this way, most of the motorneurons yield similar slopes for their frequency-force curves, as observed by Milner-Brown et al. To produce tensions greater than about 1 kg, mechanisms not included in this model must come into play, i.e. perhaps introduction of phasic motorneurons. The most important data needed to improve this model are sets of isometric frequency-force curves for muscle units of different twitch tensions.     

Antiapoptotic activity maintenance of Brain Derived Neurotrophic Factor and the C fragment of the tetanus toxin genetic fusion protein

Neurotrophic factors have been widely suggested as a treatment for multiple diseases including motorneuron pathologies, like Amyotrophic Lateral Sclerosis. However, clinical trials in which growth factors have been systematically administered to Amyotrophic Lateral Sclerosis patients have not been effective, owing in part to the short half-life of these factors and their low concentrations at target sites. A possible strategy is the use of the atoxic C fragment of the tetanus toxin as a neurotrophic factor carrier to the motorneurons. The activity of trophic factors should be tested because their genetic fusion to proteins could alter their folding and conformation, thus undermining their neuroprotective properties. For this purpose, in this paper we explored the Brain Derived Neurotrophic Factor (BDNF) activity maintenance after genetic fusion with the C fragment of the tetanus toxin. We demonstrated that BDNF fused with the C fragment of the tetanus toxin induces the neuronal survival Akt kinase pathway in mouse cortical culture neurons and maintains its antiapoptotic neuronal activity in Neuro2A cells.     

The corpus luteum of the guinea pig. IV. Fine structure of macrophages during pregnancy and postpartum luteolysis, and the phagocytosis of luteal cells.

Little information is available on the ultrastructure of macrophages in the corpus luteum or their importance in the regression of luteal tissue. In the present study, the fine structure of activated luteal macrophages during pregnancy and the postpartum period was examined by electron microscopy of guinea pig ovaries fixed by vascular perfusion. In these corpora lutea, macrophages can readily be distinguished from luteal cells. Activated macrophages typically display three prominent inclusions in their cytoplasm: (1) heterophagic vacuoles, (2) distinctive large dense inclusions, and (3) large and small electron-lucent vacuoles. In addition, they contain numerous smaller lysosome-like dense bodies. Activated macrophages in corpora lutea also characteristically show many surface protrusions, such as processes, folds or pseudopodia, which often occur in close contact with nearby luteal cells. Generally, nuclei of macrophages are irregular in shape and display a dense border of heterochromatin, thus differing from those of luteal cells. Macrophages seem to be most abundant in regressing corpora lutea, where they commonly display heterophagic vacuoles containing recognizable luteal cell fragments, evidence that these phagocytes ingest senescent luteal cells. The digestion of luteal cell components in heterophagic vacuoles presumably gives rise to the distinctive large dense inclusions typically seen in macrophages. The findings of this study indicate that macrophages play a central role in luteolysis by phagocytizing luteal cells or their remnants. They therefore appear to bring about the reduction in volume of the corpus luteum that occurs as this tissue regresses. These results taken together with those previously published (Paavola, '78) further indicate that breakdown of the corpus luteum during postpartum luteolysis in guinea pigs involves both autophagy and heterophagy.     

The kakapo Mutation Affects Terminal Arborization and Central Dendritic Sprouting of Drosophila Motorneurons

The lethal mutation l(2)CA4 causes specific defects in local growth of neuronal processes. We uncovered four alleles of l(2)CA4 and mapped it to bands 50A-C on the polytene chromosomes and found it to be allelic to kakapo (Prout et al. 1997. Genetics. 146:275– 285). In embryos carrying our kakapo mutant alleles, motorneurons form correct nerve branches, showing that long distance growth of neuronal processes is unaffected. However, neuromuscular junctions (NMJs) fail to form normal local arbors on their target muscles and are significantly reduced in size. In agreement with this finding, antibodies against kakapo (Gregory and Brown. 1998. J. Cell Biol. 143:1271–1282) detect a specific epitope at all or most Drosophila NMJs. Within the central nervous system of kakapo mutant embryos, neuronal dendrites of the RP3 motorneuron form at correct positions, but are significantly reduced in size. At the subcellular level we demonstrate two phenotypes potentially responsible for the defects in neuronal branching: first, transmembrane proteins, which can play important roles in neuronal growth regulation, are incorrectly localized along neuronal processes. Second, microtubules play an important role in neuronal growth, and kakapo appears to be required for their organization in certain ectodermal cells: On the one hand, kakapo mutant embryos exhibit impaired microtubule organization within epidermal cells leading to detachment of muscles from the cuticle. On the other, a specific type of sensory neuron (scolopidial neurons) shows defects in microtubule organization and detaches from its support cells.     

The use of chemiluminescence for assessing the physiological status of macrophages activated by pertussis preparations

The work deals with the comparative analysis of oxygen-dependent metabolic processes in the peritoneal macrophages of intact and immune, mice after their interaction with whole-cell pertussis vaccine (WCPV), dialyzed pertussis antigen (DPA) and fraction 2 of this antigen. The study revealed that WCPV, DPA and its fraction 2 modulated the production of active forms of oxygen by peritoneal macrophages, evaluated by means of luminol-dependent chemiluminescence (CL). The influence of WCPV of oxygen-dependent metabolic processes in macrophages after the first contacts with them had a dose-dependent character: low concentrations activated and higher concentrations suppressed the "respiratory" explosion. The immunization of animals abolished the effect of extinguishing CL on the contact of macrophages with high doses of WCPV and essentially increased the level of their CL response to all pertussis preparations in comparison with those for intact cells. DPA and its fraction 2 were not inferior to WCPV in their capacity for inducing the "respiratory" explosion, and in high doses they essentially surpassed WCPV doses in this capacity, especially on the first contact with macrophages.     

Unresponsive, a new behavioral mutant in Xenopus laevis

Abstract. A new recessive nonlethal behavioral mutant, unresponsive (ur), was recovered from a wild-caught Xenopus laevis female by gynogenesis and inbreeding. Mutant embryos do not move until they are three days old, just before feeding begins, in contrast to normal embryos which begin movements at one day, during tailbud stage. Recovery of mutant embryos is complete but slow, requiring another nine days. Grafting analysis suggests that sensory neuron function is normal in homozygous mutant embryos, but that both motorneurons and their target muscles derived from somites are affected by the mutation. Either muscle or motorneurons of unresponsive embryos can participate in normal movements during early development, prior to the stage at which intact mutant embryos recover, in chimerae with normal tissue. Failure of mutant muscle to respond normally to acetylcholine, along with the behavior of chimerae, suggests that mutant embryos do not move because they do not form functional neuromuscular junctions during early development and that the component process of neuromuscular junction formation affected by this mutation is normally performed by both nerve and muscle. during embryogenesis due to a defect in the muscle cells [5]. Armstrong and collaborators [I] have used the immobile mutant to demonstrate that the cholinergic stimulation- induced loss of gap junctions during development does not depend on muscle contraction. In this report, we examine a new mutant of Xenopus laevis, unresponsive (ur), which does not move voluntarily or in response to stimulation until just prior to the feeding stage, after which it recovers.