Recognising the potential of large animals for modelling neuromuscular junction physiology and disease

The aetiology and pathophysiology of many diseases of the motor unit remain poorly understood and the role of the neuromuscular junction (NMJ) in this group of disorders is particularly overlooked, especially in humans, when these diseases are comparatively rare. However, elucidating the development, function and degeneration of the NMJ is essential to uncover its contribution to neuromuscular disorders, and to explore potential therapeutic avenues to treat these devastating diseases. Until now, an understanding of the role of the NMJ in disease pathogenesis has been hindered by inherent differences between rodent and human NMJs: stark contrasts in body size and corresponding differences in associated axon length underpin some of the translational issues in animal models of neuromuscular disease. Comparative studies in large mammalian models, including examination of naturally occurring, highly prevalent animal diseases and evaluation of their treatment, might provide more relevant insights into the pathogenesis and therapy of equivalent human diseases. This review argues that large animal models offer great potential to enhance our understanding of the neuromuscular system in health and disease, and in particular, when dealing with diseases for which nerve length dependency might underly the pathogenesis

BDNF-dependent modulation of axonal transport is selectively impaired in ALS

Axonal transport ensures long-range delivery of essential cargoes between proximal and distal compartments, and is needed for neuronal development, function, and survival. Deficits in axonal transport have been detected at pre-symptomatic stages in the SOD1G93A and TDP-43M337V mouse models of amyotrophic lateral sclerosis (ALS), suggesting that impairments in this critical process are fundamental for disease pathogenesis. Strikingly, in ALS, fast motor neurons (FMNs) degenerate first whereas slow motor neurons (SMNs) are more resistant, and this is a currently unexplained phenomenon. The main aim of this investigation was to determine the effects of brain-derived neurotrophic factor (BDNF) on in vivo axonal transport in different α-motor neuron (MN) subtypes in wild-type (WT) and SOD1G93A mice. We report that despite displaying similar basal transport speeds, stimulation of wild-type MNs with BDNF enhances in vivo trafficking of signalling endosomes specifically in FMNs. This BDNF-mediated enhancement of transport was also observed in primary ventral horn neuronal cultures. However, FMNs display selective impairment of axonal transport in vivo in symptomatic SOD1G93A mice, and are refractory to BDNF stimulation, a phenotype that was also observed in primary embryonic SOD1G93A neurons. Furthermore, symptomatic SOD1G93A mice display upregulation of the classical non-pro-survival truncated TrkB and p75NTR receptors in muscles, sciatic nerves, and Schwann cells. Altogether, these data indicate that cell- and non-cell autonomous BDNF signalling is impaired in SOD1G93A MNs, thus identifying a new key deficit in ALS

Evaluation of species-specific polyclonal antibodies to detect and differentiate between Neospora caninum and Toxoplasma gondii

Neosporosis and toxoplasmosis are major causes of abortion in livestock worldwide, leading to substantial economic losses. Detection tools are fundamental to the diagnosis and management of those diseases. Current immunohistochemistry (IHC) tests, using sera raised against whole parasite lysates, have not been able to distinguish between Toxoplasma gondii and Neospora caninum. We used T. gondii and N. caninum recombinant proteins, expressed in Escherichia coli and purified using insoluble conditions, to produce specific polyclonal rabbit antisera. We aimed to develop species-specific sera that could be used in IHC on formalin-fixed, paraffin-embedded (FFPE) tissue sections to improve the diagnosis of ruminant abortions caused by protozoa. Two polyclonal rabbit sera, raised against recombinant proteins, anti–Neospora-rNcSRS2 and anti–Toxoplasma-rTgSRS2, had specificity for the parasite they were raised against. We tested the specificity for each polyclonal serum using FFPE tissue sections known to be infected with T. gondii and N. caninum. The anti–Neospora-rNcSRS2 serum labeled specifically only N. caninum–infected tissue blocks, and the anti–Toxoplasma-rTgSRS2 serum was specific to only T. gondii–infected tissues. Moreover, tissues from 52 cattle and 19 sheep previously diagnosed by lesion profiles were tested using IHC with our polyclonal sera and PCR. The overall agreement between IHC and PCR was 90.1% for both polyclonal anti-rNcSRS2 and anti-rTgSRS2 sera. The polyclonal antisera were specific and allowed visual confirmation of protozoan parasites by IHC, but they were not as sensitive as PCR testing.</p

Cultured dissociated primary dorsal root ganglion neurons from adult horses enable study of axonal transport

Neurological disorders are prevalent in horses, but their study is challenging due to anatomic constraints and the large body size; very few host-specific in vitro models have been established to study these types of diseases, particularly from adult donor tissue. Here we report the generation of primary neuronal dorsal root ganglia (DRG) cultures from adult horses: the mixed, dissociated cultures, containing neurons and glial cells, remained viable for at least 90 days. Similar to DRG neurons in vivo, cultured neurons varied in size, and they developed long neurites. The mitochondrial movement was detected in cultured cells and was significantly slower in glial cells compared to DRG-derived neurons. In addition, mitochondria were more elongated in glial cells than those in neurons. Our culture model will be a useful tool to study the contribution of axonal transport defects to specific neurodegenerative diseases in horses as well as comparative studies aimed at evaluating species-specific differences in axonal transport and survival

BDNF-regulation of in vivo axonal transport is selectively impaired in fast motor neurons in SOD1ᴳ⁹³ᴬ mice

Background: Axonal transport ensures long-range delivery of essential cargoes between proximal and distal compartments of neurons, and is needed for neuronal development, function and survival. ALS mice, including SOD1G93A and TDP- 43M337V mice, display in vivo deficits in axonal transport pre-symptomatically suggesting that impairment contributes to disease. The aim of this study is to determine the influence of brain-derived neurotrophic factor (BDNF) and α-motor neuron (MN) subtypes on axonal transport. / Methods: Signalling endosome axonal transport was visualised in vivo with intramuscular injections of a fluorescently-labelled atoxic fragment of tetanus neurotoxin (HcT). HcT was delivered into the tibialis anterior (TA) or soleus muscles of wild-type (WT) and SOD1G93A mice +/- 25ng of recombinant BDNF. 4+ hours later, sciatic nerves were exposed in live, anaesthetised animals, and imaged using time-lapse confocal-microscopy at 37°C. Retrogradely transported signalling endosomes within single axons were tracked using the TrackMate plugin (FIJI/ImageJ). BDNF, TrkB and p75NTR levels were assessed in muscles, neuromuscular junctions and sciatic nerves in WT/SOD1G93A mice. / Results: Basal axonal transport analysis reveals that signalling endosome dynamics are similar between MN/muscle subgroups. BDNF-stimulation significantly enhanced axonal transport dynamics in WT MNs innervating TA only, and this was significantly impaired in SOD1G93A mice. BDNF, TrkB and p75NTR levels were differentially affected in both muscles and sciatic nerve in SOD1G93A mice. / Discussion and Conclusions: These data indicate that different MN/muscle subgroups have distinct axonal transport features and are differentially afflicted in SOD1G93A mice, and may reveal novel clues about selective MN vulnerability in ALS

Primary cardiac malignant peripheral nerve sheath tumour in a dog

A 6-year-old, female labrador retriever was presented for acute onset abdominal distention. Clinical examination identified tachycardia, a novel grade 3/6 right basilar systolic cardiac murmur, a distended abdomen and ventral pitting oedema. Jugular pulses were not present. Echocardiography revealed an infiltrative, intramural cardiac mass, with partial occlusion of the right atrium and ventricle, resulting in isolated caudal vena cava syndrome. Postmortem examination revealed a large mass arising from the base of the right atrium and extending into the ventricular lumen. Histopathological and immunohistochemical findings were consistent with a diagnosis of malignant peripheral nerve sheath tumour, a rare primary cardiac neoplasm in dogs.</p

Cultured dissociated primary dorsal root ganglion neurons from adult horses enable study of axonal transport.

Funder: UK Horserace Betting Levy BoardNeurological disorders are prevalent in horses, but their study is challenging due to anatomic constraints and the large body size; very few host-specific in vitro models have been established to study these types of diseases, particularly from adult donor tissue. Here we report the generation of primary neuronal dorsal root ganglia (DRG) cultures from adult horses: the mixed, dissociated cultures, containing neurons and glial cells, remained viable for at least 90 days. Similar to DRG neurons in vivo, cultured neurons varied in size, and they developed long neurites. The mitochondrial movement was detected in cultured cells and was significantly slower in glial cells compared to DRG-derived neurons. In addition, mitochondria were more elongated in glial cells than those in neurons. Our culture model will be a useful tool to study the contribution of axonal transport defects to specific neurodegenerative diseases in horses as well as comparative studies aimed at evaluating species-specific differences in axonal transport and survival

Dynamics of the December 2020 Ash‐Poor Plume Formed by Lava‐Water Interaction at the Summit of Kīlauea Volcano, Hawaiʻi

Abstract On 20 December 2020, after more than 2 years of quiescence at Kīlauea Volcano, Hawaiʻi, renewed volcanic activity in the summit crater caused boiling of the water lake over a period of ∼90 min. The resulting water‐rich, electrified plume rose to 11–13 km above sea level, which is among the highest plumes on record for Kīlauea. Although conventional models would infer a high mass flux from explosive magma‐water interaction, the plume was not associated with an infrasound signal indicative of “explosive” activity, nor did it produce a measurable ash‐fall deposit. We use multisensor data to characterize lava‐water interaction and plume generation during this opening phase of the 2020–21 eruption. Satellite, weather radar, and eyewitness observations revealed that the plume was rich in water vapor and hydrometeors but transported less ash than expected from its maximum height. Volcanic lightning flashes detected by ground‐based cameras were confined to freezing altitudes of the upper cloud, suggesting that the ice formation drove the electrification of this plume. The low acoustic energy from lava‐water interaction points to a weakly explosive style of hydrovolcanism. Heat transfer calculations show that the lava to water heat flux was sufficient to boil the lake within 90 min. Limited mixing of lava and water inhibited major steam explosions and fine fragmentation. Results from one‐dimensional plume modeling suggest that the models may underpredict plume height due to overestimation of crosswind air‐entrainment. Our findings shed light on an unusual style of volcanism in which weakly explosive lava‐water interaction generated an outsized plume