Angela S. Laird

EPHA4 is a disease modifier of amyotrophic lateral sclerosis in animal models and in humans

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Disease onset and progression are variable, with survival ranging from months to decades. Factors underlying this variability may represent targets for therapeutic intervention. Here, we have screened a zebrafish model of ALS and identified Epha4, a receptor in the ephrin axonal repellent system, as a modifier of the disease phenotype in fish, rodents and humans. Genetic as well as pharmacological inhibition of Epha4 signaling rescues the mutant SOD1 phenotype in zebrafish and increases survival in mouse and rat models of ALS. Motor neurons that are most vulnerable to degeneration in ALS express higher levels of Epha4, and neuromuscular re-innervation by axotomized motor neurons is inhibited by the presence of Epha4. In humans with ALS, EPHA4 expression inversely correlates with disease onset and survival, and loss-of-function mutations in EPHA4 are associated with long survival. Furthermore, we found that knockdown of Epha4 also rescues the axonopathy induced by expression of mutant TAR DNA-binding protein 43 (TDP-43), another protein causing familial ALS, and the axonopathy induced by knockdown of survival of motor neuron 1, a model for spinomuscular atrophy. This suggests that Epha4 generically modulates the vulnerability of (motor) neurons to axonal degeneration and may represent a new target for therapeutic intervention.

Progranulin is Neurotrophic In Vivo and Protects against a Mutant TDP-43 Induced Axonopathy

Mislocalization, aberrant processing and aggregation of TAR DNA-binding protein 43 (TDP-43) is found in the neurons affected by two related diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal lobe dementia (FTLD). These TDP-43 abnormalities are seen when TDP-43 is mutated, such as in familial ALS, but also in FTLD, caused by null mutations in the progranulin gene. They are also found in many patients with sporadic ALS and FTLD, conditions in which only wild type TDP-43 is present. The common pathological hallmarks and symptomatic cross over between the two diseases suggest that TDP-43 and progranulin may be mechanistically linked. In this study we aimed to address this link by establishing whether overexpression of mutant TDP-43 or knock-down of progranulin in zebrafish embryos results in motor neuron phenotypes and whether human progranulin is neuroprotective against such phenotypes. Mutant TDP-43 (A315T mutation) induced a motor axonopathy characterized by short axonal outgrowth and aberrant branching, similar, but more severe, than that induced by mutant SOD1. Knockdown of the two zebrafish progranulin genes, grna and grnb, produced a substantial decrease in axonal length, with knockdown of grna alone producing a greater decrease in axonal length than grnb. Progranulin overexpression rescued the axonopathy induced by progranulin knockdown. Interestingly, progranulin also rescued the mutant TDP-43 induced axonopathy, whilst it failed to affect the mutant SOD1-induced phenotype. TDP-43 was found to be nuclear in all conditions described. The findings described here demonstrate that progranulin is neuroprotective in vivo and may have therapeutic potential for at least some forms of motor neuron degeneration.

Cardiovascular and temperature changes in spinal cord injured rats at rest and during autonomic dysreflexia

In patients with high spinal cord injuries autonomic dysfunction can be dangerous, leading to medical complications such as postural hypotension, autonomic dysreflexia and temperature disturbance. While animal models have been developed to study autonomic dysreflexia, associated temperature changes have not been documented. Our aim here was to use radiotelemetry and infrared thermography in rodents to record the development of cardiovascular and skin temperature changes following complete T4 transection. In adult male Wistar rats (n= 5), responses were assessed prior to spinal cord injury (intact) and for 6 weeks following injury. Statistical analysis by a repeated‐measure ANOVA revealed that following spinal cord injury (SCI), rats exhibited decreased mean arterial pressure (MAP, average decrease of 26 mmHg; P < 0.035) and elevated heart rate (HR, average increase of 65 bpm, P < 0.035) at rest. The basal core body temperature following SCI was also significantly lower than intact levels (−0.9°C; P < 0.0035). Associated with this decreased basal core temperature following SCI was an increased skin temperature of the mid‐tail and hindpaw (+5.6 and +4.0°C, respectively; P < 0.0003) consistent with decreased cutaneous vasoconstrictor tone. Autonomic dysreflexia, in response to a 1 min colorectal distension (25 mmHg), was fully developed by 4 weeks after spinal cord transection, producing increases in MAP greater than 25 mmHg (P < 0.0003). In contrast to the tachycardia seen in intact animals in response to colorectal distension, SCI animals exhibited bradycardia (P < 0.0023). During episodes of autonomic dysreflexia mid‐tail surface temperature decreased (approx. −1.7°C, P < 0.012), consistent with cutaneous vasoconstriction. This is the first study to compare cardiovascular dysfunction with temperature changes following spinal cord transection in rats.

Effect of Treadmill Training on Autonomic Dysreflexia in Spinal Cord—Injured Rats:

Background. Weight-supported treadmill training is an emerging rehabilitation method used to improve locomotor ability in patients with spinal cord injury (SCI). However, little research has been undertaken to test the effect of such training on other consequences of SCI, such as neuropathic pain and autonomic dysfunction. Objective. This study investigates the effects of chronic treadmill training on the development of autonomic dysreflexia (AD), a form of cardiovascular dysfunction common in patients with cervical or high thoracic injury. Methods. Treadmill training commenced in adult male rats (n = 11) 3 days following complete T4 transection, whereas a sedentary SCI group (n = 9) and an intact group (n = 6) had no intervention. Treadmill training (up to 0.4 m/s) lasted for 10 min/d 5 days a week, for 6 weeks. Weekly measurements of locomotor ability (BBB scale), baseline mean arterial pressure, and heart rate were made, as were cardiovascular responses to training and colorectal distension (to trigger AD). Results. Treadmill training improved BBB scores from 2 weeks post-transection onward (P = .010). However, it increased AD, resulting in augmented pressor responses from 2 to 6 weeks post-transection (P = .029). Comparison of the vascular response to phenylephrine under ganglionic blockade showed an enhanced vasoconstrictor response in the renal vasculature of trained SCI animals. Immunohistochemical comparison of the L1—L6 spinal cord segments showed an increased area of CGRP immunoreactivity in the dorsal horn (lamina III/IV) of treadmill-trained SCI compared with intact and sedentary SCI animals. Conclusions. These results suggest that treadmill training exaggerated AD responses perhaps through a combination of enhanced vascular reactivity and central plasticity.

Mutant human FUS Is ubiquitously mislocalized and generates persistent stress granules in primary cultured transgenic zebrafish cells.

FUS mutations can occur in familial amyotrophic lateral sclerosis (fALS), a neurodegenerative disease with cytoplasmic FUS inclusion bodies in motor neurons. To investigate FUS pathology, we generated transgenic zebrafish expressing GFP-tagged wild-type or fALS (R521C) human FUS. Cell cultures were made from these zebrafish and the subcellular localization of human FUS and the generation of stress granule (SG) inclusions examined in different cell types, including differentiated motor neurons. We demonstrate that mutant FUS is mislocalized from the nucleus to the cytosol to a similar extent in motor neurons and all other cell types. Both wild-type and R521C FUS localized to SGs in zebrafish cells, demonstrating an intrinsic ability of human FUS to accumulate in SGs irrespective of the presence of disease-associated mutations or specific cell type. However, elevation in relative cytosolic to nuclear FUS by the R521C mutation led to a significant increase in SG assembly and persistence within a sub population of vulnerable cells, although these cells were not selectively motor neurons.

A Simple and Efficient Protocol for the Treatment of Zebrafish Colonies Infected with Parasitic Nematodes

Abstract Our zebrafish colony experienced a period of increased mortality rate of 6.5 times more deaths per month in a colony of over 13,000 zebrafish (Danio rerio), which developed over 3 months. We observed that before death, affected fish appeared emaciated, often with an abdominal bulge. We performed dissection on 18 fish that had this appearance and found in 15 that their gut was infected with a nematode that closely resembled Pseudocapillaria tomentosa. We devised a treatment protocol for this nematode infection, which involved addition of fenbendazole, a drug used to treat nematode infections in cattle and sheep, to the fish feed. Fenbendazole produced no severe side effects in the fish and several treatments have effectively eradicated the parasite from our colony. The mortality rate of our fish has decreased to a value of 0.7%/month (p<0.001, equal to that before the infection). We propose this protocol as an inexpensive alternative to having to rederive an entire colony from bleached eggs, and as a prophylactic measure used in quarantine facilities on a regular basis.

Modeling Neurodegenerative Diseases in Zebrafish Embryos

Although the zebrafish (Danio rerio) has been used extensively for many years in neurodevelopmental studies, use of this teleost to study neurological diseases has evolved only recently. Being a vertebrate, this animal offers advantages for the study of human disease over other small animals, such as the fly or worm. Genetic, as well as nongenetic, disorders can be modeled in both the adult organism and the embryo. Genetic manipulation of the embryo to generate stable and transiently expressing transgenic fish, and to knockdown genes to study loss of their function, can be easily achieved. Because of large offspring numbers screening studies can also be readily performed. Here, we describe some of the protocols useful for modeling neurodegenerative disease in zebrafish embryos, with particular emphasis on models to study motor neuron phenotypes.

Calpain inhibition is protective in Machado-Joseph disease zebrafish due to induction of autophagy

The neurodegenerative disease Machado–Joseph disease (MJD), also known as spinocerebellar ataxin-3, affects neurons of the brain and spinal cord, disrupting control of the movement of muscles. We have successfully established the first transgenic zebrafish (Danio rerio) model of MJD by expressing human ataxin-3 protein containing either 23 glutamines (23Q, wild-type) or 84Q (MJD-causing) within neurons. Phenotypic characterization of the zebrafish (male and female) revealed that the ataxin-3-84Q zebrafish have decreased survival compared with ataxin-3-23Q and develop ataxin-3 neuropathology, ataxin-3 cleavage fragments and motor impairment. Ataxin-3-84Q zebrafish swim shorter distances than ataxin-3-23Q zebrafish as early as 6 days old, even if expression of the human ataxin-3 protein is limited to motor neurons. This swimming phenotype provides a valuable readout for drug treatment studies. Treating the EGFP-ataxin-3-84Q zebrafish with the calpain inhibitor compound calpeptin decreased levels of ataxin-3 cleavage fragments, but also removed all human ataxin-3 protein (confirmed by ELISA) and prevented the early MJD zebrafish motor phenotype. We identified that this clearance of ataxin-3 protein by calpeptin treatment resulted from an increase in autophagic flux (indicated by decreased p62 levels and increased LC3II). Cotreatment with the autophagy inhibitor chloroquine blocked the decrease in human ataxin-3 levels and the improved movement produced by calpeptin treatment. This study demonstrates that this first transgenic zebrafish model of MJD is a valuable tool for testing potential treatments for MJD. Calpeptin treatment is protective in this model of MJD and removal of human ataxin-3 through macro-autophagy plays an important role in this beneficial effect. SIGNIFICANCE STATEMENT We have established the first transgenic zebrafish model of the neurodegenerative disease MJD, and identified relevant disease phenotypes, including impaired movement from an early age, which can be used in rapid drug testing studies. We have found that treating the MJD zebrafish with the calpain inhibitor compound calpeptin produces complete removal of human ataxin-3 protein, due to induction of the autophagy quality control pathway. This improves the movement of the MJD zebrafish. Artificially blocking the autophagy pathway prevents the removal of human ataxin-3 and improved movement produced by calpeptin treatment. These findings indicate that induction of autophagy, and removal of ataxin-3 protein, plays an important role in the protective effects of calpain inhibition for the treatment of MJD.

Expression of ALS/FTD-linked mutant CCNF in zebrafish leads to increased cell death in the spinal cord and an aberrant motor phenotype

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurodegenerative disease characterised by the death of upper and lower motor neurons. Approximately 10% of cases have a known family history of ALS and disease-linked mutations in multiple genes have been identified. ALS-linked mutations in CCNF were recently reported, however the pathogenic mechanisms associated with these mutations are yet to be established. To investigate possible disease mechanisms, we developed in vitro and in vivo models based on an ALS-linked missense mutation in CCNF. Proteomic analysis of the in vitro models identified the disruption of several cellular pathways in the mutant model, including caspase-3 mediated cell death. Transient overexpression of human CCNF in zebrafish embryos supported this finding, with fish expressing the mutant protein found to have increased levels of cleaved (activated) caspase-3 and increased cell death in the spinal cord. The mutant CCNF fish also developed a motor neuron axonopathy consisting of shortened primary motor axons and increased frequency of aberrant axonal branching. Importantly, we demonstrated a significant correlation between the severity of the CCNF-induced axonopathy and a reduced motor response to a light stimulus (photomotor response). This is the first report of an ALS-linked CCNF mutation in vivo and taken together with the in vitro model identifies the disruption of cell death pathways as a significant consequence of this mutation. Additionally, this study presents a valuable new tool for use in ongoing studies investigating the pathobiology of ALS-linked CCNF mutations.

Tissue-specific models of spinal muscular atrophy confirm a critical role of SMN in motor neurons from embryonic to adult stages.

Spinal muscular atrophy (SMA) is an autosomal recessive disease linked to survival motor neuron (SMN) protein deficiency. While SMN protein is expressed ubiquitously, its deficiency triggers tissue-specific hallmarks, including motor neuron death and muscle atrophy, leading to impaired motor functions and premature death. Here, using stable miR-mediated knockdown technology in zebrafish, we developed the first vertebrate system allowing transgenic spatio-temporal control of the smn1 gene. Using this new model it is now possible to investigate normal and pathogenic SMN function(s) in specific cell types, independently or in synergy with other cell populations. We took advantage of this new system to first test the effect of motor neuron or muscle-specific smn1 silencing. Anti-smn1 miRNA expression in motor neurons, but not in muscles, reproduced SMA hallmarks, including abnormal motor neuron development, poor motor function and premature death. Interestingly, smn1 knockdown in motor neurons also induced severe late-onset phenotypes including scoliosis-like body deformities, weight loss, muscle atrophy and, seen for the first time in zebrafish, reduction in the number of motor neurons, indicating motor neuron degeneration. Taken together, we have developed a new transgenic system allowing spatio-temporal control of smn1 expression in zebrafish, and using this model, we have demonstrated that smn1 silencing in motor neurons alone is sufficient to reproduce SMA hallmarks in zebrafish. It is noteworthy that this research is going beyond SMA as this versatile gene-silencing transgenic system can be used to knockdown any genes of interest, filling the gap in the zebrafish genetic toolbox and opening new avenues to study gene functions in this organism.