CHCHD10 knock-in zebrafish display a mild ALS-like phenotype.

Mutations in the nuclear-encoded mitochondrial gene CHCHD10 have been observed in patients with a spectrum of diseases that include amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To investigate the pathogenic nature of disease-associated variants of CHCHD10 we generated a zebrafish knock-in (KI) model expressing the orthologous ALS-associated CHCHD10 variant (zebrafish: Chchd10). Larval chchd10 fish displayed reduced Chchd10 protein expression levels, motor impairment, reduced survival and abnormal neuromuscular junctions (NMJ).

Loss of mitochondrial Chchd10 or Chchd2 in zebrafish leads to an ALS-like phenotype and Complex I deficiency independent of the mitochondrial integrated stress response.

Mutations in CHCHD10 and CHCHD2, encoding two paralogous mitochondrial proteins, have been identified in cases of amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and Parkinson's disease. Their role in disease is unclear, though both have been linked to mitochondrial respiration and mitochondrial stress responses. Here, we investigated the biological roles of these proteins during vertebrate development using knockout (KO) models in zebrafish.

KHz-rate volumetric voltage imaging of the whole Zebrafish heart.

Fast volumetric imaging is essential for understanding the function of excitable tissues such as those found in the brain and heart. Measuring cardiac voltage transients in tissue volumes is challenging, especially at the high spatial and temporal resolutions needed to give insight to cardiac function. We introduce a new imaging modality based on simultaneous illumination of multiple planes in the tissue and parallel detection with multiple cameras, avoiding compromises inherent in any scanning approach.

Development of an endogenously myc-tagged TARDBP (TDP-43) zebrafish model using the CRISPR/Cas9 system and homology directed repair.

Many of the modern advances in cellular biology have been made by the expression of engineered constructs with epitope tags for subsequent biochemical investigations. While the utility of epitope tags has permitted insights in cellular and animal models, these are often expressed using traditional transgenic approaches. Using the CRISPR/Cas9 system and homology directed repair we recombine a single myc epitope sequence following the start codon of the zebrafish ortholog of TARDBP (TDP-43).

A cell-based GEF assay reveals new substrates for DENN domains and a role for DENND2B in primary ciliogenesis.

Primary cilia are sensory antennae crucial for cell and organism development, and defects in their biogenesis cause ciliopathies. Ciliogenesis involves membrane trafficking mediated by small guanosine triphosphatases (GTPases) including Rabs, molecular switches activated by guanine nucleotide exchange factors (GEFs). The largest family of Rab GEFs is the DENN domain-bearing proteins.

Efficacy of Ciprofloxacin/Celecoxib combination in zebrafish models of amyotrophic lateral sclerosis.

Objective: To evaluate the efficacy of a fixed-dose combination of two approved drugs, Ciprofloxacin and Celecoxib, as a potential therapeutic treatment for amyotrophic lateral sclerosis (ALS).

Methods: Toxicity and efficacy of Ciprofloxacin and Celecoxib were tested, each alone and in distinct ratio combinations in SOD1 G93R transgenic zebrafish model for ALS. Quantification of swimming measures following stimuli, measurements of axonal projections from the spinal cord, neuromuscular junction structure and morphometric analysis of microglia cells were performed in the combination- treated vs nontreated mutant larvae.

The Novel Small Molecule TRVA242 Stabilizes Neuromuscular Junction Defects in Multiple Animal Models of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder in which the neuromuscular junction progressively degenerates, leading to movement difficulties, paralysis, and eventually death. ALS is currently being treated by only two FDA-approved drugs with modest efficacy in slowing disease progression. Often, the translation of preclinical findings to bedside terminates prematurely as the evaluation of potential therapeutic compounds focuses on a single study or a single animal model.

Augmentation of spinal cord glutamatergic synaptic currents in zebrafish primary motoneurons expressing mutant human TARDBP (TDP-43).

Though there is compelling evidence that de-innervation of neuromuscular junctions (NMJ) occurs early in amyotrophic lateral sclerosis (ALS), defects arising at synapses in the spinal cord remain incompletely understood. To investigate spinal cord synaptic dysfunction, we took advantage of a zebrafish larval model and expressed either wild type human TARDBP (wtTARDBP) or the ALS-causing G348C variant (mutTARDBP). The larval zebrafish is ideally suited to examine synaptic connectivity between descending populations of neurons and spinal cord motoneurons as a fully intact spinal cord is preserved during experimentation.

Neuromuscular junction abnormalities in a zebrafish loss-of-function model of TDP-43.

Almost 90% of amyotrophic lateral sclerosis (ALS) cases are characterized by the presence of aggregates of insoluble, misfolded cytoplasmic TAR DNA binding protein of 43 kDa (TDP-43). Distal axonopathy with impaired neuromuscular junctions (NMJs) before motor neuron degeneration or clinical onset of symptoms has been hypothesized as an early pathology in ALS. However, synaptic defects at the NMJ caused by TDP-43 mutations have not been characterized.

Expression of C9orf72-related dipeptides impairs motor function in a vertebrate model.

Large expansions of hexanucleotide GGGGCC (G4C2) repeats (hundreds to thousands) in the first intron of the chromosome 9 open reading frame 72 (C9orf72) locus are the strongest known genetic factor associated with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Different hypotheses exist about the underlying disease mechanism including loss of function by haploinsufficiency, toxicity arising as a result of RNA or dipeptide repeats (DPRs). Five different DPRs are produced by repeat-associated non-ATG-initiated translation of the G4C2 repeats.

Augmented quantal release of acetylcholine at the vertebrate neuromuscular junction following tdp-43 depletion.

TAR DNA binding protein (TDP-43) is a 43 kD, predominately nuclear, protein involved in RNA metabolism. Of clinical significance is that the majority of amyotrophic lateral sclerosis (ALS) patients display abnormal accumulation of misfolded TDP-43 in the cytoplasm, which is coincident with a loss of nuclear localization in the afflicted regions of the central nervous system. Little is known about defects that arise in loss-of-function models, in particular synaptic defects that arise at the neuromuscular junction (NMJ).

Homology Directed Knockin of Point Mutations in the Zebrafish tardbp and fus Genes in ALS Using the CRISPR/Cas9 System.

The methodology for site-directed editing of single nucleotides in the vertebrate genome is of considerable interest for research in biology and medicine. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 type II (Cas9) system has emerged as a simple and inexpensive tool for editing genomic loci of interest in a variety of animal models. In zebrafish, error-prone non-homologous end joining (NHEJ) has been used as a simple method to disrupt gene function.

Fishing for causes and cures of motor neuron disorders.

Motor neuron disorders (MNDs) are a clinically heterogeneous group of neurological diseases characterized by progressive degeneration of motor neurons, and share some common pathological pathways. Despite remarkable advances in our understanding of these diseases, no curative treatment for MNDs exists. To better understand the pathogenesis of MNDs and to help develop new treatments, the establishment of animal models that can be studied efficiently and thoroughly is paramount.

Na+-K+-ATPase trafficking induced by heat shock pretreatment correlates with increased resistance to anoxia in locusts.

The sensitivity of insect nervous systems to anoxia can be modulated genetically and pharmacologically, but the cellular mechanisms responsible are poorly understood. We examined the effect of a heat shock pretreatment (HS) on the sensitivity of the locust (Locusta migratoria) nervous system to anoxia induced by water immersion. Prior HS made locusts more resistant to anoxia by increasing the time taken to enter a coma and by reducing the time taken to recover the ability to stand.

Loss and gain of FUS function impair neuromuscular synaptic transmission in a genetic model of ALS.

Amyotrophic lateral sclerosis (ALS) presents clinically in adulthood and is characterized by the loss of motoneurons in the spinal cord and cerebral cortex. Animal models of the disease suggest that significant neuronal abnormalities exist during preclinical stages of the disease. Mutations in the gene fused in sarcoma (FUS) are associated with ALS and cause impairment in motor function in animal models.

Calcium channel agonists protect against neuromuscular dysfunction in a genetic model of TDP-43 mutation in ALS.

TAR DNA binding protein (TDP-43, encoded by the TARDBP gene) has recently been shown to be associated with amyotrophic lateral sclerosis (ALS), but the early pathophysiological deficits causing impairment in motor function are unknown. Here we expressed the wild-type human gene (wtTARDBP) or the ALS mutation G348C (mutTARDBP) in zebrafish larvae and characterized their motor (swimming) activity and the structure and function of their neuromuscular junctions (NMJs). Of these groups only mutTARDBP larvae showed impaired swimming and increased motoneuron vulnerability with reduced synaptic fidelity, reduced quantal transmission, and more orphaned presynaptic and postsynaptic structures at the NMJ.

Cold hardening modulates K+ homeostasis in the brain of Drosophila melanogaster during chill coma.

Environmental temperature is one of the most important abiotic factors affecting insect behaviour; virtually all physiological processes, including those which regulate nervous system function, are affected. At both low and high temperature extremes insects enter a coma during which individuals do not display behaviour and are unresponsive to stimulation. We investigated neurophysiological correlates of chill and hyperthermic coma in Drosophila melanogaster.

Glial Hsp70 protects K+ homeostasis in the Drosophila brain during repetitive anoxic depolarization.

Neural tissue is particularly vulnerable to metabolic stress and loss of ion homeostasis. Repetitive stress generally leads to more permanent dysfunction but the mechanisms underlying this progression are poorly understood. We investigated the effects of energetic compromise in Drosophila by targeting the Na(+)/K(+)-ATPase.

Metabolic stress modulates motor patterning via AMP-activated protein kinase.

Neural circuits are especially vulnerable to metabolic stress. The locust (Locusta migratoria) responds to anoxia by entering a coma during which neural and muscular systems shut down. During anoxic coma, arrest of the ventilatory central pattern generator is tightly correlated with an abrupt spreading depression (SD)-like increase in extracellular potassium concentration within the metathoracic neuropile.

Coma in response to environmental stress in the locust: a model for cortical spreading depression.

Spreading depression (SD) is an interesting and important phenomenon due to its role in mammalian pathologies such as migraine, seizures, and stroke. Until recently investigations of the mechanisms involved in SD have mostly utilized mammalian cortical tissue, however we have discovered that SD-like events occur in the CNS of an invertebrate model, Locusta migratoria. Locusts enter comas in response to stress during which neural and muscular systems shut down until the stress is removed, and this is believed to be an adaptive strategy to survive extreme environmental conditions.

Inhibition of protein kinase G activity protects neonatal mouse respiratory network from hyperthermic and hypoxic stress.

In spite of considerable research attention focused on clarifying the mechanisms by which the mammalian respiratory rhythm is generated, little attention has been given to examining how this neuronal circuit can be protected from heat stress. Hyperthermia has a profound effect on neuronal circuits including the circuit that generates breathing in mammals. As temperature of the brainstem increases, respiratory frequency concomitantly rises.

Suppression of spreading depression-like events in locusts by inhibition of the NO/cGMP/PKG pathway.

Despite considerable research attention focused on mechanisms underlying neural spreading depression (SD), because of its association with important human CNS pathologies, such as stroke and migraine, little attention has been given to explaining its occurrence and regulation in invertebrates. In the locust metathoracic ganglion (MTG), an SD-like event occurs during heat and anoxia stress, which results in cessation of neuronal output for the duration of the applied stress. SD-like events were characterized by an abrupt rise in extracellular potassium ion concentration ([K(+)](o)) from a baseline concentration of approximately 8 to >30 mm, which returned to near baseline concentrations after removal of the applied stress.

Stress preconditioning of spreading depression in the locust CNS.

Cortical spreading depression (CSD) is closely associated with important pathologies including stroke, seizures and migraine. The mechanisms underlying SD in its various forms are still incompletely understood. Here we describe SD-like events in an invertebrate model, the ventilatory central pattern generator (CPG) of locusts.

Natural variation in the thermotolerance of neural function and behavior due to a cGMP-dependent protein kinase.

Although it is acknowledged that genetic variation contributes to individual differences in thermotolerance, the specific genes and pathways involved and how they are modulated by the environment remain poorly understood. We link natural variation in the thermotolerance of neural function and behavior in Drosophila melanogaster to the foraging gene (for, which encodes a cGMP-dependent protein kinase (PKG)) as well as to its downstream target, protein phosphatase 2A (PP2A). Genetic and pharmacological manipulations revealed that reduced PKG (or PP2A) activity caused increased thermotolerance of synaptic transmission at the larval neuromuscular junction.