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In JoVE (1)
Other Publications (6)
Articles by Cody Locke in JoVE
JoVE General
Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants
Department of Biological Sciences, University of Alabama
This video demonstrates how to employ two neural stimulants, aldicarb and pentylenetetrazole (PTZ), in complementary ways to study synaptic function in the nematode, C. elegans. This complementary approach may also be used to shed light on evolutionarily conserved mechanisms for modulating neuronal synchrony and has implications for epilepsy and seizures.
Other articles by Cody Locke on PubMed
Epileptic-like Convulsions Associated with LIS-1 in the Cytoskeletal Control of Neurotransmitter Signaling in Caenorhabditis Elegans
Cortical malformations are a collection of disorders affecting brain development. Mutations in the LIS1 gene lead to a disorganized and smooth cerebral cortex caused by failure in neuronal migration. Among the clinical consequences of lissencephaly are mental retardation and intractable epilepsy. It remains unclear whether the seizures result from aberrant neuronal placement, disruption of intrinsic properties of neurons, or both. The nematode Caenorhabditis elegans offers an opportunity to study such convulsions in a simple animal with a defined nervous system. Here we show that convulsions mimicking epilepsy can be induced by a mutation in a C. elegans lis-1 allele (pnm-1), in combination with a chemical antagonist of gamma-aminobutyric acid (GABA) neurotransmitter signaling. Identical convulsions were obtained using C. elegans mutants defective in GABA transmission, whereas none of these mutants or the antagonist alone caused convulsions, indicating a threshold was exceeded in response to this combination. Crosses between pnm-1 and fluorescent marker strains designed to exclusively illuminate either the processes of GABAergic neurons or synaptic vesicles surprisingly showed no deviations in neuronal architecture. Instead, presynaptic defects in GABAergic vesicle distribution were clearly evident and could be phenocopied by RNAi directed against cytoplasmic dynein, a known LIS1 interactor. Furthermore, mutations in UNC-104, a neuronal-specific kinesin, and SNB-1, a synaptic vesicle-associated protein termed synaptobrevin, exhibit similar convulsion phenotypes following chemical induction. Taken together, these studies establish C. elegans as a system to investigate subtle cytoskeletal mechanisms regulating intrinsic neuronal activity and suggest that it may be possible to dissociate the epileptic consequences of lissencephaly from the more phenotypically overt cortical defects associated with neuronal migration.
Genetic Interactions Among Cortical Malformation Genes That Influence Susceptibility to Convulsions in C. Elegans
Epilepsy is estimated to affect 1-2% of the world population, yet remains poorly understood at a molecular level. We have previously established the roundworm Caenorhabditis elegans as a model for investigating genetic susceptibilities to seizure-like convulsions in vivo. Here we investigate the behavioral consequences of decreasing the activity of nematode gene homologs within the LIS1 pathway that are associated with a human cortical malformation termed lissencephaly. Bioinformatic analysis revealed the nud-2 gene, encoding the worm homolog of mammalian effectors of LIS1, termed NDE1 and NDEL1. Phenotypic analysis of animals targeted by RNA interference (RNAi) was performed using a pentylenetetrazole (PTZ) exposure paradigm to induce convulsions. Worms depleted for LIS1 pathway components (NUD-1, NUD-2, DHC-1, CDK-5, and CDKA-1) exhibited significant convulsions following PTZ and RNAi treatment. Strains harboring fluorescent markers for GABAergic neuronal architecture and synaptic vesicle trafficking were employed to discern putative mechanisms accounting for observed convulsion behaviors. We found that depletion of LIS1 pathway components resulted in defective GABA synaptic vesicle trafficking. We also utilized combinations of specific genetic backgrounds to create a sensitized state for convulsion susceptibility and discovered that convulsion effects were significantly enhanced when LIS-1 and other pathway components were compromised within the same animals. Thus, interactions among gene products with LIS-1 may mediate intrinsic thresholds of neuronal synchrony.
Acetaminophen Attenuates Dopamine Neuron Degeneration in Animal Models of Parkinson's Disease
Parkinson's disease (PD) is the second most common neurodegenerative disorder with approximately 2% of people over age 65 suffering from this disease. Risk factors for PD involve interplay between still poorly defined genetic and non-genetic contributors, but appear to converge upon cellular pathways that mediate protein misfolding and oxidative stress that lead to dopaminergic neuron loss. The identification of either new or repurposed drugs that exhibit benefit in slowing the age-dependent neuronal damage that occurs in PD is a significant goal of much ongoing research. We have exploited the nematode Caenorhabditis elegans as a model system by which the neuroprotective capacity of acetaminophen could be rapidly evaluated for efficacy in attenuating dopamine (DA) neurodegeneration. Using three independent and established neurodegenerative models in C. elegans, we assayed for acetaminophen-dependent rescue in response to: (1) over-expression of the PD-associated protein, alpha-synuclein; (2) acute exposure to 6-hydroxydopamine (6-OHDA); (3) excess intracellular DA production due to over-expression of the DA biosynthetic enzyme, tyrosine hydroxylase (TH). These data suggest that acetaminophen significantly protected C. elegans DA neurons from stressors related to oxidative damage, but not protein misfolding. Taken together, these studies imply an activity for acetaminophen in the attenuation of DA neuron loss that, following essential corroborative analyses in mammalian systems, may represent a potential benefit for PD.
Pharmacogenetic Analysis Reveals a Post-developmental Role for Rac GTPases in Caenorhabditis Elegans GABAergic Neurotransmission
The nerve-cell cytoskeleton is essential for the regulation of intrinsic neuronal activity. For example, neuronal migration defects are associated with microtubule regulators, such as LIS1 and dynein, as well as with actin regulators, including Rac GTPases and integrins, and have been thought to underlie epileptic seizures in patients with cortical malformations. However, it is plausible that post-developmental functions of specific cytoskeletal regulators contribute to the more transient nature of aberrant neuronal activity and could be masked by developmental anomalies. Accordingly, our previous results have illuminated functional roles, distinct from developmental contributions, for Caenorhabditis elegans orthologs of LIS1 and dynein in GABAergic synaptic vesicle transport. Here, we report that C. elegans with function-altering mutations in canonical Rac GTPase-signaling-pathway members demonstrated a robust behavioral response to a GABA(A) receptor antagonist, pentylenetetrazole. Rac mutants also exhibited hypersensitivity to an acetylcholinesterase inhibitor, aldicarb, uncovering deficiencies in inhibitory neurotransmission. RNA interference targeting Rac hypomorphs revealed synergistic interactions between the dynein motor complex and some, but not all, members of Rac-signaling pathways. These genetic interactions are consistent with putative Rac-dependent regulation of actin and microtubule networks and suggest that some cytoskeletal regulators cooperate to uniquely govern neuronal synchrony through dynein-mediated GABAergic vesicle transport in C. elegans.
S6 Kinase Localizes to the Presynaptic Active Zone and Functions with PDK1 to Control Synapse Development
The dimensions of neuronal dendrites, axons, and synaptic terminals are reproducibly specified for each neuron type, yet it remains unknown how these structures acquire their precise dimensions of length and diameter. Similarly, it remains unknown how active zone number and synaptic strength are specified relative the precise dimensions of presynaptic boutons. In this paper, we demonstrate that S6 kinase (S6K) localizes to the presynaptic active zone. Specifically, S6K colocalizes with the presynaptic protein Bruchpilot (Brp) and requires Brp for active zone localization. We then provide evidence that S6K functions downstream of presynaptic PDK1 to control synaptic bouton size, active zone number, and synaptic function without influencing presynaptic bouton number. We further demonstrate that PDK1 is also a presynaptic protein, though it is distributed more broadly. We present a model in which synaptic S6K responds to local extracellular nutrient and growth factor signaling at the synapse to modulate developmental size specification, including cell size, bouton size, active zone number, and neurotransmitter release.
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.
