Disrupted autophagy and neuronal dysfunction in C. elegans knockin models of FUS amyotrophic lateral sclerosis.

How mutations in FUS lead to neuronal dysfunction in amyotrophic lateral sclerosis (ALS) patients remains unclear. To examine mechanisms underlying ALS FUS dysfunction, we generate C. elegans knockin models using CRISPR-Cas9-mediated genome editing, creating R524S and P525L ALS FUS models. Although FUS inclusions are not detected, ALS FUS animals show defective neuromuscular function and locomotion under stress. Unlike animals lacking the endogenous FUS ortholog, ALS FUS animals have impaired neuronal autophagy and increased SQST-1 accumulation in motor neurons. Loss of sqst-1, the C. elegans ortholog for ALS-linked, autophagy adaptor protein SQSTM1/p62, suppresses both neuromuscular and stress-induced locomotion defects in ALS FUS animals, but does not suppress neuronal autophagy defects. Therefore, autophagy dysfunction is upstream of, and not dependent on, SQSTM1 function in ALS FUS pathogenesis. Combined, our findings demonstrate that autophagy dysfunction likely contributes to protein homeostasis and neuromuscular defects in ALS FUS knockin animals.

Discriminating between sleep and exercise-induced fatigue using computer vision and behavioral genetics.

Following prolonged swimming, Caenorhabditis elegans cycle between active swimming bouts and inactive quiescent bouts. Swimming is exercise for C. elegans and here we suggest that inactive bouts are a recovery state akin to fatigue. It is known that cGMP-dependent kinase (PKG) activity plays a conserved role in sleep, rest, and arousal. Using C. elegans EGL-4 PKG, we first validate a novel learning-based computer vision approach to automatically analyze C. elegans locomotory behavior and an edge detection program that is able to distinguish between activity and inactivity during swimming for long periods of time. We find that C. elegans EGL-4 PKG function impacts timing of exercise-induced quiescent (EIQ) bout onset, fractional quiescence, bout number, and bout duration, suggesting that previously described pathways are engaged during EIQ bouts. However, EIQ bouts are likely not sleep as animals are feeding during the majority of EIQ bouts. We find that genetic perturbation of neurons required for other C. elegans sleep states also does not alter EIQ dynamics. Additionally, we find that EIQ onset is sensitive to age and DAF-16 FOXO function. In summary, we have validated behavioral analysis software that enables a quantitative and detailed assessment of swimming behavior, including EIQ. We found novel EIQ defects in aged animals and animals with mutations in a gene involved in stress tolerance. We anticipate that further use of this software will facilitate the analysis of genes and pathways critical for fatigue and other C. elegans behaviors.

Whole-organism behavioral profiling reveals a role for dopamine in state-dependent motor program coupling in C. elegans.

Animal behaviors are commonly organized into long-lasting states that coordinately impact the generation of diverse motor outputs such as feeding, locomotion, and grooming. However, the neural mechanisms that coordinate these distinct motor programs remain poorly understood. Here, we examine how the distinct motor programs of the nematode C. elegans are coupled together across behavioral states. We describe a new imaging platform that permits automated, simultaneous quantification of each of the main C. elegans motor programs over hours or days. Analysis of these whole-organism behavioral profiles shows that the motor programs coordinately change as animals switch behavioral states. Utilizing genetics, optogenetics, and calcium imaging, we identify a new role for dopamine in coupling locomotion and egg-laying together across states. These results provide new insights into how the diverse motor programs throughout an organism are coordinated and suggest that neuromodulators like dopamine can couple motor circuits together in a state-dependent manner.

Animals generate many different motor programs (such as moving, feeding and grooming) that they can alter in response to internal needs and environmental cues. These motor programs are controlled by dedicated brain circuits that act on specific muscle groups. However, little is known about how organisms coordinate these different motor programs to ensure that their resulting behavior is coherent and appropriate to the situation. This is difficult to investigate in large organisms with complex nervous systems, but with 302 brain cells that control 143 muscle cells, the small worm Caenorhabditis elegans provides a good system to examine this question. Here, Cermak, Yu, Clark et al. devised imaging methods to record each type of motor program in C. elegans worms over long time periods, while also dissecting the underlying neural mechanisms that coordinate these motor programs. This constitutes one of the first efforts to capture and quantify all the behavioral outputs of an entire organism at once. The experiments also showed that dopamine ­ a messenger molecule in the brain ­ links the neural circuits that control two motor programs: movement and egg-laying. A specific type of high-speed movement activates brain cells that release dopamine, which then transmits this information to the egg-laying circuit. This means that worms lay most of their eggs whilst traveling at high speed through a food source, so that their progeny can be distributed across a nutritive environment. This work opens up the possibility to study how behaviors are coordinated at the level of the whole organism ­ a departure from the traditional way of focusing on how specific neural circuits generate specific behaviors. Ultimately, it will also be interesting to look at the role of dopamine in behavior coordination in a wide range of animals.

Single copy/knock-in models of ALS SOD1 in C. elegans suggest loss and gain of function have different contributions to cholinergic and glutamatergic neurodegeneration.

Mutations in Cu/Zn superoxide dismutase 1 (SOD1) lead to Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease that disproportionately affects glutamatergic and cholinergic motor neurons. Previous work with SOD1 overexpression models supports a role for SOD1 toxic gain of function in ALS pathogenesis. However, the impact of SOD1 loss of function in ALS cannot be directly examined in overexpression models. In addition, overexpression may obscure the contribution of SOD1 loss of function in the degeneration of different neuronal populations. Here, we report the first single-copy, ALS knock-in models in C. elegans generated by transposon- or CRISPR/Cas9- mediated genome editing of the endogenous sod-1 gene. Introduction of ALS patient amino acid changes A4V, H71Y, L84V, G85R or G93A into the C. elegans sod-1 gene yielded single-copy/knock-in ALS SOD1 models. These differ from previously reported overexpression models in multiple assays. In single-copy/knock-in models, we observed differential impact of sod-1 ALS alleles on glutamatergic and cholinergic neurodegeneration. A4V, H71Y, G85R, and G93A animals showed increased SOD1 protein accumulation and oxidative stress induced degeneration, consistent with a toxic gain of function in cholinergic motor neurons. By contrast, H71Y, L84V, and G85R lead to glutamatergic neuron degeneration due to sod-1 loss of function after oxidative stress. However, dopaminergic and serotonergic neuronal populations were spared in single-copy ALS models, suggesting a neuronal-subtype specificity previously not reported in invertebrate ALS SOD1 models. Combined, these results suggest that knock-in models may reproduce the neurotransmitter-type specificity of ALS and that both SOD1 loss and gain of toxic function differentially contribute to ALS pathogenesis in different neuronal populations.

A V-to-F substitution in SK2 channels causes Ca2+ hypersensitivity and improves locomotion in a C. elegans ALS model.

Small-conductance Ca2+-activated K+ (SK) channels mediate medium afterhyperpolarization in the neurons and play a key role in the regulation of neuronal excitability. SK channels are potential drug targets for ataxia and Amyotrophic Lateral Sclerosis (ALS). SK channels are activated exclusively by the Ca2+-bound calmodulin. Previously, we identified an intrinsically disordered fragment that is essential for the mechanical coupling between Ca2+/calmodulin binding and channel opening. Here, we report that substitution of a valine to phenylalanine (V407F) in the intrinsically disordered fragment caused a ~6 fold increase in the Ca2+ sensitivity of SK2-a channels. This substitution resulted in a novel interaction between the ectopic phenylalanine and M411, which stabilized PIP2-interacting residue K405, and subsequently enhanced Ca2+ sensitivity. Also, equivalent valine to phenylalanine substitutions in SK1 or SK3 channels conferred Ca2+ hypersensitivity. An equivalent phenylalanine substitution in the Caenorhabditis elegans (C. elegans) SK2 ortholog kcnl-2 partially rescued locomotion defects in an existing C. elegans ALS model, in which human SOD1G85R is expressed at high levels in neurons, confirming that this phenylalanine substitution impacts channel function in vivo. This work for the first time provides a critical reagent for future studies: an SK channel that is hypersensitive to Ca2+ with increased activity in vivo.

LIN-12/Notch Regulates GABA Signaling at the Caenorhabditis elegans Neuromuscular Junction.

The role of Notch signaling in cell-fate decisions has been studied extensively; however, this pathway is also active in adult tissues, including the nervous system. Notch signaling modulates a wide range of behaviors and processes of the nervous system in the nematode Caenorhabditis elegans, but there is no evidence for Notch signaling directly altering synaptic strength. Here, we demonstrate Notch-mediated regulation of synaptic activity at the C. elegans neuromuscular junction (NMJ). For this, we used aldicarb, an inhibitor of the enzyme acetylcholinesterase, and assessed paralysis rates of animals with altered Notch signaling. Notch receptors LIN-12 and GLP-1 are required for normal NMJ function; they regulate NMJ activity in an opposing fashion. Complete loss of LIN-12 skews the excitation/inhibition balance at the NMJ toward increased activity, whereas partial loss of GLP-1 has the opposite effect. Specific Notch ligands and co-ligands are also required for proper NMJ function. The role of LIN-12 is independent of cell-fate decisions; manipulation of LIN-12 signaling through RNAi knockdown or overexpression of the co-ligand OSM-11 after development alters NMJ activity. We demonstrate that LIN-12 modulates GABA signaling in this paradigm, as loss of GABA signaling suppresses LIN-12 gain-of-function defects. Further analysis, in vivo and in silico, suggests that LIN-12 may modulate transcription of the GABAB receptor GBB-2 Our findings confirm a non-developmental role for the LIN-12/Notch receptor in regulating synaptic signaling and identify the GABAB receptor GBB-2 as a potential Notch transcriptional target in the C. elegans nervous system.