Thatcher, Joseph et al. (2017) International Worm Meeting "Development of a three dimensional fluorescence tracking microscope for the motional analysis and behavioral stimulation of C. elegans."

Lam, Brian, Madruga, Blake, Carmona, Javier, Shrestha, Ahis, Jin, Suying, Mendoza, Steve, Thatcher, Joseph, Arisaka, Katsushi, Niaki, Shayan

[International Worm Meeting, 2017]

Extensive advances have been made in understanding the behavior of C. elegans in two dimensional environments. However, they impose substantial constraints on the worm's motion and ultimately restrict the set of possible natural behavioral states it can demonstrate. Addressing limitations encountered by previous efforts in three dimensional imaging, we designed and built a microscope capable of tracking the motion of C. elegans via a set of motorized stages while navigating freely within a sample volume. The variation of gelatin concentration (1% - 4%) and the utilization of temporally controlled ultraviolet photo-stimulation (405 nm) were also incorporated into the system. The addition of a refractive index mismatch correction chamber and fluorescence detection enable novel opportunities for observation and categorization of motion. Preliminary data of photoavoidance response in three dimensions was acquired and demonstrates the added complexity present in an unconstrained response. A novel use of fluorescence enables the identification of C. elegans' absolute orientation with respect to the ventral nerve cord. A model of motion based on sinusoidal wave propagation was applied to C. elegans' forward locomotion, thereby categorizing a set of three dimensional body states inhabited. From this analysis, we have identified three distinct motional states: one of which is sinusoidal in the worm's ventrodorsal plane, another which is sinusoidal in their lateral plane, and a final state that is helical in shape. Fitting this parametric model allows the extraction of a variety of wave-based parameters including wavelength, frequency, wave speed and phase difference which may then be correlated with other dynamic quantities and gelatin concentrations. Namely, the phase difference acts as a direct indicator of the degree to which the worm's posture is planar or helical, allowing the ability to parameterize its general motional form with a single number. Furthermore, from pre-existing, established data of the C. elegans' connectome, we hypothesize a neuronal mechanism for rhythmic signal generation based on the SMD motor neurons which predicts the motional states observed.

Arena JP (1994) Parasitol Today "Expression of Caenorahbditis elegans messenger RNA in Xenopus oocytes: A model systems to study the mechanism of action of avermectins."

Arena JP

[Parasitol Today, 1994]

It has recently been shown that Xenopus oocytes injected with mRNA from the free-living nematode Caenorhabditis elegans express avermectin-sensitive chloride channels(1). Joseph Arena here reviews whet is known about the mechanism of action of avermectin and how these recent results relate to the mechanism in nematodes.

Teixeira-Castro A et al. (2015) Brain "Serotonergic signalling suppresses ataxin 3 aggregation and neurotoxicity in animal models of Machado-Joseph disease."

Bessa C, Duarte-Silva S, Maciel P, Bessa J, Silverman RB, Miranda A, Kang S, Summavielle T, Oliveira S, da Silva Santos L, Neto MF, Esteves S, Brielmann RM, Neves-Carvalho A, Teixeira-Castro A, Oliveira P, Morimoto RI, Silva-Fernandes A, Jalles A

[Brain, 2015]

Polyglutamine diseases are a class of dominantly inherited neurodegenerative disorders for which there is no effective treatment. Here we provide evidence that activation of serotonergic signalling is beneficial in animal models of Machado-Joseph disease. We identified citalopram, a selective serotonin reuptake inhibitor, in a small molecule screen of FDA-approved drugs that rescued neuronal dysfunction and reduced aggregation using a Caenorhabditis elegans model of mutant ataxin 3-induced neurotoxicity. MOD-5, the C. elegans orthologue of the serotonin transporter and cellular target of citalopram, and the serotonin receptors SER-1 and SER-4 were strong genetic modifiers of ataxin 3 neurotoxicity and necessary for therapeutic efficacy. Moreover, chronic treatment of CMVMJD135 mice with citalopram significantly reduced ataxin 3 neuronal inclusions and astrogliosis, rescued diminished body weight and strikingly ameliorated motor symptoms. These results suggest that small molecule modulation of serotonergic signalling represents a promising therapeutic target for Machado-Joseph disease.

Kawaguchi Y et al. (1994) Nat Genet "CAG expansions in a novel gene for Machado-Joseph disease at chromosome 14q32.1."

Kawaguchi Y, Inoue M, Nishimura M, Kawakami H, Okamoto T, Katayama S, Nakamura S, Aizawa M, Akiguchi I, Taniwaki M

[Nat Genet, 1994]

We have identified a novel gene containing CAG repeats and mapped it to chromosome 14q32.1, the genetic locus for Machado-Joseph disease (MJD). In normal individuals the gene contains between 13 and 36 CAG repeats, whereas most of the clinically diagnosed patients and all of the affected members of a family with the clinical and pathological diagnosis of MJD show expansion of the repeat-number (from 68-79). Southern blot analyses and genomic cloning demonstrates the existence of related genes. These results raise the possibility that similar abnormalities in related genes may give rise to diseases similar to MJD.

Carmona, Javier et al. (2017) International Worm Meeting "2D worm tracking line-confocal microscope with virtual reality."

Wang, Charles, Carmona, Javier, Baldo, Anthony, Madruga, Blake, Mendoza, Steve, Jin, Suying, Arisaka, Katsushi, Liu, Larry, Thatcher, Joseph

[International Worm Meeting, 2017]

Calcium dynamic imaging and free motion tracking coupled with external stimulations allow for in depth analysis of C. elegans behavior. We have developed an integrated platform for monitoring and controlling C. elegans under a variety of external stimulations, including thermal, electrical, and photo stimuli. This innovative platform combines rapid volumetric (20 volume/s) diffraction limited dual line-confocal microscopy (0.5 um x 1 um x 5 um voxel) to determine the neural pathways different external stimuli induce, while tracking worm's two dimensional motion. Never before has dynamic signal propagation, from neuron to neuron, been observed for C. elegans in free motion at such high volume scanning rate. External stimuli are computer controlled with < 10 ms resolution for precise spatio-temporal synchronization with free motion behavior and whole-brain calcium dynamics. Physical linear and circular thermal gradients were implemented using customized temperature plates with thermal fluctuations of less than 0.05 deg C. In addition, thermal stimulation was applied via a 1490 nm infrared laser to create virtual temperature conditions, synchronized with head motion. Infrared laser stimulation allows C. elegans' thermoreceptor (AFD neuron) to perceive temperature fluctuations exclusively in the time domain, thereby allowing for the complete virtual manipulation of the nematode's thermal environment. Electrical responses were induced using a technique that involves applying a linear or spatially alternating electrical field through a gelatin sample with fields ranging from 4 to 14 V/cm. Photon stimulation was implemented using a 405 nm laser with intensities ranging from 0 to 10 mW/mm^2. Volumetric Calcium imaging of QW1217 has also allowed for the complete mapping of the neurons responsible for each of the aforementioned stimuli. The microscope and software accommodate multiple simultaneous stimuli applications, such as electrical and photon simulations. Also tested were the neural pathway differences between infrared and photo avoidance behavior due to their very similar behavioral responses.

Rodrigues AJ et al. (2007) FASEB J "Functional genomics and biochemical characterization of the C. elegans orthologue of the Machado-Joseph disease protein ataxin-3."

Ailion M, Rodrigues AJ, Maciel P, Geschwind DH, Coppola G, Santos C, Costa MD, Sequeiros J

[FASEB J, 2007]

Machado-Joseph disease (MJD) is the most common dominant spinocerebellar ataxia. MJD is caused by a CAG trinucleotide expansion in the ATXN3 gene, which encodes a protein named ataxin-3. Ataxin-3 has been proposed to act as a deubiquitinating enzyme in the ubiquitin-proteasome pathway and to be involved in transcriptional repression; nevertheless, its precise biological function(s) remains unknown. To gain further insight into the function of ataxin-3, we have identified the Caenorhabditis elegans orthologue of the ATXN3 gene and characterized its pattern of expression, developmental regulation, and subcellular localization. We demonstrate that, analogous to its human orthologue, C. elegans ataxin-3 has deubiquitinating activity in vitro against polyubiquitin chains with four or more ubiquitins, the minimum ubiquitin length for proteasomal targeting. To further evaluate C. elegans ataxin-3, we characterized the first known knockout animal models both phenotypically and biochemically, and found that the two C. elegans strains were viable and displayed no gross phenotype. To identify a molecular phenotype, we performed a large-scale microarray analysis of gene expression in both knockout strains. The data revealed a significant deregulation of core sets of genes involved in the ubiquitin-proteasome pathway, structure/motility, and signal transduction. This gene identification provides important clues that can help elucidate the specific biological role of ataxin-3 and unveil some of the physiological effects caused by its absence or diminished function.--Rodrigues, A-J., Coppola, G., Santos, C., do Carmo Costa, M., Ailion, M., Sequeiros, J., Geschwind, D. H., Maciel, P. Functional genomics and biochemical characterization of the C. elegans orthologue of the Machado-Joseph disease protein ataxin-3.

Dao, Chris et al. (2017) International Worm Meeting "Development of a high-statistics, three dimensional imaging system for C. elegans under magnetic and chemical stimuli."

Arisaka, Katushi, David, Angela, Vincent, Nitin, Thatcher, Joseph, Chow, Aaron, Wang, Charles, Dao, Chris, Du, Angela, Alberto, Jasmine, Li, Lina

[International Worm Meeting, 2017]

In contrast to previous behavioral experiments limited to two dimensional surfaces, we extended the study of C. elegans into three dimensional environments to observe the resulting unconstrained motion. While research has been initiated towards single worm, high magnification tracking, this has limited practicality in terms of gathering a large number of statistics with bulk behavior of many worms simultaneously. Incorporating stimuli allows for a new, more thorough examination of motion and environmental interaction, extending inquiry beyond simple free motion. Previously performed, two dimensional results suggest hypotheses, which may serve as limiting-case projections of greater trends in three dimensional space. A novel, cost effective system was developed to image C elegans' behavior over a period of 30 to 60 minutes using time lapse acquisition. The setup consists of three, perpendicularly oriented DSLR cameras, synchronized via the open source, MIT developed DigiCam control software and illuminated with lowly-invasive, conical, red LED light sources. Samples consisted of N2 type worms embedded in the center of 2% porcine gelatin (4 to 5 cm) quartz cuvettes. Each camera had an affixed aperture to elongate its depth of field, promoting focus throughout the volume of the cubes. In addition to free motion studies, three sets of Helmholtz coils (with axes coinciding with the cameras' optical axes) were utilized to generate a uniform magnetic field at the location of the sample. NaCl concentration gradients throughout the gelatin were also simulated and created for three dimensional chemotaxis experiments. Numerous free motion trials have been conducted alongside magnetic field experiments of field magnitude ranging from 0 Gauss (cancelling the geomagnetic field) to 10 Gauss. Using this data, analysis software tools were developed in MATLAB in order to extract the center of mass trajectory of worms navigating the gelatin, enabling further quantification of motional trends such as the worms' velocities and distributions of initial and final positions. While preliminary results for free motion (with chemical attractants) appear to reflect standard biased random motion, positive verification of magnetic field trials remains elusive.

Pohl F et al. (2024) MicroPubl Biol "Pharmacological inhibition of acetylcholinesterase improves the locomotion defective phenotype of a SCA3 C. elegans model."

Maciel P, Pohl F, Teixeira-Castro A, Lindsay-McGee V, Kong Thoo Lin P

[MicroPubl Biol, 2024]

Inhibition of acetylcholinesterase (AChE) is a common used treatment option for Alzheimer's disease. However, there has been limited research on the potential use of AChE inhibitors for the treatment of Machado-Joseph disease (MJD)/Spinocerebellar Ataxia 3 (SCA3), in spite of the positive results using AChE inhibitors in patients with other inherited ataxias. MJD/SCA3, the most common form of dominant Spinocerebellar Ataxia worldwide, is caused by an expansion of the polyglutamine tract within the ataxin-3 protein, and is characterized by motor impairments. Our study shows that administration of the AChE inhibitor neostigmine is beneficial in treating the locomotion defective phenotype of a SCA3/MJD model of <i>C. elegans</i> and highlights the potential contribution of AChE enzymes to mutant ataxin-3-mediated toxicity.

Teixeira-Castro A et al. (2011) Hum Mol Genet "Neuron-specific proteotoxicity of mutant ataxin-3 in C. elegans: rescue by the DAF-16 and HSF-1 pathways."

Oliveira JF, Vilaca JL, Brignull HR, Ailion M, Teixeira-Castro A, Dias N, Neves-Carvalho A, Maciel P, Morimoto RI, Jalles A, Rodrigues P

[Hum Mol Genet, 2011]

The risk of developing neurodegenerative diseases increases with age. Although many of the molecular pathways regulating proteotoxic stress and longevity are well characterized, their contribution to disease susceptibility remains unclear. In this study, we describe a new Caenorhabditis elegans model of Machado-Joseph disease pathogenesis. Pan-neuronal expression of mutant ATXN3 leads to a polyQ-length dependent, neuron subtype-specific aggregation and neuronal dysfunction. Analysis of different neurons revealed a pattern of dorsal nerve cord and sensory neuron susceptibility to mutant ataxin-3 that was distinct from the aggregation and toxicity profiles of polyQ-alone proteins. This reveals that the sequences flanking the polyQ-stretch in ATXN3 have a dominant influence on cell-intrinsic neuronal factors that modulate polyQ-mediated pathogenesis. Aging influences the ATXN3 phenotypes which can be suppressed by the downregulation of the insulin/insulin growth factor-1-like signaling pathway and activation of heat shock factor-1.

Wu Y et al. (2023) Int J Mol Sci "Towards Understanding Neurodegenerative Diseases: Insights from Caenorhabditis elegans."

Yu X, Wu Y, Zhang M, Li Z, Chen Y

[Int J Mol Sci, 2023]

The elevated occurrence of debilitating neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD) and Machado-Joseph disease (MJD), demands urgent disease-modifying therapeutics. Owing to the evolutionarily conserved molecular signalling pathways with mammalian species and facile genetic manipulation, the nematode <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) emerges as a powerful and manipulative model system for mechanistic insights into neurodegenerative diseases. Herein, we review several representative <i>C. elegans</i> models established for five common neurodegenerative diseases, which closely simulate disease phenotypes specifically in the gain-of-function aspect. We exemplify applications of high-throughput genetic and drug screenings to illustrate the potential of <i>C. elegans</i> to probe novel therapeutic targets. This review highlights the utility of <i>C. elegans</i> as a comprehensive and versatile platform for the dissection of neurodegenerative diseases at the molecular level.