Reed E et al. (2018) J Neurochem "Animal models of Wilson disease."

Lutsenko S, Bandmann O, Reed E

[J Neurochem, 2018]

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism manifesting with hepatic, neurological and psychiatric symptoms. The limitations of the currently available therapy for WD (particularly in the management of neuropsychiatric disease), together with our limited understanding of key aspects of this illness (e.g. neurological vs hepatic presentation) justify the ongoing need to study WD in suitable animal models. Four animal models of WD have been established: the Long-Evans Cinnamon rat, the toxic-milk mouse, the Atp7b knockout mouse and the Labrador retriever. The existing models of WD all show good similarity to human hepatic WD and have been helpful in developing an improved understanding of the human disease. As mammals, the mouse, rat and canine models also benefit from high homology to the human genome. However, important differences exist between these mammalian models and human disease, particularly the absence of a convincing neurological phenotype. This review will first provide an overview of our current knowledge of the orthologous genes encoding ATP7B and the closely related ATP7A protein in C. elegans, Drosophila and zebrafish (Danio rerio) and then summarise key characteristics of rodent and larger mammalian models of ATP7B-deficiency. This article is protected by copyright. All rights reserved.

Khazma T et al. (2023) Cell Rep "Structure-function analysis of ceTIR-1/hSARM1 explains the lack of Wallerian axonal degeneration in C. elegans."

Grossman A, Dabas H, Zalk R, Sain R, Isupov MN, Feng C, Khazma T, Guez-Haddad J, Weitman M, Hammarlund M, Hons M, Opatowsky Y

[Cell Rep, 2023]

Wallerian axonal degeneration (WD) does not occur in the nematode C. elegans, in contrast to other model animals. However, WD depends on the NADase activity of SARM1, a protein that is also expressed in C. elegans (ceSARM/ceTIR-1). We hypothesized that differences in SARM between species might exist and account for the divergence in WD. We first show that expression of the human (h)SARM1, but not ceTIR-1, in C. elegans neurons is sufficient to confer axon degeneration after nerve injury. Next, we determined the cryoelectron microscopy structure of ceTIR-1 and found that, unlike hSARM1, which exists as an auto-inhibited ring octamer, ceTIR-1 forms a readily active 9-mer. Enzymatically, the NADase activity of ceTIR-1 is substantially weaker (10-fold higher Km) than that of hSARM1, and even when fully active, it falls short of consuming all cellular NAD⁺. Our experiments provide insight into the molecular mechanisms and evolution of SARM orthologs and WD across species.

Frances SP et al. (2008) Am J Trop Med Hyg "Seroconversion to filarial antigens in Australian defence force personnel in Timor-Leste."

Nissen MD, Nutman TB, Frances SP, Melrose WD, McCarthy JS, Kubofcik J, Baade LM

[Am J Trop Med Hyg, 2008]

To investigate whether Australian soldiers were exposed to filarial parasites that cause lymphatic filariasis during a 6-month deployment to Timor-Leste, antifilarial antibody levels were measured in 907 soldiers using an enzyme linked immunosorbent assay (ELISA). Initial testing using Dirofilaria immitis antigen demonstrated that 49 of 907 (5.4%) soldiers developed antifilarial antibodies of the IgG1 subclass after deployment, whereas 1 of 944 (0.1%) seroconverted to the IgG4 subclass. When a sub sample of 88 D. immitis-reactive sera was subject to testing with an antifilarial antibody test using Brugia malayi antigen, 46 had elevated IgG antibodies, whereas 5 had elevated antibodies of the IgG4 subclass. A total of 24 soldiers seroconverted to B. malayi, as measured by parasite-specific IgG, whereas 1 seroconverted to IgG4. The relatively low number of seroconversions indicates a low but measurable risk of exposure to human filarial parasites among Australian soldiers deployed to Timor-Leste. However, to reduce the risk of exposure to these parasites, soldiers deploying to endemic areas should practice strict adherence to personal protective measures against mosquito bites.

Meir JYJ et al. (1996) East Coast Worm Meeting "UNC-4 and UNC-37 function together to specify VA motor neuron identity"

Meir JYJ, Miller DM, Barnes TM, Pflugrad A

[East Coast Worm Meeting, 1996]

VA and VB motor neurons arise from a common precursor but adopt different morphologies and synapse with separate sets of interneurons. unc-4 mutants are unable to crawl backward because VA motor neurons assume synaptic inputs normally reserved for their VB sisters. The unc-4 homeoprotein is expressed in the VA motor neurons and is therefore likely to regulate target genes that distinguish VAs from VBs. Here we show that the groucho-like protein, UNC-37, is also expressed in the VA motor neurons and propose that UNC-4 function depends on specific interactions with UNC-37. Groucho is the founding member of a family of WD repeat proteins that are believed to interact with specific transcription factors to repress target genes. unc-37 encodes a 612 amino acid protein with a highly conserved C-terminal array of six WD repeats (~60% identity to fly groucho, mouse ESG, and human TLE proteins). Specific mutations in the UNC-37 WD domains modify UNC-4 function. Substitution of a conserved residue (H to Y) in the fifth WD repeat in unc-37(e262) phenocopies the Unc-4 movement defect. Conversely, an amino acid substitution (E to K) in the sixth WD repeat restores backward locomotion to unc-37(e262) and to specific unc-4 missense mutants. By immunostaining, UNC-37 protein is ubiquitously expressed and nuclear-localized. However, expression of a functional gfp-tagged UNC-37 protein in VA motor neurons using the unc-4 promoter is sufficient to restore normal movement to unc-37(e262) animals which suggests that the e262 mutation specifically disrupts UNC-37 function in the VA motor neurons. Recently, we have performed domain swapping experiments and found that a chimeric protein which contains the amino terminus of UNC-37 and the WD domain of human TLE1 rescues the unc-37(e262) phenotype, indicating that the highly conserved WD region is functionally interchangable. In Drosophila, groucho physically interacts with basic helix-loop-helix (bHLH) proteins to repress target gene expression. Similarly, TUP1, a related WD protein found in yeast, binds to the alpha-2 homeodomain protein to mediate transcriptional repression. Our findings favor a model in which the UNC-4 and UNC-37 proteins function in a common transcription complex to regulate genes that govern synaptic input to the VA motor neurons and that the fifth and sixth WD repeats of UNC-37 could mediate this interaction. Alternatively, UNC-37 and UNC-4 may act in parallel pathways that coordinately define synaptic input to the VAs. Co-immunoprecipitation experiments are currently underway to distinguish between these possibilites.

Yu L et al. (2000) Protein Sci "Thirty-plus functional families from a single motif."

Yu L, Smith TF, Neer E, Gaitatzes C

[Protein Sci, 2000]

It is now possible to identify over 30 functional subfamilies among the WD-repeat-containing proteins found in the completed genomes. The majority of these subfamilies have at least one member for which experimental data allow assignment to a cellular pathway or process. Half of the 63 WD-repeat-containing proteins in Saccharomyces cerevisiae, half of the 70 in Caenorhabditis elegans, and a third of the 100 plus predicted in Drosophila can be assigned to 23 of these functional subfamilies. Perhaps indicative of the future, 33 WD-repeat-containing proteins from the partial genome of Arabidopsis thaliana can now be assigned to 18 of these subfamilies. These assignments have been made possible by combining traditional sequence similarity with an implied common beta propeller structural context to obtain measures of protein-protein surface similarity. The beta propeller structural context is represented in the form of a Hidden Markov Model. The procedure is completely automated.

Weil GJ et al. (2011) Acta Trop "A multicenter evaluation of a new antibody test kit for lymphatic filariasis employing recombinant Brugia malayi antigen Bm-14."

Fischer PU, Joseph H, Lammie PJ, Won KY, Melrose WD, Weil GJ, Curtis KC, Brattig NW

[Acta Trop, 2011]

Antibody tests are useful for mapping the distribution of lymphatic filariasis (LF) in countries and regions and for monitoring progress in elimination programs based on mass drug administration (MDA). Prior antibody tests have suffered from poor sensitivity and/or specificity or from a lack of standardization. We conducted a multicenter evaluation of a new commercial ELISA that detects IgG4 antibodies to the recombinant filarial antigen Bm14. Four laboratories tested a shared panel of coded serum or plasma samples that included 55 samples from people with microfilaremic Wuchereria bancrofti or Brugia infections and 26 control samples. Qualitative results were identical in all four test sites. In addition, each laboratory tested samples from their own serum banks. The test detected antibodies in 32 of 36 samples (91%) from people with Brugian filariasis and in 96 of 98 samples (98%) from people with Bancroftian filariasis. Specificity testing showed that many serum or plasma samples from patients with other filarial infections such as onchocerciasis had positive antibody tests. Specificity was otherwise excellent, although 3 of 30 samples from patients with ascariasis and 4 of 51 with strongyloidiasis had positive antibody tests; it is likely that some or all of these people had previously lived in filariasis-endemic areas. Antibody test results obtained with eluates from blood dried on filter paper were similar to those obtained with plasma tested at the same dilution. This test may be helpful for diagnosing LF in patients with clinical signs of filariasis. It may also be a useful tool for use in LF endemic countries to monitor the progress of filariasis elimination programs and for post-MDA surveillance.

Catalano, Federico et al. (2021) International Worm Meeting "Characterization of a new model of Wilson disease to find innovative targets and pathways to attenuate Cu toxicity"

Santonicola, Pamela, Zampi, Giuseppina, Puchkova, Ludmila, Di Schiavi, Elia, Ilicheva, Ekaterina, Orlov, Iurii, Polishchuk, Roman, Polishchuk, Elena, Catalano, Federico

[International Worm Meeting, 2021]

Copper is an essential nutrient that is required for many vitally important processes, such as respiration, connective tissue biogenesis and iron metabolism. However, excess of copper represents a serious danger, due to its ability to induce free radical-induced oxidative damage, as well as impairments of lipid metabolism and neuronal activity. Wilson disease (WD) represents an excellent system for Cu toxicity studies. WD is caused by mutations in ATP7B pump effluxing excess Cu from hepatocytes into the bile. Loss of ATP7B leads to toxic Cu overload in liver and then in brain, causing fatal hepatic and neurologic abnormalities. Over the last years, C. elegans has emerged as an easy-to-use and highly responsive model of micronutrient metabolism. For this reason, we have generated and characterized a new C. elegans model of WD. CUA-1, the C. elegans ortholog of ATP7B, resides in lysosome-like organelles to sequester excess of copper and, therefore, represents a key component regulating copper supply and detoxification in order to maintain copper homeostasis. Using Crispr-Cas9 technology a cua-1(knu781 [H828Q]) strain was generated, that carries a substitution in conserved histidine corresponding to the most common H1069Q variant of ATP7B causing WD in European and North American population. In order to understand the effect of H828Q mutation on C. elegans phenotype we employed several assays that allow life span, motility, egg laying, larval development and mitochondria damage to be evaluated. Moreover, we are studying the colocalization of the mutant CUA-1 protein with markers of different organelles, such as lysosome, Golgi and ER, as well as with the copper sensor CF4. Our studies established that in absence of copper, cua-1(knu781) does not show any significant phenotypic aberrations. However, mutant worms exhibited very poor resistance to copper compared to the control strain. This manifested in a strong decrease in number of eggs, a delay in the larval development, a shorter lifespan, impaired motility and mitochondrial damage. Taken together our finding suggest that cua-1(knu781) represents an excellent model for Cu toxicity studies in WD. We further plan to use this model for identification and validation of the new therapeutic targets for WD. Moreover, cua-1(knu781) will be used for evaluation of the FDA-approved drugs that emerged from a high throughput screening for compounds reducing Cu toxicity in WD.

Mizushima N et al. (2003) J Cell Sci "Mouse Apg16L, a novel WD-repeat protein, targets to the autophagic isolation membrane with the Apg12-Apg5 conjugate."

Yamamoto A, Matsubae M, Kobayashi Y, Natsume T, Mizushima N, Kuma A, Takao T, Yoshimori T, Ohsumi Y

[J Cell Sci, 2003]

Macroautophagy is the major intracellular degradation system delivering cytoplasmic components to the lysosome/vacuole. We have shown that, in yeast and mammalian cells, the Apg12-Apg5 protein conjugate, which is formed by a ubiquitin-like system, is essential for autophagosome formation. In yeast, the Apg12-Apg5 conjugate interacts with a small coiled-coil protein, Apg16, to form a approximately 350 kDa multimeric complex. We demonstrate that the mouse Apg12-Apg5 conjugate forms a approximately 800 kDa protein complex containing a novel WD-repeat protein. Because the N-terminal region of this novel protein shows homology with yeast Apg16, we have designated it mouse Apg16-like protein (Apg16L). Apg16L, however, has a large C-terminal domain containing seven WD repeats that is absent from yeast Apg16. Apg16L interacts with both Apg5 and additional Apg16L monomers; neither interaction, however, depends on the WD-repeat domain. In conjunction with Apg12-Apg5, Apg16L associates with the autophagic isolation membrane for the duration of autophagosome formation. Because these features are similar to yeast Apg16, we concluded Apg16L is the functional counterpart of the yeast Apg16. We also found that membrane targeting of Apg16L requires Apg5 but not Apg12. Because WD-repeat proteins provide a platform for protein-protein interactions, the approximately 800 kDa complex is expected to function in autophagosome formation, further interacting with other proteins in mammalian cells.

Pflugrad A et al. (1997) Development "The Groucho-like transcription factor UNC-37 functions with the neural specificity gene unc-4 to govern motor neuron identity in C. elegans."

Pflugrad A, Miller DM, Barnes TM, Meir JYJ

[Development, 1997]

Groucho and Tup1 are members of a conserved family of WD repeat proteins that interact with specific transcription factors to repress target genes. Here we show that mutations in WD domains of the Groucho-like protein, UNC-37, affect a motor neuron trait that also depends on UNC-4, a homeodomain protein that controls neuronal specificity in Caenorhabditis elegans. In unc-4 mutants, VA motor neurons assume the pattern of synaptic input normally reserved for their lineal sister cells, the VB motor neurons; the loss of normal input to the VAs produces a distinctive backward movement defect. Substitution of a conserved residue (H to Y) in the fifth WD repeat in unc-37(e262) phenocopies the Unc-4 movement defect. Conversely, an amino acid change (E to K) in the sixth WD repeat of UNC-37 is a strong suppressor of unc-37(e262) and of specific unc-4 missense mutations. We have previously shown that UNC-4 expression in the VA motor neurons specifies the wild-type pattern of presynaptic input. Here we demonstrate that UNC-37 is also expressed in the VAs and that unc-37 activity in these neurons is sufficient to restore normal movement to unc-37(e262) animals. We propose that UNC-37 and UNC-4 function together to prevent expression of genes that define the VB pattern of synaptic inputs and thereby generate connections specific to the VA motor neurons. In addition, we show that the WD repeat domains of UNC-37 and of the human homolog, TLE1, are functionally interchangeable in VA motor neurons which suggests that this highly conserved protein domain may also specify motor neuron identity and synaptic choice in more complex nervous systems.

Catalano F et al. (2023) Traffic "A new Caenorhabditis elegans model to study copper toxicity in Wilson disease."

Brown AEX, Zampi G, Elia M, Romanov AA, Ilyechova EY, Indrieri A, Mekhova AA, Gallotta I, De Cegli R, Catalano F, Di Schiavi E, O'Brien TJ, Kim BE, Sepe LV, Puchkova LV, Salzano J, Petruzzelli R, Santonicola P, Polishchuk EV, Polishchuk RS, Samuseva PD

[Traffic, 2023]

Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a copper (Cu) transporting ATPase whose trafficking from the Golgi to endo-lysosomal compartments drives sequestration of excess Cu and its further excretion from hepatocytes into the bile. Loss of ATP7B function leads to toxic Cu overload in the liver and subsequently in the brain, causing fatal hepatic and neurological abnormalities. The limitations of existing WD therapies call for the development of new therapeutic approaches, which require an amenable animal model system for screening and validation of drugs and molecular targets. To achieve this objective, we generated a mutant Caenorhabditis elegans strain with a substitution of a conserved histidine (H828Q) in the ATP7B ortholog cua-1 corresponding to the most common ATP7B variant (H1069Q) that causes WD. cua-1 mutant animals exhibited very poor resistance to Cu compared to the wild-type strain. This manifested in a strong delay in larval development, a shorter lifespan, impaired motility, oxidative stress pathway activation, and mitochondrial damage. In addition, morphological analysis revealed several neuronal abnormalities in cua-1 mutant animals exposed to Cu. Further investigation suggested that mutant CUA-1 is retained and degraded in the endoplasmic reticulum, similarly to human ATP7B-H1069Q. As a consequence, the mutant protein does not allow animals to counteract Cu toxicity. Notably, pharmacological correctors of ATP7B-H1069Q reduced Cu toxicity in cua-1 mutants indicating that similar pathogenic molecular pathways might be activated by the H/Q substitution and, therefore, targeted for rescue of ATP7B/CUA-1 function. Taken together, our findings suggest that the newly generated cua-1 mutant strain represents an excellent model for Cu toxicity studies in WD.