Joseph, B.B. et al. (2019) International Worm Meeting "NEKL kinases are novel regulators of clathrin-mediated endocytosis."

Joseph, B.B., Grant, B.D., Wang, Y., Fay, D.S.

[International Worm Meeting, 2019]

We previously showed that downregulation of NEKL-2/NEK8/9 and NEKL-3/NEK6/7 causes molting defects in C. elegans. NEKL kinases are highly conserved members of NIMA-kinase family, which have been predominantly implicated in cell cycle functions and ciliogenesis. In addition, we found that a conserved set of ankyrin repeat proteins, MLT-2/ANKS6, MLT-3/ANKS3, and MLT-4/INVS, partner with the NEKLs to regulate intracellular trafficking, actin organization, and normal molting. To better understand the mechanistic functions of NEKL-MLTs we obtained genetic suppressors of molting defects in nekl-2; nekl-3 double mutants. Notably, our screen identified mutations affecting subunits of AP2, a key clathrin adapter complex acting at the plasma membrane. AP2 is a heterotetrametric complex consisting of two partially functional hemi complexes ( /? and ?/?), and downregulation of , ?, and ? subunits suppressed nekl-mlt mutant lethality. Additionally, we isolated a mutation affecting FCHO-1, an allosteric regulator of AP2 that promotes the open/active AP2 conformation. These data indicate that nekl defects can be suppressed by mutations that reduce the amount of open-active AP2. Consistent with this, mutations affecting NCAP-1, an allosteric inhibitor of AP2, strongly enhance nekl-mlt defects, as do mutations in the AP2 subunit that stabilize the open conformation. Interestingly, loss of nekl-mlt functions suppressed the Jowls phenotype associated with mutations in fcho-1 and AP2, revealing a unique phenomenon of reciprocal suppression. We previously reported that nekl-mlt arrested larvae displayed abnormal clathrin expression. To circumvent complications due to larval arrest, we generated auxin-degradable versions of the NEKLs. Auxin-treated adults exhibited increased signal density and higher expression levels of a CRISPR clathrin heavy chain reporter. Furthermore, clathrin recovery after photo bleaching was comparatively rapid in NEKL-depleted adults versus control. Likewise, similar effects on clathrin were observed in ncap-1(0) and apm-2(E306K) mutants. Conversely, fcho-1(0) and apa-2(0) mutants showed a reduction in clathrin puncta density and levels. Localization of LRP-1, a cargo required for normal molting, was also greatly perturbed in NEKL-depleted adults. Overall, our data supports a model whereby the NEKL-MLT network regulates clathrin mediated endocytosis through AP2 activity, which is necessary for the normal trafficking of cargo required for molting.

Joseph BB et al. (2020) PLoS Genet "Control of clathrin-mediated endocytosis by NIMA family kinases."

Edeen P, Lazetic V, Joseph BB, Fay DS, Wang Y, Grant BD

[PLoS Genet, 2020]

Endocytosis, the process by which cells internalize plasma membrane and associated cargo, is regulated extensively by posttranslational modifications. Previous studies suggested the potential involvement of scores of protein kinases in endocytic control, of which only a few have been validated in vivo. Here we show that the conserved NIMA-related kinases NEKL-2/NEK8/9 and NEKL-3/NEK6/7 (the NEKLs) control clathrin-mediated endocytosis in C. elegans. Loss of NEKL-2 or NEKL-3 activities leads to penetrant larval molting defects and to the abnormal localization of trafficking markers in arrested larvae. Using an auxin-based degron system, we also find that depletion of NEKLs in adult-stage C. elegans leads to gross clathrin mislocalization and to a dramatic reduction in clathrin mobility at the apical membrane. Using a non-biased genetic screen to identify suppressors of nekl molting defects, we identified several components and regulators of AP2, the major clathrin adapter complex acting at the plasma membrane. Strikingly, reduced AP2 activity rescues both nekl mutant molting defects as well as associated trafficking phenotypes, whereas increased levels of active AP2 exacerbate nekl defects. Moreover, in a unique example of mutual suppression, NEKL inhibition alleviates defects associated with reduced AP2 activity, attesting to the tight link between NEKL and AP2 functions. We also show that NEKLs are required for the clustering and internalization of membrane cargo required for molting. Notably, we find that human NEKs can rescue molting and trafficking defects in nekl mutant worms, suggesting that the control of intracellular trafficking is an evolutionarily conserved function of NEK family kinases.

Joseph BB et al. (2023) PLoS Genet "Conserved NIMA kinases regulate multiple steps of endocytic trafficking."

Robison L, Fay DS, Joseph BB, Naslavsky N, Caplan S, Grant BD, Conquest S, Homer RO, Bai G, Binti S

[PLoS Genet, 2023]

Human NIMA-related kinases have primarily been studied for their roles in cell cycle progression (NEK1/2/6/7/9), checkpoint-DNA-damage control (NEK1/2/4/5/10/11), and ciliogenesis (NEK1/4/8). We previously showed that Caenorhabditis elegans NEKL-2 (NEK8/9 homolog) and NEKL-3 (NEK6/7 homolog) regulate apical clathrin-mediated endocytosis (CME) in the worm epidermis and are essential for molting. Here we show that NEKL-2 and NEKL-3 also have distinct roles in controlling endosome function and morphology. Specifically, loss of NEKL-2 led to enlarged early endosomes with long tubular extensions but showed minimal effects on other compartments. In contrast, NEKL-3 depletion caused pronounced defects in early, late, and recycling endosomes. Consistently, NEKL-2 was strongly localized to early endosomes, whereas NEKL-3 was localized to multiple endosomal compartments. Loss of NEKLs also led to variable defects in the recycling of two resident cargoes of the trans-Golgi network (TGN), MIG-14/Wntless and TGN-38/TGN38, which were missorted to lysosomes after NEKL depletion. In addition, defects were observed in the uptake of clathrin-dependent (SMA-6/Type I BMP receptor) and independent cargoes (DAF-4/Type II BMP receptor) from the basolateral surface of epidermal cells after NEKL-2 or NEKL-3 depletion. Complementary studies in human cell lines further showed that siRNA knockdown of the NEKL-3 orthologs NEK6 and NEK7 led to missorting of the mannose 6-phosphate receptor from endosomes. Moreover, in multiple human cell types, depletion of NEK6 or NEK7 disrupted both early and recycling endosomal compartments, including the presence of excess tubulation within recycling endosomes, a defect also observed after NEKL-3 depletion in worms. Thus, NIMA family kinases carry out multiple functions during endocytosis in both worms and humans, consistent with our previous observation that human NEKL-3 orthologs can rescue molting and trafficking defects in C. elegans nekl-3 mutants. Our findings suggest that trafficking defects could underlie some of the proposed roles for NEK kinases in human disease.

Joseph BB et al. (2022) PLoS Genet "An unexpected role for the conserved ADAM-family metalloprotease ADM-2 in Caenorhabditis elegans molting."

Fay DS, Edeen PT, Meadows S, Binti S, Joseph BB

[PLoS Genet, 2022]

Molting is a widespread developmental process in which the external extracellular matrix (ECM), the cuticle, is remodeled to allow for organismal growth and environmental adaptation. Studies in the nematode Caenorhabditis elegans have identified a diverse set of molting-associated factors including signaling molecules, intracellular trafficking regulators, ECM components, and ECM-modifying enzymes such as matrix metalloproteases. C. elegans NEKL-2 and NEKL-3, two conserved members of the NEK family of protein kinases, are essential for molting and promote the endocytosis of environmental steroid-hormone precursors by the epidermis. Steroids in turn drive the cyclic induction of many genes required for molting. Here we report a role for the sole C. elegans ADAM-meltrin metalloprotease family member, ADM-2, as a mediator of molting. Loss of adm-2, including mutations that disrupt the metalloprotease domain, led to the strong suppression of molting defects in partial loss-of-function nekl mutants. ADM-2 is expressed in the epidermis, and its trafficking through the endo-lysosomal network was disrupted after NEKL depletion. We identified the epidermally expressed low-density lipoprotein receptor-related protein, LRP-1, as a candidate target of ADM-2 regulation. Whereas loss of ADM-2 activity led to the upregulation of apical epidermal LRP-1, ADM-2 overexpression caused a reduction in LRP-1 levels. Consistent with this, several mammalian ADAMs, including the meltrin ADAM12, have been shown to regulate mammalian LRP1 via proteolysis. In contrast to mammalian homologs, however, the regulation of LRP-1 by ADM-2 does not appear to involve the metalloprotease function of ADM-2, nor is proteolytic processing of LRP-1 strongly affected in adm-2 mutants. Our findings suggest a noncanonical role for an ADAM family member in the regulation of a lipoprotein-like receptor and lead us to propose that endocytic trafficking may be important for both the internalization of factors that promote molting as well as the removal of proteins that can inhibit the process.

Joseph BB et al. (2017) G3 (Bethesda) "Use of a Sibling Subtraction Method (SSM) for Identifying Causal Mutations in C. elegans by Whole-Genome Sequencing."

Joseph BB, Blouin NA, Fay DS

[G3 (Bethesda), 2017]

Whole-genome sequencing (WGS) is an indispensible tool for identifying causal mutations obtained from genetic screens. To reduce the number of causal mutation candidates typically uncovered by WGS, C. elegans researchers have developed several strategies. One involves crossing N2-background mutants to the polymorphic HA strain, which can be used to simultaneously identify mutant-strain variants and obtain high-density mapping information. This approach, however, is not well suited for uncovering mutations in complex genetic backgrounds, and HA polymorphisms can alter phenotypes. Other approaches make use of DNA variants present in the initial background or introduced by mutagenesis. This information is used to implicate genomic regions with high densities of DNA lesions that persist after backcrossing, but these methods can provide lower resolution than HA mapping. To identify suppressor mutations using WGS, we developed an approach termed the Sibling Subtraction Method (SSM). This method works by eliminating variants present in both mutants and their non-mutant siblings, thus greatly reducing the number of candidates. We used this method with two members of the C. elegans NimA-related kinase family, nekl-2 and nekl-3. Combining weak aphenotypic alleles of nekl-2 and nekl-3 leads to penetrant molting defects and larval arrest. We isolated ~35 suppressors of nekl-2; nekl-3 synthetic lethality using F1-clonal screening methods and a peel-1-based counter-selection strategy. When applied to five of the suppressors, SSM led to only one to four suppressor candidates per strain. Thus SSM is a powerful approach for identifying causal mutations in any genetic background and provides an alternative to current methods.

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.

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.

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.