Winnier AR et al. (1997) International C. elegans Meeting "RESIDUES OUTSIDE OF THE HOMEODOMAIN ARE REQUIRED FOR UNC-4 FUNCTION"

Miller DM, Saito T, Winnier AR

[International C. elegans Meeting, 1997]

Homeodomain (HD) proteins function as DNA-specific regulators of gene expression. The gene regulatory function of the HD can be augmented by sequences outside of this domain (i.e. paired, LIM, POU). We have identified residues outside of the UNC-4 HD that are required for its function: specifying synaptic input to VA motor neurons. A cluster of five missense mutations were identified C-terminal to the HD. These residues are contained within a region predicted to form four beta sheets separated by turns (BTB). We note that specific BTB mutations are suppressed by a specific missense mutation in unc-37, a groucho-like transcription factor. Therefore, one function of the BTB domain may be to mediate UNC-4/UNC-37 interactions. The vertebrate HD proteins PHD1 and uncx-4.1 share 90% identity with the UNC-4 HD but do not include an UNC-4-like BTB domain. We evaluated the ability of a vertebrate UNC-4-like HD to substitute for unc-4 function. The rat HD protein, PHD1,under unc-4 promoter control, fails to rescue unc-4(wd1) animals, suggesting that both BTB and HD residues are required to regulate synaptic specificity in C. elegans. To identify additional unc-4-interacting genes, we are performing screens for dominant suppressors of BTB mutations. One novel suppressor mutation has been identified. bkn-1(wd29) (backing again) maps to the 0.5 m.u. interval between bli-6 and unc-24 (IV). Like unc-37(gf), wd29 suppresses specific missense mutations in the BTB domain as well as in the HD. This allele-specific suppression suggests that wd29 may affect the activity of a transcription factor that functions with unc-4.

Lickteig KM et al. (1997) International C. elegans Meeting "IDENTIFYING UNC-4 HOMEODOMAIN BINDING SITES"

Lickteig KM, Miller DM

[International C. elegans Meeting, 1997]

unc-4 encodes a paired-like homeodomain (HD) protein expressed in the VA motor neurons but not in their VB sisters cells. unc-4 functions to specify synaptic inputs to the VAs; in unc-4 mutants, the VA motor neurons receive synaptic input from an interneuron that is normally reserved for VB sister cells. Therefore, we have proposed that unc-4 regulates genes in the VAs that distinguish them from VBs. In an effort to identify these UNC-4 target genes, we are determining the sequence of the UNC-4 DNA binding site. As a member of the paired-like class of HD proteins, UNC-4 is predicted to bind to a palindromic sequence (TAATYNRATTA) as a homodimer or as a heterodimer with other paired-like HDs. In gel shift assays, His-tagged UNC-4 HD binds cooperatively as a homodimer to the predicted palindromic sequence. UNC-4 HD also binds as a heterodimer with ALX4, a mammalian paired-like HD of the aristaless family. Heterodimeric binding presents the interesting possiblity that UNC-4 could regulate transcription in cooperation with other HD proteins. We are currently using the PCR based site selection assay to define an optimal UNC-4 HD DNA binding site. This lab has identified two genes, acr-4 and del-1, that are negatively regulated by unc-4 in the VAs (see Ross et al.). Therefore, acr-4 and del-1 are candidate UNC-4 target genes. Transgenic gfp constructs will be used to identify sites in the regulatory regions of the del-1 and acr-4 genes that respond to UNC-4 regulation. In addition, these regions will be scanned for UNC-4 DNA binding sites and directly tested for UNC-4 HD binding in gel shift assays.

Rudich, P. et al. (2019) International Worm Meeting "PolyQ Independent Toxicity Occurs in C. elegans models of Novel Translational Products from CAG Repeat Expansions."

Rudich, P., Lamitina, S.T.

[International Worm Meeting, 2019]

Expanded CAG nucleotide repeats are the underlying genetic cause of at least 13 incurable diseases, including Huntington's disease (HD). In HD, >37 (CAG) repeats in exon 1 of the Huntington gene (HTT) leads to the development of disease, which includes the death of neurons in several brain regions, such as the striatum and basal ganglia. The major molecular cause for the loss of these neurons is thought to be the translation of the expanded (CAG) repeats into a poly-glutamine (polyGln/polyQ) expanded HTT protein. However, several brain regions that degenerate in HD do not express polyGln aggregates, suggesting that additional polyGln-independent pathways play a significant role in HD. Recent studies discovered HD CAG repeats undergo a novel type of translation called Repeat Associated Non-AUG-dependent (RAN) Translation. RAN translation of the CAG and CUG containing sense and anti-sense RNAs produces five distinct repeat peptides: polyalanine (polyAla), polyserine (polySer), polyleucine (polyLeu), polycysteine (polyCys), and polyglutamine (polyGln). RAN polypeptides are present in HD patients and are particularly prevalent in degenerating regions that lack polyQ, suggesting RAN products are major contributors to HD pathophysiology. We developed C. elegans models for each CAG RAN peptide. To separate RAN protein toxicity from RNA-derived toxicity, we generated 'pure' protein models by synthesizing codon-varied constructs that preserve RAN protein sequence but eliminate repetitive RNA. The RAN CAG peptides were expressed individually in muscle cells or GABAergic neurons. Every RAN peptide formed protein aggregates at 90 repeats, and every RAN peptide except for polyLeu formed aggregates at a disease relevant length of 38 repeats. Surprisingly, (Leu)38 was highly toxic in multiple cell types. In GABAergic neurons, which exhibit significant neurodegeneration in HD patients, codon varied (Leu)38, but not (Gln)38, caused substantial neurodegeneration and motility defects. We performed a forward mutagenesis screen to identify polyLeu toxicity mechanisms and discovered 26 suppressors. Bioinformatic analysis of genome resequencing data from the polyLeu suppressors has identified several candidate genes. RNAi knockdown of some candidates phenocopies the mutant and suppresses polyLeu toxicity. Characterizing the various HD RAN polypeptides and determining the cellular pathways required for their toxicity is vital to fully understanding HD and developing new biomarkers and therapies.

Sebastien Holbert et al. (2001) International C. elegans Meeting "The Gln-Ala repeat transcriptional activator CA150 interacts with huntingtin: neuropathologic and genetic evidence for a role in Huntington's disease pathogenesis"

Robert J Ferrante, Isabelle Denghien, Sebastien Holbert, Jean Dausset, Tamara Kiechle, Christopher A Ross, Christian Neri, Cheryll Wellington, Michael R Hayden, Adam Rosenblatt, Russell L Margolis

[International C. elegans Meeting, 2001]

Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine (polyQ) expansion in the protein huntingtin (htt). Pathogenesis in HD appears to involve the formation of ubiquitinated neuronal intranuclear inclusions containing N-terminal mutated htt, abnormal protein interactions, and the aggregate sequestration of a variety of proteins (noticeably transcription factors). To identify novel htt-interacting proteins in a simple model system, we used a yeast two-hybrid screen with a Caenorhabditis elegans activation domain library (R. Barstead, ORMF, Oklahoma City, OK). We found a predicted WW domain protein (ZK1127.9) that interacts with N-terminal fragments of htt in two-hybrid tests. A human homologue of ZK1127.9 is CA150, a transcriptional coactivator with a N-terminal insertion that contains an unperfect (Gln-Ala)38 tract encoded by a polymorphic repeat DNA. CA150 interacted in vitro with full length htt from lymphoblastoid cells. The immunohistochemical expression of CA150 was markedly increased in human HD brain tissue, in comparison to normal age-matched human brain tissue, and showed aggregate formation with colocalization to ubiquitin-positive aggregates. In 432 HD patients, the CA150 repeat length explains a small but statistically significant amount of the variability in HD onset age. Our data suggest that abnormal expression of CA150, as mediated by interaction with polyQ-expanded htt, may alter transcription, and have a role in HD pathogenesis. Our data illustrate the value of using C. elegans for the direct identifcation of novel biochemical and genetic modifiers of human neurodegenerative disease pathogenesis [Holbert et al. (2001) Proc. Natl. Acad. Sci. USA 98 1811-1816].

Alex Parker et al. (2002) European Worm Meeting "A C. elegans model of neuronal dysfunction in Huntington's Disease: Expanded polyglutamines cause severe neuronal phenotypes without cell death."

Alex Parker, Emmanuel Lambert, Christian Neri

[European Worm Meeting, 2002]

Proteins with expanded polyglutamine repeats cause Huntington's Disease (HD) and several other neurodegenerative disorders. HD is a dominant neurodegenerative disorder caused by polyglutamine expansion in the protein huntingtin. HD pathogenesis appears to involve the production of a mutated amino terminal fragment, cytoplasmic and nuclear aggregates, and abnormal activity of huntingtin interactor proteins essential to neuronal survival. Before cell death, neuronal dysfunction may be an important stage of HD pathogenesis. The number of proteins and genetic pathways implicated in HD is large and is growing. Simple model organisms may provide a convenient setting in which to manipulate and decipher polyglutamine mediated neuronal toxicity. We have used Caenorhabditis elegans to investigate the effects of expressing normal and mutant N-terminal huntingtin fused to fluorescent marker proteins in a portion of the nematode nervous system responsible for touch reception. Mutated huntingtin (128 Glns) produced a loss of touch response at the tail at high penetrance. Similar perinuclear deposits and faint nuclear accumulation of fusion proteins was observed for normal and expanded polyglutamine containing proteins. In contrast, significant deposits and morphological abnormalities in the touch receptor PLM cell axons were observed in animals expressing 128 glutamines. Neuronal cell death was not detected. These animals indicate that significant neuronal dysfunction can occur without cell death and may be induced by expanded polyglutamines and may correlate with axonal insult and not cell body aggregates.

Ryan Keating et al. (2007) International Worm Meeting "Initial analysis of the LIM-homeodomain-encoding gene, lim-7."

Laura G. Vallier, Ryan Keating

[International Worm Meeting, 2007]

To understand the role of LIM-7, a LIM-homeodomain (LIM-HD) protein, in developmental processes, characterization of the lim-7 gene has been initiated. LIM-HD proteins are transcription factors, characterized by containing two tandem Zinc-finger-like LIM domains N-terminal to a homeodomain, that are important for specification and identity of cell types in the developing embryo. LIM-HD transcriptions factors can be grouped into six related families and have been implicated in neuronal development and identity as well as for diverse tissue types, including cardioblasts, pancreatic islet cells and pituitary gland cells. There are seven LIM-HD-encoding genes in C. elegans that describe the six different LIM-HD families. One of them, lim-7, encodes a 452-amino acid protein that is a member of the Islet family of LIM-HD transcription factors. The founding member of the Islet family of LIM-HD proteins, ISL1, was identified by its ability to bind the insulin gene enhancer in pancreatic cells; subsequently Islet family members have been implicated also in cardiac development and motor neuron identity, among others. A deletion mutation, lim-7(tm674), has been generated by the Japanese consortium and results in an in-frame deletion of amino acids 84-162 of LIM-7, which partially eliminates the first LIM-domain and completely eliminates the second LIM-domain; the homeodomain is intact. The lethality phenotype of the deletion is 100% penetrant with the majority of the animals dying in the first larval stage shortly after hatching. The lethality phenotype appears to be tightly linked to the lim-7 deletion after five backcrosses; further backcrosses are underway. The L1 larvae homozygous for tm674 arising from tm674/hT2 mothers appear Unc and have other associated defects. Partial rescue has been achieved using fosmid DNA that contains only lim-7 and its closest downstream gene. Progress on the characterization will be reported. *Contact information: biolgv@hofstra.edu.

Roumen Voutev et al. (2008) Development & Evolution Meeting "Characterization of the C. elegans Islet LIM-homeodomain Ortholog, lim-7"

E. Jane Albert Hubbard, Ryan Keating, Roumen Voutev, Laura Vallier

[Development & Evolution Meeting, 2008]

C. elegans lim-7, is one of seven LIM-homeodomain (LIM-HD) genes in the genome and the sole Islet ortholog. LIM-HD transcription factors are mainly involved in motorneuron development, pathfinding and identity across diverse phyla. Additionally, LIM-HD proteins have developmental roles in other non-neuronally derived tissues. There are six classes of LIM-HD proteins, including the Islet class, and the seven C. elegans LIM-HD are distributed among all the classes. One of the seven LIM-HD genes in C. elegans is lim-7, an Islet ortholog. Here we present an analysis of the mutant phenotype and the full-length reporter expression pattern as well as define three elements in Intron 1: one regulatory region and two that are conserved among three other Caenorhabditis species. A 1345 bp intron-intron deletion allele of lim-7, tm674, resulted in L1 lethality. The terminal phenotypes included uncoordinated movement, detached pharynx, morphological defects, and constipation. Introduction of a multicopy array carrying lim-7(+) into tm674 rescue the L1 lethality but the resulting adult mutant hermaphrodites were sterile. Beyond that observed for the fusion tnIs6 lim-7::GFP, which contain 2.3 kb upstream sequences, the first exon and intron and a small portion of the second exon, we saw an expanded localization pattern for the full-length lim-7::mCherry in its native genomic context: the expanded pattern expresses in the complete set of gonadal sheath cells and in at least ten additional cells in the pharyngeal region, near the URA neurons. Finally, using enhancer bashing, we identified three elements in Intron 1 and demonstrate that one element, a 125-bp region, within Intron 1 is both necessary and sufficient to target GFP to the gonadal sheath.

Christian Neri et al. (2007) International Worm Meeting "Longevity modulators and protection against the cytotoxicity of degenerative disease proteins: of simple models and translational research."

Cendrine Tourette, J. Alex Parker, Salima Abderhamane, Emmanuel Lambert, Sabine Cleophax, Alexandre Heissat, Beate Gerstbrein, Christian Neri

[International Worm Meeting, 2007]

Genes and gene networks at the interface between longevity/survival and neuroprotection are currently the subject of intense research. Recent reports based on the use of simple model systems have emphasized a role for these genes in protection against the effects of proteins and polypeptides associated to neurodegenerative diseases. We will present a short overview of recent data in the field. We will also present our most recent data on the study of longevity modulators in simple models of Huntingtons disease (HD) pathogenesis. HD is a neurodegenerative disease caused by expanded polyglutamines (polyQs) in huntingtin (htt). Although HD is inherited, its age-at-onset shows a significant level of variability not fully explained by expanded htt alleles, suggesting a role for modifier genes. We hypothesized HD modifier genes may belong to longevity pathways that modulate the neuronal cell response to mutant htt. To study how the neuronal cell may respond to mutant htt, we use translational research primarily based on the genetic manipulation of C. elegans transgenics at the neuronal cell level and biological assays in cellular and mouse models of HD. We found sirtuin activation is a neuroprotective mechanism against the effects of mutant htt, an effect mediated by daf-16/FOXO (Parker et al., Nature Genetics 2005), providing the first direct evidence for longevity modulators like Sir2 and FOXO to modify the neurotoxic properties of neurodegenerative disease-associated proteins like mutant htt, and defining a new strategy with therapeutic potential. DAF-16/FOXO has numerous upstream modulators and downstream targets, not all of which are likely to be important to neuronal protection. Using RNAi, we identified a subset of daf-16 targets that modify the neuronal dysfunction induced by mutant polyQs. We are extending this approach to include the microarray analysis of gene expression profiles in cell-sorted polyQ neurons from C. elegans transgenics. Data integration and classification using gene networks emphasize gene classes, pathways and biological processes that are conserved in humans and may be essential to neuronal cell activity and survival in HD, providing new information to best understand the mechanisms underlying the disease pathogenesis and residual age-at-onset. Drug discovery aspects will also be commented.

Rudich, Paige et al. (2017) International Worm Meeting "Novel translational products encoded by disease-associated GC-rich repeat expansions cause toxicity in C. elegans."

Rudich, Paige, Lamitina, Todd, Snoznik, Carley

[International Worm Meeting, 2017]

Expanded GC-rich repeats cause many age-onset neurodegenerative diseases. An emerging mechanism that may underlie disease pathology is an unusual type of protein translation called Repeat Associated non-ATG (RAN) translation, which specifically targets these GC-rich repeats. RAN translation requires extended GC-rich repeats and occurs independent of a canonical start codon, allowing translation in all three reading frames. Antisense RNA from these GC-rich repeats also gives rise to RAN protein products causing up to six distinct protein products from one repeat expansion. Most of these RAN proteins have never been studied in any biological system. Defining the role of RAN proteins in neurodegenerative diseases and their potential mechanisms of toxicity represents an exciting new translational research opportunity. We developed RAN protein models in C. elegans for two GC-rich expansion repeat diseases - C9orf72-associated Amyotrophic Lateral Sclerosis (ALS) and Huntington's Disease (HD). To separate RAN protein toxicity from RNA-derived toxicity, we generated 'pure' protein models by synthesizing codon-varied constructs that preserve RAN protein sequence but eliminate repeat-derived RNA structures. We used canonical ATG-dependent protein translation to bypass RAN translation and a C-terminal GFP or RFP tag to examine the subcellular localization. In our C. elegans ALS model, the arginine-containing dipeptides Pro-Arg (PR) and Gly-Arg (GR) exhibited age-onset toxicity when expressed in multiple cell types, including motor neurons. PR and GR exhibited nuclear localization that was necessary for toxicity. PR and GR did not form protein aggregates. Age-onset toxicity of PR, but not GR, was influenced by mutations that alter the rate of ageing, suggesting differences in the toxicity mechanisms of these related RAN peptides. In our HD model, initial studies suggest that HD RAN proteins exhibit distinct cell biological properties from the canonical polyGln (Q) HD protein. Currently, we are comparing the toxicity of these new HD RAN products to the well-described toxicity of polyQ. Understanding how different RAN peptides contribute to HD and to ALS is vital for developing appropriate treatments for these diseases. Moreover, comparing RAN proteins within a single experimental paradigm may reveal common themes that facilitate RAN protein toxicity and generate a more integrated understanding of the role of RAN-derived proteins in neurodegenerative diseases.

Vallier, Laura et al. (2015) International Worm Meeting "Life and death: discerning the role of the essential gene, lim-7."

Vallier, Laura, Mahabir, Arnold, Ramadin, Vidia

[International Worm Meeting, 2015]

LIM-homeodomain (LIM-HD) transcription factors are proteins that contain two LIM domains that reside N-terminally to a homeodomain. The LIM-HD family of transcription factors is evolutionarily conserved in all multicellular organisms and seven LIM-HD proteins are found in the worm, representing each of the six subfamilies of LIM-HD proteins. The sole C. elegans ortholog of the Islet LIM-HD subfamily is lim-7, which localizes to many neurons, the ten gonadal sheath cells and other as yet unidentified cells. Its use is mainly noted as a gonadal sheath marker but its precise role there and elsewhere is still unknown. A deletion of the LIM domains is a loss of function mutation that is fully recessive and results in 100% lethality in the early L1 larva. Prior to death there is a plethora of phenotypes including a non-penetrant Pun occurring during the late embryonic stage, which we have determined is not the cause of death, since all animals die but not all are Pun. Due to the fact that lim-7 is necessary for late embryo pharynx attachment, for early L1 larval viability, and is expressed in the somatic gonadal sheath, it appears that lim-7 may act at multiple times throughout the life of the worm. To better understand one of the roles of lim-7, we are focusing on the early essential pathway(s) that lim-7 is required in. Using the Ahringer RNAi feeding library, we have been searching for genes that are able eliminate the requirement for lim-7, thus allowing lim-7 homozygotes to live. We have a collection of candidate genes from our screening of Chromosomes I and II that we will present at the meeting along with some initial analysis. We plan to pursue how the candidates and lim-7 act in the mechanisms to promote early larval life in the worm. .