Radice AD et al. (1989) International C. elegans Meeting "expression of the transposable element Tc1."

Sherif NE, Emmons SW, Radice AD

[International C. elegans Meeting, 1989]

Radice AD et al. (1996) East Coast Worm Meeting "Glutamate transporter subtypes in C. elegans"

Radice AD, Visser JW

[East Coast Worm Meeting, 1996]

The function of the excitatory neurotransmitter L-glutamate is not well defined in invertebrates. High-affinity glutamate transport by transporter molecules is essential in modulation of synaptic transmission and regulation of extracellular glutamate concentrations. Our goal is to characterize the glutamate transporter subtypes and identify the glutamatergic cells and neurons in C. elegans. Results of screening genomic and cDNA libraries and database searching indicates there may be five subtypes of glutamate transporters in C. elegans. The five transporter subtypes map to three different chromosomes (LG II, IV and X) and are 57-34% identical at the protein level to the vertebrate glutamate transporters. Previously, we have identified and cloned the glutamate transporter glt-1 (LG X). The glt-1 mRNA is alternately spliced resulting in two mRNA species encoding proteins differing by only 11 amino acids at the amino terminus. The expression pattern of glt-1 was examined in transgenic worms that express GFP under control of the upstream glt-1 sequences. GFP was expressed in the M3 pharyngeal neuron, eight cells in the male tail and six yet unidentified cells. Surprisingly GFP was also expressed in the anterior hypodermal cells hyp1-6. We intend to confirm the GFP localizations using indirect immunofluorescence with affinity purified antibodies raised against recombinant GLT-1 protein. Our aim is to isolate the full length cDNAs from each subtype in order to study functional expression by transient expression in COS cells.

Radice AD et al. (1991) International C. elegans Meeting "THE STRUCTURE OF Tcl TRANSCRIPTS AND EXTRACHROMASOMAL Tcl MOLECULES"

Emmons SW, Radice AD

[International C. elegans Meeting, 1991]

In order to understand the rnechanism and reguladon of Tcl transposition, we are studying the structure of extrachromosomal Tcl circles and Tcl transcripts. In previous work Tcl extracluornsomal circular and linear rnolecules have been detected by Southern hybridization in DNA of the Bergerac strain We have detennined the seguence at the novel junction in circular rnolecules by PCR arnplification of undigested genomic DNA and gel purified Tcl circles. The structures of the joined ends of twenty two sequenced clones fan into the following categories: l) eight clones have junctions with two intact transposon ends separated by 1 to 6 TA dinucleotides, 2) nine clones have one intact end and one deleted end, separated by zero to three TA dinucleotides, and 3) five clones have deletions within both inverted repeats and either zero or one TA dinucleotide. A Tcl-specific probe hybridizes to a large nurnber of transcripts in a Northem hybridizadon experiment. In order to determine whether specific initiation and termination sites rnight define a unique Tcl transcript, we have sequenced Tcl cDNA's and PCR amplified first strand cDNA products (RACE protocol). A single 3' terrnination site within Tcl has been defined by three RACE products and two cDNA clones, each of which ends at position 1509. Multiple S' initiation sites are defined by five RACE products, which end at 137, 147, 382, 382 and 394 respectively. The two longest transcripts contain an additional T at position 361 when compared to the sequence of Rosenzweig and Hirsh. This extra T would bring two ATG sequences in frame if a potential intron (404468) were spliced out. Sequencing two cloned S' RACE products indicates this intron was not spliced out of these transcripts. RNase protection experiments are underway to verify that these S' ends are present in vivo, and do not result from prernature termination of reverse transcriptase during the RACE experiments. RNase protection experiments can be quantified to deterniine whether the frequency of initiation events at these start sites correlates with the level of Tcl transposition activity in several strains.

Radice AD et al. (1995) International C. elegans Meeting "MOLECULAR ANALYSIS OF THE C. ELEGANS GLUTAMATE TRANSPORTER"

Radice AD, Lustigman S

[International C. elegans Meeting, 1995]

We are investigating whether L-glutamate, the major excitatory transmitter in vertebrates, is utilized by neurons that regulate development and behavior in nematodes. In addition, we are interested in the processes regulating signalling at excitatory synapses. Initially, we identified and cloned the glutamate transporter, which is essential for removing glutamate from the synaptic clef. This transporter will be used as a marker to identify neurons that synaptically release glutamate and aspartate. The full length cDNA, CeGlu-1, is 1804 bp and contains an ORF of 1476 bp which encodes a polypeptide of 492 amino acids. The CeGlu-1 ORF is 57-50% identical to the vertebrate glutamate transporters. Southern blot analysis indicates that CeGlu-1 is a single copy gene. The cDNA hybridizes to the overlapping YACs Y43A10, Y43D5 and Y60B6 and maps to the left arm of the X chromosome close to vit-3. The cosmids R01F10, C12D12, M02D2, and B0015 were used to identify a 7 kb genomic fragment containing the CeGlu-1 gene. Flanking DNA upstream of the CeGlu-1 sequence will be used to construct GFP-reporter plasmids to analyze the spatial and temporal expression. To determine which cells express the native CeGlu-1 protein, we have raised antibodies against three distinct regions of the recombinant protein. Affinity purified antibodies recognized a 63 kDa protein on Western blots containing C. elegans extracts.

Radice AD et al. (1993) International C. elegans Meeting "Ancient origin of the Tc1 transposon family."

Fitch DHA, Radice AD, Emmons SW, Bugaj B

[International C. elegans Meeting, 1993]

Radice AD et al. (1993) International C. elegans Meeting "Cloning and characterization of neurotransporters in Caenorhabditis elegans and Onchocerca volvulus."

Radice AD, Lustigman S

[International C. elegans Meeting, 1993]

Koeppen M et al. (1996) Midwest Worm Meeting "PROGRESS TOWARDS THE CONSTRUCTION OF A GFP-MH27 ANTIGEN FUSION PROTEIN"

Radice AD, Koeppen M, Hardin JD

[Midwest Worm Meeting, 1996]

The antibody MH27 recognizes a component of adherens junctions in epithelial cells of C. elegans. It is extensively used to visualize cell boundaries in embryos and is a powerful tool in the study of the development of the worm. We are working on the construction of a functional fusion protein between Green Fluorescent Protein (GFP) and the antigen recognized by MH27. A putative cDNA clone (mh-1) was isolated in collaboration with A. Radice (New York Blood Center) by probing a lgt11 expression library with MH27 antibodies. One positive plaque was isolated, cloned into pBluescript, and sequenced. The sequence was compared with sequences generated by the St. Louis genome sequencing project and with cDNAs sequenced by Yuji Kohara and colleagues in Japan. mh-1 corresponds to genomic sequences within the cosmid C25A11, and to two Kohara cDNAs (yk28c11 and yk44h10). Sequencing of yk28c11 (about 3kb; almost identical to yk44h10) is almost completed. A large open reading frame derived from the sequence shows no significant homology to any protein available in databases. The following evidence suggests that this clone encodes the antigen recognized by the antibody MH27: 1) C25A11 is covered by the deficiency stDf5. Homozygous deficiency embryos do not stain with MH27 (Joel Rothman, personal communication). 2) Embryos injected with mh-1 anti-sense RNA arrest at the 2-3 fold stage. Immunostained anti-sense injected embryos possess either patches of MH27 staining or no staining. Using a subclone from C25A11, we are currently preparing to make GFP fusion constructs. Ultimately, we hope such constructs will prove useful for studying epithelial morphogenesis in C. elegans.

Sae-Lee, W. et al. (2017) International Worm Meeting "Synergistic patterned neurodegeneration caused by APP and APOE4 in C. elegans model of Alzheimer's disease."

Sae-Lee, W., Encanacion, A.J., Satarasinghe, P., Scott, L.S., Pierce, J.T.

[International Worm Meeting, 2017]

Alzheimer's disease (AD) is a progressive neurodegenerative disease that cannot be prevented, cured, or slowed down. Genetic and epidemiological studies have linked several genes to AD, notably the amyloid precursor protein (APP) and the e4 allele of apoliopoprotein E (APOE4). APOE4 confers the highest risk for developing AD compared to the other isoforms (APOE3 and APOE2) contributing to a 91% life-time chance of developing AD for individuals homozygous for APOE4. Moreover, APOE4 represents the most common genetic risk factor for AD; over 40% of current AD patients carry an APOE4 allele. At present, how APP and APOE4 functionally interact at the cellular and molecular level to trigger neurodegeneration in AD remains elusive. Progress has been slow using common mouse models of AD that do not display neurodegeneration. Here, we show that C. elegans can model important aspects of AD including age-related, patterned neurodegeneration that is modified by APOE. In our worm model of AD, we found that a certain subset of neurons is vulnerable to death, while other neurons survive. This is reminiscent of localized degeneration observed in the hippocampus and entorhinal cortex of brains in AD patients. Furthermore, in our worm model of AD we found that APOE4, but not its neutral risk isoform APOE3, acts synergistically with APP to hasten and expand the pattern of neurodegeneration. By combining genetic, molecular, and cell-specific proteomic approaches, we aim to uncover how these molecules synergize to kill some neurons while leaving others unaffected.

Teo, E. et al. (2015) International Worm Meeting "Characterization of a novel C. elegans Alzheimer's model with constitutive neuronal expression of Abeta1-42."

Ng, L., Gruber, J., Fong, S., Halliwell, B., Chen, C., Tsoi, S., Lakshmanan, L., Teo, E.

[International Worm Meeting, 2015]

Alzheimer's Disease (AD) is a progressive neurological disorder characterized by the deposition of amyloid beta (Abeta) in the brain, predominantly the Abeta1-42 form. While numerous transgenic C. elegans model have been generated to study AD, there is no well characterized model that has constitutive expression of Abeta1-42 in neurons. We therefore generated a novel C. elegans strain that constitutively expresses Abeta1-42 in all neurons and used it to study AD progression. Similar to the age-related onset of sporadic AD in humans, behavioral deficits including executive dysfunction and impaired learning ability were only observed in middle-aged but not in young Abeta worms. Mechanistically, Abeta worms showed altered mitochondrial metabolism, lower maximal and spare respiratory capacity at old age, but surprisingly no significant oxidative stress was observed. Abeta worms also had a decreased health- and lifespan. This novel C. elegans AD model recapitulates the progression of sporadic AD in humans and can be used for identifying potential interventions. .

Koeppen M et al. (1997) International C. elegans Meeting "ANALYSIS OF THE GENE ENCODING THE ANTIGEN RECOGNIZED BY THE MH27 ANTIBODY"

Moran AN, Radice AD, Koeppen M, Simske JS, Hardin JD

[International C. elegans Meeting, 1997]

The antibody MH27 is extensively used for the staining of epithelial junctions in developmental studies of C.elegans. The antigen recognized by the antibody localizes to adherens junctions as shown by immunogold staining (David Hall, pers. comm.). The gene encoding the antigen for the MH27 antibody (provisionally called mh27) maps to the center of the X-chromosome. A putative cDNA clone (mh-1) had been isolated in collaboration with A. Radice (New York Blood Center) by probing a lgt11 expression library with the MH27 antibody. Its sequence corresponds to genomic sequence within the cosmid C25A11 and to the Kohara cDNA yk28c11. Sequencing of yk28c11 revealed a large open reading frame (about 1.4 kb) that shows structural homology to the vertebrate proteins trichohyalin and caldesmon. C25A11 is covered by the deficiency stDf5 and homozygous deficiency embryos do not stain with MH27 (Terns et al., 1997. Genetics, in press). Embryos injected with mh-1 anti-sense RNA have been shown to arrest at the 2-3 fold stage and showed either just patches of MH27 staining or no staining; occasionally there is staining of the pharynx. We are currently generating and testing mh27-GFP fusion constructs which could prove very helpful for studying epithelial morphogenesis. In order to further characterize mh27 we are attempting to isolate a mutation in the gene using a pre-complementation screen: We will look for embryonic lethality in the absence of an array containing C25A11. Also, we are preparing to perform Northern analysis of the mh27 transcript and to clone the complete transcript using RT-PCR. Acknowledgments: We would like to thank Ahna Skop for assistance with initial antisense injection experiments.