Link, Chris et al. (2015) International Worm Meeting "Role of TDP-1 in RNA metabolism and heterochromatin formation."

Gonzales, Patrick, Dostal, Vishantie, Link, Chris, Saldi, Tassa, Roberts, Christine

[International Worm Meeting, 2015]

TDP-1 is the C. elegans ortholog of mammalian TDP-43, an RNA-binding protein believed to play a central role in Amyotrophic Lateral Sclerosis (ALS). We have previously shown that loss of TDP-1 leads to the accumulation of double-stranded RNA and altered chemotaxis. We have now found that deletion of the tdp-1 gene results in increased sensitivity to exogenous RNA interference (RNAi) in the soma by enhancing transcriptional gene silencing (nuclear RNAi). As transcriptional gene silencing involves heterochromatin formation, we used chromatin immunoprecipitation and deep sequencing (ChIP-seq) to globally assay the repressive H3K9me3 histone modification in the tdp-1 mutant. Unexpectedly, H3K9me3 was reduced at many loci throughout the genome, and this reduction could not be accounted for by increased pools of endogenous silencing RNAs. Co-immunoprecipitation studies revealed that TDP-1 directly interacts with HPL-2, a C. elegans homolog of HP1, an essential heterochromatin component. These studies identify a novel role for TDP-1 in the maintenance of heterochromatin, which may account for phenotypes previously attributed to tdp-1 mutants and molecular changes observed in ALS tissue.

Chris Link et al. (2005) International Worm Meeting "Investigating the cellular and molecular basis of beta amyloid peptide toxicity."

Maria Florez-McClure, Brian Hiester, Chris Link, John E Yerg, Gin Fonte, Wail Hassan, Jmil Ferguson

[International Worm Meeting, 2005]

The beta amyloid peptide (Abeta) is a primary component of senile plaques and is generally believed to be centrally involved in Alzheimer's disease. Although both extracellular and intracellular Abeta has been found to be toxic to cells, the mechanism(s) of this toxicity are still controversial. We have been using transgenic C. elegans strains with inducible expression of Abeta and other toxic proteins to investigate the molecular and cellular basis of Abeta and general protein aggregation toxicity. These studies have involved a combination of microarray analysis, high-throughput RNAi screening, proteomics, reporter and overexpression transgene construction, and electron microscopy. We have identified cellular responses apparently specific to Abeta toxicity, as well "generic" responses to protein aggregation toxicity . A prominent component of the generic response to protein aggregation is the upregulation of chaperone proteins, including small HSPs and HSP70s. Surprisingly, hsp-16.2 induction by Abeta is not primarily controlled by hsf-1, and upregulation of this small HSP appears to enhance Abeta-dependent pathology. Specific toxic effects of Abeta include induction of autophagy, abnormal mitochondria, and oxidative damage to a specific set of proteins. We are currently attempting to order this pathological cascade and determine which of these changes are directly related to Abeta-dependent cellular dysfunction.

Chris Link et al. (2003) International Worm Meeting "Investigating beta amyloid toxicity in a C. elegans Alzheimer's disease model."

Gin Fonte, Noel Rieder, Andrew Taft, Chris Link

[International Worm Meeting, 2003]

We have transgenically engineered C. elegans to express, in body wall muscles and neurons, the human beta amyloid peptide (Abeta) associated with Alzheimer's disease. Induction of Abeta in muscle cells (via a smg-1-dependent expression system developed in the Fire lab) leads to rapid paralysis. Using this Abeta toxicity model, we have previously identified Abeta-interacting proteins by co-immunoprecipitation/mass spectrometry, and characterized Abeta-dependent gene expression by microarray analysis. We are now undertaking a variety of approaches to sort through these identified proteins and genes to determine which are directly related to Abeta toxicity, and to understand how Abeta expression leads to cellular dysfunction. One informative approach has been to examine control transgenic strains that mimic aspects of Abeta-expressing strains. For example, strain XA1440 (kindly provided by Creg Darby) expresses the YpkA bacterial toxin using the same smg-1-dependent expression system as the Abeta-expressing strains, and shows a paralysis phenotype upon transgene induction indistinguishable from that of induced Abeta animals. Using quantitative RT-PCR, we have asked which genes upregulated in Abeta-expressing animals are also up-regulated in induced XA1440, to identify genes that are generally responsive to muscle pathology and thus not specific for Abeta toxicity. Surprisingly, many genes upregulated before the onset of paralysis in Abeta-expressing animals are also induced in XA1440, suggesting that significant muscle pathology occurs in induced Abeta animals before any cellular changes are observable by light microscopy. We have therefore used high pressure freeze electron microscopy to examine induced Abeta animals before paralysis onset. We find that induced Abeta animals have a subset of ultrastructurally abnormal mitochondria 12 hrs after Abeta induction, preceding paralysis by at least 12 hrs. These abnormal mitochondria are only observed in (Abeta-expressing) body wall muscle cells, and not in other tissues or in any control strains, including induced XA1440 animals. This specific ultrastructural defect is particularly intriguing, given the extensive literature associating Alzheimer's disease with deficits in glucose utilization and mitochondrial function.

Chris Link et al. (2007) International Worm Meeting "Using C. elegans for in vivo structure/function analysis of beta amyloid peptide toxicity."

Chris Link, Christine Roberts, David Merin, Gin Fonte

[International Worm Meeting, 2007]

Expression of human beta amyloid peptide (Abeta), causally implicated in Alzheimers disease, in C. elegans body wall muscle results in a robust paralysis phenotype that provides a convenient readout for Abeta toxicity. We have engineered C. elegans to express wild type Abeta 1-42 or single amino acid variants thereof to test models of Abeta toxicity. One such model is the formation of membrane pores via Abeta oligomerization driven by glycine zipper interactions between alpha helical regions of Abeta monomers (Kim S, Jeon TJ, Oberai A, Yang D, Schmidt JJ, Bowie JU. 2005. Proc Natl Acad Sci U S A 102: 14278-83). Transgenic worms expressing Abeta with a Gly37-to-Leu substitution, predicted to disrupt glycine zipper interactions, show a dramatic reduction in paralysis relative to worms expressing wild type Abeta. In addition to this reduction in paralysis, Abeta G37L worms did not show the muscle cell calcium perturbations or abnormal mitochondria observed in worms expressing wt Abeta. Immunoblot and immunohistochemical analysis demonstrates that the Abeta G37L strain has Abeta levels and cellular distribution indistinguishable from control wt Abeta strains, indicating that this reduced toxicity is due to a qualitative difference in the Abeta G37L peptide. Studies with transgenic worms co-expressing wt and G37L Abeta reveal that the Abeta G37L peptide can be anti-toxic, suggesting a dominant negative effect consistent with the variant peptide interfering with toxic oligomerization of wt Abeta. To test the oligomer model more directly, we engineered second site substitutions in the Abeta G37L peptide that based on the proposed structural model could potentially restore the predicted glycine zipper interface. Two of these second site mutations (N27G and M35G) restore toxicity to the G37L variant, strongly supporting the toxic oligomer model. These studies support the oligomer pore mechanism and identify the glycine zipper interface as a potential target for therapeutic drugs.

Tomberlin, Cole et al. (2017) International Worm Meeting "Cold-tolerance is a fast and easy method to identify neuronal dysfunction in C. elegans."

Link, Chris, Tomberlin, Cole, Liachko, Nichole

[International Worm Meeting, 2017]

Wild-type C. elegans raised at cooler temperatures (16 deg C) can survive a sudden shift to a cold temperature (4 deg C). However, animals raised at warmer temperatures (20-25 deg C) die following a similar sudden cold stress. A single pair of neurons, ASJL and ASJR, are required for sensing temperature. Active ASJ neurons initiate a signaling cascade that results in lipid changes in the gut and decreased cold tolerance. In the absence of signaling from these neurons, worms maintain a cold-resistant lipid profile. Mutants that affect neuronal signaling broadly or ASJ signaling specifically are resistant to cold temperatures (Ohta et al., 2014). Testing C. elegans' ability to survive a sudden drop in temperature is a rapid and robust method to detect changes in signaling from the ASJ nerve pair, and may provide a surrogate screen for broader neuronal dysfunction. We have used this assay to test a broad range of transgenic strains and single gene mutants, particularly those with relevance to neurodegenerative disease. Transgenic strains with neuronal expression of neurodegeneration-related proteins (e.g., TDP-43, Abeta) display cold resistance, while equivalent strains with muscle expression of these same proteins do not. This assay has been particularly useful in detecting neuronal dysfunction in strains lacking obvious movement or morphological defects [e.g., tdp-1(ok803), sut-2(bk741), trx-1(ok1449)]. We suggest that cold resistance can be used as a simple method for characterizing new strains or screening for mutants with neuronal dysfunction.

Julien, Carl et al. (2017) International Worm Meeting "Investigating the "toxic pore" model for beta-amyloid peptide toxicity."

Morgenstern, Joshua, Link, Chris, Julien, Carl, Tomberlin, Cole, Roberts, Christine

[International Worm Meeting, 2017]

Many lines of evidence support the view that accumulation of the betaamyloid peptide (Abeta) is central to Alzheimer's disease (AD) pathology, but the relevant molecular mechanisms remain unresolved. Abeta can convincingly make ion-conducting pores in synthetic membranes, but whether this occurs in vivo is not known. We have developed a novel assay for Abeta toxicity, based on feeding worms E. coli engineered to secrete human Abeta. We find that feeding worms E. coli secreting wild type Abeta leads to the induction of intestinal endocytosis previously observed for worms fed E. coli producing the known pore-forming toxin CRY5B. Endosome induction did not occur in parallel experiments with E. coli producing Abeta Gly37Leu, a single residue substitution that blocks pore formation in synthetic membranes. Super resolution microscopy revealed that Abeta-induced endosomes contain membrane-associated Abeta, supporting the view that endosome induction reflects a membrane repair process that removes plasma membrane-associated Abeta pores. To investigate the relevance of this phenomenon to AD, we assayed the role of worm orthologs of human genes associated with increased AD risk by genome-wide association studies ("GWAS genes") in endosome induction. We find that loss of function of either unc-11 (ortholog of human PICALM) or amph-1 (ortholog of human BIN1) significantly increase Abeta-induced endosome induction, as well as sensitize worms to CRY5B toxicity. We are currently assaying the effects of Abeta-responsive genes (identified in our gene expression analysis of Abeta transgenic worms) on endosome induction in this model.

Julien, Carl et al. (2017) International Worm Meeting "Using engineered E. coli to assay membrane damage and repair in C. elegans."

Morgenstern, Joshua, Julien, Carl, Tomberlin, Cole, Roberts, Christine, Link, Chris

[International Worm Meeting, 2017]

Multiple disease-associated proteins (Abeta, alpha-synuclein, amylin) have been proposed to form membrane-damaging oligomers, but this has not been directly observed in vivo. Following the elegant studies of the Aroian group on the toxicity of the CRY5B pore-forming toxin in C. elegans, we have generated E. coli strains that secrete disease-associated proteins, including wild type and variant Abeta. Feeding E. coli expressing wild type Abeta (but not the non-toxic single residue variant Abeta G37L) to worms results in an induction of intestinal endocytosis. This is analogous to the endocytosis induced as a membrane repair process in mammalian cells exposed to streptolysin O, another pore-forming toxin. This endocytosis can be visualized using a fluorescent reporter localized to the intestinal lumenal membrane (vha-6::mCherry) or by uptake of Texas Red-dextran. Importantly, induction of endocytosis can be suppressed by mutations in genes encoding acid sphingomeylinase, a key component of the membrane repair process in mammalian cells. Induction of endocytosis can also be modulated by mutations in worm genes orthologous to human associated with increased Alzheimer's disease risk (e.g., BIN1 and PICALM) or by genes we have previously identified as upregulated in response to transgenic Abeta expression (e.g., clp-4). Currently we are investigating the mechanistic roles of these interacting genes and developing methods to follow the fate of endocytosed toxic proteins.

Crisp A et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Age-related Cholinergic Neurodegeneration by Overexpression of apl-1 in C.elegans"

Pierce-Shimomura J, Crisp A

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Alzheimer's disease (AD) is the most common cause of dementia. It is characterized by selective degeneration of cholinergic neurons involved in memory. Dying neurons are surrounded by dense plaques primarily composed of beta-amyloid peptide (Aβ). Aβ is just one cleavage product from the protein APP. Mutations in APP that affect the processing of Aβ result in early-onset AD; however, a single additional wild-type copy of APP can also lead to AD, as seen in all individuals with Down syndrome. Aβ plaques were originally thought to be the primary cause of neurodegeneration, but new research suggests the role of the APP in AD is more complex than originally appreciated. The labs of Dr. Chris Li and Dr. Chris Link have pioneered the use of C. elegans to study APP function and dysfunction. For instance, overexpression of human Aβ in C. elegans muscle leads to the formation of Aβ aggregates(1). Overexpression of multiple copies of the APP-related gene, apl-1, in C. elegans leads to gross phenotypes, including partial lethality, arrested development, and vacuolization(2). We are testing whether expression of only one additional wild-type copy of apl-1 results in AD-related phenotypes. DIC and fluorescence microscopy were used to evaluate worms for signs of neurodegeneration. We have discovered that overexpression of apl-1 in a single copy results in the age-related degeneration of a specific subset of cholinergic neurons in C. elegans. To quantify the impact of degeneration, we track the deterioration of two natural behaviors (swimming and egg-laying) that rely on these neurons. These worms displayed no obvious defects in early adulthood but began to display defects in egg-laying and swimming by the third day of adulthood ('middle age'). In addition, ablation of these specific neurons in wild-type individuals recapitulates the behavioral defects observed in egg-laying and swimming. These results suggest that the behavioral defects observed in the overexpression strain are primarily due to the selective degeneration of these neurons. The quantifiable link between these behaviors and neurodegeneration allows us to test strategies to recover the function mediated by the degenerated cholinergic neurons. We are now studying the mechanisms by which apl-1 overexpression causes death of cholinergic neurons in the worm and in the process, generate novel hypotheses about the mechanism of Alzheimer's disease. 1. Link CD. (1995) PNAS 92, 9368-9372. 2. Hornsten A. et al. (2007) PNAS 104, 1971-1976.

Cacho-Valadez, Briseida et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "C. elegans mutants lacking the mitochondrial thioredoxin system are viable and enhance the paralysis phenotype of worm models of Alzheimer's Disease"

Munoz-Lobato, Fernando, Link, Chris D, Miranda-Vizuete, Antonio, Cacho-Valadez, Briseida, Swoboda, Peter, Fierro-Gonzalez, Juan Carlos

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

All eukaryotic organisms are equipped with a mitochondrial thioredoxin system made up of dedicated thioredoxin (TRX) and thioredoxin reductase (TRXR), which plays, among other functions, an important role as antioxidant system. In C. elegans the mitochondrial thioredoxin system consists of the ZK637.10/ trxr-2 and B0024.9/ trx -2 genes. By transcriptional and translational GFP fusions we demonstrate that both TRX-2 and TRXR-2 proteins are located in mitochondria of different worm cells and tissues as shown by a general punctuated fluorescence expression pattern. Biochemical analysis of both recombinant proteins has also confirmed that they are enzymatically active at reducing disulfide bonds in substrate proteins. By RT-PCR and western blot analysis, we show that two different trxr-2 alleles ( tm2047) and (ok2267) are null mutants while the trx-2 (tm2720) allele expresses an abnormal mRNA, which theoretically encodes a truncated TRX-2 lacking half of the C-terminus. Interestingly, trx-2 and trxr-2 single mutants are viable, in sharp contrast with the mammalian scenario where KO mice have embryonic lethality. Similarly, double mutants trxr-2

Munoz-Lobato, Fernando et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Protective role of dnj-27, a thioredoxin family member, on C. elegans models of Alzheimer's and Parkinson's Disease."

Caldwell, Guy A, Munoz-Lobato, Fernando, Miranda-Vizuete, Antonio, Caldwell, Kim A, Link, Chris D, Hamamichi, Sushei

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The incidence of aging-associated diseases such as Alzheimers (AD) and Parkinsons (PD) diseases is increasing together with greater life expectancy. It is now well established that cellular mechanisms such as protein aggregation and oxidative stress are behind the initiation and progression of many neurodegenerative diseases (ND). The thioredoxin (TRX) system is one of the most important antioxidant defenses and among their functions are a cytoprotective effect against oxidative stress and chaperone activity. To study whether the TRX system p articipates in the molecular mechanisms underlying ND we used transgenic C. elegans models of AD and PD (that show pathological phenotypes upon overexpression of ?-amyloid (A?) and ?-synuclein (Syn)) to carry out a RNAi screening with all known members of the TRX family in worms. Interestingly, one TRX family member, called dnj-27, appears to have a simultaneous protective effect in both AD and PD models. dnj-27 encodes the orthologue of the mammalian endoplasmic reticulum (ER) resident protein ERdj5, a protein required as a disulfide reductase for degradation of misfolded proteins by the ER-associated degradation (ERAD) pathway. We have found that dnj-27 expression, similarly to other genes involved in ERAD, is induced upon ER stress via IRE-1/XBP-1 pathway, one of the branches of the unfolded protein response (UPR). In addition, although dnj-27(ok2302) mutant worms develop normally and are fertile, they show increased sensitivity to ER stress conditions. Furthermore, our d ata show that A? and Syn overexpression activates the UPR and that this activation leads to increased expression of dnj-27. Together, these results indicate that ER homeostasis is altered in the two worm models of ND and that dnj-27 could be involved in the maintenance of this ER homeostasis. We are currently carrying out further analysis to confirm the protective roles of dnj-27 and decipher the mechanisms involved.