Richard Morimoto et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "The Stress of Misfolded Proteins: A Systems Approach to Heat Shock and Proteostasis in Disease and Aging"

Veena Prahlad, Richard Morimoto, Cindy Voisine, Susana Garcia, Catarina Silva, Ning Wang, Monica Beam, Elise Kikis, Anat Ben-Zvi, Tali Gidalevitz

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

The long-term health of the cell is inextricably linked to proteostasis, a complex network of molecular interactions that maintains the health of proteins. An imbalance in proteostasis, whether caused by environmental or physiological stress, ageing, or the chronic expression of disease-associated proteins (mutant SOD1, huntingtin, polyQ, or Abeta) can be restored by genes that control lifespan (Daf-16) and the heat shock response (Hsf-1). How such folding networks and stress responses are integrated at the molecular and cellular level to the organism has not been examined. The response of C. elegans to heat shock requires the AFD thermosensory neurons and animals harboring mutations in AFD function are defective for induction of the heat shock response in other somatic cells. This AFD requirement is highly selective for activation of heat shock genes by cadmium is unaffected in mutant animals. This reveals that transmission of the heat shock signal is cell non-autonomous and requires active neuronal signaling which serves to integrate temperature-dependent behavioral, metabolic, and stress-specific responses. We draw similar conclusions from a forward genetic screen for enhancers of protein misfolding in body wall muscle cells that identified a mutation in unc-30 that regulates GABAergic signaling. Whether caused by mutations in the GABAergic or cholinergic pathways or by small molecule agonists and antagonists, an imbalance in cholinergic signaling enhances misfolding of polyQ and other metastable proteins in post-synaptic body wall muscle cells.

Morimoto Richard I et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "The Proteostasis Challenge: Regulation of Cellular Stress Responses at the Organismal Level by Neuronal Networks"

Chauve Laetitia, Morimoto Richard I, Silva Catarina, Prahlad Veena

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

The stability of the proteome is an essential aspect of cellular health during development, aging, and exposure to environmental and physiological stress. This is achieved by the Proteostasis Network (PN) and the cytoprotective Heat Shock Response (HSR) and Unfolded Protein Response (UPR) that function in concert to suppress the accumulation of misfolded and damaged proteins and direct the expression of a highly functional proteome. We have observed that aging in C. elegans is associated with a sharp decline in the function and folding of metastable proteins during early adulthood. This is associated with a decline in the HSR and UPR, during the peak of fecundity. Activation of Hsf1 (or DAF-16) in early development restores youthful proteostasis revealing the potential to re-adapt, if not restore a protective state. These studies provide a basis to understand the role of Hsf1 and other stress sensors that function at a cellular and molecular level. At the organismal level, however, the HSR is regulated by the AFD thermosensory neurons that transmit the stress signal via specific neurohormones and neuropeptides to somatic tissues to regulate HS gene expression in a cell non-autonomous manner. Unexpectedly, in the presence of functional thermosensory neurons, muscle and intestinal cells accumulate toxic misfolded proteins, which is insufficient to activate the HSR. However, when the activity of thermosensory neurons is altered, the cellular response to misfolded proteins is restored and protein aggregation and toxicity is suppressed. Neuronal control of proteostasis is also regulated at yet another level by cholinergic signaling at the neuromuscular junction. Overexcitation results in enhanced misfolding in post-synaptic muscle cells, whereas increased levels of the cholinergic receptor results in the activation of the HSR, elevated levels of chaperones, and suppression of proteostatic imbalance. These results, reveal that the HSR is organized at the systems level of the organism to sense the stress signal through active neuronal activity, and together with the metabolic state, sets the proteostasis network to ensure stability of the proteome and the health of the organism.

Heather Brignull et al. (2001) International C. elegans Meeting "NEURONAL SENSITIVITY TO POLYGLUTAMINE-PROTEIN AGGREGATES IN CAENORHABDITIS ELEGANS ."

Richard Morimoto, Heather Brignull

[International C. elegans Meeting, 2001]

A number of human neurodegenerative diseases such as Huntington's, Parkinson's, and ALS, are characterized by protein aggregates and often associated with aging. The biochemical consequence of misfolded or mutant proteins that self-associate to form protein aggregates should, in principle, be independent of cell type. However, the Huntingtin's gene, for example, is expressed widely in the body, yet protein aggregates and cell death are restricted to specific subsets of neuronal cells. The expression of polyglutamine (polyQ or CAG) expansions, as occurs in Huntington's, results in the appearance of size-dependent protein aggregates in yeast, Drosophila, and Caenorhabditis elegans , thus providing alternate model systems for genetic, biochemical, and cell biological investigations. Of these, C. elegans is a particularly interesting model, as subsets of the 302 neurons in the adult hermaphrodite can be differentially targeted to express aggregation-prone proteins. We have already shown that the expression of polyQ-containing proteins in C. elegans body wall muscle cells causes the size-dependent appearance of protein aggregates. The transition to aggregate formation expansions occurs at approximately 40 polyQ repeats. To investigate the sensitivity of neurons to polyQ induced aggregation, we have made constructs containing polyQ-YFP (Q0, 19, 40, and 82) under the control of the pan-neuronal unc-119 promoter for the generation of transgenic animals. Our preliminary data reveals that Q19-YFP expressing animals exhibit a number of deficiencies including an egg laying defect and uncoordinated phenotype. In contrast, animals expressing Q40-YFP yield transgenic animals with low viability while no progeny expressing Q82-YFP were obtained. Future analysis will take advantage of the both forward and reverse genetics to screen for genes involved in the pan-neuronal, polyQ-induced phenotypes. We will also examine the role of neuronal sub-groups such as sensory or dopaminergic neurons, by generating constructs with subset-specific promoters expressing a series of polyQ-YFPs. This will enable us to determine whether neuronal subsets have differential susceptibility to polyQ induced protein aggregation. The use of subset-specific neuronal promoters will also provide a mechanism for investigating whether polyQ-induced phenotypes differ depending on the neuronal sub-group expressing polyQ.

Veena Prahlad et al. (2005) International Worm Meeting "Identification of a bacterial component that induces postembryonic sexual transformation and genomic instability in C. elegans"

Richard Morimoto, Veena Prahlad

[International Worm Meeting, 2005]

Previous work demonstrated a surprising plasticity in the sex determination mechanism of C. elegans: specific bacterial metabolites could cause XX larvae to post-embryonically change sexual fate and develop as males instead of hermaphrodites. This sexual transformation was accompanied by chromosomal instability and an apparent loss of an X chromosome. In these initial experiments conducted using crude bacterial metabolites, sexual transformation occurred only in XX cross-progeny larvae generated by mating males with hermaphrodites; XX self-progeny did not show a significant degree of sexual transformation. We have subsequently identified a component of bacterial metabolite preparations that appears responsible for the sexual transformation and apparent X chromosome loss. This component is bacterial DNA. Purified Op50 DNA not only induces the sexual transformation of XX cross-progeny, but can also cause 3.6% XX self-progeny larvae to post-embryonically change sexual fate and develop as males. Plasmid DNA, and synthetic single stranded oligonucleotides can also induce both XX cross- and self-progeny to undergo sexual transformation. This ability of DNA to induce sexual transformation appears to be, to some extent, sequence specific. Using synthetic oligonucleotides, we show that the presence of specific CpG motifs within the DNA, repetitive sequences and the oligonucleotide backbone all affect the efficiency of sexual transformation: certain sequences cause as many as 6-10% XX self-progeny larvae to undergo sexual transformation, while others are not effective. These findings are reminiscent of the ability of exogenous DNA to induce an immune response in mammals, and numerous other organisms. The mammalian immune response to specific DNA sequences is presumably a defense mechanism against bacteria and other pathogens. We are currently exploring the extent to which the sexual transformation of C. elegans larvae in response to specific exogenous DNA sequences also reflects the innate immune response of C.elegans to bacterial pathogens. Thus, we are in midst of screening mutants in candidate genes to identify the pathways involved in this process, and using RT-PCR to identify whether specific antibacterial genes known to upregulated during theC. elegans immune response are also specifically upregulated upon exposure to exogenous DNA. The observed DNA-induced generation of males is intriguing. Our studies may provide direct evidence for the Red-Queen Hypothesis that suggests that sexual reproduction in nature is advantageous due to its ability to combat the invasion of pathogens.

Janine Kirstein et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Interrelation Between Compartment Specific Aggregation And Protein Trafficking"

Richard Morimoto, Janine Kirstein

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Neurodegenerative diseases such as polyglutamine repeat disease, Lewy bodies in Parkinson disease and amyloid fibrils in Alzheimer''s disease share the accumulation and inclusion of protein aggregates as pathologic feature. The aberrant accumulation of aggregated proteins affects vital cellular processes. The proposed underlying mechanisms of the cellular toxicity range from specific protein-protein interactions to the sequestration of essential proteins by aggregation prone proteins such as polyQ. PolyQ expansions disrupt the global balance of the protein quality control (PQC) system in that the aggregation of a single protein species is sufficient to affect the folding state of metastable proteins (Gidalevitz et al., 2006).However, a previous study demonstrated that also the cellular environment plays a critical role for the aggregation. PolyQ aggregation is abolished when its expression is targeted to the ER or the mitochondrion (Rousseau et al., 2004).The restriction of polyQ aggregation to the cytoplasm and nucleus supports the hypothesis that the deleterious effects could be due to compartment specific components interfering with the aggregation of cytotoxic proteins. Therefore, the overall question I want to address is whether polyQ aggregation in the cytoplasm affects the functionality of proteins localized in the organelles.

Susana Garcia et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "The GABAergic Nervous System Negatively Regulates Protein Misfolding In Muscle Cells of C. elegans"

Susana Garcia, Margarida Amaral, Richard Morimoto

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

The appearance of misfolded protein species and aggregates is associated with pathogenesis in the CAG-repeat diseases and represents a common characteristic of many neurodegenerative diseases. To identify modifiers of polyglutamine aggregation, we employed a forward genetic screen of Caenorhabditis elegans expressing expanded polyglutamine proteins in body wall muscle cells. This screen identified unc-30, a neuron-specific transcription factor, that regulates the synthesis of g-aminobutyric acid (GABA). Animals defective in unc-30 or downstream components in GABA synthesis and signaling exhibited enhanced polyglutamine aggregation in body wall muscle cells. Moreover, stimulation with acetylcholine agonists or GABA antagonists, including nicotine or the organochlorine pesticide lindane, also enhanced polyglutamine aggregation. Our data indicates that the imbalance in muscle cell protein homeostasis is due to cholinergic overstimulation and reveals that dysregulation of neurotransmitters, whether by physiologic means or by environmental toxins, influences directly on protein folding quality control in post-synaptic cells.

Andreia Teixeira-Castro et al. (2007) International Worm Meeting "In vivo dynamics of ataxin-3 aggregation in C. elegans neurons."

Patricia Maciel, Richard Morimoto, Andreia Teixeira-Castro

[International Worm Meeting, 2007]

At least nine human neurodegenerative diseases are caused by the expansion of CAG repeats within otherwise unrelated genes. In these diseases, including Machado-Joseph disease (MJD), polyglutamine (polyQ) expansions cause the appearance of misfolded protein species, aggregates, neuronal dysfunction and cell death. Along with the pathogenic motif, all these diseases have in common the fact that the associated gene products are widely expressed but affect only specific subsets of neurons. This specificity suggests that protein misfolding and its toxic outcomes may be determined by the amino acid sequence of the particular protein. Ataxin-3 (AT3) is a polyQ protein and expansion of its repetitive glutamine tract causes MJD. MJD, like other polyQ diseases, is characterized by the formation of intraneuronal inclusions but the mechanism underlying their formation is poorly understood. Caenorhabditis elegans offers unique advantages for examining the aggregation behavior and toxic effects of polyQ proteins on individual neurons, since the transparency of all 959 cells allows easy detection of fluorescent proteins in live animals. Here, we used high-end imaging techniques, such as Fluorescence Recovery after photobleaching (FRAP) and Fluorescence Resonance Energy Transfer (FRET), to analyze the biophysical properties of YFP-tagged AT3, in live C. elegans neurons. In our novel pan-neuronal C. elegans model of AT3 aggregation, we show that expression of human pathogenic full-length AT3 alone did not cause aggregation, assessed by FRAP, but was dependent on the presence of an aggregated seed capable of initiating the nucleation events. FRAP analysis showed that when full-length AT3 is sequestered into aggregated polyQ-alone proteins, it acquires properties of immobile, aggregated protein. FRET results suggested that AT3 does not orderly interact with polyQ-only protein within these co-aggregates. Moreover, the study of the dynamics of the sequestration process of pathogenic and non-pathogenic wild-type AT3 showed that this process may occur in an ageing-dependent manner.

Andreia Teixeira-Castro et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "In vivo dynamics of human ataxin-3 aggregation and neurotoxicity in C. elegans neuronal cells is modulated by Q-length and age"

Patricia Maciel, Richard Morimoto, Andreia Teixeira-Castro

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Machado-Joseph disease, like other polyglutamine (polyQ) diseases, is a late onset neurological disorder characterized by the appearance of misfolded protein species, aggregates, neuronal dysfunction and cell death. Although the mechanism(s) underlying the formation of ataxin-3 (AT3) neuronal inclusions are poorly understood, it is becoming increasingly evident that proteolysis of full-length AT3 is a biological relevant event in the disease since it occurs and affects aggregation both in vitro and in vivo. In this study, we developed a new model for AT3 pathogenesis in Caenorhabditis elegans, in which we observed that expression of the full-length human pathogenic AT3 alone did not cause aggregation in live neuronal cells. In contrast, expression of a C-terminal fragment of mutant AT3 resulted in protein aggregation, suggesting that the aggregation-prone fragment was behaving as seed capable of initiating the nucleation events. Moreover, we studied the dynamics of the sequestration process of full-length pathogenic and wild-type AT3 into polyQ aggregates and observed that this process occurs in an age-dependent manner and that there is a tight correlation between aggregation and neuronal toxicity onset. We are currently using this model to address the molecular mechanisms of the ageing-dependence of the aggregation and neurological phenotypes, which could provide clues to the late onset of the human disease.

Andreia Teixeira-Castro et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Pathological ataxin-3 misfolding in C. elegans neurons"

Andreia Teixeira-Castro, Richard Morimoto, Patricia Maciel

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Expansion of polyglutamine (polyQ) tracts has been identified as the basis of at least nine neurodegenerative diseases, including Machado-Joseph disease (MJD). MJD is a hereditary ataxia of adult onset caused by expansion of a polyQ tract in ataxin-3 (AT3). AT3 is widely expressed and consists of an N-terminal globular domain with significant helical content, which spans the Josephin domain (JD), and a flexible C-terminal tail containing up to three Ubiquitin interacting motifs (UIM) and the polyQ tract. AT3-induced neurodegeneration affects a specific subset of neurons and is characterized by the presence of AT3- containing protein aggregates. Mutant AT3 forms mainly intranuclear inclusions in diseased human brain as well as in cell culture. Studies suggest that the pathological form of AT3 undergoes a conformational change leading to an alteration in protein homeostasis, misfolding and toxicity. To identify the factors involved in cell-specific pathogenesis observed for MJD, we generated pan-neural Caenorhabditis elegans models expressing chimeric fusion proteins of AT3, with normal and expanded polyQ lengths, tagged on the C-terminus with YFP. We are currently performing the behavioral analysis and looking at the aggregation properties of these models with particular emphasis on polyQ length-dependent aggregation and neurotoxicity. Once we have characterized our model, we will search for genetic modulators of AT3 pathogenesis thus revealing a subset of regulating genes uniquely relevant for mutant AT3 misfolding and toxicity in a metazoan. The comparison to the existing C. elegans polyQ models will contribute significantly in identifying the importance of protein context in cell-specific pathogenesis, providing a better understanding of the disease mechanisms.

Maria Catarina Silva et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Genetic Modifiers of Protein Aggregation in C. elegans"

Maria Catarina Silva, Sue Fox, Richard Morimoto

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Protein misfolding and aggregation are common characteristics of several human neurodegenerative diseases that include polyglutamine expansion disorders and Familial Amyotrophic Lateral Sclerosis. In order to identify the cellular mechanisms underlying protein sequestration into aggregates and its consequences, Caenorhabditis elegans models have been established by our lab. These express fluorescently-tagged proteins associated to each disease: PolyQ and mutant SOD1. We wished to determine whether these conformation disorders share common aggregation pathways and cellular dysfunction, or whether these are protein-specific. To address this, we performed a genome-wide RNA interference screen for the generated models, using a high throughput automated system, to identify enhancer genes of protein aggregation. Down-regulation of these genes causes a delay on the onset or reduces the number of aggregates. The polyQ model screen was performed using the threshold Q-length for aggregation, Q35, and the candidate genes were counter screened with appropriate controls (Q0, Q24 and Q40). 162 modifiers were identified, and the main classes of enhancers of aggregation include genes involved in metabolism, mitochondrial function, protein synthesis, signaling, cell cytoskeleton and vesicular trafficking. To examine whether the polyQ modifiers are common to other proteins, an RNAi screen using the SOD1 model has been initiated. Preliminary results show some overlap with the polyQ modifiers. We will use both FRAP and western blotting to examine the biochemical and biophysical properties of the YFP-tagged proteins in the RNAi-suppressor background. Furthermore, study of the modifier gene functions and pathways involved, together with elucidation of the upstream and downstream events of aggregation will reveal the mechanisms involved in general or protein-specific cellular response to aggregating proteins.