Kim A. Caldwell et al. (2007) International Worm Meeting "A translational strategy for therapeutic intervention of human movement disorders via chemical screening in C. elegans."

Yuqing Li, Kim A. Caldwell, Fumiaki Yokoi, Guy A. Caldwell, Jeffrey W. Hewett, Jun Lu, Songsong Cao, Xandra O. Breakefield

[International Worm Meeting, 2007]

Human movement disorders represent a significant societal burden. Early onset torsion dystonia (EOTD) is one such disorder characterized by sustained muscle contractions that frequently cause repetitive movements or abnormal postures. EOTD is caused by a dominant mutation in the DYT1 gene where a single glutamic acid residue of the affected protein, torsinA, is deleted in frame (<font face=symbol>D</font>E). Therapeutic options for EOTD are currently limited and involve either surgery or chemodenervation via injection of botulinum toxin. TorsinA is a member of the large AAA+ family of proteins encoding functionally diverse ATPases and molecular chaperones. We have previously established in vivo torsinA activity assays in C. elegans that reveal a putative chaperone-like activity of torsinA in suppressing protein misfolding resulting from polyglutamine-induced protein aggregation. Additionally, we have shown that torsinA prevents the degeneration of dopamine neurons resulting from <font face=symbol>a</font>-synuclein overexpression, an established cause of Parkinsons disease. In either of these experimental assays, wildtype and mutant torsinA display differential functionality, where wildtype torsinA has high activity while the activity of mutant torsinA (<font face=symbol>D</font>E) is significantly reduced. Furthermore, when expressed together, wild type torsinA and torsinA (<font face=symbol>D</font>E) exhibit reduced activity near mutant levels, thereby reflecting the dominant negative nature associated with EOTD. Here we report the use of these assays to evaluate 240 off patent and FDA-approved drugs for the potential to modulate the activity of wild type and mutant torsinA (<font face=symbol>D</font>E) in vivo. This nematode screen identified two classes of drugs that exert their effects through enhancing the activity of wildtype torsinA, specifically. Representative molecules from these two classes were assayed in EOTD patient fibroblasts for improvements in torsinA-dependent secretory function, where one drug significantly improved this readout. Importantly, a behavioral defect associated with a mouse knock-in model for EOTD was also rescued following administration of this drug. This FDA-approved molecule may be of therapeutic benefit for EOTD, a disease for which a new drug candidate has not been discovered in more than thirty years. A clinical trial for repurposing of this drug is currently in the planning stages. These results demonstrate the utility of chemical effector screening in C. elegans for the identification of novel, potentially therapeutic, applications for drugs within the context of biological assays that recapitulate mechanisms involved in disease processes.

Kim A Caldwell et al. (2001) International C. elegans Meeting "C. elegans NUD-1 and LIS-1 are Neuronally Expressed and are Required for Embryonic Development and Nuclear Positioning"

Kim A Caldwell, Guy A Caldwell, Shelli N Williams, Jenny M Whitworth

[International C. elegans Meeting, 2001]

Accurate nuclear positioning is necessary for correct segregation of genetic material in dividing cells and is critical to the vertebrate and invertebrate embryonic development. We discovered the presence of genes related to this mechanism within the genome of the nematode Caenorhabditis elegans. Our work has focused on the C. elegans orthologs of two genes, lis-1 and nud-1 , originally identified as being required for nuclear migration in the multinucleated hyphae of the filamentous fungus, Aspergillus nidulans . We have shown cross-species conservation of function for one of these genes and both are highly expressed in early embryos and neurons, in addition to other tissues. C. elegans LIS-1 protein is 68% identical to human LIS-1, a gene product linked to a severe defect in neuronal migration termed lissencephaly. We used RNA-mediated interference (RNAi) to generate mutant phenocopies of the lis-1 and nud-1 genes. These data indicate that both these genes are essential for a variety of developmental processes including proper embryonic cell division, gonad morphogenesis, and neuronal function. Using time-lapse video microscopy, we have determined that depletion of C. elegans LIS-1 and NUD-1 results in embryonic lethality and phenotypically mimics results obtained by blocking dynein and dynactin activity, respectively. Specifically, these genes exhibit defective pronuclear migration and rotation abnormalities. We are in the process of cloning candidate mutations corresponding to these genes and further defining their function cytologically. These results serve as a foundation for future investigation into the cytoskeletal mechanisms governing nuclear positioning, cell division, and neuronal migration.

Willicott, Corey et al. (2021) International Worm Meeting "The mitochondrial unfolded protein stress response is impacted by alpha-synuclein"

Thies, Jennifer, Caldwell, Guy, Willicott, Corey, Caldwell, Kim

[International Worm Meeting, 2021]

PD pathogenesis is associated with misfolding of a-synuclein (a-syn) and mitochondrial dysfunction. Notably, a-syn was shown to interact with, and disrupt, TOMM20, a mitochondrial outer membrane protein. This interaction causes a mitochondrial protein import block, reduced respiration and enhanced generation of reactive oxygen species in cell culture and mammalian brain studies. Our lab extended these findings by reporting that UPRmt stress was increased following overexpression of a-syn variants within the intestine. This was examined via ATFS-1-dependent transcriptional upregulation with the established reporter for the UPRmt, Phsp-6::GFP. These a-syn variants were wild-type human a-syn (WT), two disease variants (A53T and A30P), and a deletion disrupting an N-terminal mitochondrial targeting sequence (delta1-32). The disease variants and the N-terminal mutant induced significantly higher UPRmt activity during both larval development and in adults whereas WT a-syn only induced the UPRmt more than controls in larval development. To determine if UPRmt regulation occurs differently for various familial a-syn variants, the transgenic strains were subjected to RNAi of clpp-1, haf-1, and lonp-1 and then re-analyzed for UPRmt stress. Following knockdown of lonp-1, all the transgenic worm strains displayed significantly enhanced UPRmt activity compared to their EV RNAi controls. Additionally, all variants, except the delta1-32 line displayed enhanced UPRmt stress following knockdown of haf-1. One striking difference in these data, however, was that WT and A53T a-syn-expressing worm lines displayed increases in UPRmt stress between larval and adult stages, while the other two lines did not display increases in UPRmt stress over time. It is interesting to note that the protein pool for at least a subset of WT and A53T a-syn is likely localized to mitochondria, based on previously published work, while A30P and delta1-32 a-syn will remain in the aqueous cytoplasm. These studies were extended to neurons, where knockdown of clpp-1, haf-1, and lonp-1 was performed exclusively in dopaminergic neurons of animals overexpressing WT a-syn. Enhanced neurodegeneration was observed after depletion of clpp-1 and haf-1, whereas knockdown of lonp-1 was similar to EV control. In total, these data suggest that WT and A53T a-syn enter mitochondria where CLPP-1 mitochondrial protease and HAF-1 matrix peptide exporter are involved in their degradation and removal.

Yan, Xiaohui et al. (2011) International Worm Meeting "Identification of genetic modifiers for amyloid-beta toxicity in a C. elegans Alzheimer's disease model."

Yan, Xiaohui, Caldwell, Guy, Knight, Adam, Caldwell, Kim

[International Worm Meeting, 2011]

It is well known that insulin signaling pathways regulating longevity and healthspan are conserved from invertebrates to mammals. In humans, several age-related diseases are associated with protein misfolding or aggregation, including neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD). Others and we have shown that reduced insulin signaling attenuates the protein misfolding or cytotoxicity associated with poly-glutamine (polyQ) aggregation in HD, amyloid b (Ab) aggregation in AD, and a-synuclein aggregation in PD C. elegans models. Based on the similarity of the effect of insulin signaling on the modulation of misfolded protein toxicity, we tested the hypothesis that a subset of the aging-associated genetic modifiers of PD-related phenotypes identified in our lab will also work for AD. By using a C. elegans AD model in which temperature-inducible muscle expression of human Ab42 transgene leads to a reproducible paralysis phenotype upon temperature upshift, we identified seven candidate genes that accelerate Ab-induced paralysis upon RNAi knockdown. These modifiers include components of ER associated protein degradation (ERAD) and select metabolic factors, including two genes encoding independent but functionally related enzymes, the serine hydroxymethyltransferase (SHMT) and the aminomethyltransferase of the glycine cleavage system (GCS). The reactions catalyzed by SHMT and GCS generate one-carbon units for folate one-carbon metabolism whose normal function supports a variety of cellular activities. This study provides the first evidence of a role for SHMT and GCS in modulating a-synuclein and Ab related cellular toxicity. Interestingly, prior reports have shown that abnormal homocysteine level could affect epigenetic modulation of AD gene activity and that folate deficiency may also influence Ab-induced neurotoxicity. Taken together, these findings expand our mechanistic understanding of the well established, but poorly understood, interface between metabolism and age-associated neurodegenerative diseases.

Gaeta, Anthony et al. (2021) International Worm Meeting "Dietary Composition Modulates Neurodegeneration in a C. elegans Parkinson's Disease Model"

Caldwell, Kim, Caldwell, Guy, Keogh, Candice, McKay, Luke, Gaeta, Anthony

[International Worm Meeting, 2021]

The diet of any organism is crucial in providing the energy necessary to not only exist and persist in the environment, but to overcome stress when challenged. One such type of stress comes in the form of Parkinson's disease (PD), which is characterized by the progressive loss of dopaminergic (DA) neuron function and integrity. Although it has been reported and observed that diet type has wide ranging effects on phenotypes in both humans and animal models, the effect that diet has on the progression of DA neuron loss in PD is largely unknown. However, evidence supporting a connection between the brain and the gut microbiome has become increasingly relevant to neurodegenerative diseases. Furthermore, the effect of parental diet on attributes of PD in subsequent generations has not been adequately investigated. The findings of this study indicate that a non-standard diet for Caenorhabditis elegans (C. elegans) has an impact on PD-associated pathology in this animal, in a transgenerational manner. Specifically, feeding worms an alternative bacterial diet of HB101 E. coli, as opposed to the laboratory standard OP50 E. coli, promotes significant, transgenerational protection of DA neurons in response to the stressor and pathological hallmark of PD, a-synuclein (a-syn) overexpression and accumulation. This protection is shown to be dependent on the normal function of Systemic RNA Interference Defective (SID) proteins, including SID-1, SID-2, and SID-3. These proteins are involved in the ability of double-stranded RNAs (dsRNAs) to traverse cellular boundaries, systemically, and silence target genes in C. elegans to varying degrees. Comparative transcriptomic analysis between worms reared on OP50 vs. HB101 E. coli in an a-syn model background has revealed genes and pathways associated with a wide range of processes. These included genes encoding proteins involved in metabolism, cell signaling, development, transcription, stress responses, and transmembrane transport, all of which were observed to be differentially expressed in worms reared on HB101 E. coli. Subsequent functional analysis of select targets by RNA interference revealed the contribution of individual genes to neuroprotection against a-syn-mediated neurodegeneration. This research lays a foundation for future investigation of the subtle distinctions in diet that can impact conserved pathways modulating neuron survival in response to stress.

Ray, Arpita et al. (2011) International Worm Meeting "Characterizing an uncharacterized protein: F16A11.2, a neuroprotective gene product with a putative role in RNA localization."

DeLeon, Susan M., Caldwell, Kim A., Ray, Arpita, Caldwell, Guy A.

[International Worm Meeting, 2011]

Our studies use C. elegans as an animal model for Parkinson's disease (PD) to identify genetic factors which may impact this condition. PD is characterized by the degeneration of dopaminergic (DA) neurons and the formation of protein inclusions that contain the a-synuclein (a-syn) protein. Overexpression of human a-syn specifically in the eight DA neurons of C. elegans causes neurodegeneration in an age- and dose-dependent manner. The F16A11.2 gene product was found as one of several significant modifiers in an RNAi screen for functional effectors of a-syn toxicity (Hamamichi et al., 2008 PNAS). F16A11.2 is the worm homolog of the human HSPC117 protein (also identified as C22orf28) and shares approximately 75% sequence identity to its human homolog. Interestingly, HSPC117 was determined to be a component of RNA trafficking granules in neurons, along with other proteins, such as CGI-99 (an mRNA transcriptional modulator) and DDX1 (an RNA binding protein). Accordingly, HSPC117 has the ability to bind to AU-rich elements within mRNA. The RtcB domain in F16A11.2 is highly conserved and, based on studies of its bacterial homolog, could represent an uncharacterized RNA modification enzyme. Here, we show that overexpression of the F16A11.2 cDNA in DA neurons not only significantly protected these cells from a-syn neurotoxicity but also that neuronal-specific RNAi knockdown of this target results in enhanced neurodegeneration. Similar results were obtained when worms were exposed to the DA neurotoxin, 6-hydroxydopamine (6-OHDA). Result of yeast two-hybrid (Y2H) screen point to a possible interaction of the F16A11.2 protein with several cytoskeletal factors including actin (ACT-1) and alpha-tubulin (TBA-1). We conducted double knockdown studies between F16A11.2 and those Y2H hits that have been shown to be expressed in neurons and/or have an RNA binding domain, using dopamine neuron-specific RNAi. Our preliminary findings indicate genetic interaction with F59G1.4, a worm homolog of the ceramide glucosyltransferase, DDX-1 and mir-2. As we continue to evaluate other positives from Y2H screening, we are also investigating the possible role of F16A11.2 in RNA trafficking by analyzing P granule localization. Collectively, these studies serve to illuminate the role of this evolutionarily conserved, but previously uncharacterized protein, coincident with the goal of uncovering new pathways associated with neuroprotection.

Griffin, Edward F et al. (2013) International Worm Meeting "Functional analysis of VPS41-mediated protection from b-Amyloid cytotoxicity."

Caldwell, Guy A, Caldwell, Kim A, Griffin, Edward F, Gilmartin, Christopher

[International Worm Meeting, 2013]

According to the Alzheimer's Association 2012 report, 13% of Americans over 65 years of age suffer from Alzheimer's Disease (AD), resulting in an estimated cost of 200 billion dollars for related health care. Additionally, AD is the most prevalent dementia, and the sixth leading cause of death in the United States. Thus, investigating mechanisms of pathophysiology and identifying potential therapeutic targets for AD is significant. AD is characterized by the formation of plaques, composed primarily of b-amyloid 1-42 (Ab) in the brain, resulting in neurodegeneration. Our lab has observed that over-expression of VPS41 in C. elegans provides neuroprotection from Ab toxicity, and that deficiency of VPS41 in worms expressing Ab increases the toxicity of Ab (unpublished data). In yeast, VPS41 has been demonstrated to function in the tethering of vesicles, late endosomes, and AP-3-coated vesicles from the late Golgi, to the lysosome for degradation. Previously, our lab has shown that over-expression of human VPS41 is neuroprotective in a transgenic worm model of Parkinson's Disease, wherein dopaminergic neurodegeneration is induced by a-synuclein overexpression (Harrington et al., 2012, J. Neurosci.). VPS41-mediated neuroprotection from a-synuclein was dependent on the phosphorylation of VPS41 as well as its interactions with AP-3d, RAB7, VPS core proteins, and VPS39. Through this study, and others, we can conclude that VPS41 has a role in lysosomal trafficking. Yet, how this specifically relates to the processing and ameliorates the cellular impact of neurotoxic proteinaceous aggregates is undetermined. Here we report the results of a systematic RNAi screen whereby we knocked down the core components involved in lysosomal trafficking and categorized their requirement for Ab protein toxicity. Preliminary results indicate that Ab is trafficked to through the endosomes rather than through AP-3-coated vesicles from the late Golgi. In this regard, further analysis of functional effectors of Ab protein processing via the lysosomal pathway, along with subsequent evaluation in our worm neuronal model of AD (Treusch et al, 2011, Science), will assist in the elucidation of the underlying mechanism involving VPS-41.

Yan, Xiaohui et al. (2013) International Worm Meeting "Genetic modifiers of amyloid-beta toxicity in C. elegans Alzheimer's disease models."

Knight, Adam L., Caldwell, Kim A., Yan, Xiaohui, Caldwell, Guy A.

[International Worm Meeting, 2013]

The insulin/insulin-like growth factor 1 signaling (IIS) pathway regulates both aging and proteotoxicity and is conserved from invertebrates to mammals. In humans, several age-related neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD) are associated with protein misfolding or aggregation. Others and we have shown that the reduction in IIS ameliorates the proteotoxicity associated with amyloid b (Ab) aggregation in AD, and a-synuclein (a-syn) aggregation in PD C. elegans models. Therefore, we hypothesized that there could be common cellular mechanisms to maintain the proteostasis for counteracting a-syn and Ab misfolding during aging. In this regard, we screened sixty age-related genetic factors that modify a-syn aggregation, in a C. elegans AD model in which temperature-inducible muscle expression of human Ab42 leads to a reproducible paralysis phenotype. We identified seven genes that accelerate Ab-induced paralysis upon RNAi. These modifiers include components of the protein clearance and select metabolic factors, including two genes encoding independent but functionally related enzymes, the serine hydroxymethyltransferase (SHMT) and the aminomethyltransferase (AMT) of the glycine cleavage system (GCS). The reactions catalyzed by SHMT and GCS generate one-carbon units for folate-dependent one-carbon metabolism whose normal function supports a variety of cellular activities. When AMT was overexpressed in our C. elegans neuronal AD model, where Ab42 accumulation induces an age-dependent degeneration of glutamatergic neurons (Treusch et al., 2011, Science), the neuronal loss was significantly rescued. This study provides the first evidence of a role for SHMT and GCS in modulating a-syn and Ab related proteotoxicity. Interestingly, alterations in one-carbon metabolism have been linked to epidemiological and genetic association studies for AD risk, yet, the underlying cellular mechanisms are largely unknown. Taken together, these findings will potentially expand our mechanistic understanding of the well established, but poorly understood, interaction between metabolism, especially the folate-dependent one-carbon metabolism, and age-associated neurodegenerative diseases.

Berkowitz, Laura A. et al. (2009) International Worm Meeting "Dopamine Signal Transduction Components Alter Neurodegeneration in a Parkinson's Disease Model."

Hamamichi, Shu, Caldwell, Kim A., Berkowitz, Laura A., Caldwell, Guy A.

[International Worm Meeting, 2009]

Improvements to the diagnosis and treatment of Parkinson''s disease (PD) are contingent upon knowledge about susceptibility factors that render populations at risk. Our work has focused on a functional analysis that exploits the experimental attributes of C. elegans to accelerate the translational path toward identification of effectors of dopamine (DA) neuron degeneration in vivo. C. elegans has only 8 DA neurons, providing a simple system in which to study PD-like neurodegeneration with unparalleled accuracy in quantitation of neuron survival in statistically substantial numbers of test animals. Our prior application of C. elegans in this directed manner has been successful in rapidly predicting genes that have significant consequences for neuronal survival in mammalian systems (Gitler et al., 2008; PNAS; Gitler et al., 2009, Nat. Genetics) Human a-synuclein, a protein found in the PD-associated aggregates called Lewy bodies, when overexpressed in C. elegans DA neurons, causes them to degenerate in an age- and dose-dependent manner. GAIP Interacting Protein C-terminus (GIPC) was identified in a large-scale RNAi screen for genes that alter both a-synuclein aggregation and DA neuron degeneration (Hamamichi et al., 2008, PNAS). GIPC interacts with the RGS-GAP (GTPase Activating Protein) GAIP, which attenuates G-protein signals. Additionally, GIPC interacts with a number of G-coupled receptors including the dopamine receptor, where it has been proposed to play multiple roles in DA signal transduction such as: 1) membrane recruitment of GAIP (which down-regulates DA signaling); 2) DA receptor internalization; and 3) DA receptor recycling. Here we report that genetic loss of GIPC enhances neurodegeneration, whereas overexpression (OEx) is neuroprotective. To determine what GIPC-dependent function(s) and possible interactor(s) is involved in its neuroprotective capacity, proteins that act in the above pathways are being analyzed via genetic knockouts and DA neuron-specific overexpression. This analysis has revealed that loss of presynaptic dopamine D2 receptors (dop-2) increases degeneration. Moreover, the neuroprotective capacity of GIPC OEx is eliminated in a dop-2 mutant background, suggesting that GIPC protects through the DA receptor, as opposed to acting via any number of other potential receptors. Loss of the C. elegans homolog of GAIP, rgs-2, is also protective while overexpression of this GIPC interactor enhances DA neuron loss. Taken together, these results suggest that components of the DA signaling cascade contribute to neuron survival. Further analysis of GIPC activity may provide mechanistic insights into PD.

Kim, Do-Young et al. (2019) International Worm Meeting "FMRFamide-related neuropeptide FLP-12 regulates head locomotion of C. elegans."

Kim, Kyuhyung, Li, Chris, Kim, Do-Young, Park, Cheon-Gyu, Suh, Byung-Chang, Kim, Jinmahn

[International Worm Meeting, 2019]

Neuropeptides regulate a multitude of behaviors in many organisms (Li et al., 2014). However, mechanisms by which functions of neuropeptides are coordinated to generate proper behaviors are not fully understood. In C. elegans, the SMB motor neurons consist of two pair of neurons that innervate head/neck muscles (White et al, 1986) and shape the amplitude of sinusoidal movement (Gray et al, 2005, Kim et al., 2014), indicating a role of SMB in head locomotion. The SMB neurons have shown to express FMRFamide-related neuropeptide FLP-12 (Kim and Li, 2004, Kim et al., 2014). To identify roles of FLP-12 neuropeptide in head locomotive behavior, we first observed phenotypic consequences by loss of FLP-12. We measured five locomotive parameters including omega turn, reversal, head lift, foraging and frequency of head movement. We found that all five locomotive parameters are increased in flp-12 mutants. We fully rescued defects in head locomotion of flp-12 mutants by expressing flp-12cDNA specifically in SMB. These results indicate that FLP-12 may act in the SMB to regulate head movement. To identify receptors for FLP-12, we performed RNAi screening against a total of 16 putative neuropeptide receptor genes. We found that knock-down of several genes including frpr-8 exhibits increases foraging behavior similarly to flp-12 mutants and frpr-8 mutants. Expressing frpr-8 cDNA with its own promoter successfully rescues foraging phenotype of frpr-8. Heterogously expressed FRPR-8 in HEK cells are sufficient to confer Ca2+ response upon FLP-12 exposure. Altogether, our data expand our understanding of how a single neuropeptide and its receptor modulate behaviors.