Cox HD et al. (2008) Methods Mol Biol "Purification and proteomic analysis of synaptic vesicles."

Thompson CM, Cox HD

[Methods Mol Biol, 2008]

Synaptic vesicles store and subsequently release neurotransmitters into the synaptic cleft thereby regulating chemical neurotransmission in the brain. Proteins present in synaptic vesicles vary greatly in structure and function and have been identified primarily by genetic knock-out analysis in C. elegans, Drosophila, and mice (1,2). However, knock-out methods are not useful for the identification of proteins when a detectable phenotype is not created. Further, certain knocked-out proteins have function(s) that could be compensated for by another protein or cause a lethal phenotype when deleted. Additionally, some transporters and enzymes that appear to copurify with synaptic vesicles have not been characterized and confirmed (3-5). We have determined the proteins associated with purified synaptic vesicles using 2-D polyacrylamide gel electrophoresis (PAGE) protein separation followed by identification by mass spectrometry (6). Some of the new proteins identified were evaluated by western blot and confocal immunofluorescence analysis.

Varma H et al. (2007) Nat Chem Biol "Selective inhibitors of death in mutant huntingtin cells."

Stockwell BR, DeMarco CT, Hart AC, Varma H, Lo DC, Voisine C, Cattaneo E

[Nat Chem Biol, 2007]

Huntington disease (HD) is an inherited neurodegenerative disorder with unclear pathophysiology. We developed a high-throughput assay in a neuronal cell culture model of HD, screened 43,685 compounds and identified 29 novel selective inhibitors of cell death in mutant huntingtin-expressing cells. Four compounds were active in diverse HD models, which suggests a role for cell death in HD; these compounds are mechanistic probes and potential drug leads for HD.

Machiela E et al. (2016) Neurobiol Dis "Oxidative stress is increased in C. elegans models of Huntington's disease but does not contribute to polyglutamine toxicity phenotypes."

Senchuk MM, Machiela E, Dues DJ, Van Raamsdonk JM

[Neurobiol Dis, 2016]

Huntington's disease (HD) is an adult onset neurodegenerative disorder for which there is currently no cure. While HD patients and animal models of the disease exhibit increased oxidative damage, it is currently uncertain to what extent oxidative stress contributes to disease pathogenesis. In this work, we use a genetic approach to define the role of oxidative stress in HD. We find that a C. elegans model of HD expressing a disease-length polyglutamine tract in the body wall muscle is hypersensitive to oxidative stress and shows an upregulation of antioxidant defense genes, indicating that the HD worm model has increased levels of oxidative stress. To determine whether this increase in oxidative stress contributes to the development of polyglutamine-toxicity phenotypes in this HD model, we examined the effect of deleting individual superoxide dismutase (sod) genes in the HD worm model. As predicted, we found that deletion of sod genes in the HD worm model resulted in a clear increase in sensitivity to oxidative stress. However, we found that increasing oxidative stress in the HD worm model did not exacerbate deficits caused by polyglutamine toxicity. We confirmed these observations in two worm models expressing disease-length polyglutamine tracts in neurons. Furthermore, we found that treatment antioxidants failed to rescue movement deficits or decrease aggregation in HD worm models. Combined, this suggests that the increase in oxidative stress in worm models of HD does not contribute to the phenotypic deficits observed in these worms, and provides a possible explanation for the failure of antioxidants in HD clinical trials.

Cong W et al. (2019) ACS Appl Mater Interfaces "Selenium Nanoparticles as an Efficient Nanomedicine for the Therapy of Huntington's Disease."

Cong W, Bai R, Chen C, Li YF, Wang L

[ACS Appl Mater Interfaces, 2019]

Huntington's disease (HD) is an incurable disease with progressive loss of neural function, which is influenced by epigenetic, oxidative stress, metabolic, and nutritional factors. Targeting inhibition of huntingtin protein aggregation is a strategy for HD therapy, but the efficacy is unsatisfactory. Studies found that selenium (Se) levels in the brain is insufficient for HD disease, while improvement in Se homeostasis in the brain may attenuate neuronal loss and dysfunction. In this study, we applied selenium nanoparticles (NPs) (Nano-Se) for the HD disease therapy by regulating HD-related neurodegeneration and cognitive decline based on transgenic HD models of Caenorhabditis elegans (C. elegans). At low dosages, Nano-Se NPs significantly reduced neuronal death, relieved behavioral dysfunction, and protected C. elegans from damages in stress conditions. Molecular mechanism also revealed that Nano-Se attenuated oxidative stress, inhibited the aggregation of huntingtin proteins, and down regulated the expression of histone deacetylase family members at mRNA levels. The results suggested that Nano-Se has great potential for Huntington's disease therapy. In conclusion, mechanism about how Nano-Se NPs protect from damages in stress conditions and repair neural functions will benefit to HD disease therapy. This study will also guide rational design of Nano-Se NPs or other selenium compounds to improve HD therapy in the future.

Suthammarak W et al. (2009) J Biol Chem "Complex I function is defective in complex IV-deficient Caenorhabditis elegans."

Suthammarak W, Morgan PG, Sedensky MM, Yang YY

[J Biol Chem, 2009]

Cytochrome c oxidase (COX) is hypothesized to be an important regulator of oxidative phosphorylation. However, no animal phenotypes have been described due to genetic defects in nuclear encoded subunits of COX. We knocked down predicted homologues of COX IV and COX Va in the nematode Caenorhabditis elegans. Animals treated with W09C5.8 (COX IV) or Y37D8A.14 (COX Va) RNAi had shortened lifespans and severe defects in mitochondrial respiratory chain (MRC) function. Amount and activity of complex IV, as well as supercomplexes that included complex IV, were decreased in COX-deficient worms. The formation of supercomplex I:III was not dependent on COX. We found that COX deficiencies decreased intrinsic complex I enzymatic activity, as well as complex I-III enzymatic activity. However, overall amounts of complex I were not decreased in these animals. Surprisingly, intrinsic complex I enzymatic activity is dependent on the presence of complex IV, despite no overall decrease in amount of complex I. Presumably the association of complex I with complex IV within the supercomplex I:III:IV enhances electron flow through complex I. Our results indicate that reduction of a single subunit within the ETC can affect multiple enzymatic steps of electron transfer, including movement within a different protein complex. Patients presenting with multiple defects of electron transport may, in fact, harbor a single genetic defect.

Strogolova V et al. (2012) Mol Cell Biol "Rcf1 and Rcf2, members of the hypoxia-induced gene 1 protein family, are critical components of the mitochondrial cytochrome bc1-cytochrome c oxidase supercomplex."

Stuart RA, Furness A, Strogolova V, Garlich J, Robb-McGrath M

[Mol Cell Biol, 2012]

We report that Rcf1 (formerly Aim31), a member of the conserved hypoxia-induced gene 1 (Hig1) protein family, represents a novel component of the yeast cytochrome bc(1)-cytochrome c oxidase (COX) supercomplex. Rcf1 (respiratory supercomplex factor 1) partitions with the COX complex, and evidence that it may act as a bridge to the cytochrome bc(1) complex is presented. Rcf1 interacts with the Cox3 subunit and can do so prior to their assembly into the COX complex. A close proximity of Rcf1 and members of the ADP/ATP carrier (AAC) family was also established. Rcf1 displays overlapping function with another Hig1-related protein, Rcf2 (formerly Aim38), and their joint presence is required for optimal COX enzyme activity and the correct assembly of the cytochrome bc(1)-COX supercomplex. Rcf1 and Rcf2 can independently associate with the cytochrome bc(1)-COX supercomplex, indicating that at least two forms of this supercomplex exist within mitochondria. We provide evidence that the association with the cytochrome bc(1)-COX supercomplex and regulation of the COX complex are a conserved feature of Hig1 family members. Based on our findings, we propose a model where the Hig1 proteins regulate the COX enzyme activity through Cox3 and associated Cox12 protein, in a manner that may be influenced by the neighboring AAC proteins.

Lejeune F et al. (2012) BMC Genomics "Large-scale functional RNAi screen in C. elegans identifies genes that regulate the dysfunction of mutant polyglutamine neurons."

Vert JP, Lejeune F, Parmentier F, Mesrob L, Parker A, Vazquez R, Bicep C, Tourette C, Neri C

[BMC Genomics, 2012]

BACKGROUND: A central goal in Huntington's disease (HD) research is to identify and prioritize candidate targets for neuroprotective intervention, which requires genome-scale information on the modifiers of early-stage neuron injury in HD. RESULTS: Here, we performed a large-scale RNA interference screen in C. elegans strains that express N-terminal huntingtin (htt) in touch receptor neurons. These neurons control the response to light touch. Their function is strongly impaired by expanded polyglutamines (128Q) as shown by the nearly complete loss of touch response in adult animals, providing an in vivo model in which to manipulate the early phases of expanded-polyQ neurotoxicity. In total, 6034 genes were examined, revealing 662 gene inactivations that either reduce or aggravate defective touch response in 128Q animals. Several genes were previously implicated in HD or neurodegenerative disease, suggesting that this screen has effectively identified candidate targets for HD. Network-based analysis emphasized a subset of high-confidence modifier genes in pathways of interest in HD including metabolic, neurodevelopmental and pro-survival pathways. Finally, 49 modifiers of 128Q-neuron dysfunction that are dysregulated in the striatum of either R/2 or CHL2 HD mice, or both, were identified. CONCLUSIONS: Collectively, these results highlight the relevance to HD pathogenesis, providing novel information on the potential therapeutic targets for neuroprotection in HD.

Cordeiro LM et al. (2025) Methods Cell Biol "Evaluating polyglutamine protein aggregation and toxicity in transgenic Caenorhabditis elegans models of Huntington's disease."

Arantes LP, Soares FAA, Cordeiro LM

[Methods Cell Biol, 2025]

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by a repeat of the cytosine-adenine-guanine trinucleotide (CAG) in the huntingtin gene (HTT). This results in the translation of a mutant huntingtin (mHTT) protein with an abnormally long polyglutamine (polyQ) repeat. The pathology of HD leads to neuronal cell loss, motor abnormalities, and dementia. Currently, the pathogenesis of HD remains incompletely understood, and available treatments only address symptoms. Caenorhabditis elegans has been used as a model for neurodegenerative diseases, enabling the exploration of the molecular, cellular, and physiological mechanisms underlying HD pathogenesis. It also facilitates the investigation of potential therapeutic targets and interventions. Here, we describe common experiments employed to assess polyQ aggregation and toxicity in transgenic C. elegans models of HD, utilizing fluorescent markers to detect protein aggregation and neuron degeneration, in addition to specific behavioral assays (thrash frequency, nose touch response, and octanol response).

Cerqua C et al. (2018) Biochim Biophys Acta "COX16 is required for assembly of cytochrome c oxidase in human cells and is involved in copper delivery to COX2."

Frasson C, Basso G, Salviati L, Sacconi S, Doimo M, Baldoin MC, Morbidoni V, Sartori G, Desbats MA, Trevisson E, Cerqua C

[Biochim Biophys Acta, 2018]

Cytochrome c oxidase (COX), complex IV of the mitochondrial respiratory chain, is comprised of 14 structural subunits, several prosthetic groups and metal cofactors, among which copper. Its biosynthesis involves a number of ancillary proteins, encoded by the COX-assembly genes that are required for the stabilization and membrane insertion of the nascent polypeptides, the synthesis of the prosthetic groups, and the delivery of the metal cofactors, in particular of copper. Recently, a modular model for COX assembly has been proposed, based on the sequential incorporation of different assembly modules formed by specific subunits. We have cloned and characterized the human homologue of yeast COX16. We show that human COX16 encodes a small mitochondrial transmembrane protein that faces the intermembrane space and is highly expressed in skeletal and cardiac muscle. Its knockdown in C. elegans produces COX deficiency, and its ablation in HEK293 cells impairs COX assembly. Interestingly, COX16 knockout cells retain significant COX activity, suggesting that the function of COX16 is partially redundant. Analysis of steady-state levels of COX subunits and of assembly intermediates by Blue-Native gels shows a pattern similar to that reported in cells lacking COX18, suggesting that COX16 is required for the formation of the COX2 subassembly module. Moreover, COX16 co-immunoprecipitates with COX2. Finally, we found that copper supplementation increases COX activity and restores normal steady state levels of COX subunits in COX16 knockout cells, indicating that, even in the absence of a canonical copper binding motif, COX16 could be involved in copper delivery to COX2.

Vela M et al. (2021) ACS Chem Neurosci "Neuroprotective Effect of IND1316, an Indole-Based AMPK Activator, in Animal Models of Huntington Disease."

Cumella J, Castro A, Vela M, Sanchis A, Perez C, Lagartera L, Bono-Yague J, Garcia-Gimeno MA, Priego EM, Sanz P, Vazquez-Manrique RP, Campos A

[ACS Chem Neurosci, 2021]

Aggregation of mutant huntingtin, because of an expanded polyglutamine track, underlies the cause of neurodegeneration in Huntington disease (HD). However, it remains unclear how some alterations at the cellular level lead to specific structural changes in HD brains. In this context, the neuroprotective effect of the activation of AMP-activated protein kinase (AMPK) appears to be a determinant factor in several neurodegenerative diseases, including HD. In the present work, we describe a series of indole-derived compounds able to activate AMPK at the cellular level. By using animal models of HD (both worms and mice), we demonstrate the in vivo efficacy of one of these compounds (IND1316), confirming that it can reduce the neuropathological symptoms of this disease. Taken together, in vivo results and in silico studies of druggability, allow us to suggest that IND1316 could be considered as a promising new lead compound for the treatment of HD and other central nervous system diseases in which the activation of AMPK results in neuroprotection.