Functional analysis of epilepsy-associated GABAA receptor mutations using Caenorhabditis elegans.

OBJECTIVE: GABAA receptor subunit mutations pose a significant risk for genetic generalized epilepsy; however, there are over 150 identified variants, many with unknown or unvalidated pathogenicity. We aimed to develop in vivo models for testing GABAA receptor variants using the model organism, Caenorhabditis elegans. METHODS: CRISPR-Cas9 gene editing was used to create a complete deletion of unc-49, a C. elegans GABAA receptor, and to create homozygous epilepsy-associated mutations in the endogenous unc-49 gene. The unc-49 deletion strain was rescued with transgenes for either the C. elegans unc-49B subunit or the α1, ß3, and γ2 subunits for the human GABAA receptor. All newly created strains were analyzed for phenotype and compared against existing unc-49 mutations. RESULTS: Nematodes with a full genetic deletion of the entire unc-49 locus were compared with existing unc-49 mutations in three separate phenotypic assays-coordinated locomotion, shrinker frequency and seizure-like convulsions. The full unc-49 deletion exhibited reduced locomotion and increased shrinker frequency and PTZ-induced convulsions, but were not found to be phenotypically stronger than existing unc-49 mutations. Rescue with the unc-49B subunit or creation of humanized worms for the GABAA receptor both showed partial phenotypic rescue for all three phenotypes investigated. Finally, two epilepsy-associated variants were analyzed and deemed to be loss of function, thus validating their pathogenicity. SIGNIFICANCE: These findings establish C. elegans as a genetic model to investigate GABAA receptor mutations and delineate a platform for validating associated variants in any epilepsy-associated gene. PLAIN LANGUAGE SUMMARY: Epilepsy is a complex human disease that can be caused by mutations in specific genes. Many possible mutations have been identified, but it is unknown for most of them whether they cause the disease. We tested the role of mutations in one specific gene using a small microscopic worm as an animal model. Our results establish this worm as a model for epilepsy and confirm that the two unknown mutations are likely to cause the disease.

Tissue distribution of cysteine string protein/DNAJC5 in C. elegans analysed by CRISPR/Cas9-mediated tagging of endogenous DNJ-14.

Cysteine string protein (CSP) is a member of the DnaJ/Hsp40 family of molecular chaperones. CSP is enriched in neurons, where it mainly localises to synaptic vesicles. Mutations in CSP-encoding genes in flies, worms, mice and humans result in neuronal dysfunction, neurodegeneration and reduced lifespan. Most attention has therefore focused on CSP's neuronal functions, although CSP is also expressed in non-neuronal cells. Here, we used genome editing to fluorescently tag the Caenorhabditis elegans CSP orthologue, dnj-14, to identify which tissues preferentially express CSP and hence may contribute to the observed mutant phenotypes. Replacement of dnj-14 with wrmScarlet caused a strong chemotaxis defect, as seen with other dnj-14 null mutants. In contrast, inserting the reporter in-frame to create a DNJ-14-wrmScarlet fusion protein had no effect on chemotaxis, indicating that C-terminal tagging does not impair DNJ-14 function. WrmScarlet fluorescence appeared most obvious in the intestine, head/pharynx, spermathecae and vulva/uterus in the reporter strains, suggesting that DNJ-14 is preferentially expressed in these tissues. Crossing the DNJ-14-wrmScarlet strain with GFP marker strains confirmed the intestinal and pharyngeal expression, but only a partial overlap with neuronal GFP was observed. DNJ-14-wrmScarlet fluorescence in the intestine was increased in response to starvation, which may be relevant to mammalian CSPα's role in microautophagy. DNJ-14's enrichment in worm reproductive tissues (spermathecae and vulva/uterus) parallels the testis-specific expression of CSPß and CSPγ isoforms in mammals. Furthermore, CSPα messenger RNA is highly expressed in the human proximal digestive tract, suggesting that CSP may have a conserved, but overlooked, function within the gastrointestinal system.

Proximity labelling reveals effects of disease-causing mutation on the DNAJC5/cysteine string protein α interactome.

Cysteine string protein α (CSPα), also known as DNAJC5, is a member of the DnaJ/Hsp40 family of co-chaperones. The name derives from a cysteine-rich domain, palmitoylation of which enables localization to intracellular membranes, notably neuronal synaptic vesicles. Mutations in the DNAJC5 gene that encodes CSPα cause autosomal dominant, adult-onset neuronal ceroid lipofuscinosis (ANCL), a rare neurodegenerative disease. As null mutations in CSP-encoding genes in flies, worms and mice similarly result in neurodegeneration, CSP is evidently an evolutionarily conserved neuroprotective protein. However, the client proteins that CSP chaperones to prevent neurodegeneration remain unclear. Traditional methods for identifying protein-protein interactions such as yeast 2-hybrid and affinity purification approaches are poorly suited to CSP, due to its requirement for membrane anchoring and its tendency to aggregate after cell lysis. Therefore, we employed proximity labelling, which enables identification of interacting proteins in situ in living cells via biotinylation. Neuroendocrine PC12 cell lines stably expressing wild type or L115R ANCL mutant CSP constructs fused to miniTurbo were generated; then the biotinylated proteomes were analysed by liquid chromatographymass spectrometry (LCMS) and validated by western blotting. This confirmed several known CSP-interacting proteins, such as Hsc70 and SNAP-25, but also revealed novel binding proteins, including STXBP1/Munc18-1. Interestingly, some protein interactions (such as Hsc70) were unaffected by the L115R mutation, whereas others (including SNAP-25 and STXBP1/Munc18-1) were inhibited. These results define the CSP interactome in a neuronal model cell line and reveal interactions that are affected by ANCL mutation and hence may contribute to the neurodegeneration seen in patients.

A Caenorhabditis elegans model of autosomal dominant adult-onset neuronal ceroid lipofuscinosis identifies ethosuximide as a potential therapeutic.

Autosomal dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is a rare neurodegenerative disorder characterized by progressive dementia and premature death. Four ANCL-causing mutations have been identified, all mapping to the DNAJC5 gene that encodes cysteine string protein α (CSPα). Here, using Caenorhabditis elegans, we describe an animal model of ANCL in which disease-causing mutations are introduced into their endogenous chromosomal locus, thereby mirroring the human genetic disorder. This was achieved through CRISPR/Cas9-mediated gene editing of dnj-14, the C. elegans ortholog of DNAJC5. The resultant homozygous ANCL mutant worms exhibited reduced lifespans and severely impaired chemotaxis, similar to isogenic dnj-14 null mutants. Importantly, these phenotypes were also seen in balanced heterozygotes carrying one wild-type and one ANCL mutant dnj-14 allele, mimicking the heterozygosity of ANCL patients. We observed a more severe chemotaxis phenotype in heterozygous ANCL mutant worms compared with haploinsufficient worms lacking one copy of CSP, consistent with a dominant-negative mechanism of action. Additionally, we provide evidence of CSP haploinsufficiency in longevity, as heterozygous null mutants exhibited significantly shorter lifespan than wild-type controls. The chemotaxis phenotype of dnj-14 null mutants was fully rescued by transgenic human CSPα, confirming the translational relevance of the worm model. Finally, a focused compound screen revealed that the anti-epileptic drug ethosuximide could restore chemotaxis in dnj-14 ANCL mutants to wild-type levels. This suggests that ethosuximide may have therapeutic potential for ANCL and demonstrates the utility of this C. elegans model for future larger-scale drug screening.

Cysteine string protein alpha accumulates with early pre-synaptic dysfunction in Alzheimer's disease.

In Alzheimer's disease, synapse loss causes memory and cognitive impairment. However, the mechanisms underlying synaptic degeneration in Alzheimer's disease are not well understood. In the hippocampus, alterations in the level of cysteine string protein alpha, a molecular co-chaperone at the pre-synaptic terminal, occur prior to reductions in synaptophysin, suggesting that it is a very sensitive marker of synapse degeneration in Alzheimer's. Here, we identify putative extracellular accumulations of cysteine string alpha protein, which are proximal to beta-amyloid deposits in post-mortem human Alzheimer's brain and in the brain of a transgenic mouse model of Alzheimer's disease. Cysteine string protein alpha, at least some of which is phosphorylated at serine 10, accumulates near the core of beta-amyloid deposits and does not co-localize with hyperphosphorylated tau, dystrophic neurites or glial cells. Using super-resolution microscopy and array tomography, cysteine string protein alpha was found to accumulate to a greater extent than other pre-synaptic proteins and at a comparatively great distance from the plaque core. This indicates that cysteine string protein alpha is most sensitive to being released from pre-synapses at low concentrations of beta-amyloid oligomers. Cysteine string protein alpha accumulations were also evident in other neurodegenerative diseases, including some fronto-temporal lobar dementias and Lewy body diseases, but only in the presence of amyloid plaques. Our findings are consistent with suggestions that pre-synapses are affected early in Alzheimer's disease, and they demonstrate that cysteine string protein alpha is a more sensitive marker for early pre-synaptic dysfunction than traditional synaptic markers. We suggest that cysteine string protein alpha should be used as a pathological marker for early synaptic disruption caused by beta-amyloid.

Proteomic analysis of dietary restriction in yeast reveals a role for Hsp26 in replicative lifespan extension.

Dietary restriction (DR) has been shown to increase lifespan in organisms ranging from yeast to mammals. This suggests that the underlying mechanisms may be evolutionarily conserved. Indeed, upstream signalling pathways, such as TOR, are strongly linked to DR-induced longevity in various organisms. However, the downstream effector proteins that ultimately mediate lifespan extension are less clear. To shed light on this, we used a proteomic approach on budding yeast. Our reasoning was that analysis of proteome-wide changes in response to DR might enable the identification of proteins that mediate its physiological effects, including replicative lifespan extension. Of over 2500 proteins we identified by liquid chromatography-mass spectrometry, 183 were significantly altered in expression by at least 3-fold in response to DR. Most of these proteins were mitochondrial and/or had clear links to respiration and metabolism. Indeed, direct analysis of oxygen consumption confirmed that mitochondrial respiration was increased several-fold in response to DR. In addition, several key proteins involved in mating, including Ste2 and Ste6, were down-regulated by DR. Consistent with this, shmoo formation in response to α-factor pheromone was reduced by DR, thus confirming the inhibitory effect of DR on yeast mating. Finally, we found that Hsp26, a member of the conserved small heat shock protein (sHSP) family, was up-regulated by DR and that overexpression of Hsp26 extended yeast replicative lifespan. As overexpression of sHSPs in Caenorhabditis elegans and Drosophila has previously been shown to extend lifespan, our data on yeast Hsp26 suggest that sHSPs may be universally conserved effectors of longevity.

A multiorganism pipeline for antiseizure drug discovery: Identification of chlorothymol as a novel γ-aminobutyric acidergic anticonvulsant.

OBJECTIVE: Current medicines are ineffective in approximately one-third of people with epilepsy. Therefore, new antiseizure drugs are urgently needed to address this problem of pharmacoresistance. However, traditional rodent seizure and epilepsy models are poorly suited to high-throughput compound screening. Furthermore, testing in a single species increases the chance that therapeutic compounds act on molecular targets that may not be conserved in humans. To address these issues, we developed a pipeline approach using four different organisms. METHODS: We sequentially employed compound library screening in the zebrafish, Danio rerio, chemical genetics in the worm, Caenorhabditis elegans, electrophysiological analysis in mouse and human brain slices, and preclinical validation in mouse seizure models to identify novel antiseizure drugs and their molecular mechanism of action. RESULTS: Initially, a library of 1690 compounds was screened in an acute pentylenetetrazol seizure model using D rerio. From this screen, the compound chlorothymol was identified as an effective anticonvulsant not only in fish, but also in worms. A subsequent genetic screen in C elegans revealed the molecular target of chlorothymol to be LGC-37, a worm γ-aminobutyric acid type A (GABAA ) receptor subunit. This GABAergic effect was confirmed using in vitro brain slice preparations from both mice and humans, as chlorothymol was shown to enhance tonic and phasic inhibition and this action was reversed by the GABAA receptor antagonist, bicuculline. Finally, chlorothymol exhibited in vivo anticonvulsant efficacy in several mouse seizure assays, including the 6-Hz 44-mA model of pharmacoresistant seizures. SIGNIFICANCE: These findings establish a multiorganism approach that can identify compounds with evolutionarily conserved molecular targets and translational potential, and so may be useful in drug discovery for epilepsy and possibly other conditions.

Functional analysis of epilepsy-associated variants in STXBP1/Munc18-1 using humanized Caenorhabditis elegans.

OBJECTIVE: Genetic variants in STXBP1, which encodes the conserved exocytosis protein Munc18-1, are associated with a variety of infantile epilepsy syndromes. We aimed to develop an in vivo Caenorhabditis elegans model that could be used to test the pathogenicity of such variants in a cost-effective manner. METHODS: The CRISPR/Cas9 method was used to introduce a null mutation into the unc-18 gene (the C. elegans orthologue of STXBP1), thereby creating a paralyzed worm strain. We subsequently rescued this strain with transgenes encoding the human STXBP1/Munc18-1 protein (wild-type and eight different epilepsy-associated missense variants). The resulting humanized worm strains were then analyzed via behavioral, electrophysiological, and biochemical approaches. RESULTS: Transgenic expression of wild-type human STXBP1 protein fully rescued locomotion in both solid and liquid media to the same level as the standard wild-type worm strain, Bristol N2. Six variant strains (E59K, V84D, C180Y, R292H, L341P, R551C) exhibited impaired locomotion, whereas two (P335L, R406H) were no different from worms expressing wild-type STXBP1. Electrophysiological recordings revealed that all eight variant strains displayed less frequent and more irregular pharyngeal pumping in comparison to wild-type STXBP1-expressing strains. Four strains (V84D, C180Y, R292H, P335L) exhibited pentylenetetrazol-induced convulsions in an acute assay of seizure-like activity, in contrast to worms expressing wild-type STXBP1. No differences were seen between wild-type and variant STXBP1 strains in terms of mRNA abundance. However, STXBP1 protein levels were reduced to 20%-30% of wild-type in all variants, suggesting that the mutations result in STXBP1 protein instability. SIGNIFICANCE: The approach described here is a cost-effective in vivo method for establishing the pathogenicity of genetic variants in STXBP1 and potentially other conserved neuronal proteins. Furthermore, the humanized strains we created could potentially be used in the future for high-throughput drug screens to identify novel therapeutics.

The contribution of multicellular model organisms to neuronal ceroid lipofuscinosis research.

The NCLs (neuronal ceroid lipofuscinosis) are forms of neurodegenerative disease that affect people of all ages and ethnicities but are most prevalent in children. Commonly known as Batten disease, this debilitating neurological disorder is comprised of 13 different subtypes that are categorized based on the particular gene that is mutated (CLN1-8, CLN10-14). The pathological mechanisms underlying the NCLs are not well understood due to our poor understanding of the functions of NCL proteins. Only one specific treatment (enzyme replacement therapy) is approved, which is for the treating the brain in CLN2 disease. Hence there remains a desperate need for further research into disease-modifying treatments. In this review, we present and evaluate the genes, proteins and studies performed in the social amoeba, nematode, fruit fly, zebrafish, mouse and large animals pertinent to NCL. In particular, we highlight the use of multicellular model organisms to study NCL protein function, pathology and pathomechanisms. Their use in testing novel therapeutic approaches is also presented. With this information, we highlight how future research in these systems may be able to provide new insight into NCL protein functions in human cells and aid in the development of new therapies.

A Caenorhabditis elegans assay of seizure-like activity optimised for identifying antiepileptic drugs and their mechanisms of action.

BACKGROUND: Epilepsy affects around 1% of people, but existing antiepileptic drugs (AEDs) only offer symptomatic relief and are ineffective in approximately 30% of patients. Hence, new AEDs are sorely needed. However, a major bottleneck is the low-throughput nature of early-stage AED screens in conventional rodent models. This process could potentially be expedited by using simpler invertebrate systems, such as the nematode Caenorhabditis elegans. NEW METHOD: Head-bobbing convulsions were previously reported to be inducible by pentylenetetrazol (PTZ) in C. elegans with loss-of-function mutations in unc-49, which encodes a GABAA receptor. Given that epilepsy-linked mutations in human GABAA receptors are well documented, this could represent a clinically-relevant system for early-stage AED screens. However, the original agar plate-based assay is unsuited to large-scale screening and has not been validated for identifying AEDs. Therefore, we established an alternative streamlined, higher-throughput approach whereby mutants were treated with PTZ and AEDs via liquid-based incubation. RESULTS: Convulsions induced within minutes of PTZ exposure in unc-49 mutants were strongly inhibited by the established AED ethosuximide. This protective activity was independent of ethosuximide's suggested target, the T-type calcium channel, as a null mutation in the worm cca-1 ortholog did not affect ethosuximide's anticonvulsant action. COMPARISON WITH EXISTING METHOD: Our streamlined assay is AED-validated, feasible for higher throughput compound screens, and can facilitate insights into AED mechanisms of action. CONCLUSIONS: Based on an epilepsy-associated genetic background, this C. elegans unc-49 model of seizure-like activity presents an ethical, higher throughput alternative to conventional rodent seizure models for initial AED screens.

α-Methyl-α-phenylsuccinimide ameliorates neurodegeneration in a C. elegans model of TDP-43 proteinopathy.

The antiepileptic drug ethosuximide has recently been shown to be neuroprotective in various Caenorhabditis elegans and rodent neurodegeneration models. It is therefore a promising repurposing candidate for the treatment of multiple neurodegenerative diseases. However, high concentrations of the drug are required for its protective effects in animal models, which may impact on its translational potential and impede the identification of its molecular mechanism of action. Therefore, we set out to develop more potent neuroprotective lead compounds based on ethosuximide as a starting scaffold. Chemoinformatic approaches were used to identify compounds with structural similarity to ethosuximide and to prioritise these based on good predicated blood-brain barrier permeability and C. elegans bioaccumulation properties. Selected compounds were initially screened for anti-convulsant activity in a C. elegans pentylenetetrazol-induced seizure assay, as a rapid primary readout of bioactivity; and then assessed for neuroprotective properties in a C. elegans TDP-43 proteinopathy model based on pan-neuronal expression of human A315T mutant TDP-43. The most potent compound screened, α-methyl-α-phenylsuccinimide (MPS), ameliorated the locomotion defects and extended the shortened lifespan of TDP-43 mutant worms. MPS also directly protected against neurodegeneration by reducing the number of neuronal breaks and cell body losses in GFP-labelled GABAergic motor neurons. Importantly, optimal neuroprotection was exhibited by external application of 50 µM MPS, compared to 8 mM for ethosuximide. This greater potency of MPS was not due to bioaccumulation to higher internal levels within the worm, based on 1H-nuclear magnetic resonance analysis. Like ethosuximide, the activity of MPS was abolished by mutation of the evolutionarily conserved FOXO transcription factor, daf-16, suggesting that both compounds act via the same neuroprotective pathway(s). In conclusion, we have revealed a novel neuroprotective activity of MPS that is >100-fold more potent than ethosuximide. This increased potency will facilitate future biochemical studies to identify the direct molecular target(s) of both compounds, as we have shown here that they share a common downstream DAF-16-dependent mechanism of action. Furthermore, MPS is the active metabolite of another approved antiepileptic drug, methsuximide. Therefore, methsuximide may have repurposing potential for treatment of TDP-43 proteinopathies and possibly other human neurodegenerative diseases.

Analysis of the brain palmitoyl-proteome using both acyl-biotin exchange and acyl-resin-assisted capture methods.

Palmitoylation is a reversible post-translational protein modification in which palmitic acid is added to cysteine residues, allowing association with different cellular membranes and subdomains. Recently, techniques have been developed to identify palmitoylation on a proteome-wide scale in order to reveal the full cellular complement of palmitoylated proteins. However, in the studies reported to date, there is considerable variation between the sets of identified palmitoyl-proteins and so there remains some uncertainty over what constitutes the definitive complement of palmitoylated proteins even in well-studied tissues such as brain. To address this issue, we used both acyl-biotin exchange and acyl-resin-assisted capture approaches using rat brain as a common protein source. The palmitoyl-proteins identified from each method by mass spectrometry were then compared with each other and previously published studies. There was generally good agreement between the two methods, although many identifications were unique to one method, indicating that at least some of the variability in published palmitoyl proteomes is due to methodological differences. By combining our new data with previous publications using mammalian cells/tissues, we propose a high confidence set of bona fide palmitoylated proteins in brain and provide a resource to help researchers prioritise candidate palmitoyl-proteins for investigation.

New links between SOD1 and metabolic dysfunction from a yeast model of amyotrophic lateral sclerosis.

A number of genes have been linked to familial forms of the fatal motor neuron disease amyotrophic lateral sclerosis (ALS). Over 150 mutations within the gene encoding superoxide dismutase 1 (SOD1) have been implicated in ALS, but why such mutations lead to ALS-associated cellular dysfunction is unclear. In this study, we identify how ALS-linked SOD1 mutations lead to changes in the cellular health of the yeast Saccharomyces cerevisiae We find that it is not the accumulation of aggregates but the loss of Sod1 protein stability that drives cellular dysfunction. The toxic effect of Sod1 instability does not correlate with a loss of mitochondrial function or increased production of reactive oxygen species, but instead prevents acidification of the vacuole, perturbs metabolic regulation and promotes senescence. Central to the toxic gain-of-function seen with the SOD1 mutants examined was an inability to regulate amino acid biosynthesis. We also report that leucine supplementation results in an improvement in motor function in a Caenorhabditis elegans model of ALS. Our data suggest that metabolic dysfunction plays an important role in Sod1-mediated toxicity in both the yeast and worm models of ALS.

Phosphorylation of Cysteine String Protein Triggers a Major Conformational Switch.

Cysteine string protein (CSP) is a member of the DnaJ/Hsp40 chaperone family that localizes to neuronal synaptic vesicles. Impaired CSP function leads to neurodegeneration in humans and model organisms as a result of misfolding of client proteins involved in neurotransmission. Mammalian CSP is phosphorylated in vivo on Ser10, and this modulates its protein interactions and effects on neurotransmitter release. However, there are no data on the structural consequences of CSP phosphorylation to explain these functional effects. We show that Ser10 phosphorylation causes an order-to-disorder transition that disrupts CSP's extreme N-terminal α helix. This triggers the concomitant formation of a hairpin loop stabilized by ionic interactions between phosphoSer10 and the highly conserved J-domain residue, Lys58. These phosphorylation-induced effects result in significant changes to CSP conformation and surface charge distribution. The phospho-switch revealed here provides structural insight into how Ser10 phosphorylation modulates CSP function and also has potential implications for other DnaJ phosphoproteins.

Using C. elegans to discover therapeutic compounds for ageing-associated neurodegenerative diseases.

Age-associated neurodegenerative disorders such as Alzheimer's disease are a major public health challenge, due to the demographic increase in the proportion of older individuals in society. However, the relatively few currently approved drugs for these conditions provide only symptomatic relief. A major goal of neurodegeneration research is therefore to identify potential new therapeutic compounds that can slow or even reverse disease progression, either by impacting directly on the neurodegenerative process or by activating endogenous physiological neuroprotective mechanisms that decline with ageing. This requires model systems that can recapitulate key features of human neurodegenerative diseases that are also amenable to compound screening approaches. Mammalian models are very powerful, but are prohibitively expensive for high-throughput drug screens. Given the highly conserved neurological pathways between mammals and invertebrates, Caenorhabditis elegans has emerged as a powerful tool for neuroprotective compound screening. Here we describe how C. elegans has been used to model various human ageing-associated neurodegenerative diseases and provide an extensive list of compounds that have therapeutic activity in these worm models and so may have translational potential.

Erratum to: Ethosuximide ameliorates neurodegenerative disease phenotypes by modulating DAF-16/FOXO target gene expression.

The original version of this article [1] unfortunately contained a mistake. The author list contained a spelling error for the author Hannah V. McCue. The original article has been corrected for this error. The corrected author list is given below:Xi Chen, Hannah V. McCue, Shi Quan Wong, Sudhanva S. Kashyap, Brian C. Kraemer, Jeff W. Barclay, Robert D. Burgoyne and Alan Morgan

Ethosuximide ameliorates neurodegenerative disease phenotypes by modulating DAF-16/FOXO target gene expression.

BACKGROUND: Many neurodegenerative diseases are associated with protein misfolding/aggregation. Treatments mitigating the effects of such common pathological processes, rather than disease-specific symptoms, therefore have general therapeutic potential. RESULTS: Here we report that the anti-epileptic drug ethosuximide rescues the short lifespan and chemosensory defects exhibited by C. elegans null mutants of dnj-14, the worm orthologue of the DNAJC5 gene mutated in autosomal-dominant adult-onset neuronal ceroid lipofuscinosis. It also ameliorates the locomotion impairment and short lifespan of worms expressing a human Tau mutant that causes frontotemporal dementia. Transcriptomic analysis revealed a highly significant up-regulation of DAF-16/FOXO target genes in response to ethosuximide; and indeed RNAi knockdown of daf-16 abolished the therapeutic effect of ethosuximide in the worm dnj-14 model. Importantly, ethosuximide also increased the expression of classical FOXO target genes and reduced protein aggregation in mammalian neuronal cells. CONCLUSIONS: We have revealed a conserved neuroprotective mechanism of action of ethosuximide from worms to mammalian neurons. Future experiments in mouse neurodegeneration models will be important to confirm the repurposing potential of this well-established anti-epileptic drug for treatment of human neurodegenerative diseases.

Expression profile of a Caenorhabditis elegans model of adult neuronal ceroid lipofuscinosis reveals down regulation of ubiquitin E3 ligase components.

Cysteine string protein (CSP) is a chaperone of the Dnaj/Hsp40 family of proteins and is essential for synaptic maintenance. Mutations in the human gene encoding CSP, DNAJC5, cause adult neuronal ceroid lipofucinosis (ANCL) which is characterised by progressive dementia, movement disorders, seizures and premature death. CSP null models in mice, flies and worms have been shown to also exhibit similar neurodegenerative phenotypes. Here we have explored the mechanisms underlying ANCL disease progression using Caenorhaditis elegans mutant strains of dnj-14, the worm orthologue of DNAJC5. Transcriptional profiling of these mutants compared to control strains revealed a broad down-regulation of ubiquitin proteasome system (UPS)-related genes, in particular, components of multimeric RING E3 ubiquitin ligases including F-Box, SKR and BTB proteins. These data were supported by the observation that dnj-14 mutant worm strains expressing a GFP-tagged ubiquitin fusion degradation substrate exhibited decreased ubiquitylated protein degradation. The results indicate that disruption of an essential synaptic chaperone leads to changes in expression levels of UPS-related proteins which has a knock-on effect on overall protein degradation in C. elegans. The specific over-representation of E3 ubiquitin ligase components revealed in our study, suggests that proteins and complexes upstream of the proteasome itself may be beneficial therapeutic targets.