In this chapter, I explore Mieke Bal's notion of "preposterous history" as a way to illuminate the particular historical and representational questions posed by They Shall Not Grow Old (2018), Peter Jackson's extraordinary remediation of First World War documentary footage. Although much of the praise as well as the criticism of the film centres on its use of—and for some writers, its misuse of—documentary sources, I consider whether the film might be better seen not in terms of archival restoration, but rather as a work of quotation; not in terms of its fidelity to original archival sources, but rather as a form of mimesis; not as revealing a past reality, now renewed with natural movement, voice, sound, and colour, but as producing a reality effect, along the lines discussed by Roland Barthes. Seen in this light, They Shall Not Grow Old, rather than simply adhering to or deviating from the original "truth" of the archive, becomes an enriched intertextual project that brings several different discursive frameworks into view, an act of reframing that links the cinematic traces of the past to perspectives that have consequences in the present.

Mitosis, the accurate segregation of duplicated genetic material into what will become two new daughter cells, is accompanied by extensive membrane remodelling and membrane trafficking activities. Early in mitosis, adherent cells partially detach from the substratum, round up and their surface area decreases. This likely results from an endocytic uptake of plasma membrane material. As cells enter cytokinesis they re-adhere, flatten and exhibit an associated increase in surface area. The identity of the membrane donor for this phase of mitosis remains unclear. In this paper we demonstrate how lysosomes dynamically redistribute during mitosis and exocytose. Antagonism of lysosomal exocytosis by pharmacological and genetic approaches causes mitosis failure in a significant proportion of cells. We speculate that either lysosomal membrane or luminal content release, possibly both, are therefore required for normal mitosis progression. These findings are important as they reveal a new process required for successful cell division.

Calcium signaling in neurons as in other cell types can lead to varied changes in cellular function. Neuronal Ca2+ signaling processes have also become adapted to modulate the function of specific pathways over a wide variety of time domains and these can have effects on, for example, axon outgrowth, neuronal survival, and changes in synaptic strength. Ca2+ also plays a key role in synapses as the trigger for fast neurotransmitter release. Given its physiological importance, abnormalities in neuronal Ca(2+)signaling potentially underlie many different neurological and neurodegenerative diseases. The mechanisms by which changes in intracellular Ca2+ concentration in neurons can bring about diverse responses is underpinned by the roles of ubiquitous or specialized neuronal Ca(2+)sensors. It has been established that synaptotagmins have key functions in neurotransmitter release, and, in addition to calmodulin, other families of EF-hand-containing neuronal Ca2+ sensors, including the neuronal calcium sensor (NCS) and the calcium-binding protein (CaBP) families, play important physiological roles in neuronal Ca2+ signaling. It has become increasingly apparent that these various Ca(2+)sensors may also be crucial for aspects of neuronal dysfunction and disease either indirectly or directly as a direct consequence of genetic variation or mutations. An understanding of the molecular basis for the regulation of the targets of the Ca2+ sensors and the physiological roles of each protein in identified neurons may contribute to future approaches to the development of treatments for a variety of human neuronal disorders.

Mitosis defects can lead to premature ageing and cancer. Understanding mitosis regulation therefore has important implications for human disease. Early data suggested that calcium (Ca 2+ ) signals could influence mitosis, but these have hitherto not been observed in mammalian cells. Here, we reveal a prolonged yet spatially restricted Ca 2+ signal at the centrosomes of actively dividing cells. Local buffering of the centrosomal Ca 2+ signals, by flash photolysis of the caged Ca 2+ chelator diazo‐2‐acetoxymethyl ester, arrests mitosis. We also provide evidence that this Ca 2+ signal emanates from the endoplasmic reticulum. In summary, we characterize a unique centrosomal Ca 2+ signal as a functionally essential input into mitosis.—Helassa, N., Nugues, C., Rajamanoharan, D., Burgoyne, R. D., Haynes, L. P. A centrosome‐localized calcium signal is essential for mammalian cell mitosis. FASEB J. 33, 14602‐14610 (2019). www.fasebj.org

Dystonia is a neurological movement disorder that provokes muscle spasms and contractions. It is characterized by sustained or intermittent muscle contractions causing abnormal, often repetitive movements and painful postures. Recently, mutations at positions T71N and A190T in the neuronal calcium-binding protein hippocalcin, have been shown to be critical in development of DYT2 dystonia. However, the effect of these mutations on the physiological role of hippocalcin has not yet been elucidated. Using a multidisciplinary approach, we showed that mutations T71N and A190T in hippocalcin did not affect stability, calcium-binding affinity, translocation to cellular membranes (Ca2+/myristoyl switch) and three-dimensional structure of the protein. However, the disease-associated mutations caused a defect in calcium-induced oligomerisation of hippocalcin. In KCl-stimulated SH-SY5Y cells expressing mutated hippocalcin, we observed an increased calcium influx, mostly driven by N-type voltage-gated calcium channels. Our data demonstrate that the dystonia-associated mutations strongly affect hippocalcin cellular functions which suggest a central role for perturbed calcium signalling in DYT2 dystonia. This work has been funded by the Leverhulme Trust RPG-2014-194.

To generate a complex multicellular organism like a human requires enormous expansion in cell numbers and this is achieved predominantly through mitosis. Defects in mitosis can lead to premature ageing and cancer so understanding how it is regulated has important implications for human disease. Early data from plant and invertebrate model systems indicated that calcium (Ca2+) could influence mitosis. Here we explore this key question in the cell biology of mammalian cells by targeting high affinity genetically encoded Ca2+ sensors to mitosis specific subcellular locations. We reveal a prolonged yet spatially restricted Ca2+ signal at the centrosomes of mitotic cells using an actin-targeted Ca2+ sensor. Local depletion of Ca2+ at centrosomes using flash-photolysis of the caged Ca2+ chelator diazo-2 arrests mitosis and we provide evidence that this signal emanates from the endoplasmic reticulum. In summary, we characterize a centrosomal Ca2+ signal as a functionally essential input into mitosis. This extends our understanding of the complex regulatory network controlling cell division and pinpoints Ca2+ as an important controller of this fundamental process.

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.

This chapter explores some of the implications of computer generated imagery for the cinematic representation of the past. It focuses on the most contested and controversial area of contemporary fiction cinema's representation of the past, the use of documentary images as a mode of imaginative reconstruction or re-enactment. The chapter presents an argument that has been made by Alison Landsberg, who has coined the term 'prosthetic memory' to describe the way mass cultural technologies of memory enable individuals to experience events through which they themselves did not live. These arguments appear to have a particular salience for understanding the popularity and the larger cultural significance of films such as Forrest Gump, JFK, Glory, The Hurricane and Saving Private Ryan. The most striking uses of digital compositing and morphing in film is found in Forrest Gump, which digitally rewrites some of the most sensitive scenes of the American past.

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.

Alcohol is a potent pharmacological agent when consumed acutely at sufficient quantities and repeated overuse can lead to addiction and deleterious effects on health. Alcohol is thought to modulate neuronal function through low-affinity interactions with proteins, in particular with membrane channels and receptors. Paradoxically, alcohol acts as both a stimulant and a sedative. The exact molecular mechanisms for the acute effects of ethanol on neurons, as either a stimulant or a sedative, however remain unclear. We investigated the role that the heat shock transcription factor HSF-1 played in determining a stimulatory phenotype of Caenorhabditis elegans in response to physiologically relevant concentrations of ethanol (17 mM; 0.1% v/v). Using genetic techniques, we demonstrate that either RNA interference of hsf-1 or use of an hsf-1(sy441) mutant lacked the enhancement of locomotion in response to acute ethanol exposure evident in wild-type animals. We identify that the requirement for HSF-1 in this phenotype was IL2 neuron-specific and required the downstream expression of the α-crystallin ortholog HSP-16.48 Using a combination of pharmacology, optogenetics, and phenotypic analyses we determine that ethanol activates a Gαs-cAMP-protein kinase A signaling pathway in IL2 neurons to stimulate nematode locomotion. We further implicate the phosphorylation of a specific serine residue (Ser322) on the synaptic protein UNC-18 as an end point for the Gαs-dependent signaling pathway. These findings establish and characterize a distinct neurosensory cell signaling pathway that determines the stimulatory action of ethanol and identifies HSP-16.48 and HSF-1 as novel regulators of this pathway.

Dystonia is a neurological movement disorder that forces the body into twisting, repetitive movements or sometimes painful abnormal postures. With the advent of next-generation sequencing technologies, the homozygous mutations T71N and A190T in the neuronal calcium sensor (NCS) hippocalcin were identified as the genetic cause of primary isolated dystonia (DYT2 dystonia). However, the effect of these mutations on the physiological role of hippocalcin has not yet been elucidated. Using a multidisciplinary approach, we demonstrated that hippocalcin oligomerises in a calcium-dependent manner and binds to voltage-gated calcium channels. Mutations T71N and A190T in hippocalcin did not affect stability, calcium-binding affinity or translocation to cellular membranes (Ca2+/myristoyl switch). We obtained the first crystal structure of hippocalcin and alignment with other NCS proteins showed significant variability in the orientation of the C-terminal part of the molecule, the region expected to be important for target binding. We demonstrated that the disease-causing mutations did not affect the structure of the protein, however both mutants showed a defect in oligomerisation. In addition, we observed an increased calcium influx in KCl-depolarised cells expressing mutated hippocalcin, mostly driven by N-type voltage-gated calcium channels. Our data demonstrate that the dystonia-causing mutations strongly affect hippocalcin cellular functions which suggest a central role for perturbed calcium signalling in DYT2 dystonia.

Alcohol is a potent pharmacological agent when cons umed acutely at sufficient quantities and repeated overuse can lead to addiction and deleteri ous effects on health. Alcohol is thought to modulate neuronal function through low affinity interactions with proteins, in particular with membrane channels and receptors. Paradoxicall y, alcohol acts as both a stimulant and a sedative. The exact molecular mechanisms for the a cute effects of ethanol on neurons, as either a stimulant or a sedative, however remain un clear. We investigated the role that the heat shock transcription factor, HSF-1, played in d etermining a stimulatory phenotype of Caenorhabditis elegans in response to physiologically relevant concentrat ions of ethanol (17 mM; 0.1% v/v). Using genetic techniques we demonst rate that either RNAi of hsf-1 or use of an hsf-1(sy441) mutant lacked the enhancement of locomotion in res pon e to acute ethanol exposure evident in wild-type animals. We identify hat the requirement for HSF-1 in this phenotype was IL2-neuron specific and required the downstream expression of the αcrystallin orthologue HSP-16.48. Using a combinati on of pharmacology, optogenetics and phenotypic analyses we determine that ethanol activ ates a Gαs – cAMP – protein kinase A signalling pathway in IL2 neurons to stimulate nema tode locomotion. We further implicate the phosphorylation of a specific serine residue (S er322) on the synaptic protein UNC-18 as an end-point for the G αs-dependent signalling pathway. These findings esta bli h and characterise a distinct neurosensory cell signallin g pathway that determines the stimulatory action of ethanol and identifies HSP-16.48 and HSF1 as novel regulators of this pathway.

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.

Neuronal calcium sensor-1 (NCS-1) mediates changes in cellular function by regulating various target proteins. Many potential targets have been identified but the physiological significance of only a few has been established. Upon temperature elevation, Caenorhabditis elegans exhibits reversible paralysis. In the absence of NCS-1, worms show delayed onset and a shorter duration of paralysis. This phenotype can be rescued by re-expression of ncs-1 in AIY neurons. Mutants with defects in four potential NCS-1 targets (arf-1.1, pifk-1, trp-1 and trp-2) showed qualitatively similar phenotypes to ncs-1 null worms, although the effect of pifk-1 mutation on time to paralysis was considerably delayed. Inhibition of pifk-1 also resulted in a locomotion phenotype. Analysis of double mutants showed no additive effects between mutations in ncs-1 and trp-1 or trp-2. In contrast, double mutants of arf-1.1 and ncs-1 had an intermediate phenotype, consistent with NCS-1 and ARF-1.1 acting in the same pathway. Over-expression of arf-1.1 in the AIY neurons was sufficient to rescue partially the phenotype of both the arf-1.1 and the ncs-1 null worms. These findings suggest that ARF-1.1 interacts with NCS-1 in AIY neurons and potentially pifk-1 in the Ca(2+) signaling pathway that leads to inhibited locomotion at an elevated temperature.

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.

NSF (N-ethylmaleimide-sensitive fusion protein) is a hexameric ATPase essential for all intracellular membrane traffic steps. In its role in membrane traffic, NSF is recruited to membranes by SNAPs (soluble NSF attachment proteins), which in turn are recruited by a complex of SNARE (SNAP receptor) proteins formed during membrane fusion. SNAP-stimulated NSF ATPase activity then disassembles the SNARE complex, recycling SNAREs for further fusion reactions. In addition to this ubiquitous function, NSF directly interacts with and affects the trafficking of various receptors, notably synaptic glutamate receptors. SNAPs are also thought to have moonlighting roles, notably in store-operated calcium entry. NSF activity can be modulated by S-nitrosylation, oxidation, and phosphorylation, thus enabling sophisticated regulation of its cellular functions.

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.