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Protein aggregation and abnormal lipid homeostasis are both implicated in neurodegeneration through unknown mechanisms. Here we demonstrate that aggregate-membrane interaction is critical to induce a form of cell death called ferroptosis. Importantly, the aggregate-membrane interaction that drives ferroptosis depends both on the conformational structure of the aggregate, as well as the oxidation state of the lipid membrane. We generated human stem cell-derived models of synucleinopathy, characterized by the intracellular formation of α-synuclein aggregates that bind to membranes. In human iPSC-derived neurons with SNCA triplication, physiological concentrations of glutamate and dopamine induce abnormal calcium signaling owing to the incorporation of excess α-synuclein oligomers into membranes, leading to altered membrane conductance and abnormal calcium influx. α-synuclein oligomers further induce lipid peroxidation. Targeted inhibition of lipid peroxidation prevents the aggregate-membrane interaction, abolishes aberrant calcium fluxes, and restores physiological calcium signaling. Inhibition of lipid peroxidation, and reduction of iron-dependent accumulation of free radicals, further prevents oligomer-induced toxicity in human neurons. In summary, we report that peroxidation of polyunsaturated fatty acids underlies the incorporation of β-sheet-rich aggregates into the membranes, and that additionally induces neuronal death. This suggests a role for ferroptosis in Parkinson’s disease, and highlights a new mechanism by which lipid peroxidation causes cell death.

Long-term changes in synaptic strength form the basis of learning and memory. These changes rely upon energy-demanding mechanisms, which are regulated by local Ca2+ signalling. Mitochondria are optimised for providing energy and buffering Ca2+. However, our understanding of the role of mitochondria in regulating synaptic plasticity is incomplete. Here, we have used optical and electrophysiological techniques in cultured hippocampal neurons and ex vivo hippocampal slices from mice with haploinsufficiency of the mitochondrial Ca2+ uniporter (MCU+/−) to address whether reducing mitochondrial Ca2+ uptake alters synaptic transmission and plasticity. We found that cultured MCU+/− hippocampal neurons have impaired Ca2+ clearance, and consequently enhanced synaptic vesicle fusion at presynapses occupied by mitochondria. Furthermore, long-term potentiation (LTP) at mossy fibre (MF) synapses, a process which is dependent on presynaptic Ca2+ accumulation, is enhanced in MCU+/− slices. Our results reveal a previously unrecognised role for mitochondria in regulating presynaptic plasticity of a major excitatory pathway involved in learning and memory.

α-Synuclein is strongly linked to Parkinson’s disease but the molecular targets for its toxicity are not fully clear. However, many neuronal functions damaged in Parkinson’s disease are regulated by signalling between the endoplasmic reticulum (ER) and mitochondria. This signalling involves close physical associations between the two organelles that are mediated by binding of the integral ER protein vesicle-associated membrane protein-associated protein B (VAPB) to the outer mitochondrial membrane protein, protein tyrosine phosphatase-interacting protein 51 (PTPIP51). VAPB and PTPIP51 thus act as a scaffold to tether the two organelles. Here we show that α-synuclein binds to VAPB and that overexpression of wild-type and familial Parkinson’s disease mutant α-synuclein disrupt the VAPB-PTPIP51 tethers to loosen ER–mitochondria associations. This disruption to the VAPB-PTPIP51 tethers is also seen in neurons derived from induced pluripotent stem cells from familial Parkinson’s disease patients harbouring pathogenic triplication of the α-synuclein gene. We also show that the α-synuclein induced loosening of ER–mitochondria contacts is accompanied by disruption to Ca2+ exchange between the two organelles and mitochondrial ATP production. Such disruptions are likely to be particularly damaging to neurons that are heavily dependent on correct Ca2+ signaling and ATP.

Protein aggregation and abnormal lipid homeostasis are both implicated in neurodegeneration through unknown mechanisms. Here we demonstrate that aggregate-membrane interaction is critical to induce a form of cell death called ferroptosis. Importantly, the aggregate-membrane interaction that drives ferroptosis depends both on the conformational structure of the aggregate, as well as the oxidation state of the lipid membrane. We generated human stem cell-derived models of synucleinopathy, characterized by the intracellular formation of α-synuclein aggregates that bind to membranes. In human iPSC-derived neurons with SNCA triplication, physiological concentrations of glutamate and dopamine induce abnormal calcium signaling owing to the incorporation of excess α-synuclein oligomers into membranes, leading to altered membrane conductance and abnormal calcium influx. α-synuclein oligomers further induce lipid peroxidation. Targeted inhibition of lipid peroxidation prevents the aggregate-membrane interaction, abolishes aberrant calcium fluxes, and restores physiological calcium signaling. Inhibition of lipid peroxidation, and reduction of iron-dependent accumulation of free radicals, further prevents oligomer-induced toxicity in human neurons. In summary, we report that peroxidation of polyunsaturated fatty acids underlies the incorporation of β-sheet-rich aggregates into the membranes, and that additionally induces neuronal death. This suggests a role for ferroptosis in Parkinson's disease, and highlights a new mechanism by which lipid peroxidation causes cell death.

Mitochondrial dysfunction is linked to many neurological diseases; therefore, the ability to measure mitochondrial function is of great use for researching disease and testing potential therapeutics. Here we describe a high-content assay to simultaneously measure mitochondrial membrane potential, morphology and cell viability in iPSC-derived neurons. Neurons are seeded into plates suitable for fluorescent microscopy, stained with the mitochondrial membrane potential-dependent dye TMRM, cytoplasmic dye Calcein AM, and nuclear stain Hoechst 33342. Images are acquired in live cells and analyzed using automated image analysis software.

Autophagy is the process by which cellular proteins and organelles are degraded and recycled and is essential to the survival of cells. Defective autophagic degradation has been linked to many neurodegenerative diseases and in particular lysosomal storage diseases. Here we describe a high-content assay to detect defects in the autophagy pathway in induced pluripotent stem cell-derived neurons. This assay utilizes immunofluorescence to stain autophagosomes and uses automated image analysis to measure changes in autophagosome levels in response to modulators of autophagy.

Induced pluripotent stem cell (iPSC) derived neurons are an excellent in vitro model of neurological diseases that are often used in early stage drug discovery projects. Thus far, the use of iPSC-derived cells in small molecule drug screening has been limited, and one of the reasons for this has been the challenge of miniaturization of iPSC culture and differentiation in low volume microwell plate formats. Here we describe a method of seeding iPSC-derived neurons into 384-well plates towards the end of the differentiation procedure. This method covers coating the plates with substrates to aid attachment, dissociation of the cells into a single cell suspension, and seeding onto 384-well plates to give an even distribution of neurons. This method facilitates the use of iPSC-derived neurons for high-content imaging, whole-well assays, and small-molecule drug screening.

Much of the functionality of multicellular systems arises from the spatial organization and dynamic behaviours within and between cells. Current single-cell genomic methods only provide a transcriptional ‘snapshot’ of individual cells. The real-time analysis and perturbation of living cells would generate a step change in single-cell analysis. Here we describe minimally invasive nanotweezers that can be spatially controlled to extract samples from living cells with single-molecule precision. They consist of two closely spaced electrodes with gaps as small as 10–20 nm, which can be used for the dielectrophoretic trapping of DNA and proteins. Aside from trapping single molecules, we also extract nucleic acids for gene expression analysis from living cells without affecting their viability. Finally, we report on the trapping and extraction of a single mitochondrion. This work bridges the gap between single-molecule/organelle manipulation and cell biology and can ultimately enable a better understanding of living cells. A nanotweezer system based on dielectrophoresis can trap, manipulate and extract biomolecules and organelles in living cells at physiological conditions with high spatiotemporal resolution and minimal invasiveness.

Mitochondrial dysfunction is linked to many neurological diseases; therefore, the ability to measure mitochondrial function is of great use for researching disease and testing potential therapeutics. Here we describe a high-content assay to simultaneously measure mitochondrial membrane potential, morphology and cell viability in iPSC-derived neurons. Neurons are seeded into plates suitable for fluorescent microscopy, stained with the mitochondrial membrane potential-dependent dye TMRM, cytoplasmic dye Calcein AM, and nuclear stain Hoechst 33342. Images are acquired in live cells and analyzed using automated image analysis software.

Induced pluripotent stem cell (iPSC) derived neurons are an excellent in vitro model of neurological diseases that are often used in early stage drug discovery projects. Thus far, the use of iPSC-derived cells in small molecule drug screening has been limited, and one of the reasons for this has been the challenge of miniaturization of iPSC culture and differentiation in low volume microwell plate formats. Here we describe a method of seeding iPSC-derived neurons into 384-well plates towards the end of the differentiation procedure. This method covers coating the plates with substrates to aid attachment, dissociation of the cells into a single cell suspension, and seeding onto 384-well plates to give an even distribution of neurons. This method facilitates the use of iPSC-derived neurons for high-content imaging, whole-well assays, and small-molecule drug screening.

Autophagy is the process by which cellular proteins and organelles are degraded and recycled and is essential to the survival of cells. Defective autophagic degradation has been linked to many neurodegenerative diseases and in particular lysosomal storage diseases. Here we describe a high-content assay to detect defects in the autophagy pathway in induced pluripotent stem cell-derived neurons. This assay utilizes immunofluorescence to stain autophagosomes and uses automated image analysis to measure changes in autophagosome levels in response to modulators of autophagy.

The long, asymmetric and specialised architecture of neuronal processes necessitates a properly regulated transport network of molecular motors and cytoskeletal tracks. This allows appropriate distribution of cargo for correct formation and activity of the synapse, and thus normal neuronal communication. This communication is impaired in psychiatric disease, and ongoing studies have proposed that Disrupted in schizophrenia 1 (DISC1) is an important genetic risk factor for these disorders. The mechanisms by which DISC1 dysfunction might increase propensity to psychiatric disease are not completely understood; however, an emerging theme is that DISC1 can function as a key regulator of neuronal intracellular trafficking. Transport of a wide range of potential cargoes - including mRNAs, neurotransmitter receptors, vesicles and mitochondria - can be modulated by DISC1, and therefore is susceptible to DISC1 dysfunction. This theme highlights the importance of understanding precisely how DISC1 can regulate intracellular trafficking, and suggests that a novel approach to the treatment of psychiatric disorders could be provided by targeting this protein and the trafficking machinery with which it interacts.

The misfolding and aggregation of proteins into amyloid fibrils characterizes many neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. We report here a method, termed SAVE (single aggregate visualization by enhancement) imaging, for the ultrasensitive detection of individual amyloid fibrils and oligomers using single-molecule fluorescence microscopy. We demonstrate that this method is able to detect the presence of amyloid aggregates of α-synuclein, tau, and amyloid-β. In addition, we show that aggregates can also be identified in human cerebrospinal fluid (CSF). Significantly, we see a twofold increase in the average aggregate concentration in CSF from Parkinson's disease patients compared to age-matched controls. Taken together, we conclude that this method provides an opportunity to characterize the structural nature of amyloid aggregates in a key biofluid, and therefore has the potential to study disease progression in both animal models and humans to enhance our understanding of neurodegenerative disorders.

Induced pluripotent stem cells and their derivatives have become an important tool for researching disease mechanisms. It is hoped that they could be used to discover new therapies by providing the most reliable and relevant human in vitro disease models for drug discovery. This review will summarize recent efforts to use stem cell–derived neurons for drug screening. We also explain the current hurdles to using these cells for high-throughput pharmaceutical screening and developments that may help overcome these hurdles. Finally, we critically discuss whether induced pluripotent stem cell–derived neurons will come to fruition as a model that is regularly used to screen for drugs to treat neurological diseases.

Mitochondrial dysfunction is implicated in many neurodegenerative diseases including Parkinson’s disease (PD). Induced pluripotent stem cells (iPSCs) provide a unique cell model for studying neurological diseases. We have established a high-content assay that can simultaneously measure mitochondrial function, morphology and cell viability in iPSC-derived dopaminergic neurons. iPSCs from PD patients with mutations in SNCA and unaffected controls were differentiated into dopaminergic neurons, seeded in 384-well plates and stained with the mitochondrial membrane potential dependent dye TMRM, alongside Hoechst-33342 and Calcein-AM. Images were acquired using an automated confocal screening microscope and single cells were analysed using automated image analysis software. PD neurons displayed reduced mitochondrial membrane potential and altered mitochondrial morphology compared to control neurons. This assay demonstrates that high content screening techniques can be applied to the analysis of mitochondria in iPSC-derived neurons. This technique could form part of a drug discovery platform to test potential new therapeutics for PD and other neurodegenerative diseases.

Synapses enable neurons to communicate with each other and are therefore a prerequisite for normal brain function. Presynaptically, this communication requires energy and generates large fluctuations in calcium concentrations. Mitochondria are optimized for supplying energy and buffering calcium, and they are actively recruited to presynapses. However, not all presynapses contain mitochondria; thus, how might synapses with and without mitochondria differ? Mitochondria are also increasingly recognized to serve additional functions at the presynapse. Here, we discuss the importance of presynaptic mitochondria in maintaining neuronal homeostasis and how dysfunctional presynaptic mitochondria might contribute to the development of disease.

Highlights • Mutations in the ROC, COR and Kinase domain of LRRK2 alter the autophagic response to starvation. • LC3-I/II ratio following starvation is altered by mutations, as well as p62 and WIPI2 positive puncta. • This occurs independently of any alteration in downstream targets of mTORC1., LRRK2 is one of the most important genetic contributors to Parkinson’s disease (PD). Point mutations in this gene cause an autosomal dominant form of PD, but to date no cellular phenotype has been consistently linked with mutations in each of the functional domains (ROC, COR and Kinase) of the protein product of this gene. In this study, primary fibroblasts from individuals carrying pathogenic mutations in the three central domains of LRRK2 were assessed for alterations in the autophagy/lysosomal pathway using a combination of biochemical and cellular approaches. Mutations in all three domains resulted in alterations in markers for autophagy/lysosomal function compared to wild type cells. These data highlight the autophagy and lysosomal pathways as read outs for pathogenic LRRK2 function and as a marker for disease, and provide insight into the mechanisms linking LRRK2 function and mutations.

α-Synuclein is strongly linked to Parkinson’s disease but the molecular targets for its toxicity are not fully clear. However, many neuronal functions damaged in Parkinson’s disease are regulated by signalling between the endoplasmic reticulum (ER) and mitochondria. This signalling involves close physical associations between the two organelles that are mediated by binding of the integral ER protein vesicle-associated membrane protein-associated protein B (VAPB) to the outer mitochondrial membrane protein, protein tyrosine phosphatase-interacting protein 51 (PTPIP51). VAPB and PTPIP51 thus act as a scaffold to tether the two organelles. Here we show that α-synuclein binds to VAPB and that overexpression of wild-type and familial Parkinson’s disease mutant α-synuclein disrupt the VAPB-PTPIP51 tethers to loosen ER–mitochondria associations. This disruption to the VAPB-PTPIP51 tethers is also seen in neurons derived from induced pluripotent stem cells from familial Parkinson’s disease patients harbouring pathogenic triplication of the α-synuclein gene. We also show that the α-synuclein induced loosening of ER–mitochondria contacts is accompanied by disruption to Ca2+ exchange between the two organelles and mitochondrial ATP production. Such disruptions are likely to be particularly damaging to neurons that are heavily dependent on correct Ca2+ signaling and ATP. Electronic supplementary material The online version of this article (doi:10.1007/s00401-017-1704-z) contains supplementary material, which is available to authorized users.

We examined emotional responses to standard affective pictures in 18 psychogenic nonepileptic seizure (PNES) patients. Given reports of trauma and posttraumatic stress symptoms (PTS) in many PNES patients, comparison groups were seizure-free individuals high and low in PTS (PTS-high, PTS-low; n=18 per group). Patients with psychogenic nonepileptic seizures (1) reported more emotional intensity to neutral and pleasant pictures than PTS-low and more intensity to neutral pictures than PTS-high, and (2) showed less positive emotional behavior to pleasant pictures than PTS-high. Groups did not differ in pleasantness/unpleasantness ratings, negative emotional behavior, cardiac interbeat interval, or respiratory sinus arrhythmia (RSA) reactivity to the pictures. Patients with psychogenic nonepileptic seizures reported more general emotion regulation difficulties and showed lower baseline RSA than PTS-low but not PTS-high. In sum, intense emotional experience and diminished positive emotional behavior characterized PNES patients' emotional responses.

Emotion research historically has adopted a fairly homogeneous view of positive emotions. The aim of the current study was to explore how two positive emotions, amusement and joy, differ in subjective, behavioral, cardiovascular, and respiratory characteristics. Thirty-nine participants viewed two film clips, each selected to elicit amusement or joy. As predicted, participants reported more amusement, showed more positive facial expressions and laughter, and exhibited less heart rate deceleration and a larger increase in respiratory amplitude in response to the amusement clip than in response to the joy clip. In addition, subjective, behavioral, and physiological indicators were more closely related in amusement than joy, which was largely attributable to laughter during amusement. The current study adds to a growing literature suggesting the importance of adopting a more nuanced conceptualization of positive emotion.

The last decade has seen clear links emerge between the genetic determinants and neuropathological hallmarks of parkinsonism and dementia, notably with the discovery of mutations in alpha-synuclein and tau. Following the description of mutations in LRRK2 linked to Parkinson's disease, characterized by variable pathology including either alpha-synuclein or tau deposition, it has been suggested that LRRK2 functions as an upstream regulator of Parkinson's disease pathogenesis. This minireview explores this model, in the context of our current understanding of the biochemistry of LRRK2, alpha-synuclein and tau.

Neurons are highly polarised cells with an elaborate and diverse cytoarchitecture. But this complex architecture presents a major problem: how to appropriately distribute metabolic resources where they are most needed within the cell. The solution comes in the form of mitochondria: highly dynamic organelles subject to a repertoire of trafficking, fission/fusion and quality control systems which work in concert to orchestrate a precisely distributed and healthy mitochondrial network. Mitochondria are critical for maintaining local energy supply and buffering Ca(2+) flux within neurons, and are increasingly recognised as being essential for healthy neuronal function. Mitochondrial movements are facilitated by their coupling to microtubule-based transport via kinesin and dynein motors. Adaptor proteins are required for this coupling and the mitochondrial Rho GTPases Miro1 and Miro2 are core components of this machinery. Both Miros have Ca(2+)-sensing and GTPase domains, and are therefore ideally suited to coordinating mitochondrial dynamics with intracellular signalling pathways and local energy turnover. In this review, we focus on Miro's role in mediating mitochondrial transport in neurons, and the relevance of these mechanisms to neuronal health and disease.

The alternative splicing of the tau gene, MAPT, generates six protein isoforms in the adult human central nervous system (CNS). Tau splicing is developmentally regulated and dysregulated in disease. Mutations in MAPT that alter tau splicing cause frontotemporal dementia (FTD) with tau pathology, providing evidence for a causal link between altered tau splicing and disease. The use of induced pluripotent stem cell (iPSC)-derived neurons has revolutionized the way we model neurological disease in vitro. However, as most tau mutations are located within or around the alternatively spliced exon 10, it is important that iPSC–neurons splice tau appropriately in order to be used as disease models. To address this issue, we analyzed the expression and splicing of tau in iPSC-derived cortical neurons from control patients and FTD patients with the 10 + 16 intronic mutation in MAPT. We show that control neurons only express the fetal tau isoform (0N3R), even at extended time points of 100 days in vitro. Neurons from FTD patients with the 10 + 16 mutation in MAPT express both 0N3R and 0N4R tau isoforms, demonstrating that this mutation overrides the developmental regulation of exon 10 inclusion in our in vitro model. Further, at extended time points of 365 days in vitro, we observe a switch in tau splicing to include six tau isoforms as seen in the adult human CNS. Our results demonstrate the importance of neuronal maturity for use in in vitro modeling and provide a system that will be important for understanding the functional consequences of altered tau splicing.

Astrocytes play a critical role during the development and the maintenance of the CNS in health and disease. Yet, their lack of accessibility from fetuses and from the brain of diseased patients has hindered our understanding of their full implication in developmental and pathogenic processes. Human pluripotent stem cells (PSCs) are an alternative source to obtain large quantities of astrocytes in vitro, for mechanistic studies of development and disease. However, these studies often require highly pure populations of astrocytes, which are not always achieved, depending on the PSC lines and protocols used. Here, we describe the generation and characterization of human PSC reporter lines expressing TagRFP driven by the ABC1D region of the human GFAP promoter, as new cellular model for generating homogenous population of astrocytes generated from CNS regionally defined PSC-derived neural progenitors. GFA(ABC1D)::TagRFP-expressing astrocytes can be purified by fluorescent-activated cell sorting and maintain a bright expression for several additional weeks. These express canonical astrocyte markers NF1A, S100 beta, CX43, GLAST, GS and CD44. These new cellular models, from which highly pure populations of fluorescence-expressing astrocytes can be obtained, provide a new platform for studies where pure or fluorescently labeled astrocyte populations are necessary, for example to assess pro-inflammatory cytokine and chemokine release in response to specific treatment, and uptake and degradation of fluorescently labeled pathogenic proteins, as reported in this study. (C) 2015 The Authors. Published by Elsevier B.V.

Bone marrow contains many cellular elements that may contribute to fracture repair. We used a pluripotential stromal cell in a mouse model to demonstrate the presence of transplanted cells in fracture hematoma and subsequently in maturing fracture callus. Cells were transduced with traceable genes (lac Z and neomycin resistance) and traced in vivo after intravenous injection into syngeneic mice. These transduced cells home to bone marrow, suggesting that they might be detected in fracture callus. Cells were injected intravenously into mice and stabilized femoral shaft fractures were induced. Control mice received intravenous lactated-Ringer's solution prior to fracture. Callus tissue and marrow were examined histologically from I to 10 weeks after fracture to detect transplanted cells. Transplanted cells were detected in fracture callus in areas, and at times, of most active bone formation. Control specimens showed minimal staining of the callus tissue. Levels of the traceable gene in fracture callus increased, reached a peak between 3 and 4 weeks after fracture, then diminished and disappeared by 10 weeks post-fracture as woven bone at the fracture site was replaced by lamellar bone with cells from the host mouse. The results show that pluripotent bone marrow cells home to the marrow after systemic injection and localize in fracture callus.

Alpha-synuclein aggregation and mitochondrial dysfunction are central to the pathogenesis of Parkinson's disease (PD). This study investigates the structure specific effects of α-synuclein on mitochondrial function and demonstrates the mechanism by which oligomerisation of the protein results in a toxic gain of function within mitochondria.We have previously shown that monomeric α-synuclein enters mitochondria where it interacts with ATP synthase, and aids its efficiency. Employing a proximity ligation assay, we show an interaction between oligomers and ATP synthase, a proposed key component of the mPTP. We demonstrate that beta sheet rich α-synuclein oligomers uniquely induce opening of the mitochondrial permeability transition pore (mPTP) in whole cells and isolated mitochondria. We report that oligomers, but not monomers, generate reactive oxygen species and mitochondrial membrane lipid peroxidation, and these trigger mPTP opening. This oligomer-induced effects lead to neuronal cell death, which could be abrogated by preincubation of the cells with the mPTP inhibitor Cyclosporin A.We validated our findings in an iPS derived neuronal model with an α-synuclein triplication in which increased levels of aggregated α-synuclein cause early onset PD. In this model we report that oligomers also have a strong interaction with the ATP synthase. Furthermore, we demonstrate a low threshold for mPTP opening on exposure to high laser and calcium. mPTP opening in this model led to cell death which could again be prevented by mPTP inhibition. We were thus able to conclude that the mitochondrial abnormalities seen in the iPS cells with high levels of α-synuclein were likely to be mediated by the beta-sheet oligomeric form of the protein.This study provides evidence of a direct effect of oligomers on mitochondria and is the first to specifically link oligomeric α-synuclein to ATP synthase leading to neuronal death.

The transfer of α-synuclein (α-syn) between cells has been proposed to be the primary mechanism of disease spreading in Parkinson's disease. Several cellular models exist that monitor the uptake of recombinant α-syn from the culture medium. Here we established a more physiologically relevant model system in which α-syn is produced and transferred between mammalian neurons. We generated cell lines expressing either α-syn tagged with fluorescent proteins or fluorescent tags alone then we co-cultured these cell lines to measure protein uptake. We used live-cell imaging to demonstrate intercellular α-syn transfer and used flow cytometry and high content analysis to quantify the transfer. We then successfully inhibited intercellular protein transfer genetically by down-regulating dynamin or pharmacologically using dynasore or heparin. In addition, we differentiated human induced pluripotent stem cells carrying a triplication of the α-syn gene into dopaminergic neurons. These cells secreted high levels of α-syn, which was taken up by neighboring neurons. Collectively, our co-culture systems provide simple but physiologically relevant tools for the identification of genetic modifiers or small molecules that inhibit α-syn cell-to-cell transfer.

As A GRADUATE STUDENT IN THE EARLY 1970s, I had no thought of working in any occupation other than university teaching. In 1974, the year I completed my doctoral degree, I held only a vague sense of historians working in archives and special collection libraries, and the term "public historian" had yet to appear in the professional literature. The dreadful job market in academe, coupled with a strange set of circumstances, led me into my first administrative job with a public history institution, the Ohio

A 43-year-old British man, who had spent the last 18 months working on road building projects in rural Rwanda, presented to hospital in Kigali with a 2-day diarrheal illness, 1 month after swimming in the western shores of Lake Muhazi near Rwesero. Two days later he developed left-sided numbness and weakness and unsteadiness on his feet. HIV testing was negative and CSF examination was normal. No neuroimaging was performed. Dexamethasone, sulfadiazine, pyrimethamine, and broad-spectrum antibiotics were commenced. However, over the following 48 hours he developed worsening headache, diplopia, dysarthria, ataxia, and left hemiparesis. He was repatriated to the United Kingdom. On arrival he was drowsy, orientated, and afebrile. He had profound cerebellar dysarthria. Pupils and fundi were normal. Eye movements were abnormal with a left VIth nerve palsy and multidirectional gaze-evoked nystagmus. There were also left Vth and lower motor neuron VIIth nerve palsies, moderate left hemiparesis, bilateral ataxia, left extensor plantar response, and left hemisensory impairment. Functionally he was able to stand but unable to walk. Examination was otherwise unremarkable. MRI brain demonstrated extensive signal change with marked swelling throughout the pons and lower midbrain (figure, A and B) and patchy enhancement (figure, C and D). Figure MRI and histologic findings (A) Axial FLAIR sequence and (B) …

α-synuclein(α-syn) plays a prominent role in the degeneration of midbrain dopaminergic (mDA) neurons in Parkinson disease (PD). However, only a few studies on α-syn have been carried out in the mDA neurons in vivo, which may be attributed to a lack of α-syn transgenic mice that develop PD-like severe degeneration of mDA neurons. To gain mechanistic insights into the α-syn-induced mDA neurodegeneration, we generated a new line of tetracycline-regulated inducible transgenic mice that overexpressed the PD-related α-syn A53T missense mutation in the mDA neurons. Here we show that the mutant mice developed profound motor disabilities and robust mDA neurodegeneration, resembling some key motor and pathological phenotypes of PD. We further systematically examined the subcellular abnormalities appeared in the mDA neurons of mutant mice, and observed a profound decrease of dopamine release, the fragmentation of Golgi apparatus, and impairments of autophagy/lysosome degradation pathways in these neurons. To further understand the specific molecular events leading to the α-syn-dependent degeneration of mDA neurons, we found that over-expression of α-syn promoted a proteasome-dependent degradation of nuclear receptor related 1 protein (Nurr1); while inhibition of Nurr1 degradation ameliorated the α-syn-induced loss of mDA neurons. Given that Nurr1 plays an essential role in maintaining the normal function and survival of mDA neurons, our studies suggest that the α-syn-mediated suppression of Nurr1 protein expression may contribute to the preferential vulnerability of mDA neurons in the pathogenesis of PD.

Parkinson's disease is caused by the premature death of neurons in the midbrain. By contrast, cancer spawns from cells that refuse to die. We would therefore expect their pathogenic mechanisms to be very different. However, recent genetic studies and emerging functional work show that strikingly similar and overlapping pathways are involved in both diseases. We consider these areas of convergence and discuss how insights from one disease can inform us about, and possibly help us to treat, the other.

Genetic studies of Parkinson's disease over the last decade or more have revolutionized our understanding of this condition. α-Synuclein was the first gene to be linked to Parkinson's disease, and is arguably the most important: the protein is the principal constituent of Lewy bodies, and variation at its locus is the major genetic risk factor for sporadic disease. Intriguingly, duplications and triplications of the locus, as well as point mutations, cause familial disease. Therefore, subtle alterations of α-synuclein expression can manifest with a dramatic phenotype. We outline the clinical impact of α-synuclein locus multiplications, and the implications that this has for Parkinson's disease pathogenesis. Finally, we discuss potential strategies for disease-modifying therapies for this currently incurable disorder.

A major barrier to research on Parkinson's disease is inaccessibility of diseased tissue for study. One solution is to derive induced pluripotent stem cells from patients and differentiate them into neurons affected by disease. Triplication of SNCA, encoding α-synuclein, causes a fully penetrant, aggressive form of Parkinson's disease with dementia. α-Synuclein dysfunction is the critical pathogenic event in Parkinson's disease, multiple system atrophy and dementia with Lewy bodies. Here we produce multiple induced pluripotent stem cell lines from an SNCA triplication patient and an unaffected first-degree relative. When these cells are differentiated into midbrain dopaminergic neurons, those from the patient produce double the amount of α-synuclein protein as neurons from the unaffected relative, precisely recapitulating the cause of Parkinson's disease in these individuals. This model represents a new experimental system to identify compounds that reduce levels of α-synuclein, and to investigate the mechanistic basis of neurodegeneration caused by α-synuclein dysfunction., Pluripotent stem cells can be generated from the somatic cells of humans and are a useful model to study disease. Here, pluripotent stem cells are made from a patient with familial Parkinson's disease, and the resulting neurons exhibit elevated levels of α-synuclein, recapitulating the molecular features of the patient's disease.

This family has autosomal dominant parkinsonism due to α-synuclein triplication. Dopamine-responsive parkinsonism, cognitive difficulties, RBD, and dysautonomia are common. End-stage illness includes contractures, myoclonus, and severe motor and cognitive impairment. Of note, there was pre-symptomatic amphetamine/cocaine use in two of the cases described.

Objective.To report a large outbreak ofClostridium difficileinfection (CDI; ribotype 027) between June 2007 and August 2008, describe infection control measures, and evaluate the impact of restricting the use of fluoroquinolones in controlling the outbreak.Design.Outbreak investigation in 3 acute care hospitals of the Northern Health and Social Care Trust in Northern Ireland.Interventions.Implementation of a series of CDI control measures that targeted high-risk antibiotic agents (ie, restriction of fluoroquinolones), infection control practices, and environmental hygiene.Results.A total of 318 cases of CDI were identified during the outbreak, which was the result of the interaction betweenC. difficileribotype 027 being introduced into the affected hospitals for the first time and other predisposing risk factors (ranging from host factors to suboptimal compliance with antibiotic guidelines and infection control policies). The 30-day all-cause mortality rate was 24.5%; however, CDI was the attributable cause of death for only 2.5% of the infected patients. Time series analysis showed that restricting the use of fluoroquinolones was associated with a significant reduction in the incidence of CDI (coefficient, —0.054; lag time, 4 months;P= .003).Conclusion.These findings provide additional evidence to support the value of antimicrobial stewardship as an essential element of multifaceted interventions to control CDI outbreaks. The present CDI outbreak was ended following the implementation of an action plan improving communication, antibiotic stewardship, infection control practices, environmental hygiene, and surveillance.

Point mutations in LRRK2 cause autosomal dominant Parkinson's disease. Despite extensive efforts to determine the mechanism of cell death in patients with LRRK2 mutations, the aetiology of LRRK2 PD is not well understood. To examine possible alterations in gene expression linked to the presence of LRRK2 mutations, we carried out a case versus control analysis of global gene expression in three systems: fibroblasts isolated from LRRK2 mutation carriers and healthy, non-mutation carrying controls; brain tissue from G2019S mutation carriers and controls; and HEK293 inducible LRRK2 wild type and mutant cell lines. No significant alteration in gene expression was found in these systems following correction for multiple testing. These data suggest that any alterations in basal gene expression in fibroblasts or cell lines containing mutations in LRRK2 are likely to be quantitatively small. This work suggests that LRRK2 is unlikely to play a direct role in modulation of gene expression, although it remains possible that this protein can influence mRNA expression under pathogenic cicumstances.