CCT complex restricts neuropathogenic protein aggregation via autophagy

Aberrant protein aggregation is controlled by various chaperones, including CCT (chaperonin containing TCP-1)/TCP-1/TRiC. Mutated CCT4/5 subunits cause sensory neuropathy and CCT5 expression is decreased in Alzheimer's disease. Here, we show that CCT integrity is essential for autophagosome degradation in cells or Drosophila and this phenomenon is orchestrated by the actin cytoskeleton. When autophagic flux is reduced by compromise of individual CCT subunits, various disease-relevant autophagy substrates accumulate and aggregate. The aggregation of proteins like mutant huntingtin, ATXN3 or p62 after CCT2/5/7 depletion is predominantly autophagy dependent, and does not further increase with CCT knockdown in autophagy-defective cells/organisms, implying surprisingly that the effect of loss-of-CCT activity on mutant ATXN3 or huntingtin oligomerization/aggregation is primarily a consequence of autophagy inhibition rather than loss of physiological anti-aggregation activity for these proteins. Thus, our findings reveal an essential partnership between two key components of the proteostasis network and implicate autophagy defects in diseases with compromised CCT complex activity.We are grateful to the Wellcome Trust (Principal Research Fellowship to DCR (095317/Z/11/Z)), a Strategic Grant to Cambridge Institute for Medical Research (100140/Z/12/Z), NIHR Biomedical Research Unit in Dementia at Addenbrooke’s Hospital, the Treat PolyQ project (European community’s Seventh Framework Programme under grant agreement No 264508) and the Wellcome Trust/MRC strategic grant for neurodegeneration (D.C.R. and C.J.O.'K.) for funding. DCC was supported by an Alzheimer’s Research U.K. Senior Research Fellowship (ART-SRF2010-2) and by the Wellcome Trust (082604/2/07/Z)

Combined rotation scarf and Akin osteotomies for hallux valgus: a patient focussed 9 year follow up of 50 patients

<p>Abstract</p> <p>Background</p> <p>The Cochrane review of hallux valgus surgery has disputed the scientific validity of hallux valgus research. Scoring systems and surrogate measures such as x-ray angles are commonly reported at just one year post operatively but these are of dubious relevance to the patient. In this study we extended the follow up to a minimum of 8 years and sought to address patient specific concerns with hallux valgus surgery. The long term follow up also allowed a comprehensive review of the complications associated with the combined rotation scarf and Akin osteotomies.</p> <p>Methods</p> <p>Between 1996 and 1999, 101 patients underwent rotation scarf and Akin osteotomies for the treatment of hallux valgus. All patients were contacted and asked to participate in this study. 50 female participants were available allowing review of 73 procedures. The average follow up was over 9 years and the average age at the time of surgery was 57. The participants were physically examined and interviewed.</p> <p>Results</p> <p>Post-operatively, in 86% of the participants there were no footwear restrictions. Stiffness of the first metatarsophalangeal joint was reported in 8% (6 feet); 10% were unhappy with the cosmetic appearance of their feet, 3 feet had hallux varus, and 2 feet had recurrent hallux valgus. There were no foot-related activity restrictions in 92% of the group. Metatarsalgia occurred in 4% (3 feet). 96% were better than before surgery and 88% were completely satisfied with their post-operative result. Hallux varus was the greatest single cause of dissatisfaction. The most common adverse event in the study was internal fixation irritation. Hallux valgus surgery is not without risk and these findings could be useful in the informed consent process.</p> <p>Conclusions</p> <p>When combined the rotation scarf and Akin osteotomies are an effective treatment for hallux valgus that achieves good long-term correction with a low incidence of recurrence, footwear restriction or metatarsalgia. The nature of the osteotomies allows early return to normal shoes and activity without the need for postoperative immobilisation in a plaster cast.</p

Modeling of axonal endoplasmic reticulum network by spastic paraplegia proteins

Axons contain a smooth tubular endoplasmic reticulum (ER) network that is thought to be continuous with ER throughout the neuron; the mechanisms that form this axonal network are unknown. Mutations affecting reticulon or REEP proteins, with intramembrane hairpin domains that model ER membranes, cause an axon degenerative disease, hereditary spastic paraplegia (HSP). We show that Drosophila axons have a dynamic axonal ER network, which these proteins help to model. Loss of HSP hairpin proteins causes ER sheet expansion, partial loss of ER from distal motor axons, and occasional discontinuities in axonal ER. Ultrastructural analysis reveals an extensive ER network in axons, which shows larger and fewer tubules in larvae that lack reticulon and REEP proteins, consistent with loss of membrane curvature. Therefore HSP hairpin-containing proteins are required for shaping and continuity of axonal ER, thus suggesting roles for ER modeling in axon maintenance and function.RCUK | Biotechnology and Biological Sciences Research Council (BBSRC): Lu Zhao, Cahir J O'Kane, BB/L021706/1; Wellcome: Martin Stofanko, Cahir J O'Kane, 08136; European Commission (EC): Lu Zhao, Niamh C O'Sullivan, Sophie Zaessinger, Olivier Blard, MCSA fellowships 220851,220874,236777,660516; Yousef Jameel Foundation: Belgin Yalçın; Singapore A*STAR Scholarship: Zi Han Kang, BM/RES/07/005; Cambridge Commonwealth, European and International Trust (Cambridge Commonwealth, European & International Trust): Belgin Yalçın, Anood Sohail; Pakistan Higher Education Council Scholarship: Anood Sohail; Motor Neurone Disease Association (MNDA): Alex L Patto, Studentship 861-792 The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication

Genetic Labeling of Neuronal Subsets through Enhancer Trapping in Mice

The ability to label, visualize, and manipulate subsets of neurons is critical for elucidating the structure and function of individual cell types in the brain. Enhancer trapping has proved extremely useful for the genetic manipulation of selective cell types in Drosophila. We have developed an enhancer trap strategy in mammals by generating transgenic mice with lentiviral vectors carrying single-copy enhancer-detector probes encoding either the marker gene lacZ or Cre recombinase. This transgenic strategy allowed us to genetically identify a wide variety of neuronal subpopulations in distinct brain regions. Enhancer detection by lentiviral transgenesis could thus provide a complementary method for generating transgenic mouse libraries for the genetic labeling and manipulation of neuronal subsets

Revised Selection Criteria for Candidate Restriction Enzymes in Genome Walking

A new method to improve the efficiency of flanking sequence identification by genome walking was developed based on an expanded, sequential list of criteria for selecting candidate enzymes, plus several other optimization steps. These criteria include: step (1) initially choosing the most appropriate restriction enzyme according to the average fragment size produced by each enzyme determined using in silico digestion of genomic DNA, step (2) evaluating the in silico frequency of fragment size distribution between individual chromosomes, step (3) selecting those enzymes that generate fragments with the majority between 100 bp and 3,000 bp, step (4) weighing the advantages and disadvantages of blunt-end sites vs. cohesive-end sites, step (5) elimination of methylation sensitive enzymes with methylation-insensitive isoschizomers, and step (6) elimination of enzymes with recognition sites within the binary vector sequence (T-DNA and plasmid backbone). Step (7) includes the selection of a second restriction enzyme with highest number of recognition sites within regions not covered by the first restriction enzyme. Step (8) considers primer and adapter sequence optimization, selecting the best adapter-primer pairs according to their hairpin/dimers and secondary structure. In step (9), the efficiency of genomic library development was improved by column-filtration of digested DNA to remove restriction enzyme and phosphatase enzyme, and most important, to remove small genomic fragments (<100 bp) lacking the T-DNA insertion, hence improving the chance of ligation between adapters and fragments harbouring a T-DNA. Two enzymes, NsiI and NdeI, fit these criteria for the Arabidopsis thaliana genome. Their efficiency was assessed using 54 T3 lines from an Arabidopsis SK enhancer population. Over 70% success rate was achieved in amplifying the flanking sequences of these lines. This strategy was also tested with Brachypodium distachyon to demonstrate its applicability to other larger genomes

P-Element Homing Is Facilitated by engrailed Polycomb-Group Response Elements in Drosophila melanogaster

P-element vectors are commonly used to make transgenic Drosophila and generally insert in the genome in a nonselective manner. However, when specific fragments of regulatory DNA from a few Drosophila genes are incorporated into P-transposons, they cause the vectors to be inserted near the gene from which the DNA fragment was derived. This is called P-element homing. We mapped the minimal DNA fragment that could mediate homing to the engrailed/invected region of the genome. A 1.6 kb fragment of engrailed regulatory DNA that contains two Polycomb-group response elements (PREs) was sufficient for homing. We made flies that contain a 1.5kb deletion of engrailed DNA (enΔ1.5) in situ, including the PREs and the majority of the fragment that mediates homing. Remarkably, homing still occurs onto the enΔ1. 5 chromosome. In addition to homing to en, P[en] inserts near Polycomb group target genes at an increased frequency compared to P[EPgy2], a vector used to generate 18,214 insertions for the Drosophila gene disruption project. We suggest that homing is mediated by interactions between multiple proteins bound to the homing fragment and proteins bound to multiple areas of the engrailed/invected chromatin domain. Chromatin structure may also play a role in homing

Alcohol and cannabis use as risk factors for injury – a case-crossover analysis in a Swiss hospital emergency department

BACKGROUND: There is sufficient and consistent evidence that alcohol use is a causal risk factor for injury. For cannabis use, however, there is conflicting evidence; a detrimental dose-response effect of cannabis use on psychomotor and other relevant skills has been found in experimental laboratory studies, while a protective effect of cannabis use has also been found in epidemiological studies. METHODS: Implementation of a case-crossover design study, with a representative sample of injured patients (N = 486; 332 men; 154 women) from the Emergency Department (ED) of the Lausanne University Hospital, which received treatment for different categories of injuries of varying aetiology. RESULTS: Alcohol use in the six hours prior to injury was associated with a relative risk of 3.00 (C.I.: 1.78, 5.04) compared with no alcohol use, a dose-response relationship also was found. Cannabis use was inversely related to risk of injury (RR: 0.33; C.I.: 0.12, 0.92), also in a dose-response like manner. However, the sample size for people who had used cannabis was small. Simultaneous use of alcohol and cannabis did not show significantly elevated risk. CONCLUSION: The most surprising result of our study was the inverse relationship between cannabis use and injury. Possible explanations and underlying mechanisms, such as use in safer environments or more compensatory behavior among cannabis users, were discussed

Tolerance and cross-tolerance to neurocognitive effects of THC and alcohol in heavy cannabis users

and cross-tolerance to neurocognitive effects of THC and alcohol in heavy cannabis user

CNF1 Improves Astrocytic Ability to Support Neuronal Growth and Differentiation In vitro

Modulation of cerebral Rho GTPases activity in mice brain by intracerebral administration of Cytotoxic Necrotizing Factor 1 (CNF1) leads to enhanced neurotransmission and synaptic plasticity and improves learning and memory. To gain more insight into the interactions between CNF1 and neuronal cells, we used primary neuronal and astrocytic cultures from rat embryonic brain to study CNF1 effects on neuronal differentiation, focusing on dendritic tree growth and synapse formation, which are strictly modulated by Rho GTPases. CNF1 profoundly remodeled the cytoskeleton of hippocampal and cortical neurons, which showed philopodia-like, actin-positive projections, thickened and poorly branched dendrites, and a decrease in synapse number. CNF1 removal, however, restored dendritic tree development and synapse formation, suggesting that the toxin can reversibly block neuronal differentiation. On differentiated neurons, CNF1 had a similar effacing effect on synapses. Therefore, a direct interaction with CNF1 is apparently deleterious for neurons. Since astrocytes play a pivotal role in neuronal differentiation and synaptic regulation, we wondered if the beneficial in vivo effect could be mediated by astrocytes. Primary astrocytes from embryonic cortex were treated with CNF1 for 48 hours and used as a substrate for growing hippocampal neurons. Such neurons showed an increased development of neurites, in respect to age-matched controls, with a wider dendritic tree and a richer content in synapses. In CNF1-exposed astrocytes, the production of interleukin 1β, known to reduce dendrite development and complexity in neuronal cultures, was decreased. These results demonstrate that astrocytes, under the influence of CNF1, increase their supporting activity on neuronal growth and differentiation, possibly related to the diminished levels of interleukin 1β. These observations suggest that the enhanced synaptic plasticity and improved learning and memory described in CNF1-injected mice are probably mediated by astrocytes