Young children’s understanding of photo self-representations

This experiment examined delayed self-recognition in 24 2.5-year-old and 24 3-year-old children. Children were marked covertly with a sticker on their forehead while playing a game, after which their photograph was taken. When show this photograph, the 3- but not the 2.5-year-olds reached to remove this sticker reliably. However, the older children reached reliably only when first shown how a recently taken photograph can be used to guide their search for an object in the testing room that was not directly visible to the unaided eye. Implications of the findings in terms of the development of a temporally extended sense of self are discussed.<br /

Synucleins in the midbrain dopaminergic system: the role in health and disease

Synucleinopathies are a group of diseases characterised by the presence of insoluble aggregated forms of α-synuclein. The most common of these diseases is Parkinson’s disease (PD) which affects approximately 1% of the UK population over the age of 60. Alpha-synuclein has also been linked to the disease through familial mutations and genome wide association studies as well as by its presence in sporadic cases. Although solid evidence exists for a role of α-synuclein in PD, it remains unclear as to how this protein exerts its toxicity on neurons and exactly how this leads to the cell death characteristic of this neurodegenerative disease. Alpha-synuclein belongs to a family of three proteins which also includes β- and γ-synuclein. These three proteins are highly homologous and evolutionarily conserved, however none of them have a well defined function. Evidence suggests a role for these proteins in synaptic vesicle dynamics but a more specific function remains to be unveiled. However, due to the considerable degree of homology across these three proteins, knockout models have been considered to allow functional compensation of the missing synuclein protein through one of the remaining family members. This has hindered studies from elucidating not only the role of α-synuclein but also β- and γ-synuclein. To overcome this problem triple synuclein knockout mice have been produced and characterised, as described in this thesis. As expected studies of these animals revealed no alterations in the number of dopaminergic neurons in either the substantia nigra pars compacta or ventral tegmental area. Despite this, a significant deficit in striatal dopamine concentrations was detected, regardless of the fact that the levels and function of tyrosine hydroxylase being normal. As well as this triple synuclein null mice were demonstrated to be hyperdopaminergic through various behavioural tests. Work employing physcostimulants and, through a collaboration, using fast scan cyclic voltametry suggested a role for these proteins in normal dopamine release dynamics at the level of the synaptic vesicle. A previous body of work has indicated that the loss of α- and/or γ-synuclein is able to provide a degree of resistance against the toxic affects of the dopaminergic neurotoxin MPTP. It was therefore hypothesised that the triple synuclein null animals would also display resistance to this toxin. However, these animals were shown to be more sensitive than wild type controls. Importantly it was apparent that animals lacking β-synuclein alone or in combination with other synucleins were the most sensitive to this toxin. Further work revealed a significant deficit in the ability of triple synuclein null mice to store dopamine in their synaptic vesicles. This may explain the sensitivity to MPP+, the active metabolite of MPTP, due to the fact it cannot be efficiently stored in synaptic vesicles, which restricts the toxins access to the mitochondria where it normally inhibits complex I, thus leading to cell death. When recombinant β-synuclein was reintroduced the deficit in synaptic vesicle dopamine uptake could be restored. However, β-synuclein can not do this alone and requires incubation with cytosolic factors, suggesting it acts as a chaperone in this role. This may explain why lines of synuclein null mice that specifically have the absence of β-synuclein apparently fair least well when exposed to MPTP. Finally, in order to assess the extent to which a loss of function role of α-synuclein leads to pathological alteration at the synapse an entirely novel conditional α-synuclein knockout mouse model was produced. Currently no ideal model exists to answer this question as conventional knockout models are based on the knockout of the protein in development. This may allow functional compensatory mechanisms to be established which can be overcome with a conditional knockout approach. As well as this it is important to assess this loss in an aged nervous system, as PD is a disease of aging. It is likely that, as α-synuclein forms insoluble Lewy bodies and undergoes abnormal posttranslational modifications, the amount of normally functioning protein at the synapse is depleted, therefore allowing a loss of function effect to develop. It is hoped this model will allow new insight into the early disease process. Overall this work further contributes to a body of evidence that suggests the synucleins play an important role in synaptic dopamine handling, particularly at the synaptic vesicle level. It is hoped that the newly established conditional α-synuclein knockout model will produce a new perspective on the loss of function role of α-synuclein in early disease development, an avenue that has yet to be fully explored

Combinational losses of synucleins reveal their differential requirements for compensating age-dependent alterations in motor behavior and dopamine metabolism

Synucleins are involved in multiple steps of the neurotransmitter turnover but the largely normal synaptic function in young adult animals completely lacking synucleins suggests their roles are dispensable for execution of these processes. Instead, they may be utilized for boosting the efficiency of certain molecular mechanisms in presynaptic terminals, with a deficiency of synuclein proteins sensitizing to or exacerbating synaptic malfunction caused by accumulation of mild alterations, which are commonly associated with aging. Although functional redundancy within the family has been reported, it is unclear whether the remaining synucleins can fully compensate for the deficiency of a lost family member, or whether some functions are specific for a particular member. We assessed several structural and functional characteristics of the nigrostriatal system of mice lacking members of the synuclein family in every possible combination and demonstrated that stabilization of the striatal dopamine level depends on the presence of α-synuclein and cannot be compensated for by other family members, whereas β-synuclein is required for efficient maintenance of animal’s balance and coordination in old age

LRRK2: Autophagy and Lysosomal Activity

It has been 15 years since the Leucine-rich repeat kinase 2 (LRRK2) gene was identified as the most common genetic cause for Parkinson’s disease (PD). The two most common mutations are the LRRK2-G2019S, located in the kinase domain, and the LRRK2-R1441C, located in the ROC-COR domain. While the LRRK2-G2019S mutation is associated with increased kinase activity, the LRRK2-R1441C exhibits a decreased GTPase activity and altered kinase activity. Multiple lines of evidence have linked the LRRK2 protein with a role in the autophagy pathway and with lysosomal activity in neurons. Neurons rely heavily on autophagy to recycle proteins and process cellular waste due to their post-mitotic state. Additionally, lysosomal activity decreases with age which can potentiate the accumulation of α-synuclein, the pathological hallmark of PD, and subsequently lead to the build-up of Lewy bodies (LBs) observed in this disorder. This review provides an up to date summary of the LRRK2 field to understand its physiological role in the autophagy pathway in neurons and related cells. Careful assessment of how LRRK2 participates in the regulation of phagophore and autophagosome formation, autophagosome and lysosome fusion, lysosomal maturation, maintenance of lysosomal pH and calcium levels, and lysosomal protein degradation are addressed. The autophagy pathway is a complex cellular process and due to the variety of LRRK2 models studied in the field, associated phenotypes have been reported to be seemingly conflicting. This review provides an in-depth discussion of different models to assess the normal and disease-associated role of the LRRK2 protein on autophagic function. Given the importance of the autophagy pathway in Parkinson’s pathogenesis it is particularly relevant to focus on the role of LRRK2 to discover novel therapeutic approaches that restore lysosomal protein degradation homeostasis

Agreement of Pain Assessment Using the Short Form of the Canine Glasgow Composite Measure Pain Scale between Veterinary Students, Veterinary Nurses, Veterinary Surgeons, and ECVAA-Diplomates

Several pain scoring systems have been validated to measure pain in dogs. However, pain may not be adequately assessed since these tools are associated with high-level inter-observer variation. The aim of this study is to evaluate the agreement of pain assessment using the CMPS-SF between veterinary students, veterinary nurses, veterinary surgeons, and European College of Veterinary Anaesthesia and Analgesia (ECVAA) diplomates. Forty-five client-owned dogs presented to a teaching hospital were enrolled in this prospective, observational study. All dogs were pain-scored in vivo, while a video of the assessment was recorded and subsequently evaluated by twenty assessors, with five per group. Mean scores between groups were compared, and agreement within groups and agreement of the average scores between groups were assessed by calculating the intraclass correlation coefficient (ICC). The intervention point at which dogs were deemed to require additional analgesia was also evaluated. Overall agreement of pain assessment was poor (ICC = 0.494). Nurses had the best inter-observer agreement (ICC = 0.656), followed by ECVAA diplomates (ICC = 0.540), veterinary surgeons (ICC = 0.478), and veterinary students (ICC = 0.432). The best inter-group agreement was between veterinary surgeons and nurses (ICC = 0.951) and between ECVAA diplomates and nurses (ICC = 0.951). Students were more likely to determine that additional analgesia was required compared to other groups. Pain assessment is key for animal welfare, and training in this area should be reinforced to improve consistency.</jats:p

Cell type-specific functions of Alzheimer's disease endocytic risk genes

Endocytosis is a key cellular pathway required for the internalization of cellular nutrients, lipids and receptor-bound cargoes. It is also critical for the recycling of cellular components, cellular trafficking and membrane dynamics. The endocytic pathway has been consistently implicated in Alzheimer's disease (AD) through repeated genome-wide association studies and the existence of rare coding mutations in endocytic genes. BIN1 and PICALM are two of the most significant late-onset AD risk genes after APOE and are both key to clathrin-mediated endocytic biology. Pathological studies also demonstrate that endocytic dysfunction is an early characteristic of late-onset AD, being seen in the prodromal phase of the disease. Different cell types of the brain have specific requirements of the endocytic pathway. Neurons require efficient recycling of synaptic vesicles and microglia use the specialized form of endocytosis—phagocytosis—for their normal function. Therefore, disease-associated changes in endocytic genes will have varied impacts across different cell types, which remains to be fully explored. Given the genetic and pathological evidence for endocytic dysfunction in AD, understanding how such changes and the related cell type-specific vulnerabilities impact normal cellular function and contribute to disease is vital and could present novel therapeutic opportunities. This article is part of a discussion meeting issue ‘Understanding the endo-lysosomal network in neurodegeneration’

Hypoxia-induced nitric oxide production and tumour perfusion is inhibited by pegylated arginine deiminase (ADI-PEG20).

The hypoxic tumour microenvironment represents an aggressive, therapy-resistant compartment. As arginine is required for specific hypoxia-induced processes, we hypothesised that arginine-deprivation therapy may be useful in targeting hypoxic cancer cells. We explored the effects of the arginine-degrading agent ADI-PEG20 on hypoxia-inducible factor (HIF) activation, the hypoxia-induced nitric oxide (NO) pathway and proliferation using HCT116 and UMUC3 cells and xenografts. The latter lack argininosuccinate synthetase (ASS1) making them auxotrophic for arginine. In HCT116 cells, ADI-PEG20 inhibited hypoxic-activation of HIF-1α and HIF-2α, leading to decreased inducible-nitric oxide synthase (iNOS), NO-production, and VEGF. Interestingly, combining hypoxia and ADI-PEG20 synergistically inhibited ASS1. ADI-PEG20 inhibited mTORC1 and activated the unfolded protein response providing a mechanism for inhibition of HIF and ASS1. ADI-PEG20 inhibited tumour growth, impaired hypoxia-associated NO-production, and decreased vascular perfusion. Expression of HIF-1α/HIF-2α/iNOS and VEGF were reduced, despite an increased hypoxic tumour fraction. Similar effects were observed in UMUC3 xenografts. In summary, ADI-PEG20 inhibits HIF-activated processes in two tumour models with widely different arginine biology. Thus, ADI-PEG20 may be useful in the clinic to target therapy-resistant hypoxic cells in ASS1-proficient tumours and ASS1-deficient tumours.Thanks to Dr John Bomalaski, (Polaris Pharmaceuticals, Inc) for supplying the ADI-PEG20, to Dr Simon S Hoer for useful discussions and to members of Histopathology/ISH (CRUK Cambridge Institute, UK) for IHC and imaging assistance. This work was supported by the Wellcome Trust and the NIHR Cambridge Biomedical Research Centre Senior Investigator Awards (to P.H.M., supporting N.B.), EU FP7 Metoxia Grant agreement no. 222741 (to P.H.M., supporting G.C.), UCL Cancer Research UK Centre (to M.R.), King’s College London and UCL Comprehensive Cancer Imaging Centre, Cancer Research UK and EPSRC in association with the Medical Research Council (MRC), the DoH (England: to R.B.P.), MRC Cancer Unit Core Funding (to C.F., supporting E.G.).This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/srep2295

Chlorpyrifos Accumulation Patterns for Child-Accessible Surfaces and Objects and Urinary Metabolite Excretion by Children for 2 Weeks after Crack-and-Crevice Application

The Children’s Post-Pesticide Application Exposure Study (CPPAES) was conducted to look at the distribution of chlorpyrifos within a home environment for 2 weeks after a routine professional crack-and-crevice application and to determine the amount of the chlorpyrifos that is absorbed by a child living within the home. Ten residential homes with a 2- to 5-year-old child in each were selected for study, and the homes were treated with chlorpyrifos. Pesticide measurements were made from the indoor air, indoor surfaces, and plush toys. In addition, periodic morning urine samples were collected from each of the children throughout the 2-week period. We analyzed the urine samples for 3,5,6-trichloropyridinol, the primary urinary metabolite of chlorpyrifos, and used the results to estimate the children’s absorbed dose. Average chlorpyrifos levels in the indoor air and surfaces were 26 (pretreatment)/120 (posttreatment) ng/m(3) and 0.48 (pretreatment)/2.8 (posttreatment) ng/cm(2), respectively, reaching peak levels between days 0 and 2; subsequently, concentrations decreased throughout the 2-week period. Chlorpyrifos in/on the plush toys ranged from 7.3 to 1,949 ng/toy postapplication, with concentrations increasing throughout the 2-week period, demonstrating a cumulative adsorption/absorption process indoors. The daily amount of chlorpyrifos estimated to be absorbed by the CPPAES children postapplication ranged from 0.04 to 4.8 μg/kg/day. During the 2 weeks after the crack-and-crevice application, there was no significant increase in the amount of chlorpyrifos absorbed by the CPPAES children