Protection of cortical cells by equine estrogens against glutamate-induced excitotoxicity is mediated through a calcium independent mechanism

BACKGROUND: High concentrations of glutamate can accumulate in the brain and may be involved in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease. This form of neurotoxicity involves changes in the regulation of cellular calcium (Ca(2+)) and generation of free radicals such as peroxynitrite (ONOO(-)). Estrogen may protect against glutamate-induced cell death by reducing the excitotoxic Ca(2+ )influx associated with glutamate excitotoxicity. In this study, the inhibition of N-methyl-D-aspartate (NMDA) receptor and nitric oxide synthase (NOS) along with the effect of 17β-estradiol (17β-E(2)) and a more potent antioxidant Δ(8), 17β-estradiol (Δ(8), 17β-E(2)) on cell viability and intracellular Ca(2+ )([Ca(2+)](i)), following treatment of rat cortical cells with glutamate, was investigated. RESULTS: Primary rat cortical cells were cultured for 7–12 days in Neurobasal medium containing B27 supplements. Addition of glutamate (200 μM) decreased cell viability to 51.3 ± 0.7% compared to control. Treatment with the noncompetitive NMDAR antagonist, MK-801, and the NOS inhibitor, L-NAME, completely prevented cell death. Pretreatment (24 hrs) with 17β-E(2 )and Δ(8), 17β-E(2 )(0.01 to 10 μM) significantly reduced cell death. 17β-E(2 )was more potent than Δ(8), 17β-E(2). Glutamate caused a rapid 2.5 fold increase in [Ca(2+)](i). Treatment with 0.001 to 10 μM MK-801 reduced the initial Ca(2+ )influx by 14–41% and increased cell viability significantly. Pretreatment with 17β-E(2 )and Δ(8), 17β-E(2 )had no effect on Ca(2+ )influx but protected the cortical cells against glutamate-induced cell death. CONCLUSION: Glutamate-induced cell death in cortical cultures can occur through NMDAR and NOS-linked mechanisms by increasing nitric oxide and ONOO(-). Equine estrogens: 17β-E(2 )and Δ(8), 17β-E(2), significantly protected cortical cells against glutamate-induced excitotoxicity by a mechanism that appears to be independent of Ca(2+ )influx. To our knowledge, this is a first such observation. Whether the decrease in NOS related products such as ONOO(-), is a mechanism by which estrogens protect against glutamate toxicity, remains to be investigated. Estrogen replacement therapy in healthy and young postmenopausal women may protect against neurodegenerative diseases by these mechanisms

Glutamate-induced apoptosis in primary cortical neurons is inhibited by equine estrogens via down-regulation of caspase-3 and prevention of mitochondrial cytochrome c release

BACKGROUND: Apoptosis plays a key role in cell death observed in neurodegenerative diseases marked by a progressive loss of neurons as seen in Alzheimer's disease. Although the exact cause of apoptosis is not known, a number of factors such as free radicals, insufficient levels of nerve growth factors and excessive levels of glutamate have been implicated. We and others, have previously reported that in a stable HT22 neuronal cell line, glutamate induces apoptosis as indicated by DNA fragmentation and up- and down-regulation of Bax (pro-apoptotic), and Bcl-2 (anti-apoptotic) genes respectively. Furthermore, these changes were reversed/inhibited by estrogens. Several lines of evidence also indicate that a family of cysteine proteases (caspases) appear to play a critical role in neuronal apoptosis. The purpose of the present study is to determine in primary cultures of cortical cells, if glutamate-induced neuronal apoptosis and its inhibition by estrogens involve changes in caspase-3 protease and whether this process is mediated by Fas receptor and/or mitochondrial signal transduction pathways involving release of cytochrome c. RESULTS: In primary cultures of rat cortical cells, glutamate induced apoptosis that was associated with enhanced DNA fragmentation, morphological changes, and up-regulation of pro-caspase-3. Exposure of cortical cells to glutamate resulted in a time-dependent cell death and an increase in caspase-3 protein levels. Although the increase in caspase-3 levels was evident after 3 h, cell death was only significantly increased after 6 h. Treatment of cells for 6 h with 1 to 20 mM glutamate resulted in a 35 to 45% cell death that was associated with a 45 to 65% increase in the expression of caspase-3 protein. Pretreatment with caspase-3-protease inhibitor z-DEVD or pan-caspase inhibitor z-VAD significantly decreased glutamate-induced cell death of cortical cells. Exposure of cells to glutamate for 6 h in the presence or absence of 17β-estradiol or Δ(8), 17β-estradiol (10 nM-10 μM) resulted in the prevention of cell death and was associated with a significant dose-dependent decrease in caspase-3 protein levels, with Δ(8), 17β-E(2 )being more potent than 17β-E(2). Protein levels of Fas receptor remained unchanged in the presence of glutamate. In contrast, treatment with glutamate induced, in a time-dependent manner, the release of cytochrome c into the cytosol. Cytosolic cytochrome c increased as early as 1.5 h after glutamate treatment and these levels were 5 fold higher after 6 h, compared to levels in the untreated cells. Concomitant with these changes, the levels of cytochrome c in mitochondria decreased significantly. Both 17β-E(2 )and Δ(8), 17β-E(2 )reduced the release of cytochrome c from mitochondria into the cytosol and this decrease in cytosolic cytochrome c was associated with inhibition of glutamate-induced cell death. CONCLUSION: In the primary cortical cells, glutamate-induced apoptosis is accompanied by up-regulation of caspase-3 and its activity is blocked by caspase protease inhibitors. These effects of glutamate on caspase-3 appear to be independent of changes in Fas receptor, but are associated with the rapid release of mitochondrial cytochrome c, which precedes changes in caspase-3 protein levels leading to apoptotic cell death. This process was differentially inhibited by estrogens with the novel equine estrogen Δ(8), 17β-E(2 )being more potent than 17β-E(2). To our knowledge, this is the first study to demonstrate that equine estrogens can prevent glutamate-induced translocation of cytochrome c from mitochondria to cytosol in rat primary cortical cells

Glutamate-induced apoptosis in neuronal cells is mediated via caspase-dependent and independent mechanisms involving calpain and caspase-3 proteases as well as apoptosis inducing factor (AIF) and this process is inhibited by equine estrogens

BACKGROUND: Glutamate, a major excitatory amino acid neurotransmitter, causes apoptotic neuronal cell death at high concentrations. Our previous studies have shown that depending on the neuronal cell type, glutamate-induced apoptotic cell death was associated with regulation of genes such as Bcl-2, Bax, and/or caspase-3 and mitochondrial cytochrome c. To further delineate the intracellular mechanisms, we have investigated the role of calpain, an important calcium-dependent protease thought to be involved in apoptosis along with mitochondrial apoptosis inducing factor (AIF) and caspase-3 in primary cortical cells and a mouse hippocampal cell line HT22. RESULTS: Glutamate-induced apoptotic cell death in neuronal cells was associated with characteristic DNA fragmentation, morphological changes, activation of calpain and caspase-3 as well as the upregulation and/or translocation of AIF from mitochondria into cytosol and nuclei. Our results reveal that primary cortical cells and HT22 cells display different patterns of regulation of these genes/proteins. In primary cortical cells, glutamate induces activation of calpain, caspase-3 and translocation of AIF from mitochondria to cytosol and nuclei. In contrast, in HT22 cells, only the activation of calpain and upregulation and translocation of AIF occurred. In both cell types, these processes were inhibited/reversed by 17β-estradiol and Δ(8),17β-estradiol with the latter being more potent. CONCLUSION: Depending upon the neuronal cell type, at least two mechanisms are involved in glutamate-induced apoptosis: a caspase-3-dependent pathway and a caspase-independent pathway involving calpain and AIF. Since HT22 cells lack caspase-3, glutamate-induced apoptosis is mediated via the caspase-independent pathway in this cell line. Kinetics of this apoptotic pathway further indicate that calpain rather than caspase-3, plays a critical role in the glutamate-induced apoptosis. Our studies further indicate that glutamate- induced changes of these proteins can be inhibited by estrogens, with Δ(8),17β-estradiol, a novel equine estrogen being more potent than 17β-estradiol. To our knowledge, this is the first demonstration that glutamate-induced apoptosis involves regulation of multiple apoptotic effectors that can be inhibited by estrogens. Whether these observations can help in the development of novel therapeutic approaches for the prevention of neurodegenerative diseases with estrogens and calpain inhibitors remains to be investigated

Equine estrogens differentially inhibit DNA fragmentation induced by glutamate in neuronal cells by modulation of regulatory proteins involved in programmed cell death

BACKGROUND: Recent data indicate that excitotoxicity of high levels of neurotransmitter glutamate may be mediated via programmed cell death (apoptosis) and that it can be prevented in HT22 mouse hippocampal cells by various equine estrogens with Δ(8),17β-estradiol (Δ(8),17β-E(2)) being the most potent. In order to delineate the mechanism(s), glutamate-induced cell death of HT22 cells was assessed by measuring (a) DNA fragmentation in the presence or absence of 11 equine estrogens (components of the drug CEE); (b) cell death and (c) levels of anti-apoptotic (Bcl-2) and proapoptotic (Bax) proteins in the presence or absence of two equine estrogens, Δ(8),17β-E(2 )and 17β-estradiol (17β-E(2)) by LDH release assay and Western blot analysis respectively. RESULTS: Glutamate treatment induced cell death was time and dose-dependent. After 18 to 24 h, glutamate induced DNA fragmentation and morphological characteristics of apoptotic cell death. DNA fragmentation and morphological changes induced by 10 mM glutamate were completely inhibited by some equine estrogens. Exposure of cells to various concentrations of glutamate, resulted in a significant increase in cell death associated LDH release that was time-dependent. Both Δ(8),17β-E(2 )and 17β-E(2 )inhibited the glutamate-induced LDH release and cell death in a dose-dependent manner with Δ(8),17β-E(2 )being 10 times more potent than 17β-E(2). Western blot analysis indicated that glutamate also significantly decreased the levels of Bcl-2 and increased Bax levels. This glutamate-induced change in the ratio of Bcl-2 to Bax was reversed by estrogens with Δ(8),17β-E(2 )being more potent. CONCLUSIONS: In HT22 mouse hippocampal cells, glutamate induced apoptosis that was associated with DNA fragmentation, morphological changes and up-regulation of the pro-apoptotic protein Bax and down-regulation of the anti-apoptotic protein Bcl-2. This apoptotic process was differentially prevented by some equine estrogens with Δ(8),17β-E(2 )being more potent than 17β-E(2). Since HT22 cells lacked both glutamate and estrogen receptors, the neuroprotective effects of estrogens most likely involve both genomic and non-genomic mechanisms. Since Δ(8)-estrogens are less feminizing estrogens than 17β-E(2), further chemical modifications of these Δ(8)-estrogens may provide more selective estrogens that will be useful in the prevention of neurodegenerative diseases such as Alzheimer's and Parkinson's in both aging men and women

Caregiver Perceptions of Autism and Neurodevelopmental Disabilities in New Delhi, India

Evidence suggests that parenting an autistic child or a child with neurodevelopmental disabilities can be more challenging than parenting a child meeting their developmental milestones, especially when there is a dearth of support services, such as in low- and middle-income countries (LMICs). Despite the majority of the world’s children residing in LMICs, there are limited studies examining the understanding of developmental disorders and autism in these regions. We therefore aim to investigate perceptions of autism and developmental disabilities in caregivers of children in an urban setting in New Delhi, India. Thirteen semi-structured interviews with parents/caregivers of children were conducted in three groups: (1) caregivers with a child with a diagnosis of autism spectrum disorder (ASD); (2) caregivers with a child with a diagnosis of intellectual disability (ID); (3) and caregivers with children meeting their developmental milestones. Transcripts were analysed using framework analysis. Three themes on the impact of cultural and contextual factors on the recognition, interpretation, and reporting of autistic symptoms are discussed, and additional themes focus on the impact of diagnosis and family support. Our findings highlighted a vital need for greater community awareness and recognition of autism in India, for example through community and healthcare training, which may help to reduce stigma and facilitate wider family support

Crystal growth furnace safety system validation

The findings are reported regarding the safe operation of the NASA crystal growth furnace (CGF) and potential methods for detecting containment failures of the furnace. The main conclusions are summarized by ampoule leak detection, cartridge leak detection, and detection of hazardous species in the experiment apparatus container (EAC)

Attention control in autism: Eye-tracking findings from pre-school children in a low- and middle-income country setting

Alterations in the development of attention control and learning have been associated with autism and can be measured using the ‘antisaccade task’, which assesses a child’s ability to make an oculomotor response away from a distracting stimulus, and learn to instead anticipate a later reward. We aimed to assess these cognitive processes using portable eye-tracking in an understudied population of pre-school children with and without a diagnosis of autism spectrum disorder in community settings in New Delhi, India. The eye-tracking antisaccade task was presented to children in three groups (n = 104) (children with a clinical diagnosis of autism spectrum disorder or intellectual disability and children meeting developmental milestones). In accordance with findings from high-income, laboratory-based environments, children learnt to anticipate looks towards a reward, as well as inhibit eye-movements towards a distractor stimulus. We also provide novel evidence that while differences in inhibition responses might be applicable to multiple developmental conditions, a reduced learning to anticipate looks towards a target in this age group may be specific to autism. This eye-tracking task may, therefore, have the potential to identify and assess autism specific traits across development, and be used in longitudinal research studies such as investigating response to intervention in low-resource settings

Exploring the psychological rewards of a familiar semirural landscape: connecting to local nature through a mindful approach

This study analyses a 53,000 word diary of a year engaging with nature through over 200 trips to a semi-rural landscape. Thematic analysis revealed two themes; the transition from observer to nature connectedness and the ways in which the natural environment was experienced once a connection was made. These themes are discussed in relation to theories that seek to explain the positive effect of nature and nature connectedness. The findings are important as they suggest that repeated engagement with local semi-rural countryside can lead to a mindful approach and psychological rewards that do not require travel into the wilderness. The work informs further research into outcomes and processes of nature based interventions such as: trip frequency, duration and diary keeping

Exploratory investigation of drivers of attainment in ethnic minority adult learners

There is evidence that ethnic minority learners in further education in England either under-achieve or are under-represented because they face various inhibitors connected to their ethnicity. Motivators may be in place, however, which increase attainment specifically for some ethnic groups. This exploratory study intends to examine what works and what does not among South Asian (Pakistani and Bangladeshi heritage) females and black male adult learners in FE. A mixed-method study was carried out using questionnaires and focus groups with 68 ethnic minority students in three further education colleges in England. The combination of the results showed that being a member of a minority culture and/or religion may increase feelings of isolation in academic settings; teaching staff who are knowledgeable about the student’s culture increase feelings of inclusion; and role models are crucially important. Results are discussed in light of British data of school experiences of ethnic minority learners

Uninterested youth? Young people's attitudes towards party politics in Britain

Following the outcome of the 2001 and 2005 General Elections, when the numbers of abstainers outweighed the numbers of Labour voters on both occasions, much attention has focused upon the state of British democracy and how to enthuse the electorate, especially young people. While the government is exploring ways to make the whole process of voting easier, it may be failing to tackle the real problem - that youth appear to find the business of politics uninviting and irrelevant. This paper examines data derived from a nationwide survey of over 700 young people in order to shed light on what lies at the heart of young people's apparent disengagement from formal politics in Britain - political apathy or a sense of political alienation. The findings reveal that they support the democratic process, but are sceptical of the way the British political system is organised and led, and are turned off by politicians and the political parties. However, there is no uniform youth orientation to politics, and the data indicate that views differ according to social class, educational history, and also gender. However both ethnicity and region of the country in which young people live seem to have little influence in structuring political attitudes and behaviour