Parkinson's disease cerebrovascular reactivity pattern: A feasibility study.

A mounting body of research points to cerebrovascular dysfunction as a fundamental element in the pathophysiology of Parkinson's disease (PD). In the current feasibility study, blood-oxygen-level-dependent (BOLD) MRI was used to measure cerebrovascular reactivity (CVR) in response to hypercapnia in 26 PD patients and 16 healthy controls (HC), and aimed to find a multivariate pattern specific to PD. Whole-brain maps of CVR amplitude (i.e., magnitude of response to CO2) and latency (i.e., time to reach maximum amplitude) were computed, which were further analyzed using scaled sub-profile model principal component analysis (SSM-PCA) with leave-one-out cross-validation. A meaningful pattern based on CVR latency was identified, which was named the PD CVR pattern (PD-CVRP). This pattern was characterized by relatively increased latency in basal ganglia, sensorimotor cortex, supplementary motor area, thalamus and visual cortex, as well as decreased latency in the cerebral white matter, relative to HC. There were no significant associations with clinical measures, though sample size may have limited our ability to detect significant associations. In summary, the PD-CVRP highlights the importance of cerebrovascular dysfunction in PD, and may be a potential biomarker for future clinical research and practice.

Exposure to an environmentally relevant concentration of 17α-ethinylestradiol disrupts craniofacial development of juvenile zebrafish.

Endocrine disrupting chemicals (EDCs) can interact with native hormone receptors to interfere with and disrupt hormone signalling that is necessary for a broad range of developmental pathways. EDCs are pervasive in our environment, in particular in our waterways, making aquatic wildlife especially vulnerable to their effects. Many of these EDCs are able to bind to and activate oestrogen receptors, causing aberrant oestrogen signalling. Craniofacial development is an oestrogen-sensitive process, with oestrogen receptors expressed in chondrocytes during critical periods of development. Previous studies have demonstrated a negative effect of high concentrations of oestrogen on early craniofacial patterning in the aquatic model organism, the zebrafish (Danio rerio). In order to determine the impacts of exposure to an oestrogenic EDC, we exposed zebrafish larvae and juveniles to either a high concentration to replicate previous studies, or a low, environmentally relevant concentration of the oestrogenic contaminant, 17α-ethinylestradiol. The prolonged / chronic exposure regimen was used to replicate that seen by many animals in natural waterways. We observed changes to craniofacial morphology in all treatments, and most strikingly in the larvae-juveniles exposed to a low concentration of EE2. In the present study, we have demonstrated that the developmental stage at which exposure occurs can greatly impact phenotypic outcomes, and these results allow us to understand the widespread impact of oestrogenic endocrine disruptors. Given the conservation of key craniofacial development pathways across vertebrates, our model can further be applied in defining the risks of EDCs on mammalian organisms.

Estrogen-induced immune changes within the normal mammary gland.

Breast cancer (BCa) incidence increases following aberrant hormone exposure, which has been linked to direct effects on estrogen receptor (ER)+ mammary epithelium. While estrogen exposure during mammary involution has been shown to drive tumour growth via neutrophils, the potential for the ER + immune microenvironment to mediate part (in addition to mammary epithelial cells) of hormonally controlled BCa risk during normal development has not been assessed. We collected mammary tissue, lymph nodes and blood from tumour naïve mice treated with, oophorectomy, estrogen (17ß estradiol) or Fulvestrant. Flow cytometry was used to examine the impact on the frequency of innate and adaptive immune cells. Oophorectomy and fulvestrant decreased the proportion of macrophages, particularly pro-tumour polarized M2 macrophages and neutrophils. Conversely, dendritic cells were increased by these therapies, as were eosinophils. Estrogen increased the proportion of M2 macrophages and to a lesser extent CD4-CD8- double negative and FoxP3+ regulatory T cells but decreased CD8 + T cells and B cells. Excluding eosinophils, these changes were restricted to the mammary tissue. This suggests that inhibiting estrogen action lowers the immune suppressive myeloid cells, increases in antigen presentation and eosinophil-mediated direct or indirect cytotoxic effects. In contrast, estrogen exposure, which drives BCa risk, increases the suppressive myeloid cells and reduces anti-tumour cytotoxic T cells. The impact of hormonal exposure on BCa risk, may in part be linked to its immune modulatory activity.

Oestrogen Activates the MAP3K1 Cascade and ß-Catenin to Promote Granulosa-like Cell Fate in a Human Testis-Derived Cell Line.

Sex determination triggers the differentiation of the bi-potential gonad into either an ovary or testis. In non-mammalian vertebrates, the presence or absence of oestrogen dictates gonad differentiation, while in mammals, this mechanism has been supplanted by the testis-determining gene SRY. Exogenous oestrogen can override this genetic trigger to shift somatic cell fate in the gonad towards ovarian developmental pathways by limiting the bioavailability of the key testis factor SOX9 within somatic cells. Our previous work has implicated the MAPK pathway in mediating the rapid cellular response to oestrogen. We performed proteomic and phosphoproteomic analyses to investigate the precise mechanism through which oestrogen impacts these pathways to activate ß-catenin-a factor essential for ovarian development. We show that oestrogen can activate ß-catenin within 30 min, concomitant with the cytoplasmic retention of SOX9. This occurs through changes to the MAP3K1 cascade, suggesting this pathway is a mechanism through which oestrogen influences gonad somatic cell fate. We demonstrate that oestrogen can promote the shift from SOX9 pro-testis activity to ß-catenin pro-ovary activity through activation of MAP3K1. Our findings define a previously unknown mechanism through which oestrogen can promote a switch in gonad somatic cell fate and provided novel insights into the impacts of exogenous oestrogen exposure on the testis.

Oestrogen regulates SOX9 bioavailability by rapidly activating ERK1/2 and stabilising microtubules in a human testis-derived cell line.

Nuclear SOX9 is essential for Sertoli cell differentiation and the development of a testis. Exposure of Sertoli cells to exogenous oestrogen causes cytoplasmic retention of SOX9, inhibiting testis development and promoting ovarian development. The cytoplasmic localisation of SOX9 requires a stabilised microtubule network and a key MAPK complex, ERK1/2, is responsive to oestrogen and known to affect the microtubule network. We hypothesised that oestrogen could stabilise microtubules through the activation of ERK1/2 to promote the cytoplasmic retention of SOX9. Treatment of human testis-derived NT2/D1 cells for 30 min with oestrogen rapidly activated ERK1/2, stabilised the microtubule network and increased cytoplasmic localisation of SOX9. The effects of oestrogen on SOX9 and tubulin were blocked by the ERK1/2 inhibitor U0126, demonstrating that ERK1/2 mediates the stabilisation of microtubules and cytoplasmic retention of SOX9 by oestrogen. Together, these data revealed a previously unknown mechanism for oestrogen in impacting MAPK signalling to block SOX9 bioavailability and the differentiation of Sertoli cells.

Exogenous Oestrogen Impacts Cell Fate Decision in the Developing Gonads: A Potential Cause of Declining Human Reproductive Health.

The increasing incidence of testicular dysgenesis syndrome-related conditions and overall decline in human fertility has been linked to the prevalence of oestrogenic endocrine disrupting chemicals (EDCs) in the environment. Ectopic activation of oestrogen signalling by EDCs in the gonad can impact testis and ovary function and development. Oestrogen is the critical driver of ovarian differentiation in non-mammalian vertebrates, and in its absence a testis will form. In contrast, oestrogen is not required for mammalian ovarian differentiation, but it is essential for its maintenance, illustrating it is necessary for reinforcing ovarian fate. Interestingly, exposure of the bi-potential gonad to exogenous oestrogen can cause XY sex reversal in marsupials and this is mediated by the cytoplasmic retention of the testis-determining factor SOX9 (sex-determining region Y box transcription factor 9). Oestrogen can similarly suppress SOX9 and activate ovarian genes in both humans and mice, demonstrating it plays an essential role in all mammals in mediating gonad somatic cell fate. Here, we review the molecular control of gonad differentiation and explore the mechanisms through which exogenous oestrogen can influence somatic cell fate to disrupt gonad development and function. Understanding these mechanisms is essential for defining the effects of oestrogenic EDCs on the developing gonads and ultimately their impacts on human reproductive health.

Estrogen suppresses SOX9 and activates markers of female development in a human testis-derived cell line.

BACKGROUND: The increasing incidence of reproductive disorders in humans has been attributed to in utero exposure to estrogenic endocrine disruptors. In particular, exposure of the developing testis to exogenous estrogen can negatively impact male reproductive health. To determine how estrogens impact human gonad function, we treated the human testis-derived cell line NT2/D1 with estrogen and examined its impact on SOX9 and the expression of key markers of granulosa (ovarian) and Sertoli (testicular) cell development. RESULTS: Estrogen successfully activated its cognate receptor (estrogen receptor alpha; ESR1) in NT2/D1 cells. We observed a significant increase in cytoplasmic SOX9 following estrogen treatment. After 48 h of estrogen exposure, mRNA levels of the key Sertoli cell genes SOX9, SRY, AMH, FGF9 and PTGDS were significantly reduced. This was followed by a significant increase in mRNA levels for the key granulosa cell genes FOXL2 and WNT4 after 96 h of estrogen exposure. CONCLUSIONS: These results are consistent with estrogen's effects on marsupial gonads and show that estrogen has a highly conserved impact on gonadal cell fate decisions that has existed in mammals for over 160 million years. This effect of estrogen presents as a potential mechanism contributing to the significant decrease in male fertility and reproductive health reported over recent decades. Given our widespread exposure to estrogenic endocrine disruptors, their effects on SOX9 and Sertoli cell determination could have considerable impact on the adult testis.

In utero exposure to both high- and low-dose diethylstilbestrol disrupts mouse genital tubercle development.

Exposure to estrogenic endocrine disrupting chemicals (EDCs) during in utero development has been linked to the increasing incidence of disorders of sexual development. Hypospadias, the ectopic placement of the urethra on the ventral aspect of the penis, is one of the most common DSDs affecting men, and can also affect women by resulting in the misplacement of the urethra. This study aimed to comprehensively assess the resulting hypospadias phenotypes in male and female mice exposed in utero from embryonic day 9.5 to 19.5 to the potent estrogenic endocrine disruptor, diethylstilbestrol, at a high, clinically relevant dose, and a low, previously untested dose, administered via water. The anogenital distance of male pups was significantly reduced and hypospadias was observed in males at a high frequency. Females exhibited hypospadias and urethral-vaginal fistula. These results demonstrate the ability of an estrogen receptor agonist to disrupt sexual development in both male and female mice, even at a low dose, administered via drinking water.

A new state of cardiac myosin with very slow ATP turnover: a potential cardioprotective mechanism in the heart.

The mechanisms that control cardiac contractility are complex. Recent work we conducted in vertebrate skeletal muscle identified a new state of myosin, the super-relaxed state (SRX), which had a very low metabolic rate. To determine whether this state also exists in cardiac muscle we used quantitative epi-fluorescence to measure single nucleotide turnovers by myosin in bundles of relaxed permeable rabbit ventricle cells. We measured two turnover times--one compatible with the normal relaxed state, and one much slower which was shown to arise from myosin heads in the SRX. In both skeletal and cardiac muscle, the SRX appears to play a similar role in relaxed cells, providing a state with a very low metabolic rate. However, in active muscle the properties of the SRX differ dramatically. We observed a rapid transition of myosin heads out of the SRX in active skeletal fibers, whereas the population of the SRX remained constant in active cardiac cells. This property allows the SRX to play a very different role in cardiac muscle than in skeletal muscle. The SRX could provide a mechanism for decreasing the metabolic load on the heart, being cardioprotective, particularly in time of stress such as ischemia.

Myosin ATP turnover rate is a mechanism involved in thermogenesis in resting skeletal muscle fibers.

Thermogenesis by resting muscle varies with conditions and plays an active role in homeostasis of body weight. The low metabolic rate of living resting muscles requires that ATP turnover by myosin be inhibited relative to the purified protein in vitro. This inhibition has not been previously seen in in vitro systems. We used quantitative epifluorescence microscopy of fluorescent nucleotides to measure single nucleotide turnovers in relaxed, permeable skeletal muscle fibers. We observed two lifetimes for nucleotide release by myosin: a fast component with a lifetime of approximately 20 s, similar to that of purified myosin, and a slower component with a lifetime of 230 +/- 24 s. We define the latter component to be the "super relaxed state." The fraction of myosins in the super relaxed state was decreased at lower temperatures, by substituting GTP for ATP or by increased levels of myosin phosphorylation. All of these conditions have also been shown to cause increased disorder in the structure of the thick filament. We propose a model in which the structure of the thick filament modulates the nucleotide turnover rates of myosin in relaxed fibers. Modulation of the relative populations of the super relaxed and conventional relaxed states could have a profound effect on muscle thermogenesis, with the capacity to also significantly alter whole-body metabolic rate.

Myosin regulatory light chain phosphorylation inhibits shortening velocities of skeletal muscle fibers in the presence of the myosin inhibitor blebbistatin.

Phosphorylation of skeletal myosin regulatory light chain (RLC) occurs in fatigue and may play a role in the inhibition of shortening velocities observed in vivo. Forces and shortening velocities were measured in permeabilized rabbit psoas fibers with either phosphorylated or dephosphorylated RLCs and in the presence or absence of the myosin inhibitor blebbistatin. Addition of 20 microM blebbistatin decreased tensions by approximately 80% in fibers, independent of phosphorylation. In blebbistatin maximal shortening velocities (V(max)) at 30 degrees C, were decreased by 45% (3.2 +/- 0.34 vs. 5.8 +/- 0.18 lengths/s) in phosphorylated fibers but were not inhibited in dephosphorylated fibers (6.0 +/- 0.30 vs. 5.4 +/- 0.30). In the presence of 20 microM blebbistatin, K(m) for V(max) as a function of [ATP] was lower for phosphorylated fibers than for dephosphorylated fibers (50 +/- 20 vs. 330 +/- 84 microM) indicating that the apparent binding of ATP is stronger in these fibers. Phosphorylation of RLC in situ during fiber preparation or by addition of myosin light chain kinase yielded similar data. RLC phosphorylation inhibited velocity in blebbistatin at both 30 and 10 degrees C, unlike previous reports where RLC phosphorylation only affected shortening velocities at higher temperatures.

Decreasing lead bioaccessibility in industrial and firing range soils with phosphate-based amendments.

In-situ stabilization using phosphate (P) amendments, such as P-based fertilizers and rock, are a potentially cost-effective and minimally disruptive alternative for stabilizing Pb in soils. We examined the effect of time (0-365 d), in vitro extraction pH (1.5 vs. 2.3), and dosage of three P-based amendments on the bioaccessibility (as a surrogate for oral bioavailability) of Pb in 10 soils from U.S. Department of Defense facilities. Initial untreated soil bioaccessibility consistently exceeded the U.S. Environmental Protection Agency default value of 60% relative bioavailability, with higher bioaccessibility consistently observed at an in vitro extraction pH of 1.5 vs. 2.3. Although P-based amendments statistically (P < 0.05) reduced bioaccessibility in many instances, with reductions dependent on the amendment and dosage, large amendment dosages (approximately 20-25% by mass to yield 5% P by mass) were required to reduce average bioaccessibility by approximately 25%. For most amendment combinations, reductions continued to occur for periods up to 1 yr, indicating that the observed reductions were not merely experimental artifacts of the in vitro extraction procedure. Although our results indicated that reductions in Pb bioaccessibility with P amendments are technically feasible, relatively large amendment masses were required to achieve relatively modest reductions in bioaccessibility. The cost and potential environmental implications of adding such large amounts of P may limit the practicality of in situ immobilization for some Pb-contaminated soils, industrial and firing range soils in particular.

Decreasing arsenic bioaccessibility/bioavailability in soils with iron amendments.

We investigated the use of various iron amendments (metallic Fe and soluble Fe(II)- and Fe(III)-halide salts) to reduce arsenic (As) bioaccessibility (as a surrogate for oral bioavailability) in contaminated soils. Soluble Fe(II)- and Fe(III)-salts were more effective than metallic Fe in reducing As bioaccessibility. Adding soluble Fe(III)-salts to soil reduces As bioaccessibility in two ways, by increasing the Fe(III) (hydr)oxide content and by lowering the soil pH. A detailed investigation into the effect of soil moisture when adding Fe(III) amendments indicated that the reaction can occur in situ if sufficient (>or=30% moisture) is added. If the amendments are added to the soil without moisture, a reduction in bioaccessibility will occur in the extraction fluid itself (i.e., an experimental artifact not reflecting a true in situ reduction in bioaccessibility). Adding Fe (III)-salts to nine As-contaminated soils at a Fe:As molar ratio of 100:1 reduced the average bioaccessibility in the soils by approximately a factor of two. Greater reductions in As bioaccessibility can be achieved by increasing the Fe:As molar ratio. These results suggest decreasing As bioaccessibility and bioavailability in soil by adding Fe amendments may be an effective strategy to remediate As-contaminated soils.