The E46K mutation modulates α-synuclein prion replication in transgenic mice.

In multiple system atrophy (MSA), the α-synuclein protein misfolds into a self-templating prion conformation that spreads throughout the brain, leading to progressive neurodegeneration. While the E46K mutation in α-synuclein causes familial Parkinson's disease (PD), we previously discovered that this mutation blocks in vitro propagation of MSA prions. Recent studies by others indicate that α-synuclein adopts a misfolded conformation in MSA in which a Greek key motif is stabilized by an intramolecular salt bridge between residues E46 and K80. Hypothesizing that the E46K mutation impedes salt bridge formation and, therefore, exerts a selective pressure that can modulate α-synuclein strain propagation, we asked whether three distinct α-synuclein prion strains could propagate in TgM47+/- mice, which express human α-synuclein with the E46K mutation. Following intracranial injection of these strains, TgM47+/- mice were resistant to MSA prion transmission, whereas recombinant E46K preformed fibrils (PFFs) transmitted neurological disease to mice and induced the formation of phosphorylated α-synuclein neuropathology. In contrast, heterotypic seeding following wild-type (WT) PFF-inoculation resulted in preclinical α-synuclein prion propagation. Moreover, when we inoculated TgM20+/- mice, which express WT human α-synuclein, with E46K PFFs, we observed delayed transmission kinetics with an incomplete attack rate. These findings suggest that the E46K mutation constrains the number of α-synuclein prion conformations that can propagate in TgM47+/- mice, expanding our understanding of the selective pressures that impact α-synuclein prion replication.

HTR7 Mediates Serotonergic Acute and Chronic Itch.

Chronic itch is a prevalent and debilitating condition for which few effective therapies are available. We harnessed the natural variation across genetically distinct mouse strains to identify transcripts co-regulated with itch behavior. This survey led to the discovery of the serotonin receptor HTR7 as a key mediator of serotonergic itch. Activation of HTR7 promoted opening of the ion channel TRPA1, which in turn triggered itch behaviors. In addition, acute itch triggered by serotonin or a selective serotonin reuptake inhibitor required both HTR7 and TRPA1. Aberrant serotonin signaling has long been linked to a variety of human chronic itch conditions, including atopic dermatitis. In a mouse model of atopic dermatitis, mice lacking HTR7 or TRPA1 displayed reduced scratching and skin lesion severity. These data highlight a role for HTR7 in acute and chronic itch and suggest that HTR7 antagonists may be useful for treating a variety of pathological itch conditions.

The epithelial cell-derived atopic dermatitis cytokine TSLP activates neurons to induce itch.

Atopic dermatitis (AD) is a chronic itch and inflammatory disorder of the skin that affects one in ten people. Patients suffering from severe AD eventually progress to develop asthma and allergic rhinitis, in a process known as the "atopic march." Signaling between epithelial cells and innate immune cells via the cytokine thymic stromal lymphopoietin (TSLP) is thought to drive AD and the atopic march. Here, we report that epithelial cells directly communicate to cutaneous sensory neurons via TSLP to promote itch. We identify the ORAI1/NFAT calcium signaling pathway as an essential regulator of TSLP release from keratinocytes, the primary epithelial cells of the skin. TSLP then acts directly on a subset of TRPA1-positive sensory neurons to trigger robust itch behaviors. Our results support a model whereby calcium-dependent TSLP release by keratinocytes activates both primary afferent neurons and immune cells to promote inflammatory responses in the skin and airways.

The ion channel TRPA1 is required for chronic itch.

Chronic itch is a debilitating condition that affects one in 10 people. Little is known about the molecules that mediate chronic itch in primary sensory neurons and skin. We demonstrate that the ion channel TRPA1 is required for chronic itch. Using a mouse model of chronic itch, we show that scratching evoked by impaired skin barrier is abolished in TRPA1-deficient animals. This model recapitulates many of the pathophysiological hallmarks of chronic itch that are observed in prevalent human diseases such as atopic dermatitis and psoriasis, including robust scratching, extensive epidermal hyperplasia, and dramatic changes in gene expression in sensory neurons and skin. Remarkably, TRPA1 is required for both transduction of chronic itch signals to the CNS and for the dramatic skin changes triggered by dry-skin-evoked itch and scratching. These data suggest that TRPA1 regulates both itch transduction and pathophysiological changes in the skin that promote chronic itch.

Stimulation of murine intestinal secretion by daily genistein injections: gender-dependent differences.

BACKGROUND/AIMS: The effect of daily injections with genistein (naturally occurring phytoestrogen) on intestinal chloride (Cl(-)) secretion was measured with Ussing chamber short circuit current (I(sc), µA/cm(2)), in C57BL/6J male and female mice, using 600 mg/kg genistein/day (600G), 300 mg/kg genistein/day (300G), 150 mg/kg genistein/day (150G) or genistein-free vehicle control (0G) for 1- or 2-weeks. METHODS AND RESULTS: Injecting with 600G elicited significant increases in basal I(sc) in females after 1-week (ñ70 µA/cm(2), n=15, p < 0.05) and in males after 2-weeks (ñ80 µA/cm(2), n=5, p < 0.05) compared to their 0G counterparts. Chloride-free ringer significantly reduced basal I(sc) by 65% in 600G males and 72% in 600G females, suggesting that Cl(-) was the major anion comprising the genistein-stimulated secretion. The forskolin-stimulated (10 µM) I(sc) was significantly inhibited by the CFTR chloride channel inhibitors, glibenclamide (500 µM) and CFTR(inh)-172 (100 µM) in 600G males and females, suggesting some contribution by genistein-dependent CFTR-mediated Cl(-) secretion. We found no associated changes in intestinal morphology, nor change in total CFTR protein with 600G. There was a 5% increase in apical/subapical ratio in 600G males compared to controls (no change in females). CONCLUSION: These data suggest that male and female mice both exhibit increased Cl- secretion with 600G, however, the mechanisms mediating this are gender-dependent.

Genistein induces estrogen-like effects in ovariectomized rats but fails to increase cardiac GLUT4 and oxidative stress.

This study aimed to determine whether a 2-week genistein treatment induced estrogen-like effects in ovariectomized (OVX) Sprague-Dawley rats, after 2 weeks of subcutaneous genistein injections (250 mg/kg of body weight/day). Uterine weight, uterine-to-body weight ratio, femur weight, and femur-to-body weight ratio were all significantly increased with genistein in OVX rats. Body weight was significantly decreased with genistein in OVX rats. Genistein had no effect on the weights of heart, heart-to-body ratio, and fat pad but significantly decreased heart rate and pulse pressure. Genistein had no effect on cardiac GLUT4 protein, oxidative stress, plasma glucose, nonesterified fatty acids, or low-density lipoprotein levels; however, plasma insulin levels were significantly increased. Our results show that a 2-week genistein treatment produced favorable estrogen-like effects on some physical and physiological characteristics in OVX rats. However, based on our experimental conditions, the effects of genistein were not associated with changes in cardiac GLUT4 or oxidative stress.

Effects of acute and 2-day genistein treatment on cardiac function and ischemic tolerance in ovariectomized rats.

BACKGROUND: Genistein, a naturally occurring isoflavonic phytoestrogen associated with reduced incidence of heart disease, may be a possible alternative treatment for postmenopausal women with heart disease. OBJECTIVE: This study examined the effects of genistein on in vitro heart function and ischemic tolerance in ovariectomized (OVX) Sprague-Dawley rats. METHODS: To examine the acute effects of genistein on cardiac function, isolated working hearts were perfused under aerobic conditions with increasing concentrations of genistein (10-150 microM). A separate group of OVX rats was used to assess ischemic tolerance: treated rats received genistein (250 mg/kg, dissolved in 200 microL dimethyl sulfoxide [DMSO]) injected once daily for 2 days, and control rats received DMSO only. After treatment, hearts were perfused for 30 minutes under aerobic conditions and then subjected to 20 minutes of global no-flow ischemia by clamping the preload and afterload lines, followed by 30 minutes of reperfusion. RESULTS: Genistein was associated with improvements in mechanical function in OVX rat hearts (n = 5) with maximum increases in contractility (259 mm Hg/sec above baseline) and cardiac output (7 mL/min above baseline) observed with 30 microM of genistein (both, P < 0.05). Relative to baseline, genistein-treated hearts (n = 5) also had greater ischemic tolerance than did control hearts (n = 6) and significant improvements in mean (SEM) recovery of contractility (to 75.0% [9.7%] of preischemic function; P < 0.05) and cardiac output (to 48.8% [12.3%] of preischemic function; P < 0.05) after reperfusion. These effects occurred without significant changes in myocardial levels of nonprotein thiols or thiobarbituric acid reactive substances, although a reduction in mean glucose transporter protein 4 content (13.2% [2.7%]; P < 0.05) was observed in genistein-treated hearts. No significant changes in blood pressure were observed with genistein. CONCLUSIONS: Despite the lack of significant changes in physical characteristics, 2-day treatment with genistein was associated with significant cardioprotective effects in OVX rats, suggesting a potential therapeutic role in postmenopausal women.

Effect of gestational ethanol exposure on parvalbumin and calretinin expressing hippocampal neurons in a chick model of fetal alcohol syndrome.

Fetal alcohol syndrome (FAS), a condition occurring in some children of mothers who have consumed alcohol during pregnancy, is characterized by physical deformities and learning and memory deficits. The chick hippocampus, whose functions are controlled by interneurons expressing calcium-binding proteins parvalbumin (PV) and calretinin (CR), is involved in learning and memory mechanisms. Effects on growth and development and hippocampal morphology were studied in chick embryos exposed to 5% and 10% ethanol volume/volume (vol/vol) for 2 or 8 days of gestation. There was a significant dose-dependent reduction (P<.05) in body weight and mean number per section of PV and CR expressing hippocampal neurons in ethanol-exposed chicks, without alterations in neuronal nuclear size or hippocampal volume, compared appropriate controls. Moreover, when chicks exposed to 5% ethanol for 2 and 8 days of gestation were compared, no significant differences were found in body parameters or neuronal counts. Similarly, exposure to 10% ethanol did not induce any significant changes in chicks exposed for 2 or 8 gestational days. Thus, these results suggest that gestational ethanol exposure induces a reduction in the mean number per section of PV and CR expressing hippocampal neurons, and could be a possible mechanism responsible for learning and memory disorders in FAS.

The pesticide methoxychlor decreases myotube formation in cell culture by slowing myoblast proliferation.

We studied the effect of the estrogenic pesticide methoxychlor (MXC) on skeletal muscle development using C2C12 cell culture. Myoblast cultures were exposed to various concentrations of MXC at various times during the process of myoblast fusion into myotubes. We observed that MXC exposure decreased myotube formation. In addition, we observed myoblasts with cytoplasmic vacuoles in cultures exposed to MXC. Because cytoplasmic vacuoles can be characteristic of cell death, apoptosis assays and trypan blue exclusion assays were performed. We found no difference in the frequency of apoptosis or in the frequency of cell death for cultures exposed to MXC and untreated cultures. Collectively, these results indicate that MXC exposure decreases myotube formation without causing cell death. In contrast, when cell proliferation was assessed, untreated cultures had a myoblast proliferation rate 50% greater than cultures exposed to MXC. We conclude that MXC decreases myotube formation at least in part by slowing myoblast proliferation. Furthermore, we suggest that direct exposure to MXC could affect skeletal muscle development in animals or humans, in addition to the defects in reproductive development that have previously been reported.

Magnesium sulfate effectively reduces blood pressure in an animal model of preeclampsia.

OBJECTIVE: We tested the ability of magnesium sulfate to reduce hypertension and neonatal growth retardation in an animal model of preeclampsia. STUDY DESIGN: On day 17 of pregnancy, osmotic minipumps were inserted subcutaneously to continuously deliver either vehicle (saline control group), or N-nitro-L-arginine methyl ester (L-NAME) (50 mg/kg/day), or L-NAME (50 mg/kg/day) in combination with magnesium sulfate (60 mg/kg/day). Prior to insertion, blood pressure and heart rate were monitored with a pneumatic tail cuff device. Blood pressure measurements were repeated on days 18, 20, and 21 of pregnancy. Blood was obtained on days 17 and 21, along with urine, to assess magnesium levels and degree of proteinuria. Pups were weighed and measured at 48 hours postpartum. RESULTS: Rats receiving L-NAME developed hypertension within 24 hours of implantation (108 +/- 3.9 vs. 123 +/- 3.4 mmHg, p < 0.05). Magnesium sulfate, given along with L-NAME did not prevent mean blood pressure from increasing, but reduced it by day 21 compared to L-NAME given alone (107 +/- 3.4 vs. 122 +/- 8.7 mmHg, respectively, p < 0.05). Magnesium sulfate reduced neonatal growth retardation by improving the weight of the pups compared to pups from maternal rats given L-NAME alone (6.1 +/- 0.1 vs. 5.2 +/- 0.3 grams, respectively, p < 0.05). CONCLUSION: Maternal magnesium sulfate reduces blood pressure and increases neonatal size compared to L-NAME without magnesium. These findings support a beneficial effect of magnesium in preeclampsia.

MgSO4 prevents left ventricular dysfunction in an animal model of preeclampsia.

OBJECTIVE: We hypothesized that cardiac function would be reduced in a pregnant rat model of preeclampsia induced by L-NAME, a NOS inhibitor, and be reversed with magnesium sulfate prophylaxis. STUDY DESIGN: Female Sprague-Dawley rats were bred in-house. On gestational day 17, rats were anesthetized and osmotic minipumps were implanted to continuously deliver saline, L-NAME, or L-NAME and MgSO4. On gestational day 21, hearts were isolated and perfused in the working mode using Krebs Henseleit buffer. RESULTS: Pregnant rats treated with L-NAME displayed significant hypertension compared to the saline-treated controls (P < 0.05). Moreover, cardiac output and cardiac work were significantly reduced in the L-NAME-treated rats compared to controls (P < 0.05). In the L-NAME-treated rats given MgSO4, cardiac function remained normal. CONCLUSION: Cardiac function is depressed in an animal model of preeclampsia induced by L-NAME infusion. MgSO4 prevented the reduction in cardiac function and is clearly beneficial in preserving normal heart function in preeclampsia.

In vitro biophysical strain model for understanding mechanisms of osteopathic manipulative treatment.

CONTEXT: Normal physiologic movement, pathologic conditions, and osteopathic manipulative treatment (OMT) are believed to produce effects on the shape and proliferation of human fibroblasts. Studies of biophysically strained fibroblasts would be useful in producing a model of the cellular mechanisms underlying OMT. OBJECTIVE: To investigate the effects of acyclic in vitro biophysical strain on normal human dermal fibroblasts and observe potential changes in cellular shape and proliferation, as well as potential changes in cellular production of nitric oxide, interleukin (IL) 1beta, and IL-6. DESIGN AND METHODS: Randomized controlled trial. Human fibroblasts were subjected in vitro to control conditions (no strain) or biophysical strain of various magnitudes (10%-30% beyond resting length) and durations (12-72 hours). After control or strain stimuli, fibroblasts were analyzed for potential changes in cell shape, proliferative capacity, nitric oxide secretion, and cytokine (IL-1beta, IL-6) secretion. RESULTS: Low strain magnitudes (<20%) induced mild cellular rounding and pseudopodia truncation. High strain magnitudes (>20%) decreased overall cell viability and the mitogenic response, and induced cell membrane decomposition and pseudopodia loss. No basal or strain-induced secretion of IL-1beta was observed. Interleukin 6 concentrations increased two-fold, while nitric oxide levels increased three-fold, in cells strained at 10% magnitude for 72 hours (P<.05). CONCLUSION: Human fibroblasts respond to in vitro strain by secreting inflammatory cytokines, undergoing hyperplasia, and altering cell shape and alignment. The in vitro biophysical strain model developed by the authors is useful for simulating a variety of injuries, determining in vivo mediators of somatic dysfunction, and investigating the underlying mechanisms of OMT.