Neuronal Glud1 (Glutamate Dehydrogenase 1) Over-Expressing Mice: Increased Glutamate Formation and Synaptic Release, Loss of Synaptic Activity, and Adaptive Changes in Genomic Expression

Glutamate dehydrogenase 1 (GLUD1) is a mitochondrial enzyme expressed in all tissues, including brain. Although this enzyme is expressed in glutamatergic pathways, its function as a regulator of glutamate neurotransmitter levels is still not well defined. In order to gain an understanding of the role of GLUD1 in the control of glutamate levels and synaptic release in mammalian brain, we generated transgenic (Tg) mice that over-express this enzyme in neurons of the central nervous system. The Tg mice have increased activity of GLUD, as well as elevated levels and increased synaptic and depolarization-induced release of glutamate. These mice suffer age-associated losses of dendritic spines, nerve terminals, and neurons. The neuronal losses and dendrite structural changes occur in select regions of the brain. At the transcriptional level in the hippocampus, cells respond by increasing the expression of genes related to neurite growth and synapse formation, indications of adaptive or compensatory responses to the effects of increases in the release and action of glutamate at synapses. Because these Tg mice live to a relatively old age they are a good model of the effects of a “hyperglutamatergic” state on the aging process in the nervous system. The mice are also useful in defining the molecular pathways affected by the over-activation of GLUD in glutamatergic neurons of the brain and spinal cord

Transgenic Expression of Glud1 (Glutamate Dehydrogenase 1) in Neurons: In Vivo Model of Enhanced Glutamate Release, Altered Synaptic Plasticity, and Selective Neuronal Vulnerability

This is the published version. Copyright 2009 Society for Neuroscience.The effects of lifelong, moderate excess release of glutamate (Glu) in the CNS have not been previously characterized. We created a transgenic (Tg) mouse model of lifelong excess synaptic Glu release in the CNS by introducing the gene for glutamate dehydrogenase 1 (Glud1) under the control of the neuron-specific enolase promoter. Glud1 is, potentially, an important enzyme in the pathway of Glu synthesis in nerve terminals. Increased levels of GLUD protein and activity in CNS neurons of hemizygous Tg mice were associated with increases in the in vivo release of Glu after neuronal depolarization in striatum and in the frequency and amplitude of miniature EPSCs in the CA1 region of the hippocampus. Despite overexpression of Glud1 in all neurons of the CNS, the Tg mice suffered neuronal losses in select brain regions (e.g., the CA1 but not the CA3 region). In vulnerable regions, Tg mice had decreases in MAP2A labeling of dendrites and in synaptophysin labeling of presynaptic terminals; the decreases in neuronal numbers and dendrite and presynaptic terminal labeling increased with advancing age. In addition, the Tg mice exhibited decreases in long-term potentiation of synaptic activity and in spine density in dendrites of CA1 neurons. Behaviorally, the Tg mice were significantly more resistant than wild-type mice to induction and duration of anesthesia produced by anesthetics that suppress Glu neurotransmission. The Glud1 mouse might be a useful model for the effects of lifelong excess synaptic Glu release on CNS neurons and for age-associated neurodegenerative processes

Hemodiafiltration maintains a sustained improvement in blood pressure compared to conventional hemodialysis in children-the HDF, heart and height (3H) study

BACKGROUND: Hypertension is prevalent in children on dialysis and associated with cardiovascular disease. We studied the blood pressure (BP) trends and the evolution of BP over 1 year in children on conventional hemodialysis (HD) vs. hemodiafiltration (HDF). METHODS: This is a post hoc analysis of the "3H - HDF-Hearts-Height" dataset, a multicenter, parallel-arm observational study. Seventy-eight children on HD and 55 on HDF who had three 24-h ambulatory BP monitoring (ABPM) measures over 1 year were included. Mean arterial pressure (MAP) was calculated and hypertension defined as 24-h MAP standard deviation score (SDS) ≥95th percentile. RESULTS: Poor agreement between pre-dialysis systolic BP-SDS and 24-h MAP was found (mean difference - 0.6; 95% limits of agreement -4.9-3.8). At baseline, 82% on HD and 44% on HDF were hypertensive, with uncontrolled hypertension in 88% vs. 25% respectively; p < 0.001. At 12 months, children on HDF had consistently lower MAP-SDS compared to those on HD (p < 0.001). Over 1-year follow-up, the HD group had mean MAP-SDS increase of +0.98 (95%CI 0.77-1.20; p < 0.0001), whereas the HDF group had a non-significant increase of +0.15 (95%CI -0.10-0.40; p = 0.23). Significant predictors of MAP-SDS were dialysis modality (β = +0.83 [95%CI +0.51 - +1.15] HD vs. HDF, p < 0.0001) and higher inter-dialytic-weight-gain (IDWG)% (β = 0.13 [95%CI 0.06-0.19]; p = 0.0003). CONCLUSIONS: Children on HD had a significant and sustained increase in BP over 1 year compared to a stable BP in those on HDF, despite an equivalent dialysis dose. Higher IDWG% was associated with higher 24-h MAP-SDS in both groups

Hemodiafiltration Is Associated With Reduced Inflammation and Increased Bone Formation Compared With Conventional Hemodialysis in Children: The HDF, Hearts and Heights (3H) Study

BACKGROUND: Patients on dialysis have a high burden of bone-related comorbidities, including fractures. We report a post hoc analysis of the prospective cohort study HDF, Hearts and Heights (3H) to determine the prevalence and risk factors for chronic kidney disease-related bone disease in children on hemodiafiltration (HDF) and conventional hemodialysis (HD). METHODS: The baseline cross-sectional analysis included 144 children, of which 103 (61 HD, 42 HDF) completed 12-month follow-up. Circulating biomarkers of bone formation and resorption, inflammatory markers, fibroblast growth factor-23, and klotho were measured. RESULTS: Inflammatory markers interleukin-6, tumor necrosis factor-α, and high-sensitivity C-reactive protein were lower in HDF than in HD cohorts at baseline and at 12 months (P < .001). Concentrations of bone formation (bone-specific alkaline phosphatase) and resorption (tartrate-resistant acid phosphatase 5b) markers were comparable between cohorts at baseline, but after 12-months the bone-specific alkaline phosphatase/tartrate-resistant acid phosphatase 5b ratio increased in HDF (P = .004) and was unchanged in HD (P = .44). On adjusted analysis, the bone-specific alkaline phosphatase/tartrate-resistant acid phosphatase 5b ratio was 2.66-fold lower (95% confidence interval, −3.91 to −1.41; P < .0001) in HD compared with HDF. Fibroblast growth factor-23 was comparable between groups at baseline (P = .52) but increased in HD (P < .0001) and remained unchanged in HDF (P = .34) at 12 months. Klotho levels were similar between groups and unchanged during follow-up. The fibroblast growth factor-23/klotho ratio was 3.86-fold higher (95% confidence interval, 2.15–6.93; P < .0001) after 12 months of HD compared with HDF. CONCLUSION: Children on HDF have an attenuated inflammatory profile, increased bone formation, and lower fibroblast growth factor-23/klotho ratios compared with those on HD. Long-term studies are required to determine the effects of an improved bone biomarker profile on fracture risk and cardiovascular health

In vitro human growth hormone increases human chorionic gonadotropin and progesterone secretion by human placenta at term: evidence of a modulatory role by opioids

We examined the in vitro effect of human growth hormone (hGH) on hormone placental production and the modulation by opioids of this function. Small placental fragments from 12 term placentas were incubated at 37 degrees C in a 95% air and 5% CO2 atmosphere for 4 h with various concentrations of hGH (1-1000 ng/ml) or naloxone (3-500 ng/ml). Both hGH and naloxone increased the concentrations of human chorionic gonadotropin (hCG) and progesterone in the media. The effect of the hGH was dose-dependent and statistically significant at 10 ng/ml, while naloxone was able to increase hCG and progesterone production only at the highest doses (250-500 ng/ml). The concomitant treatment with ineffective doses of naloxone and hGH was able to enhance hCG and progesterone secretion reaching levels similar to those obtained with the highest doses of hGH alone. High naloxone concentrations significantly decreased both hCG and progesterone secretion induced by high doses of hGH. This study confirms the relevance of growth hormone in sustaining placental endocrine activities and indicates an effect of opioids in modulating these function

The Krüppel-like factor 9 (KLF9) network in HEC-1-A endometrial carcinoma cells suggests the carcinogenic potential of dys-regulated KLF9 expression

<p>Abstract</p> <p>Background</p> <p>Krüppel-like factor 9 (KLF9) is a transcriptional regulator of uterine endometrial cell proliferation, adhesion and differentiation; processes essential for pregnancy success and which are subverted during tumorigenesis. The network of endometrial genes controlled by KLF9 is largely unknown. Over-expression of KLF9 in the human endometrial cancer cell line HEC-1-A alters cell morphology, proliferative indices, and differentiation, when compared to KLF9 under-expressing HEC-1-A cells. This cell line provides a unique model for identifying KLF9 downstream gene targets and signaling pathways.</p> <p>Methods</p> <p>HEC-1-A sub-lines differing in relative levels of KLF9 were subjected to microarray analysis to identify differentially-regulated RNAs.</p> <p>Results</p> <p>KLF9 under-expression induced twenty four genes. The KLF9-suppressed mRNAs encode protein participants in: aldehyde metabolism (AKR7A2, ALDH1A1); regulation of the actin cytoskeleton and cell motility (e.g., ANK3, ITGB8); cellular detoxification (SULT1A1, ABCC4); cellular signaling (e.g., ACBD3, FZD5, RAB25, CALB1); and transcriptional regulation (PAX2, STAT1). Sixty mRNAs were more abundant in KLF9 over-expressing sub-lines. The KLF9-induced mRNAs encode proteins which participate in: regulation and function of the actin cytoskeleton (COTL1, FSCN1, FXYD5, MYO10); cell adhesion, extracellular matrix and basement membrane formation (e.g., AMIGO2, COL4A1, COL4A2, LAMC2, NID2); transport (CLIC4); cellular signaling (e.g., BCAR3, MAPKAPK3); transcriptional regulation [e.g., KLF4, NR3C1 (glucocorticoid receptor), RXRα], growth factor/cytokine actions (SLPI, BDNF); and membrane-associated proteins and receptors (e.g., CXCR4, PTCH1). In addition, the abundance of mRNAs that encode hypothetical proteins (KLF9-inhibited: C12orf29 and C1orf186; KLF9-induced: C10orf38 and C9orf167) were altered by KLF9 expression. Human endometrial tumors of high tumor grade had decreased KLF9 mRNA abundance.</p> <p>Conclusion</p> <p>KLF9 influences the expression of uterine epithelial genes through mechanisms likely involving its transcriptional activator and repressor functions and which may underlie altered tumor biology with aberrant KLF9 expression.</p

Genomic and biochemical approaches in the discovery of mechanisms for selective neuronal vulnerability to oxidative stress

<p>Abstract</p> <p>Background</p> <p>Oxidative stress (OS) is an important factor in brain aging and neurodegenerative diseases. Certain neurons in different brain regions exhibit selective vulnerability to OS. Currently little is known about the underlying mechanisms of this selective neuronal vulnerability. The purpose of this study was to identify endogenous factors that predispose vulnerable neurons to OS by employing genomic and biochemical approaches.</p> <p>Results</p> <p>In this report, using <it>in vitro </it>neuronal cultures, <it>ex vivo </it>organotypic brain slice cultures and acute brain slice preparations, we established that cerebellar granule (CbG) and hippocampal CA1 neurons were significantly more sensitive to OS (induced by paraquat) than cerebral cortical and hippocampal CA3 neurons. To probe for intrinsic differences between <it>in vivo </it>vulnerable (CA1 and CbG) and resistant (CA3 and cerebral cortex) neurons under basal conditions, these neurons were collected by laser capture microdissection from freshly excised brain sections (no OS treatment), and then subjected to oligonucleotide microarray analysis. GeneChip-based transcriptomic analyses revealed that vulnerable neurons had higher expression of genes related to stress and immune response, and lower expression of energy generation and signal transduction genes in comparison with resistant neurons. Subsequent targeted biochemical analyses confirmed the lower energy levels (in the form of ATP) in primary CbG neurons compared with cortical neurons.</p> <p>Conclusion</p> <p>Low energy reserves and high intrinsic stress levels are two underlying factors for neuronal selective vulnerability to OS. These mechanisms can be targeted in the future for the protection of vulnerable neurons.</p

Activity dependent stimulation increases synaptic efficacy in spared pathways in an anesthetized rat model of spinal cord contusion injury

Background: Closed-loop neuromodulation systems have received increased attention in recent years as potential therapeutic approaches for treating neurological injury and disease. Objective: The purpose of this study was to assess the ability of intraspinal microstimulation (ISMS), triggered by action potentials (spikes) recorded in motor cortex, to alter synaptic efficacy in descending motor pathways in an anesthetized rat model of spinal cord injury (SCI). Methods: Experiments were carried out in adult, male, Sprague Dawley rats with a moderate contusion injury at T8. For activity-dependent stimulation (ADS) sessions, a recording microelectrode was used to detect neuronal spikes in motor cortex that triggered ISMS in the spinal cord grey matter. SCI rats were randomly assigned to one of four experimental groups differing by: a) cortical spike-ISMS stimulus delay (10 or 25 ms) and b) number of ISMS pulses (1 or 3). Four weeks after SCI, ADS sessions were conducted in three consecutive 1-hour conditioning bouts for a total of 3 hours. At the end of each conditioning bout, changes in synaptic efficacy were assessed using intracortical microstimulation (ICMS) to examine the number of spikes evoked in spinal cord neurons during 5-minute test bouts. A multichannel microelectrode recording array was used to record cortically-evoked spike activity from multiple layers of the spinal cord. Results: The results showed that ADS resulted in an increase in cortically-evoked spikes in spinal cord neurons at specific combinations of spike-ISMS delays and numbers of pulses. Efficacy in descending motor pathways was increased throughout all dorsoventral depths of the hindlimb spinal cord. Conclusions: These results show that after an SCI, ADS can increase synaptic efficacy in spared pathways between motor cortex and spinal cord. This study provides further support for the potential of ADS therapy as an effective method for enhancing descending motor control after SCI