Regional distribution of the prostaglandin E2 receptor EP1 in the rat brain: accumulation in Purkinje cells of the cerebellum

Prostaglandin E2 (PGE2), is a major prostanoid produced by the activity of cyclooxygenases (COX) in response to various physiological and pathological stimuli. PGE2 exerts its effects by activating four specific E-type prostanoid receptors (EP1, EP2, EP3 and EP4). In the present study, we analyzed the expression of the PGE2 receptor EP1 (mRNA and protein) in different regions of the adult rat brain (hippocampus, hypothalamus, striatum, prefrontal cerebral cortex, parietal cortex, brain stem and cerebellum) using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemical methods. On a regional basis, levels of EP1 mRNA were the highest in parietal cortex and cerebellum. At the protein level, we found a very strong expression of EP1 in cerebellum as revealed by Western blotting experiments. Furthermore, the present study provides for the first time evidence that the EP1 receptor is highly expressed in the cerebellum, where the Purkinje cells displayed a very high immunolabeling of their perikaryon and dendrites as observed in the immunohistochemical analysis. Results from the present study indicate that the EP1 prostanoid receptor is expressed in specific neuronal populations, which possibly determine the region specific response to PGE2

Ascorbic acid enhances the inhibitory effect of aspirin on neuronal cyclooxygenase-2-mediated prostaglandin E2 production.

Inhibition of neuronal cyclooxygenase-2 (COX-2) and hence prostaglandin E2 (PGE2) synthesis by non-steroidal anti-inflammatory drugs has been suggested to protect neuronal cells in a variety of pathophysiological situations including Alzheimer's disease and ischemic stroke. Ascorbic acid (vitamin C) has also been shown to protect cerebral tissue in a variety of experimental conditions, which has been attributed to its antioxidant capacity. In the present study, we show that ascorbic acid dose-dependently inhibited interleukin-1beta (IL-1beta)-mediated PGE2 synthesis in the human neuronal cell line, SK-N-SH. Furthermore, in combination with aspirin, ascorbic acid augmented the inhibitory effect of aspirin on PGE2 synthesis. However, ascorbic acid had no synergistic effect along with other COX inhibitors (SC-58125 and indomethacin). The inhibition of IL-1beta-mediated PGE2 synthesis by ascorbic acid was not due to the inhibition of the expression of COX-2 or microsomal prostaglandin E synthase (mPGES-1). Rather, ascorbic acid dose-dependently (0.1-100 microM) produced a significant reduction in IL-1beta-mediated production of 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha), a reliable indicator of free radical formation, suggesting that the effects of ascorbic acid on COX-2-mediated PGE2 biosynthesis may be the result of the maintenance of the neuronal redox status since COX activity is known to be enhanced by oxidative stress. Our results provide in vitro evidence that the neuroprotective effects of ascorbic acid may depend, at least in part, on its ability to reduce neuronal COX-2 activity and PGE2 synthesis, owing to its antioxidant properties. Further, these experiments suggest that a combination of aspirin with ascorbic acid constitutes a novel approach to render COX-2 more sensitive to inhibition by aspirin, allowing an anti-inflammatory therapy with lower doses of aspirin, thereby avoiding the side effects of the usually high dose aspirin treatment

Hypoxia Alters Cell Cycle Regulatory Protein Expression and Induces Premature Maturation of Oligodendrocyte Precursor Cells

Periventricular white matter injury (PWMI) is a common form of brain injury sustained by preterm infants. A major factor that predisposes to PWMI is hypoxia. Because oligodendrocytes (OLs) are responsible for myelination of axons, abnormal OL development or function may affect brain myelination. At present our understanding of the influences of hypoxia on OL development is limited. To examine isolated effects of hypoxia on OLs, we examined the influences of hypoxia on OL development in vitro.Cultures of oligodendrocyte precursor cells (OPCs) were prepared from mixed glial cultures and were 99% pure. OPCs were maintained at 21% O(2) or hypoxia (1% or 4% O(2)) for up to 7 days. We observed that 1% O(2) lead to an increase in the proportion of myelin basic protein (MBP)-positive OLs after 1 week in culture, and a decrease in the proportion of platelet-derived growth factor receptor alpha (PDGFRalpha)-positive cells suggesting premature OL maturation. Increased expression of the cell cycle regulatory proteins p27(Kip1) and phospho-cdc2, which play a role in OL differentiation, was seen as well.These results show that hypoxia interferes with the normal process of OL differentiation by inducing premature OPC maturation

Increased Mitochondrial Calcium Sensitivity and Abnormal Expression of Innate Immunity Genes Precede Dopaminergic Defects in Pink1-Deficient Mice

BACKGROUND: PTEN-induced kinase 1 (PINK1) is linked to recessive Parkinsonism (EOPD). Pink1 deletion results in impaired dopamine (DA) release and decreased mitochondrial respiration in the striatum of mice. To reveal additional mechanisms of Pink1-related dopaminergic dysfunction, we studied Ca²+ vulnerability of purified brain mitochondria, DA levels and metabolism and whether signaling pathways implicated in Parkinson\u27s disease (PD) display altered activity in the nigrostriatal system of Pink1⁻/⁻ mice. METHODS AND FINDINGS: Purified brain mitochondria of Pink1⁻/⁻ mice showed impaired Ca²+ storage capacity, resulting in increased Ca²+ induced mitochondrial permeability transition (mPT) that was rescued by cyclosporine A. A subpopulation of neurons in the substantia nigra of Pink1⁻/⁻ mice accumulated phospho-c-Jun, showing that Jun N-terminal kinase (JNK) activity is increased. Pink1⁻/⁻ mice 6 months and older displayed reduced DA levels associated with increased DA turnover. Moreover, Pink1⁻/⁻ mice had increased levels of IL-1β, IL-12 and IL-10 in the striatum after peripheral challenge with lipopolysaccharide (LPS), and Pink1⁻/⁻ embryonic fibroblasts showed decreased basal and inflammatory cytokine-induced nuclear factor kappa-β (NF-κB) activity. Quantitative transcriptional profiling in the striatum revealed that Pink1⁻/⁻ mice differentially express genes that (i) are upregulated in animals with experimentally induced dopaminergic lesions, (ii) regulate innate immune responses and/or apoptosis and (iii) promote axonal regeneration and sprouting. CONCLUSIONS: Increased mitochondrial Ca²+ sensitivity and JNK activity are early defects in Pink1⁻/⁻ mice that precede reduced DA levels and abnormal DA homeostasis and may contribute to neuronal dysfunction in familial PD. Differential gene expression in the nigrostriatal system of Pink1⁻/⁻ mice supports early dopaminergic dysfunction and shows that Pink1 deletion causes aberrant expression of genes that regulate innate immune responses. While some differentially expressed genes may mitigate neurodegeneration, increased LPS-induced brain cytokine expression and impaired cytokine-induced NF-κB activation may predispose neurons of Pink1⁻/⁻ mice to inflammation and injury-induced cell death

Basal and inflammatory signal-induced NF-κB activity is reduced in <i>Pink1^−/−</i> embryonic fibroblasts.

<p>Wildtype and <i>Pink1^−/−</i> MEF were transfected with plasmid pNF-κB-luc (Clontech). Twenty-four hours after transfection the cells were incubated for 8 hours with 30 ng/ml TNF-α, 10 ng/ml IL-1β, 100 ng/ml LPS or remained untreated (control) and luciferase activity was measured as described in the Methods. (A) NF-κB-dependent luciferase activity, expressed as relative light units (RLU) per mg protein. Data represent pooled values from two independent experiments with similar results. In each experiment luciferase activity was measured in five wells per condition. Non-transfected cells (NT) showed no luciferase activity. (B) Wildtype and <i>Pink1^−/−</i> fibroblasts were transfected with the same plasmid/lipofectamine mixture to ensure equal transfection efficiency, which was confirmed to be the case with an EGFP expression plasmid as described in the Methods. ** <i>P</i><0.01.</p

Analysis and quantification of <i>Pink1</i> mRNA expression.

<p>Total RNA isolated from the brain of mice was converted to cDNA. (A) PCR with a forward primer located in exon 3 and reverse primers located in exon 6, 7 or 8, generating expected PCR products of 480, 624 and 1001 bp for the <i>Pink1</i> wildtype allele in wildtype and <i>Pink1^+/−</i> samples. In contrast, PCR products of 133, 149 and 289 that would arise if exon 3 were directly spliced to exon 6, 7, or 8 in transcripts derived from the mutant <i>Pink1</i> allele were absent in both <i>Pink1^+/−</i> and <i>Pink1^−/−</i> samples. This shows that alternatively spliced transcripts are <i>not</i> generated from the disrupted <i>Pink1</i> allele. (B) Quantitative real-time PCR with primers located in exon 1 (forward) and exon 3 (reverse) showing that <i>Pink1^−/−</i> mice express at most 6.8% of a truncated <i>Pink1</i> mRNA encompassing exons 1–3, compared to wildtype mice. However, such a truncated mRNA would not give rise to any PINK1 protein with kinase activity (see main text).</p

Inactivation of the mouse <i>Pink1</i> locus by gene targeting in ES cells.

<p>(A) Mouse <i>Pink1</i> gene structure, (B) targeting vector and (C) mutated <i>Pink1</i> gene lacking exons 4 and 5 after homologous recombination with the targeting vector. The PINK1 kinase domain is encoded within exons 2–8, with exons 4 and 5 specifying amino acids 257–374. Active site Asp362 and at least 15 familial PD-associated <i>Pink1</i> mutations cluster in exons 4 and 5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016038#pone.0016038-Mills1" target="_blank">[143]</a>.</p

Dopamine levels, dopamine turnover and dopamine neuron counts.

<p>(A) Decreased DA levels in the striatum of <i>Pink1^−/−</i> mice aged 6 months and older. (B) Normal counts of dopaminergic neurons in the substantia nigra pars compacta (SNc) of 1-year old <i>Pink1^−/−</i> mice. (C) Increased DA turnover in <i>Pink1^−/−</i> mice. Eight mice per genotype were used for catecholamine analysis (A and C). Five wildtype and six <i>Pink1^−/−</i> mice were used to determine nigral DA neuron numbers by unbiased stereology (B). * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001.</p

Cytokine expression in the striatum and isolated microglial cultures.

<p>(A) Wildtype and <i>Pink1^−/−</i> mice (n = 4 mice/genotype) were injected ip with 0.33 µg LPS/g body weight. Cytokines in striatal homogenates (corresponding to 100 µg total protein) were measured eight hours later by ELISA as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016038#s4" target="_blank">Materials and Methods</a>. * <i>P</i><0.05, compared to wildtype mice. Basal cytokine levels, measured in a separate experiment, were not statistically different between wildtype and <i>Pink1^−/−</i> mice. (B) Microglial cultures derived from the forebrain of neonatal wildtype and <i>Pink1^−/−</i> mice were incubated with 100 ng/ml LPS for 24 hours in 24-well plates and the cytokines were measured with an ELISA as described in the Methods (n = 8 wells per condition). (C) Strong induction of IL-6, TNF-α and G-CSF in wildtype and <i>Pink1^−/−</i> microglia cells demonstrates that the cells were capable of responding to the LPS stimulus. In panels B and C, background-corrected absorbance is plotted (OD450 minus OD570). (D) Real-time PCR expression analysis of CD3 mRNA (specific T cell marker) in the striatum of control and LPS-treated wildtype and <i>Pink1^−/−</i> mice, showing that the T cell marker is barely detectable (Ct values of 39.67 and 38.46) and not increased by LPS treatment.</p