Intermittent hypoxia regulates RNA polymerase II in hippocampus and prefrontal cortex.

Intermittent hypoxia (IH) is a major pathological factor in the development of neural deficits associated with sleep-disordered breathing. Here we demonstrate that IH lasting 2 or 30 days, but not sustained hypoxia (SH) of the same duration, was accompanied by several posttranslational modifications of the large subunit of RNA polymerase II, Rpb1, including hydroxylation of proline 1465, phosphorylation of serine 5 residues within the C-terminal domain, and nondegradative ubiquitylation. These modifications were found to occur in two regions of the brain, hippocampal region CA1 and the prefrontal cortex, but not in neocortex, brainstem and CA3 region of hippocampus. We also found that mice exposed to 14 or 30 days of IH, but not SH, demonstrated cognitive deficits in behavioral assays. Furthermore, by using the pheochromocytoma-derived PC12 cell line, we showed that, under in vitro IH conditions, induction of Rpb1 hydroxylation, phosphorylation, and ubiquitylation required that the von Hippel-Lindau protein be present. We hypothesize that the observed modifications of Rpb1 participate in regulating the expression of genes involved in mediating cognitive deficits evoked by chronic IH.

High fat/refined carbohydrate diet enhances the susceptibility to spatial learning deficits in rats exposed to intermittent hypoxia.

BACKGROUND: Intermittent hypoxia during sleep (IH), as occurs in sleep disordered breathing (SDB), induces spatial learning deficits associated with regulation of transcription factors associated with learning and memory in the hippocampal CA1 region in rats. high fat refined carbohydrate diet (HF/RC) can induce similar deficits and associated changes in signaling pathways under normoxic conditions. METHODS: Sprague-Dawley adult male rats were fed either with (HF/RC) or low fat/complex carbohydrate diet (LF/CC) starting at post-natal day 30 for 90 days, and were then exposed for 14 days during light phase (12 h/day) to either normoxia (RA) or IH (21% and 10% O2 alternations every 90 s). Place-training reference memory task deficits were assessed in the Morris water maze. Total and ser-133 phosphorylated CREB were assessed in different brain regions by Western blotting and immunostaining in rats exposed to normoxia or IH and to LF/CC or HF/RC. RESULTS: Substantial decreases in CREB phosphorylation occurred in CA1 but not in motor cortex following either IH, HF/RC, and HF/RC + IH. Place-training reference memory task deficits were observed in rats exposed to IH and to HF/RC, and to a much greater extent in rats exposed to HF/RC + IH. CONCLUSIONS: Nutritional factors alter recruitment of transcription factors, possibly via oxidative-related pathways, and modulate the vulnerability of the CA1 region of the hippocampus to the episodic hypoxia that characterizes SDB, thereby enhancing neurocognitive susceptibility in SDB patients.

Increased oxidative stress is associated with chronic intermittent hypoxia-mediated brain cortical neuronal cell apoptosis in a mouse model of sleep apnea.

Chronic intermittent hypoxia (CIH), as occurs in obstructive sleep apnea (SA), is associated with substantial cortico-hippocampal damage leading to impairments of neurocognitive, respiratory and cardiovascular functions. Previous studies in a rat model have shown that CIH increases brain cortical neuronal cell death. However, the molecular events leading to CIH-mediated neuronal cell death remain largely undefined. The oscillation of O2 concentrations during CIH remarkably mimics the processes of ischemia/re-oxygenation and could therefore increase cellular production of reactive oxygen species (ROS). We extended the CIH paradigm to a mouse model of SA to identify the molecular mechanisms underlying cortical neuronal cell death. A significant increase of ROS production in mouse brain cortex and cortical neuronal cells was detected by fluorescent oxidation assays upon exposure of mice to CIH, followed by increased expression of oxidative stress response markers, c-Fos, c-Jun and NF-kappaB in mouse brain cortex, as revealed by immunohistochemical and LacZ reporter assays respectively. Long-term exposure of mice to CIH increased the levels of protein oxidation, lipid peroxidation and nucleic acid oxidation in mouse brain cortex. Furthermore, exposure of mice to CIH induced caspase-3 activation and increased some cortical neuronal cell apoptosis. On the other hand, transgenic mice overexpressing Cu,Zn-superoxide dismutase exposed to CIH conditions had a lower level of steady-state ROS production and reduced neuronal apoptosis in brain cortex compared with that of normal control mice. Taken together, these findings suggest that the increased ROS production and oxidative stress propagation contribute, at least partially, to CIH-mediated cortical neuronal apoptosis and neurocognitive dysfunction.

Intermittent hypoxic exposure during light phase induces changes in cAMP response element binding protein activity in the rat CA1 hippocampal region: water maze performance correlates.

Intermittent hypoxia (IH) during sleep, a characteristic feature of sleep-disordered breathing (SDB) is associated with time-dependent apoptosis and spatial learning deficits in the adult rat. The mechanisms underlying such neurocognitive deficits remain unclear. Activation of the cAMP-response element binding protein (CREB) transcription factor mediates critical components of neuronal survival and memory consolidation in mammals. CREB phosphorylation and DNA binding, as well as the presence of apoptosis in the CA1 region of the hippocampus were examined in Sprague-Dawley male rats exposed to IH. Spatial reference task learning was assessed with the Morris water maze. IH induced significant decreases in Ser-133 phosphorylated CREB (pCREB) without changes in total CREB, starting as early as 1 h IH, peaking at 6 h-3 days, and returning toward normoxic levels by 14-30 days. Double-labeling immunohistochemistry for pCREB and Neu-N (a neuronal marker) confirmed these findings. The expression of cleaved caspase 3 (cC3) in the CA1, a marker of apoptosis, peaked at 3 days and returned to normoxic values at 14 days. Initial IH-induced impairments in spatial learning were followed by partial functional recovery starting at 14 days of IH exposure. We postulate that IH elicits time-dependent changes in CREB phosphorylation and nuclear binding that may account for decreased neuronal survival and spatial learning deficits in the adult rat. We suggest that CREB changes play an important role in the neurocognitive morbidity of SDB patients.

Developmental differences in cortical and hippocampal vulnerability to intermittent hypoxia in the rat.

Obstructive sleep apnea is characterized by intermittent hypoxic events during sleep, and is associated with substantial neurocognitive morbidity, particularly in children. Intermittent hypoxia (IH) leads to increases in apoptosis in the cortex and hippocampus of the adult rat, peaking at 48 h of exposure. To examine whether the susceptibility to IH exhibits developmental differences, rats were exposed to 48 h of IH at ages 2, 5, 10, 15, 20, 25, 30, 60, and 120-day postnatally, and apoptosis was determined by terminal deoxy-nucleotidyl transferase-mediated in situ end labeling and immunohistochemical staining for single-stranded DNA. Although IH induced apoptosis at all postnatal ages, smaller increases were apparent in 2 and 5-day old (P < 0.01 vs. any other age) while peak apoptosis occurred at 10-25 days (P < 0.001 vs. 30, 60, and 120 days). We conclude that a unique window of vulnerability to IH is present in the cortex and hippocampus during post-natal maturation, and may underlie the high frequency of neurobehavioral deficits associated with obstructive sleep apnea in children.

G proteins in rat prefrontal cortex (PFC) are differentially activated as a function of oxygen status and PFC region.

This study tested the hypothesis that activation of guanine nucleotide binding (G) proteins in rat prefrontal cortex (PFC) is altered by hypoxia. G protein activation by the cholinergic agonist carbachol and the opioid agonist DAMGO was quantified using [(35)S]GTPgammaS autoradiography. G protein activation was expressed as nCi/g tissue in the PFC of 18 rats exposed for 14 consecutive days to sustained hypoxia (10% O(2)), intermittent hypoxia (10% and 21% O(2) alternating every 90 s), or room air (21% O(2)). Relative to basal levels of G protein activation, carbachol and DAMGO increased G protein activation by approximately 70% across all oxygen concentrations. Compared to the room air condition, sustained hypoxia caused a significant increase in G protein activation in frontal association (FrA) region of the PFC. Region-specific comparisons revealed that intermittent and sustained hypoxia caused greater DAMGO-stimulated G protein activation in the FrA than in the pre-limbic (PrL). The data show for the first time that hypoxia increased G protein activation in PFC. The results suggest the potential for hypoxia-induced enhancements in G protein activation to alter PFC function.

Hypoxia modulates cholinergic but not opioid activation of G proteins in rat hippocampus.

Intermittent hypoxia, such as that associated with obstructive sleep apnea, can cause neuronal death and neurobehavioral dysfunction. The cellular and molecular mechanisms through which hypoxia alter hippocampal function are incompletely understood. This study used in vitro [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate ([(35)S]GTP gamma S) autoradiography to test the hypothesis that carbachol and DAMGO activate hippocampal G proteins. In addition, this study tested the hypothesis that in vivo exposure to different oxygen (O(2)) concentrations causes a differential activation of G proteins in the CA1, CA3, and dentate gyrus (DG) regions of the hippocampus. G protein activation was quantified as nCi/g tissue in CA1, CA3, and DG from rats housed for 14 days under one of three different oxygen conditions: normoxic (21% O(2)) room air, or hypoxia (10% O(2)) that was intermittent or sustained. Across all regions of the hippocampus, activation of G proteins by the cholinergic agonist carbachol and the mu opioid agonist [D-Ala(2), N-Met-Phe(4), Gly(5)] enkephalin (DAMGO) was ordered by the degree of hypoxia such that sustained hypoxia > intermittent hypoxia > room air. Carbachol increased G protein activation during sustained hypoxia (38%), intermittent hypoxia (29%), and room air (27%). DAMGO also activated G proteins during sustained hypoxia (52%), intermittent hypoxia (48%), and room air (43%). Region-specific comparisons of G protein activation revealed that the DG showed significantly less activation by carbachol following intermittent hypoxia and sustained hypoxia than the CA1. Considered together, the results suggest the potential for hypoxia to alter hippocampal function by blunting the cholinergic activation of G proteins within the DG.