Intermittent hypoxia regulates RNA polymerase II in hippocampus and prefrontal cortex

Abstract

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.

Figure 1

Chronic IH, but not SH, induced P1465 hydroxylation, Ser-5 phosphorylation, and ubiquitylation of Rpb1 in nuclear extracts from the hippocampal CA1 region. Western blot analysis of hydroxylated (HP, Rpb1-P(OH)), Ser-5–phosphorylated (H14, Rpb1-pS5), and total Rpb1 (N20), as well as of the components of the pVHL complex, VHL, cullin 2, and the RING finger protein Rbx1. Each lane represents an individual animal. RA, room air; IH, intermittent hypoxia; SH, sustained hypoxia. Arrowheads point towards the Rpb1 bands detected with all three different antibodies. IB: immunoblot. HP antibody was used at a concentration of 1:500; H14 antibody was used at 1:2000, and N20 at 1:500.

Figure 2

Quantification of IH- and SH-induced modulation of different forms of Rpb1 and components of the pVHL complex in CA1 region of hippocampus. Data presented as means±SEM; * P<0.05, ** P<0.01; *** P< 0.001 as compared to controls; n=6 per group.

Figure 3

Chronic IH induces P1465 hydroxylation, Ser5 phosphorylation, and increased total Rpb1 in nuclear extracts from prefrontal cortex. A. Western blot analysis using indicated antibody; B. Quantification of averaged results; n=6 per group. Labeling as in Figs. 1 and 2.

Figure 4

(A) In extracts obtained from cortex, unlike in the hippocampus, there is no difference in hydroxylation, phosphorylation, or ubiquitylation of Rpb1 in mice exposed to IH or SH for 2 days as compared to mice exposed to room air. However, phosphorylation of Rpb1 is inhibited after 30 days of exposure to either IH or SH. Western blot analysis of nuclear extracts probed with the indicated antibodies. (B) Western blot analysis comparing constitutive levels of pVHL, Cullin2, and Rbx 1 in extracts from different parts of the brain. Labeling as in Fig1.

Figure 5

Spatial learning deficits in mice exposed to IH but not to SH. Top panel shows schematics of place-training session schedules for 6 days, with filled areas representing 12-hour periods of darkness, open areas corresponding to 12-hour daylight periods, and arrows to timing of water maze sessions in early darkness period. Mice were exposed to room air (filled squares, dashed line), IH (open circles, solid line), or SH (closed triangles, double-dashed line) for 14 (A) or 30 days (B). Mean latencies (sec, left) and swim distances (cm, right) to locate the target platform during place training. Data are expressed as mean ± SEM; * P<0.01 vs. controls; n=18 per group; each block represents 3 training sessions.

Figure 6

P1465 hydroxylation and ubiquitylation of Rpb1 correlate with the status of pVHL in pheochromocytoma cells (PC12). (A) Schematic representation of the fluctuations in O₂ level in the environmental chamber (left) and in the cell culture medium (right) during exposure of cells to the IH paradigm. (B) Western blot (and quantification of the optical density in arbitrary units) of indicated extracts shows that IH stimulates P1465 hydroxylation, Ser5 phosphorylation, and ubiquitylation of Rpb1 in PC12 cells expressing pVHL (VHL-WT), but not in cells with pVHL knockdown (VHL-AS). (C) Hydroxylation of the Rpb1 peptide, but not the HIF-1α peptide, is induced upon incubation of peptides with extract from VHL-WT cells exposed to IH. Lane 1, input corresponding to 1% of [³⁵S]pVHL; lanes 2 and 3, capture of [³⁵S]pVHL after the peptides were incubated with cytoplasmic extracts (cyt) from control (C) or IH-exposed cells; lanes 4 and 5, capture of [³⁵S]pVHL after the peptides were incubated with nuclear extracts (nuc) from control (C) or IH-exposed cells. In B and C, a representative example of at least three independent experiments is shown.

Figure 7

pVHL is necessary for inhibition of P1465 hydroxylation and Ser5 phosphorylation by SH in PC12 cells. Exposure to the indicated duration of 1% O2 repressed P1465 hydroxylation and Ser5 phosphorylation in PC12 cells expressing wild-type pVHL, but not in cells with diminished levels of pVHL due to the expression of VHL antisense mRNA. Western blots were probed with the indicated antibodies.