Muscleblind-like 2: circadian expression in the mammalian pineal gland is controlled by an adrenergic-cAMP mechanism

Abstract

Muscleblind-like 2 (Mbnl2) is a zinc finger protein first identified in Drosophila. It appears to be essential for photoreceptor development and to be involved in RNA splicing. Here we report that Mbnl2 is strongly expressed in the rat pineal gland. The abundance of pineal Mbnl2 transcripts follows a marked circadian rhythm with peak levels approximately sevenfold higher at night than day levels. Mbnl2 protein exhibits a similar rhythm. In vitro studies indicate that the abundance of Mbnl2 transcripts and protein are controlled by an adrenergic/cAMP mechanism.

Fig. 1. Tissue distribution of Mbnl2 mRNA

A. Northern blot analysis of Mbnl2 mRNA in selective tissues at night and during day. Rats were housed in a controlled lighting environment (LD 14:10). Total RNA was obtained from day tissues removed at ZT7 and night tissues removed at ZT19 under dim red light. RNA preparation and Northern blot analysis were performed as described in Materials and Methods. The blot was hybridized with a rat Mbnl2 probe. To normalize for RNA loading, blots were stripped and reprobed for 18S rRNA. Abbreviations are HIPPO, hippocampus; HYPO, hypothalamus; CTX cerebral cortex; CB, cerebellum; AD, adrenal gland; PG, pineal gland; PIT, pituitary gland; Muscle, skeletal muscle. Results are presented as the mean ± SE of three replicates. B. Radiochemical in situ hybridization histology of Mbnl2 mRNA in the rat brain. Animals were housed in a controlled lighting environment for two weeks (LD 12:12). The day sample is from an animal killed during daytime (ZT6) and the night one is from an animal killed during nighttime (ZT18). Scale bars, 1 mm. CB, cerebellImmuum; PG, pineal gland; CTX, cortex. For further details see the Materials and Methods section.

Fig. 2. Daily rhythm in rat pineal Mbnl2 mRNA and protein

Animals were housed in a controlled lighting environment for two weeks (LD 14:10 for panels A and B). Pineal glands were obtained at the indicated times. A. Mbnl2 mRNA: Each lane was loaded with 8 μg of total RNA. The blot was hybridized with a rat Mbnl2 probe. To normalize for RNA loading, the blot was reprobed for 18S rRNA. The ZT1, 7, and 13 data points are double plotted. Results are presented as the mean ± SE of three replicates. *, p < 0.01 vs. ZT7 group. B. Mbnl2 protein: Each lane was loaded with 60 μg protein from a pineal extract. Immunodetection was performed with a polyclonal rabbit anti-Mbnl2 serum at a dilution of 1:10,000. The Mbnl2 band appears around 40 kDa. The ZT1, 7, and 13 data points are double plotted. Results are presented as the mean ± SE of three replicates. *, p < 0.01 vs. ZT15 group. C. Pineal Mbnl2 mRNA. Animals were housed for two weeks in LD 14:10 (ZT7 and ZT9) or nine days in LD 14:10 followed by 5 days in constant darkness (DD)(Circadian time [CT] 7 and 19). Each lane was loaded with 8 μg of total RNA. The blot was hybridized with a rat Mbnl2 probe. To normalize for RNA loading, the blot was reprobed for 18S rRNA. Results are presented as the mean ± SE of three replicates. *, p < 0.01 vs. ZT7 group. The insert is a typical Northern blot image. For further details see the Materials and Methods section.

Fig. 3. Immunohistochemical detection of Mbnl2 protein in the rat pineal gland

Animals were housed in a controlled lighting environment for two weeks (LD12:12). Pineal glands were obtained at ZT6 and ZT18. Immunohistochemical detection was done with the polyclonal rabbit anti-Mbnl2 used in Fig. 2B. at a dilution of 1:1,000. A pre-immunization negative control (ZT18) is displayed. Note the universal staining of the cells in the pineal parenchyma and a few intensively stained cells (arrows in ZT6). Scale bar, 50 μm. For further details see the Materials and Methods section.

Fig. 4. Regulation of pineal Mbnl2 transcripts by a photoneural mechanism

Each lane contains 3 μg total RNA obtained from a pool of two pineal glands. Northern blots were probed with a rat Mbnl2 probe. To normalize for variations in RNA loading, the blot was reprobed for 18S rRNA. Results are presented as the mean ± SE of three replicates. For further details see the Materials and Methods section. A. The nocturnal increase in Mbnl2 mRNA is driven by neural input from the SCG. Pineal RNA was obtained from Sham, DCNT, and SCGX rats killed during the day (ZT7) or at night (ZT19). In addition, one group of SCGX rats was injected with 1 mg/kg NE at ZT16 and killed 3 hours later with the other nighttime experimental group. *, p < 0.01 vs. Sham Night group. B. Effect of light exposure at night on Mbnl2 mRNA level in the rat pineal gland. Rats were housed in a controlled lighting environment (LD 14:10). Pineal glands were obtained during the day (ZT7), at night (ZT19) in the dark, after 1 hr light pulse (LP) or after 5 hr of prolonged light (PL). *, p < 0.01 vs. Night group. For further details see the Materials and Methods section.

Fig. 5. Isoproterenol (ISO) elevates Mbnl2 transcript levels in vivo

Rats were injected subcutaneously with isoproterenol (20 mg/kg) at ZT4. All pineal glands were obtained at ZT7 and glands were removed and stored on solid CO₂. Each lane was loaded with 8 μg total RNA obtained from a pool of two pineal glands. Northern blots were probed with a rat Mbnl2 probe. To normalize for variations in RNA loading, the blot was reprobed for 18S rRNA. The results were confirmed in three experiments. Results are mean ± SE of three replicates. *, p < 0.01 vs. CONT group. For further details see the Materials and Methods section.

Fig. 6. Evidence that NE and cAMP control Mbnl2 mRNA and protein

A. Mbnl2 mRNA: Cultured pineal glands were pre-incubated with 3 μM KT5720 alone for 1 hr; NE (1 μM) was then added and incubation was continued for 6 hours. Each lane was loaded 8 μg of total RNA from pools of three or four glands. Northern blots were probed with a rat Mbnl2 probe. To normalize for RNA loading, the blot was reprobed for 18S rRNA. Results are presented as the mean ± SE of three replicates. *, p < 0.01 vs. NE group. B. Mbnl2 protein: Using pineal glands from the same experiment shown in panel A, each lane was loaded with 60 μg protein from a pineal extract. Immunodetection was performed with an anti-Mbnl2 serum at a dilution of 1:10,000. Results are presented as the mean ± SE of three replicates. *, p < 0.01 vs. NE group. C. Pineal glands were cultured for 6 hours with 500 μM 8-Br cAMP (8-Br), 500 μM dibutyryl cAMP (DB) or 1 μM NE; control (CONT). *, p < 0.01 vs. CONT group. For further technical details see 5.A. and the Materials and Methods section.

Fig. 7. NE-induced elevation in Mbnl2 mRNA requires transcription but not translation

Pineal glands were treated with 30 μg/ml actinomycin D (ACTD) or 50 μg/ml puromycin (PURO) for 1 hour and during the subsequent 6 hours treatment with 1 μM NE. Each lane was loaded 8 μg total RNA obtained from a pool of four pineal glands. After hybridization with the Mbnl2 probe, the blot was stripped and reprobed with an 18S rRNA probes as a control. Results were confirmed in three independent experiments. The values are mean ± SE of three replicates. For further details see the Materials and Methods section.

Fig. 8