Clock genes and behavioral responses to light are altered in a mouse model of diabetic retinopathy.

There is increasing evidence that melanopsin-expressing ganglion cells (ipRGCs) are altered in retinal pathologies. Using a streptozotocin-induced (STZ) model of diabetes, we investigated the impact of diabetic retinopathy on non-visual functions by analyzing ipRGCs morphology and light-induced c-Fos and Period 1-2 clock genes in the central clock (SCN). The ability of STZ-diabetic mice to entrain to light was challenged by exposure animals to 1) successive light/dark (LD) cycle of decreasing or increasing light intensities during the light phase and 2) 6-h advance of the LD cycle. Our results show that diabetes induces morphological changes of ipRGCs, including soma swelling and dendritic varicosities, with no reduction in their total number, associated with decreased c-Fos and clock genes induction by light in the SCN at 12 weeks post-onset of diabetes. In addition, STZ-diabetic mice exhibited a reduction of overall locomotor activity, a decrease of circadian sensitivity to light at low intensities, and a delay in the time to re-entrain after a phase advance of the LD cycle. These novel findings demonstrate that diabetes alters clock genes and behavioral responses of the circadian timing system to light and suggest that diabetic patients may show an increased propensity for circadian disturbances, in particular when they are exposed to chronobiological challenges

BRCA2 deep intronic mutation causing activation of a cryptic exon: opening toward a new preventive therapeutic strategy.

International audiencePURPOSE: Diagnostic screening of the BRCA1/2 genes in breast cancer families is mostly done on genomic DNA. For families with a very strong family history and no mutation identified in the coding sequences or the exon-intron boundaries, BRCA1/2 transcripts' analysis is an efficient approach to uncover gene inversion and pre-mRNA splicing defaults missed by conventional DNA-based protocols. EXPERIMENTAL DESIGN: We analyzed RNA from patients of negative BRCA families by reverse transcriptase PCR and identified an insertion in one family that we characterized by sequencing and by using a minigene splicing assay. More than 2,000 additional BRCA1/2 negative families were subsequently screened for this mutation using a dedicated PCR approach. RESULTS: Nine families were found to harbor a BRCA2 mutant transcript containing a 95-nucleotide cryptic exon between exons 12 and 13. This cryptic exon results from a new mutation located deep into intron 12, c.6937+594T > G, which reinforces the strength of a preexisting 5' splice site, turning it into a perfect consensus sequence. It is systematically included in transcripts produced by the mutant allele in cells from mutation carriers or produced by a mutant splicing reporter minigene. The inclusion of the cryptic exon was prevented when we cotransfected the minigene with antisense oligonucleotides complementary to the 3' or mutated 5' splice sites. CONCLUSION: This first deep intronic BRCA mutation emphasizes the importance of analyzing RNA to provide comprehensive BRCA1/2 diagnostic tests and opens the possibility of using antisense therapy in the future as an alternative strategy for cancer prevention

Comparison of the amount of locomotor activity (mean counts per day) and the night-day ratio in control and diabetic animals submitted to two light schedules.

<p>(A) Left Panel. Mice were exposed to 3 successive 12L/12D cycle with increases of light intensity (100, 300 lux and 1000 lux). STZ-diabetic mice show a significant reduced locomotor activity at 100 (controls: 1681.9±91.6 counts and STZ-diabetic: 1097.5±119.5 counts; p≤0.001) and 300 lux (controls: 2006±108.2 counts and STZ-diabetic: 1620.6±140.7 counts; p≤0.001), whereas at 1000 lux, no significant difference is shown between the two groups (controls: 1833.2±89.9 counts and STZ-diabetic: 1842±108.5 counts). Right Panel: Percentage of activity during the dark (dotted line  = 50%). STZ-diabetic mice are significantly less nocturnal at 100 and 300 lux. (B) In the second light schedule, animals were submitted to successive decreases in light intensity. These SZT-diabetic mice exhibited a reduction in their total locomotor activity at the three light intensities (at 1000 lux: controls: 2099.5±122.1 counts; STZ-diabetic: 1704.7±125.9 counts; p≤0.01; at 300 lux: controls: 2658.7±112.9 counts; STZ-diabetic: 2124.4±0.1 counts; p≤0.001 and at 100 lux: controls: 2864.7±58.4 counts; STZ-diabetic: 2508.7±47.5 counts; p≤0.05), with a significant decreased ratio of night-day activity at 300 lux and 100 lux. Data are presented as means ± SEM (n = 12 for controls and n = 12 for diabetic for each light schedule), *p≤0.05** p≤0.01 and *** p≤0.001 compared with control.</p

Average weight and blood glucose concentrations of control and STZ-diabetic mice.

<p>Data are expressed as mean ± SEM (n = 15–25 for each group).</p><p>***p≤0.001.</p

Mean daily activity onsets following a 6/12D cycle for control and STZ-diabetic mice.

<p>(<b>A</b>) One representative actogram of locomotor activity for control and two for STZ-diabetic mice, double-plotted on a 24 h timescale. The numbered lines represent successive days and the bar above the actograms indicates the original light/dark cycle. After 17 days of entrainment under a 12L/12D cycle at 1000 lux, control and STZ-diabetic mice were exposed to a 6 h advanced light/dark cycle (black dot in the actograms) at 12 weeks post-diabetes. (<b>B</b>) Phase angle of photic entrainment. Control mice achieve stable re-entrainment after 8±0.2 days, whereas STZ-diabetic mice need 10.6±0.3 days (Diabetic-case 1; n = 6; p≤0.001). Certain STZ-diabetic mice (n = 4) display a less stable entrainment even after 20 days (Diabetic-case 2). Data are presented as means ± SEM (n = 10 for each group).</p

Relative light-induced <i>c-Fos</i>, <i>Per1</i>, and <i>Per2</i> mRNA levels in the SCN and opsin mRNA expression in the retina of control and diabetic mice at 12 weeks post-diabetes.

<p>(A) Induction of <i>c-Fos</i>, <i>Per1</i> and <i>Per2</i> in the SCN of control and STZ-diabetic mice by 480 nm monochromatic light (constant irradiance of 1.17×10¹⁴ photons/cm²/s, 15 min) at CT16 using real time RT-PCR. White bars represent animals which received a light pulse (L, n = 7 for each group). Dark controls (D, black bars), were handled in the same way but did not receive a light pulse (n = 4–5 for each group). (B) Relative expression of short wavelength cones (SW), mid-wavelength cones (MW) and rhodopsin mRNAs expression in the retina of the same animals (n = 4–5 for each group). The relative level of opsin mRNAs is equivalent in both groups. Data are presented as means ± SEM. Asterisk indicates a statistically significant difference (*: <i>p</i>≤0.05; **: <i>p</i>≤0.01 and ***: p≤0.001).</p

Rods contribute to the light-induced phase shift of the retinal clock in mammals

International audienceWhile rods, cones, and intrinsically photosensitive melanopsin-containing ganglion cells (ipRGCs) all drive light entrainment of the master circadian pacemaker of the suprachiasmatic nucleus, recent studies have proposed that entrainment of the mouse retinal clock is exclusively mediated by a UV-sensitive photopigment, neuropsin (OPN5). Here, we report that the retinal circadian clock can be phase shifted by short duration and relatively low-irradiance monochromatic light in the visible part of the spectrum, up to 520 nm. Phase shifts exhibit a classical photon dose-response curve. Comparing the response of mouse models that specifically lack middle-wavelength (MW) cones, melanopsin, and/or rods, we found that only the absence of rods prevented light-induced phase shifts of the retinal clock, whereas light-induced phase shifts of locomotor activity are normal. In a "rod-only" mouse model, phase shifting response of the retinal clock to light is conserved. At shorter UV wavelengths, our results also reveal additional recruitment of short-wavelength (SW) cones and/or OPN5. These findings suggest a primary role of rod photoreceptors in the light response of the retinal clock in mammals

Comparison of the average phase angle of activity and variability onset in control and STZ-diabetic animals submitted to two light schedules.

<p>(<b>A</b>) Mice were maintained under a 12L/12D cycle and exposed to 3 successive 12L/12D cycle with increases of light intensity (100, 300 lux and 1000 lux; n = 12 for control and for STZ-diabetic mice). Left panel: Phase angle differences between times of lights-off and onset of activity. Positive values indicate an advanced activity onset. At 100 lux, STZ-diabetic mice are entrained with a significant phase advance in relation to the time of lights-OFF compared to the age-matched control animals (p≤0.001). This difference in the phase angle of activity between the two groups is not observed when animals are exposed to 300 lux and at 1000 lux. The activity onsets were also significantly less precise at 100 and 300 lux (right panel). (<b>B</b>) In the second light schedule, mice were exposed to 3 successive 12L/12D cycle with decreases of light intensity (1000, 300 lux and 100 lux; n = 12 for control and for STZ-diabetic mice). At 1000 lux, controls and STZ-diabetic animals are entrained with a normal phase angle of activity and with variability around the onset of activity similar to controls (left panel). When the light intensity is decreased (300 and 100 lux), STZ-diabetic animals began their locomotor activity earlier than controls with increased onset variability for STZ-diabetic mice at 300 lux (p≤0.001). Data are presented as means ± SEM, ** p≤0.01 and *** p≤0.001 compared with control.</p

Total number and morphology of ipRGCs in control and STZ-diabetic mice at 12 weeks post-onset of diabetes.

<p>(<b>A</b>) Representative flat-mounted retinas from a control and a STZ-diabetic animal, immunostained with melanopsin antibody. (<b>B</b>) Total number of ipRGCs. No difference was observed between groups. (<b>C</b>) Representative photomicrographs of flat-mounted retinas immunostained by melanopsin antibody from control and STZ- diabetic animal at 12 weeks post-diabetes. No morphological abnormalities of ipRGCs were shown in the control retina and in the central retina of STZ-diabetic mouse. Soma swelling and increased varicosities in the dendrites of ipRGCs were noted in the peripheral retina. Enlarged image of a melanopsin-positive cell (Insert: magnification ×100). Data represent means ± SEM (n = 6 for controls and n = 5 for diabetic). Scale bar  = 50 µm. N =  nasal; T =  temporal; S = superior; I = inferior.</p

Rods contribute to the light-induced phase shift of the retinal clock in mammals

10.1371/journal.pbio.2006211PLOS BIOLOGY17