Rapid Eye Movement Sleep Deprivation Induces Neuronal Apoptosis by Noradrenaline Acting on Alpha1 Adrenoceptor and by Triggering Mitochondrial Intrinsic Pathway

Abstract

Many neurodegenerative disorders are associated with rapid eye movement sleep (REMS) loss; however, the mechanism was unknown. As REMS loss elevates noradrenaline (NA) level in the brain as well as induces neuronal apoptosis and degeneration, in this study, we have delineated the intracellular molecular pathway involved in REMS deprivation (REMSD)-associated NA-induced neuronal apoptosis. Rats were REMS deprived for 6 days by the classical flower pot method; suitable controls were conducted and the effects on apoptosis markers evaluated. Further, the role of NA was studied by one, intraperitoneal (i.p.) injection of NA-ergic alpha1 adrenoceptor antagonist prazosin (PRZ) and two, by downregulation of NA synthesis in locus coeruleus (LC) neurons by local microinjection of tyrosine hydroxylase siRNA (TH-siRNA). Immunoblot estimates showed that the expressions of proapoptotic proteins viz. Bcl2-associated death promoter protein, apoptotic protease activating factor-1 (Apaf-1), cytochrome c, caspase9, caspase3 were elevated in the REMS-deprived rat brains, while caspase8 level remained unaffected; PRZ treatment did not allow elevation of these proapoptotic factors. Further, REMSD increased cytochrome c expression, which was prevented if the NA synthesis from the LC neurons was blocked by microinjection of TH-siRNA in vivo into the LC during REMSD in freely moving normal rats. Mitochondrial damage was re-confirmed by transmission electron microscopy, which showed distinctly swollen mitochondria with disintegrated cristae, chromosomal condensation, and clumping along the nuclear membrane, and all these changes were prevented in PRZ-treated rats. Combining findings of this study along with earlier reports, we propose that upon REMSD NA level increases in the brain as the LC, NA-ergic REM-OFF neurons do not cease firing and TH is upregulated in those neurons. This elevated NA acting on alpha1 adrenoceptors damages mitochondria causing release of cytochrome c to activate intrinsic pathway for inducing neuronal apoptosis in REMS-deprived rat brain.

Keywords: BAD; caspases; cytochrome c; locus coeruleus; neuronal degeneration.

Figure 1

Schematic representation of microinjection of TH-siRNA and control siRNA in vivo bilaterally into the LC of freely moving normally behaving surgically prepared chronic rats.

Figure 2

BAD expression in the brain homogenate of rats under different conditions. Upper panel shows a representative Western blot of BAD expressions in the brain of rats exposed to various control and experimental (REMSD) conditions. Histogram in the lower panel shows percent changes in the mean (±SEM) band densities of the blots as compared to FMC taken as 100% in five sets of experiments. Abbreviations are as in the text. Significance levels are between the treatments of connecting horizontal bars; ***p < 0.001 and ^###p < 0.001, * as compared to FMC and ^# as compared to REMSD.

Figure 3

Cytochrome c expression in the brain homogenate of rats under different conditions. Upper panel shows a representative Western blot of cytochrome c. Histogram in the lower panel shows percent changes in the mean (±SEM) band densities of the blots as compared to FMC taken as 100% in five sets of experiments. Abbreviations are as in the text. Significance levels are between the treatments of connecting horizontal bars; ***p < 0.001 and ^##p < 0.01, * as compared to FMC and ^# as compared to REMSD.

Figure 4

Apaf-1 expression in the brain homogenate of rats under various conditions. Upper panel shows a representative Western blot of Apaf-1. Histogram in the lower panel shows percent changes in the mean (±SEM) band densities of the blots as compared to FMC taken as 100% in five sets of experiments. Abbreviations are as in the text. Significance levels are between the treatments of connecting horizontal bars; ***p < 0.001 and ^##p < 0.01, * as compared to FMC and ^# as compared to REMSD.

Figure 5

Caspase9 expression in the brain homogenate of rats under different conditions. Upper panel shows a representative Western blot of caspase9. Histogram in the lower panel shows percent changes in the mean (±SEM) band densities of the blots as compared to FMC taken as 100% in five sets of experiments. Abbreviations are as in the text. Significance levels are between the treatments of connecting horizontal bars; ***p < 0.001 and ^##p < 0.01, * as compared to FMC and ^# as compared to REMSD.

Figure 6

Caspase3 expressions in the brain homogenate of rats under different conditions. Upper panel shows a representative Western blot showing the presence of procaspase3 and cleaved caspase3. Histogram in the lower panel shows percent changes in the mean (±SEM) band densities, which were sum of both the Procaspase3 and cleaved caspase3 as compared to FMC taken as 100% in five sets of experiments. Abbreviations are as in the text. Significance levels are between the treatments of connecting horizontal bars; ***p < 0.001, * as compared to FMC and # as compared to REMSD.

Figure 7

pAkt expression in the brain homogenate of rats maintained under various conditions. Upper panel shows a representative Western blot of pAkt. Histogram in the lower panel shows percent changes in the mean (±SEM) band densities of the blots as compared to FMC taken as 100% in five sets of experiments. Abbreviations are as in the text. Significance levels are between the treatments of connecting horizontal bars; ***p < 0.001 and ^#p < 0.05, * as compared to FMC and ^# as compared to REMSD.

Figure 8

Caspase8 expression in the brain homogenate of rats maintained under different conditions. Upper panel shows a representative Western blot of caspase8. Histogram in the lower panel shows percent changes in the mean (±SEM) band densities of the blots as compared to FMC taken as 100% in five sets of experiments. Abbreviations are as in the text.

Figure 9

Tyrosine hydroxylase expression in the brain homogenate of rats maintained under different conditions. Upper panel shows a representative Western blot of tyrosine hydroxylase. Histogram in the lower panel shows percent changes in the mean (±SEM) band densities of the blots as compared to FMC taken as 100% in five sets of experiments. Abbreviations are as in the text. Significance levels are between the treatments of connecting horizontal bars; ***p < 0.001 and ^#p < 0.05, * as compared to FMC and ^# as compared to REMSD.

Figure 10

Expressions of TH in homogenate of REMSD rat brains after microinjection of TH-siRNA or control siRNA bilaterally into LC. Upper panel shows a representative Western blot of tyrosine hydroxylase. Histogram in the lower panel shows percent changes in the mean (±SEM) band densities of the blots as compared to FMC (without injection) taken as 100% in five sets of experiments. Abbreviations are as in the text. Significance levels are between the treatments of connecting horizontal bars; ***p < 0.001 and ^###p < 0.001, * as compared to FMC and ^# as compared to TH-siRNA + REMSD.

Figure 11

Expressions of cytochrome c in homogenate REMSD rat brain after microinjection of TH-siRNA or control siRNA bilaterally into LC. Upper panel shows a representative Western blot of cytochrome c. Histogram in the lower panel shows percent changes in the mean (±SEM) band densities of the blots as compared to FMC taken as 100% in five sets of experiments. Abbreviations are as in the text. Significance levels are between the treatments of connecting horizontal bars; significance levels – ***p < 0.001 and ^###p < 0.001, * as compared to TH-siRNA and ^# as compared to FMC.

Figure 12

Electron photo-micrograph (10,000×) showing mitochondrial ultrastructural changes in LC neurons of rats treated under various conditions. Normal elongated mitochondria can be seen in neurons from FMC and LPC rat brains. Images of two different rat brains after REMSD are shown as compared to one each from other control rat brains. Mitochondria appeared swollen with cristae disintegrated or reduced with transparent matrix in the REMSD rat brains. Cristae appeared less distorted in REC rat brain. In PRZ-treated group, although swelling appeared, the inner cristae of mitochondria appeared intact. M – mitochondria; scale bar: 100 nm.

Figure 13

Electron photo-micrograph (2,500×) of nucleus in LC neurons of rat brains treated under different conditions. Nucleus and chromosomes appear normal in FMC and LPC. Images of two different rat brains after REMSD are shown as compared to one each from other control rat brains. Neuron with chromatin condensation can be seen in the REMSD rat brains. The dark arrows point to areas near nuclear membrane with increased chromatin condensation. Reduced degenerative changes of nucleus were seen in the REC- and PRZ-treated rat brains. N – nucleus, NM – nuclear membrane; scale bar: 500 nm.

Figure 14

We have shown earlier that REMSD increases apoptosis in the rat brain and that is mediated by REMSD-associated elevated NA level in the brain (1). We have also shown that REMSD reduces intracellular Ca²⁺ (68) and NA reduces Ca²⁺ influx (69). Combining those findings with the results of this study that the REMSD-associated neuronal apoptosis follows mitochondrial intrinsic pathway, we have traced the molecular pathway, which has been schematically represented in this figure.