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RNA polymerase III drives alternative splicing of the potassium channel-interacting protein contributing to brain complexity and neurodegeneration.

Massone S, Vassallo I, Castelnuovo M, Fiorino G, Gatta E, Robello M, Borghi R, Tabaton M, Russo C, Dieci G, Cancedda R, Pagano A - J. Cell Biol. (2011)

Bottom Line: We found that IL1-α-dependent up-regulation of 38A, a small ribonucleic acid (RNA) polymerase III-transcribed RNA, drives the synthesis of an alternatively spliced form of the potassium channel-interacting protein (KCNIP4).Notably, synthesis of the variant KCNIP4 isoform is also detrimental to brain physiology, as it results in the concomitant blockade of the fast kinetics of potassium channels.This alternative splicing shift is observed at high frequency in tissue samples from Alzheimer's disease patients, suggesting that RNA polymerase III cogenes may be upstream determinants of alternative splicing that significantly contribute to homeostasis and pathogenesis in the brain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, Biology, and Genetics, National Institute for Cancer Research, 16132 Genoa, Italy.

ABSTRACT
Alternative splicing generates protein isoforms that are conditionally or differentially expressed in specific tissues. The discovery of factors that control alternative splicing might clarify the molecular basis of biological and pathological processes. We found that IL1-α-dependent up-regulation of 38A, a small ribonucleic acid (RNA) polymerase III-transcribed RNA, drives the synthesis of an alternatively spliced form of the potassium channel-interacting protein (KCNIP4). The alternative KCNIP4 isoform cannot interact with the γ-secretase complex, resulting in modification of γ-secretase activity, amyloid precursor protein processing, and increased secretion of β-amyloid enriched in the more toxic Aβ x-42 species. Notably, synthesis of the variant KCNIP4 isoform is also detrimental to brain physiology, as it results in the concomitant blockade of the fast kinetics of potassium channels. This alternative splicing shift is observed at high frequency in tissue samples from Alzheimer's disease patients, suggesting that RNA polymerase III cogenes may be upstream determinants of alternative splicing that significantly contribute to homeostasis and pathogenesis in the brain.

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Modulation of 38A promoter activity by inflammatory and antiinflammatory agents. (A) Induction by IL1-α treatment and recovery by IL1ra of 38A expression. The results were normalized to the untreated samples. The effects of different concentrations of IL1-α treatment on 38A expression are shown in the inset. (B) KCNIP4 splice variant shift induction and prevention by IL1-α and IL1ra, respectively. (C) Induction by IL1-α treatment of IL1-α receptor synthesis in SHSY5Y cells. HeLa and HEK293 cells were used as positive controls. (D) Inhibition of IL1-α–induced 38A overexpression by Diclofenac treatment. Experiments were performed in triplicate and repeated three times. Data are reported as mean values ± SD. Statistical significance was examined using the unpaired Student’s t test. * and ** indicate statistical significance at P < 0.05 and 0.01, respectively.
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fig9: Modulation of 38A promoter activity by inflammatory and antiinflammatory agents. (A) Induction by IL1-α treatment and recovery by IL1ra of 38A expression. The results were normalized to the untreated samples. The effects of different concentrations of IL1-α treatment on 38A expression are shown in the inset. (B) KCNIP4 splice variant shift induction and prevention by IL1-α and IL1ra, respectively. (C) Induction by IL1-α treatment of IL1-α receptor synthesis in SHSY5Y cells. HeLa and HEK293 cells were used as positive controls. (D) Inhibition of IL1-α–induced 38A overexpression by Diclofenac treatment. Experiments were performed in triplicate and repeated three times. Data are reported as mean values ± SD. Statistical significance was examined using the unpaired Student’s t test. * and ** indicate statistical significance at P < 0.05 and 0.01, respectively.

Mentions: As the aforementioned findings demonstrate a role of 38A in brain function and, possibly, in neurodegeneration associated with AD, we focused on the investigation of possible biochemical stimuli that may promote 38A transcription in neurodegenerative disorders. Because several studies support the hypothesis of a relevant contribution of inflammatory stimuli to AD onset (Griffin et al., 1989; McGeer et al., 2006; Wyss-Coray, 2006), we tested the effect on 38A expression of treatments with different proinflammatory molecules in SHSY5Y cells. We found that 12 h after treatment, 0.5 ng/ml IL1-α strongly up-regulates 38A expression, whereas the addition of IL1 receptor antagonist (IL1ra, a natural inhibitor of IL1-α proinflammatory effect) prevents the IL1-α–dependent increase of 38A transcription, demonstrating further the specificity of IL1-α action (Fig. 9, A and B). We then measured the expression of IL1-α receptor by real-time RT-PCR in the same samples. IL1-α receptor expression was found to be abundant in HeLa and, at a lesser extent in HEK293 cells, not detected in SHSY5Y-untreated cells, where it was induced by the treatment with the inflammatory agent, supporting the effects of IL1-α on Aβ processing described in this paper (Fig. 9 C). Next, we repeated the induction of 38A expression with IL1-α in samples previously treated with Diclofenac to test a possible protective effect of a nonsteroidal antiinflammatory drug on 38A activation. Under these conditions, IL1-α–dependent 38A activation was abolished, thus demonstrating that the inhibition of the COX-2 (Cyclooxygenase-2)–dependent inflammatory cascade is sufficient to prevent IL1-α–dependent up-regulation of 38A and its possible detrimental consequences (Fig. 9 D). Altogether, these results demonstrate that 38A expression is induced by IL1-α and that the harmful effects of its activation (such as the increased processing of Aβ and the impairment of A-type potassium current) might be triggered in vivo by this inflammatory stimulus.


RNA polymerase III drives alternative splicing of the potassium channel-interacting protein contributing to brain complexity and neurodegeneration.

Massone S, Vassallo I, Castelnuovo M, Fiorino G, Gatta E, Robello M, Borghi R, Tabaton M, Russo C, Dieci G, Cancedda R, Pagano A - J. Cell Biol. (2011)

Modulation of 38A promoter activity by inflammatory and antiinflammatory agents. (A) Induction by IL1-α treatment and recovery by IL1ra of 38A expression. The results were normalized to the untreated samples. The effects of different concentrations of IL1-α treatment on 38A expression are shown in the inset. (B) KCNIP4 splice variant shift induction and prevention by IL1-α and IL1ra, respectively. (C) Induction by IL1-α treatment of IL1-α receptor synthesis in SHSY5Y cells. HeLa and HEK293 cells were used as positive controls. (D) Inhibition of IL1-α–induced 38A overexpression by Diclofenac treatment. Experiments were performed in triplicate and repeated three times. Data are reported as mean values ± SD. Statistical significance was examined using the unpaired Student’s t test. * and ** indicate statistical significance at P < 0.05 and 0.01, respectively.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105541&req=5

fig9: Modulation of 38A promoter activity by inflammatory and antiinflammatory agents. (A) Induction by IL1-α treatment and recovery by IL1ra of 38A expression. The results were normalized to the untreated samples. The effects of different concentrations of IL1-α treatment on 38A expression are shown in the inset. (B) KCNIP4 splice variant shift induction and prevention by IL1-α and IL1ra, respectively. (C) Induction by IL1-α treatment of IL1-α receptor synthesis in SHSY5Y cells. HeLa and HEK293 cells were used as positive controls. (D) Inhibition of IL1-α–induced 38A overexpression by Diclofenac treatment. Experiments were performed in triplicate and repeated three times. Data are reported as mean values ± SD. Statistical significance was examined using the unpaired Student’s t test. * and ** indicate statistical significance at P < 0.05 and 0.01, respectively.
Mentions: As the aforementioned findings demonstrate a role of 38A in brain function and, possibly, in neurodegeneration associated with AD, we focused on the investigation of possible biochemical stimuli that may promote 38A transcription in neurodegenerative disorders. Because several studies support the hypothesis of a relevant contribution of inflammatory stimuli to AD onset (Griffin et al., 1989; McGeer et al., 2006; Wyss-Coray, 2006), we tested the effect on 38A expression of treatments with different proinflammatory molecules in SHSY5Y cells. We found that 12 h after treatment, 0.5 ng/ml IL1-α strongly up-regulates 38A expression, whereas the addition of IL1 receptor antagonist (IL1ra, a natural inhibitor of IL1-α proinflammatory effect) prevents the IL1-α–dependent increase of 38A transcription, demonstrating further the specificity of IL1-α action (Fig. 9, A and B). We then measured the expression of IL1-α receptor by real-time RT-PCR in the same samples. IL1-α receptor expression was found to be abundant in HeLa and, at a lesser extent in HEK293 cells, not detected in SHSY5Y-untreated cells, where it was induced by the treatment with the inflammatory agent, supporting the effects of IL1-α on Aβ processing described in this paper (Fig. 9 C). Next, we repeated the induction of 38A expression with IL1-α in samples previously treated with Diclofenac to test a possible protective effect of a nonsteroidal antiinflammatory drug on 38A activation. Under these conditions, IL1-α–dependent 38A activation was abolished, thus demonstrating that the inhibition of the COX-2 (Cyclooxygenase-2)–dependent inflammatory cascade is sufficient to prevent IL1-α–dependent up-regulation of 38A and its possible detrimental consequences (Fig. 9 D). Altogether, these results demonstrate that 38A expression is induced by IL1-α and that the harmful effects of its activation (such as the increased processing of Aβ and the impairment of A-type potassium current) might be triggered in vivo by this inflammatory stimulus.

Bottom Line: We found that IL1-α-dependent up-regulation of 38A, a small ribonucleic acid (RNA) polymerase III-transcribed RNA, drives the synthesis of an alternatively spliced form of the potassium channel-interacting protein (KCNIP4).Notably, synthesis of the variant KCNIP4 isoform is also detrimental to brain physiology, as it results in the concomitant blockade of the fast kinetics of potassium channels.This alternative splicing shift is observed at high frequency in tissue samples from Alzheimer's disease patients, suggesting that RNA polymerase III cogenes may be upstream determinants of alternative splicing that significantly contribute to homeostasis and pathogenesis in the brain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, Biology, and Genetics, National Institute for Cancer Research, 16132 Genoa, Italy.

ABSTRACT
Alternative splicing generates protein isoforms that are conditionally or differentially expressed in specific tissues. The discovery of factors that control alternative splicing might clarify the molecular basis of biological and pathological processes. We found that IL1-α-dependent up-regulation of 38A, a small ribonucleic acid (RNA) polymerase III-transcribed RNA, drives the synthesis of an alternatively spliced form of the potassium channel-interacting protein (KCNIP4). The alternative KCNIP4 isoform cannot interact with the γ-secretase complex, resulting in modification of γ-secretase activity, amyloid precursor protein processing, and increased secretion of β-amyloid enriched in the more toxic Aβ x-42 species. Notably, synthesis of the variant KCNIP4 isoform is also detrimental to brain physiology, as it results in the concomitant blockade of the fast kinetics of potassium channels. This alternative splicing shift is observed at high frequency in tissue samples from Alzheimer's disease patients, suggesting that RNA polymerase III cogenes may be upstream determinants of alternative splicing that significantly contribute to homeostasis and pathogenesis in the brain.

Show MeSH
Related in: MedlinePlus