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Novel telomerase-increasing compound in mouse brain delays the onset of amyotrophic lateral sclerosis.

Eitan E, Tichon A, Gazit A, Gitler D, Slavin S, Priel E - EMBO Mol Med (2012)

Bottom Line: Hence, we and others hypothesized that increasing telomerase expression by pharmaceutical compounds may protect brain cells from death caused by damaging agents.The survival of telomerase-expressing cells (i.e. motor neurons), but not telomerase-deficient cells, exposed to oxidative stress was increased by AGS-499 treatment, suggesting that the AGS-499 effects are telomerase-mediated.Therefore, a controlled and transient increase in telomerase expression and activity in the brain by AGS-499 may exert neuroprotective effects.

View Article: PubMed Central - PubMed

Affiliation: The Shraga Segal Department of Immunology and Microbiology, Ben-Gurion University of the Negev, Beer-Sheva, Israel.

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AGS-499 increases TERT protein level and TERT mRNA expression in the FB, BS and the SC in a time-dependent mannerA-C. Mice were injected s.c. with AGS-499 (6 mg/kg) or its vehicle DMSO and cytoplasmic and nuclear protein extract was prepared from the FB (A,C) or whole cell protein extract from the BS and SC (B), at various time after the compound injection. Protein extracts (30 µg) were analysed by Western Blot assay using anti-TERT antibody (Rockland) or anti-β-actin antibody (A,B). Quantification of the Western blot results (depicted in A) by densitometric analysis using the EZquant software are shown in (C). (mean ± s.e.m.; n = 10) Student's t-test p = *10.033, *20.035, *30.014,*40.013, *50.024, *60.004.D-E. Total RNA extract was prepared from mouse FB that were injected with 6 mg/kg AGS-499, 12 and 24 h after injection. (D) Twenty micrograms RNA was subjected to Northern blot analysis using a specific [P32] labelled TERT probe. (E) Real-time PCR was performed for quantification of TERT mRNA in mouse FB, normalized to β-actin mRNA and the increase in TERT transcripts was calculated as fold of vehicle (mean ± s.e.m.; n = 3, Student's t-test p = : *10.033, *20.021). Symbols: UT, untreated mice; V, vehicle.
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fig03: AGS-499 increases TERT protein level and TERT mRNA expression in the FB, BS and the SC in a time-dependent mannerA-C. Mice were injected s.c. with AGS-499 (6 mg/kg) or its vehicle DMSO and cytoplasmic and nuclear protein extract was prepared from the FB (A,C) or whole cell protein extract from the BS and SC (B), at various time after the compound injection. Protein extracts (30 µg) were analysed by Western Blot assay using anti-TERT antibody (Rockland) or anti-β-actin antibody (A,B). Quantification of the Western blot results (depicted in A) by densitometric analysis using the EZquant software are shown in (C). (mean ± s.e.m.; n = 10) Student's t-test p = *10.033, *20.035, *30.014,*40.013, *50.024, *60.004.D-E. Total RNA extract was prepared from mouse FB that were injected with 6 mg/kg AGS-499, 12 and 24 h after injection. (D) Twenty micrograms RNA was subjected to Northern blot analysis using a specific [P32] labelled TERT probe. (E) Real-time PCR was performed for quantification of TERT mRNA in mouse FB, normalized to β-actin mRNA and the increase in TERT transcripts was calculated as fold of vehicle (mean ± s.e.m.; n = 3, Student's t-test p = : *10.033, *20.021). Symbols: UT, untreated mice; V, vehicle.

Mentions: To examine the time-dependent activation of telomerase in the brain following AGS treatment, AGS-499-treated mice were sacrificed at 3, 6, 12, 24 and 48 h after treatment. Telomerase protein level was examined in the cytoplasmic and nuclear fractions derived from the mouse FB. As can be seen in Fig 3A and C, telomerase protein level gradually increased with time in both the nucleus and cytoplasm (up to three- and twofold, respectively; p < 0.01) following AGS treatment, peaking at 12 h, decreasing to 1.5- and 2-fold (p < 0.05) activation at 24 h, and reaching the basal level at 48 h post treatment. Examination of the effect of AGS treatment on telomerase protein in the BS and in the lumbar region of the SC demonstrates a significant increase in TERT protein 12 and 24 h after AGS injection (Fig 3B). In addition, the effect of AGS-499 injection on the TERT mRNA levels was determined by Northern blot analysis of total mRNA derived from the FB of AGS-treated and untreated mice. The results depicted in Fig 3D demonstrate the identification of the full-length mTERT mRNA (3.4 kb) and two additional unknown transcripts of 2.1 and 7 kb. In AGS-499-treated mice, a significant increase in the full-length TERT mRNA and the 2.1 kb transcript was observed. No change in the GAPDH transcript was detected, suggesting that AGS-499 specifically increases mTERT RNA expression. Quantification of the increase in mTERT RNA transcripts by real-time PCR analysis revealed a time-dependent increase (up to 3.87 ± 0.74, p < 0.05 compared to vehicle) peaking at 12 h after AGS injection (Fig 3E), which is compatible with the mTERT protein results (Fig 3A and C).


Novel telomerase-increasing compound in mouse brain delays the onset of amyotrophic lateral sclerosis.

Eitan E, Tichon A, Gazit A, Gitler D, Slavin S, Priel E - EMBO Mol Med (2012)

AGS-499 increases TERT protein level and TERT mRNA expression in the FB, BS and the SC in a time-dependent mannerA-C. Mice were injected s.c. with AGS-499 (6 mg/kg) or its vehicle DMSO and cytoplasmic and nuclear protein extract was prepared from the FB (A,C) or whole cell protein extract from the BS and SC (B), at various time after the compound injection. Protein extracts (30 µg) were analysed by Western Blot assay using anti-TERT antibody (Rockland) or anti-β-actin antibody (A,B). Quantification of the Western blot results (depicted in A) by densitometric analysis using the EZquant software are shown in (C). (mean ± s.e.m.; n = 10) Student's t-test p = *10.033, *20.035, *30.014,*40.013, *50.024, *60.004.D-E. Total RNA extract was prepared from mouse FB that were injected with 6 mg/kg AGS-499, 12 and 24 h after injection. (D) Twenty micrograms RNA was subjected to Northern blot analysis using a specific [P32] labelled TERT probe. (E) Real-time PCR was performed for quantification of TERT mRNA in mouse FB, normalized to β-actin mRNA and the increase in TERT transcripts was calculated as fold of vehicle (mean ± s.e.m.; n = 3, Student's t-test p = : *10.033, *20.021). Symbols: UT, untreated mice; V, vehicle.
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fig03: AGS-499 increases TERT protein level and TERT mRNA expression in the FB, BS and the SC in a time-dependent mannerA-C. Mice were injected s.c. with AGS-499 (6 mg/kg) or its vehicle DMSO and cytoplasmic and nuclear protein extract was prepared from the FB (A,C) or whole cell protein extract from the BS and SC (B), at various time after the compound injection. Protein extracts (30 µg) were analysed by Western Blot assay using anti-TERT antibody (Rockland) or anti-β-actin antibody (A,B). Quantification of the Western blot results (depicted in A) by densitometric analysis using the EZquant software are shown in (C). (mean ± s.e.m.; n = 10) Student's t-test p = *10.033, *20.035, *30.014,*40.013, *50.024, *60.004.D-E. Total RNA extract was prepared from mouse FB that were injected with 6 mg/kg AGS-499, 12 and 24 h after injection. (D) Twenty micrograms RNA was subjected to Northern blot analysis using a specific [P32] labelled TERT probe. (E) Real-time PCR was performed for quantification of TERT mRNA in mouse FB, normalized to β-actin mRNA and the increase in TERT transcripts was calculated as fold of vehicle (mean ± s.e.m.; n = 3, Student's t-test p = : *10.033, *20.021). Symbols: UT, untreated mice; V, vehicle.
Mentions: To examine the time-dependent activation of telomerase in the brain following AGS treatment, AGS-499-treated mice were sacrificed at 3, 6, 12, 24 and 48 h after treatment. Telomerase protein level was examined in the cytoplasmic and nuclear fractions derived from the mouse FB. As can be seen in Fig 3A and C, telomerase protein level gradually increased with time in both the nucleus and cytoplasm (up to three- and twofold, respectively; p < 0.01) following AGS treatment, peaking at 12 h, decreasing to 1.5- and 2-fold (p < 0.05) activation at 24 h, and reaching the basal level at 48 h post treatment. Examination of the effect of AGS treatment on telomerase protein in the BS and in the lumbar region of the SC demonstrates a significant increase in TERT protein 12 and 24 h after AGS injection (Fig 3B). In addition, the effect of AGS-499 injection on the TERT mRNA levels was determined by Northern blot analysis of total mRNA derived from the FB of AGS-treated and untreated mice. The results depicted in Fig 3D demonstrate the identification of the full-length mTERT mRNA (3.4 kb) and two additional unknown transcripts of 2.1 and 7 kb. In AGS-499-treated mice, a significant increase in the full-length TERT mRNA and the 2.1 kb transcript was observed. No change in the GAPDH transcript was detected, suggesting that AGS-499 specifically increases mTERT RNA expression. Quantification of the increase in mTERT RNA transcripts by real-time PCR analysis revealed a time-dependent increase (up to 3.87 ± 0.74, p < 0.05 compared to vehicle) peaking at 12 h after AGS injection (Fig 3E), which is compatible with the mTERT protein results (Fig 3A and C).

Bottom Line: Hence, we and others hypothesized that increasing telomerase expression by pharmaceutical compounds may protect brain cells from death caused by damaging agents.The survival of telomerase-expressing cells (i.e. motor neurons), but not telomerase-deficient cells, exposed to oxidative stress was increased by AGS-499 treatment, suggesting that the AGS-499 effects are telomerase-mediated.Therefore, a controlled and transient increase in telomerase expression and activity in the brain by AGS-499 may exert neuroprotective effects.

View Article: PubMed Central - PubMed

Affiliation: The Shraga Segal Department of Immunology and Microbiology, Ben-Gurion University of the Negev, Beer-Sheva, Israel.

Show MeSH
Related in: MedlinePlus