Dissociation between telomerase activity and expression during mice cortical development due to a DNA bound telomerase inhibitor
Telomerase, the enzyme that elongate telomeres during DNA replication, is expressed in the neonatal brain and in distinct regions of adult brain. The roles of telomerase in post mitotic cells such as neurons are mostly unrelated to telomere elongation but rather associated with the promotion of cell survival. Overexpression of TERT, the protein component of telomerase, protects neurons from various insults in vitro and in vivo. Here we confirm the dissociation between the drastic reduction in telomerase activity during postnatal development and the sustained levels of TERT mRNA and proteins. TERT can interact with the DNA and can be found in the cytoplasm, where it extracts some of its anti apoptotic function. We measured TERT protein and telomerase activity in cytoplasm, nucleoplasm, and DNA bound protein fractions during mice cortical development and found a strong dissociation between TERT protein levels and telomerase activity in embryonic DNA bound protein fraction. Adding the embryonic DNAbound protein fraction but not an adult DNA bound protein to telomerase activity reaction significantly inhibited telomerase activity. Thus, we suggest the existence of a yet unidentified embryo specific DNA bound telomerase inhibitor, which may regulate telomerase activity in the developed cortex. Introduction Telomerase is a ribonucleoprotein complex that elongates telomeres; the active enzyme consists of telomerase reverse transcriptase protein (TERT) and an RNA subunit (TERC) [1] [2] The activity of telomerase in vivo requires several additional proteins that are essential for the holoenzyme biogenesis and its trafficking to telomeres [3] Telomerase is expressed in more than 80% of cancer cell types, in embryonic tissue, and stem cells, but is highly restricted in most mature somatic cells [2] Telomerase activity was reported in mice embryonic and young neonatal brain, but significantly reduced to undetectable levels at around postnatal day 13. Surprisingly, TERT mRNA and protein are detected in adult and old mice, and recently, it was also found in postmortem human brain [4] [5] [6] [7] It has been shown that TERT possesses telomere unrelated functions that can protect neurons from various insults [7] [8] Thus, TERT may play a role both in brain development and in its response to insults during neurodegenerative disease and acute injuries [9] Here, we confirm the expression and activity pattern of telomerase in the developing mice cortex and report for the first time that in embryonic cortex TERT is mostly bound to the DNA. Moreover, we observed that this fraction that contains high levels of TERT protein does not support telomerase activity in vitro and even abolishes its activity when added to the reaction mixture. These observations suggest the existence of a yet undefined telomerase inhibitor in the embryonic cortex DNA bound protein extract. Objective Here we aimed to characterize telomerase expression, activity, and location during mice cortical development. Dissociation between telomerase activity and expression during mice cortical development due to a DNA bound telomerase inhibitor DOI: 10.19185/matters.201604000008 Matters Select (ISSN: 2297 9239) 2 a Figure Legend Figure 1. TERT expression and telomerase activity are reduced in the mouse brain cortex with development and aging. (A) Whole cell protein extracts were prepared from embryo (E17) postnatal days 2, 5, 14 (P2, P5, P14, respectively) adult (3 5 months) and old (20 22 months) mouse cortex. Protein (25 mg) was analyzed by western blot using anti TERT antibody. b Actin was used as a loading control. (B) Quantification of the western blot results (depicted in A) by densitometric analysis using the EZquant software. The results are mean SEM of three independent experiments. (C) Telomere elongation activity in vitro was measured in the same protein extract as in A by the telomere repeat amplification protocol (TRAP) assay using 1.5 mg of protein extract and 32 PCR cycles. U251 glioblastoma cell line was used as a positive control for telomerase activity. NG means negative control. This is a representative experiment of n 3. (D) Cytoplasmic (C) nucleoplasmic (N) and DNA bound protein (D) fractions were prepared from embryo (E17) postnatal days 2, 5, 14 (P2, P5, P14, respectively) adult (3 5 months) and old (20 22 months) mouse cortex. Protein (25 mg) was analyzed by western blot using anti TERT antibody, anti topo1 as a DNA bound marker, anti lamin as nuclear marker, anti tublin as a cytoplasmic marker, and anti VDAC as a mitochondria marker. b Actin was used as a loading control. This is a representative experiment of n 3. (E) Telomere elongation activity in vitro was measured in the same protein extract as in D by the telomere repeat amplification protocol (TRAP) assay using 1.5 mg of proteins extract and 32 PCR cycles. (F)Quantification of the western blot and TRAP results (depicted in D and E) by densitometric analysis using the EZquant software. The fraction of TERT in each compartment was calculated by dividing its level in that compartment by the level in all compartments (equation: C or N or D/(C+N+D) (G) Telomerase activity was assayed by TRAP using a combination of the embryonic nucleoplasm extract and embryonic DNA bound extract or with the addition of various concentrations of NaCl at the indicated ratio, total protein 1.5 mg, 32 PCR cycles. The final concentrations in the TRAP reaction are 25 fold lower. Dissociation between telomerase activity and expression during mice cortical development due to a DNA bound telomerase inhibitor DOI: 10.19185/matters.201604000008 Matters Select (ISSN: 2297 9239) 3 (H) Quantification of the TRAP results (depicted in G) by densitometric analysis using the EZquant software. The inhibition levels were calculated by dividing the activity level in the combined extract with the activity level of the nuclear extract alone. The x axis represents the ratio between the nuclear extract and the DNA bound extract. (I) DNA bound protein extract derived from mice at the following ages: embryonic day 17 (E17) postnatal day 3 (P3) adult (2 3 months) (adu) and old (20 22 months) were added to embryonic nucleoplasm protein extract in a ratio of 1/1 and telomerase activity was measured by TRAP assay. Animals CD 1 male mice were used for the investigation of telomerase in the mouse brain. All animal procedures were approved by the animal experimentation ethics committee of our institute. Preparation of nuclear and cytoplasmic proteins extracts Brains were quickly removed from the skull, washed, and placed in a Ringer solution at 4degC.These were homogenized using a manual homogenizer (pestle B) The homogenates were centrifuged at 500 g at 4degC for 7 min and the pellets were subjected to nuclear and cytoplasmic extractions. Cytoplasmic extract The cytoplasmic extract was prepared as previously described [12] Briefly, cells were resuspended in buffer A (10 mM Tris HCl pH 7.4, 10 mM NaCl, 1 mM EDTA) containing a mixture of protease inhibitors [final concentrations: 2 mg/ml aprotinin, 2 mg/ml antipain, 2 mg/ml leupeptin, 1 mg/ml pepstatin A, 2 mg/ml PMSF (phenylmethylsulfonyl fluoride) and kept on ice for 15 min. Lysis was performed by passing the solution 10 15 times through a syringe with a 21G needle. The cytosolic fraction was obtained by centrifugation at 9,300 g at 4oC for 7 min and the supernatant was collected. Nuclear extract The pellet from the cytoplasmic preparation was resuspended in CHAPS buffer, containing 10 mM Tris HCl, 5 mM 2 b Mercaptoethanol pH 7, 1 mM MgCl2, 1 mM EDTA, 0.1 mM PMSF, 0.5% CHAPS (3[(3Cholamidopropyl)dimethylammonio] propanesulfonic acid) and 10% glycerol, followed by incubation at 4oC for 30 min. The nuclear extract was centrifuged for 30 min at 9,300 g at 4oC, and the supernatant was collected. DNA bound protein extract The pellet of the nuclear extract was washed with CHAPS twice, each time incubating for 20 min in ice and centrifuged for 20 min at 9,300 g in 4oC. Then CHAPS with 1 M NaCl was added and the sample was incubated on ice for 30 min and centrifuged for 20 min at 9,300 g in 4oC. The supernatant contained the DNA bound proteins. Total protein concentration was determined using the BIO Rad protein assay kit (Bio Rad Laboratories) Separation of proteins by gel electrophoresis and detection Protein extracts (as indicated by various experiments) derived from nuclear and cytoplasmic fractions were analyzed by polyacrylamide gel electrophoresis and western blotting as previously described [13] using either anti hTERT mice IgG hTERT monoclonal antibody (1:100