Telomerase increasing compound protects hippocampal neurons from amyloid beta toxicity by enhancing the expression of neurotrophins and plasticity related genes

The telomerase reverse transcriptase protein, TERT, is expressed in the adult brain and its exogenic expression protects neurons from oxidative stress and from the cytotoxicity of amyloid beta (Aβ). We previously showed that telomerase increasing compounds (AGS) protected neurons from oxidative stress. Therefore, we suggest that increasing TERT by AGS may protect neurons from the Aβ-induced neurotoxicity by influencing genes and factors that participate in neuronal survival and plasticity. Here we used a primary hippocampal cell culture exposed to aggregated Aβ and hippocampi from adult mice. AGS treatment transiently increased TERT gene expression in hippocampal primary cell cultures in the presence or absence of Aβ and protected neurons from Aβ induced neuronal degradation. An increase in the expression of Growth associated protein 43 (GAP43), and Feminizing locus on X-3 genes (NeuN), in the presence or absence of Aβ, and Synaptophysin (SYP) in the presence of Aβ was observed. GAP43, NeuN, SYP, Neurotrophic factors (NGF, BDNF), beta-catenin and cyclin-D1 expression were increased in the hippocampus of AGS treated mice. This data suggests that increasing TERT by pharmaceutical compounds partially exerts its neuroprotective effect by enhancing the expression of neurotrophic factors and neuronal plasticity genes in a mechanism that involved Wnt/beta-catenin pathway.

Immunofluorescent staining. Primary cell culture. Immunofluorescence analysis was performed with anti-β tubulin 3 (Sigma, Rehovot) mouse antibody as the first antibody, to stain microtubules and with anti-NeuN (Millipore) mouse antibody as the first antibody, to stain neuronal cell bodies. Cy3 anti mouse-immunoglobulin G (IgG; Jackson ImmunoResearch) was used as the second antibody in both cases. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI).
The amount of NeuN positive and DAPI positive cells was quantified separately using the IdentifyPrimaryObjects pipeline. The amount of NeuN positive cells was then divided by the amount of DAPI positive cells and normalized to 100 percent. NeuN positive cell size was determined using MeasureObjectSizeShape pipeline. The object size list was then processed using GraphPad Prism to obtain a distribution of cell size. Cells were then categorized into 3 size groups.
The IF slides of the hippocampal sections were analyzed by the CaseViewer 2.3 software. The auto-fluorescence button was applied and all the images were adjusted to the same parameters of: Black = 0, Gamma = 1.60, White = 65535.
Real time pcR. RNA extract was prepared with "Bio Tri RNA" (Bio-Labs) kit per the manufacturer instructions.
cDNA was prepared using the qScript cDNA synthesis kit (QuantaBio). Real time PCR was performed using the following primers:

Results
Telomerase increasing compound (AGS) enhanced TERT expression in primary hippocampal cell culture in a dose and time dependent manner and protected neurons from amyloid beta toxicity. The primary hippocampal cell cultures were stained with anti GFAP and anti-beta 3 tubulin antibodies and DAPI for nuclei. The results show that the culture consist of the various cell types of the hippocampus: it contained neurons stained by the beta3 tubulin and many astrocytes and astroglial cells stained by GFAP (Supl. 1) and the % of neurons in the culture was also estimated (20%) by NeuN staining as described later in the text.
The culture was treated with varying concentrations of AGS-499 from 20 nM to 200 nM for 12 hrs (Fig. 1a). An increase in TERT gene expression was observed up to 2.8-fold with 200 nM of AGS (compared to UT or vehicle treated cells). A transient increase in TERT gene expression (up to 5 fold, 12-hrs. post treatment) was detected in the AGS-499 treated primary hippocampal cell culture, which returns to its basal level 24 hrs post treatment (Fig. 1b). Primary hippocampal cell cultures were treated with oligomerized Aβ protein at 5 μM and 10 μM with and without AGS treatment (200 nM) for 48 hrs. The AGS compound (AGS-499) was administered every 24 hrs. The cells were subjected to an immunofluorescence procedure with anti-β3-tubulin antibodies (for staining of neurites) and with DAPI as a nuclear staining for all the cell types in the primary culture (Fig. 2). As can be seen, treatment with the Aβ protein at 5 μM and 10 μM caused significant neuronal degradation demonstrated by the fragmentation of axons and neurites and the presence of smaller cell bodies. The degradation effect of Aβ protein on neurons was visibly higher at 10 μM compared to 5 μM. In the AGS treated culture a significant visible increase in neuronal survival was observed which is demonstrated by the presence of neurons with long axons, neurites, and normal cell body (soma) size, suggesting that AGS treatments conferred partial protection of neurons from the Aβ induced damage at both 5 μM and 10 μM Aβ protein treatments. the expression of Gap43 and NeuN genes is enhanced in primary hippocampal cell cultures treated with telomerase increasing compound in the presence or absence of Aβ. To elucidate the mechanism by which the telomerase increasing compound enhanced the survival of neurons in the presence of Aβ protein, we first examined the effect of AGS alone on the expression of neuronal genes that are involved in the morphology of neuronal cell body and axonal growth. The hippocampal cell culture was treated with and without various AGS-499 concentrations for 12 hrs. and the expression of the following genes was determined: NeuN for neuronal soma 33 , Growth Associated Protein 43 (GAP43) for axonal growth 34 , and Synpatophysin (SYP) a major synaptic protein p38 for synapses formation and quantification 35 . The results depicted in Fig. 3 demonstrate that AGS treatment significantly increased Gap43 gene expression up to1.95 fold (Fig. 3a) NeuN   Next, we examined the effect of telomerase increasing compound on the expression of the aforementioned neuronal genes and the expression of TERT gene in hippocampal cells that were exposed to oligomers of Aβ protein. Cell cultures were incubated with 5 μM Aβ for 36 hrs, in the presence or absence of the AGS-499 compound, which was added to the cell culture every 24 hours. The results depicted in Fig (Fig. 4d) genes were not significantly altered by Aβ protein treatment, but were significantly increased by AGS treatment in the presence of Aβ protein: up to 2-fold for NeuN (Fig. 4b), up to 1.5-fold for Gap43 (Fig. 4c) and up to 2.5-fold for SYP ( Fig. 4d) genes. It should be noted that AGS treatment increased the expression of SYP gene only in hippocampal cell culture that was exposed to Aβ protein (Fig. 4d), while the other neuronal genes NeuN and GAP43 were also significantly increased in cell cultures that were treated with AGS-499 alone.
The expression of neurotrophins in primary hippocampal cell cultures is increased following treatment with AGS compound. NGF and BDNF, the two main neurotrophins affect the expression of neuronal genes such as GAP43 and SYP 36,37 and demonstrate AD related neuroprotective effects 38,39 . Therefore we examined the effect of AGS treatment on the expression of NGF, and BDNF genes. Cell cultures were incubated with 5 μM Aβ for 36hrs with and without AGS-499 compound that was added to the cell culture every 24 hours. The results in Fig. 5 show that AGS-499 significantly increased the expression of BDNF gene (up to 3-fold) in the presence of Aβ oligomers (Fig. 5a) while eliciting no effect on NGF gene expression (Fig. 5b).  www.nature.com/scientificreports www.nature.com/scientificreports/ stained cultures were quantified for both the size of NeuN positive cells, and the relative quantity of NeuN positive cells was calculated as percentage of all the DAPI stained cells in culture (Fig. 6b,c). The results demonstrate that the hippocampal cell culture contained up to 20% NeuN positive cells. Treatment with Aβ alone significantly decreased the percentage of NeuN positive cells, and the remaining NeuN positive cells also decreased in size.  (a) Immunofluorescent staining using DAPI and NeuN after 48 hours of the indicated various treatments compared to an untreated control. Slides were visualized using a panoramic slide scanner (Panoramic Midi II scanner 3DHISTECH, Budapest, Hungary). Scale bar: 500 μm.The enlarged images were obtained using the sniping tool software for the selected area in the pictures (b) Cell size distribution. Cell size was determined using CellProfiler software. Cell sizes were divided into 3 groups of arbitrary size. Cells with a size smaller than 100 pixels (<100), a size of between 100 and 200 pixels (100-200) and cells larger than 200 pixels (>200). www.nature.com/scientificreports www.nature.com/scientificreports/ However, AGS treatments, both alone and in combination with Aβ, significantly increased the percent of NeuN positive cells (Up to 1.35-fold and 1.8-fold compared to the untreated cultures and the Aβ treated cultures respectively). In addition, an increase in the size of the NeuN expressing cells in the presence of Αβ oligomers following AGS treatment, compared to cultures treated with Aβ alone was detected (Fig. 6b) In Vivo: the expression of TERT and neuronal genes increased in hippocampi of AGS treated mice. The significant effect of the AGS compound on the expression of neuronal genes in the primary hippocampal cell cultures was tested in vivo. Six-week-old ICR mice were injected with the compound as previously described 11 and the expression of TERT, NeuN, Gap43, and SYP genes in the hippocampus was determined. A significant increase in the expression of these genes was detected: TERT up to 1.5-fold (Fig. 7a), NeuN up to 1.5-fold (7b), GAP43 up to 2.5-fold (7c), and SYP up to 1.5-fold (7d), suggesting that a single treatment with AGS compound increased the expression of these genes in the mouse hippocampus in vivo. The effect of the AGS treatment on the expression of neurotrophins in the hippocampus in vivo was also examined and the results depicted in Fig. 8 revealed that the expression of BDNF gene was significantly increased up to 1.5-fold in AGS-499 treated mice compared to untreated or vehicle treated mice (Fig. 8a). Immunofluorescence analysis of hippocampal perfusion-fixed frozen sections using specific anti BDNF antibody, revealed an increase in BDNF staining in the hippocampus (the dentate gyrus granule layers the hilus and the interneurons outlines) in AGS treated mice compared to vehicle treated mice (Fig. 8b). NGF gene expression was also significantly increased in AGS0499 treated mice compared to vehicle treated mice (Fig. 8c) Telomerase also regulates the transcriptional activity of the β-catenin dependent Wnt signaling pathway 29 . Using our previously injected mice, we determined the expression of β-catenin gene and protein as a measure of WNT signal activity as was previously demonstrated 42 . The results depicted in Fig. 9a show that AGS-499 treated mice expressed elevated levels of β-catenin gene -of up to 4-fold in the hippocampus, compared to untreated or vehicle treated mice. Immunohistofluoresence analysis of hippocampal perfusion-fixed frozen sections using specific anti beta-catenin antibody, revealed a significant increase of beta-catenin staining in the hippocampus of AGS treated mice compared to untreated mice (Fig. 9b).

AGS treatment increased
To confirm that the Wnt/beta catenin pathway is active we examined the expression of cyclin D1 gene that its level is regulated by the Wnt-beta catenin signaling 43 .Indeed the expression of cyclin D1 in the hippocampus of AGS treated mice significantly increased compared to vehicle treated mice (Fig. 9c). www.nature.com/scientificreports www.nature.com/scientificreports/ AGS treatment of the primary hippocampal cell culture in the presence or absence of Aβ significantly increased the relative amount of NeuN expressing cells. This increase could be due to a neurogenesis process. We examined this possibility in vivo by investigating the expression of genes known to be expressed in various stages of the neuro-differentiation pathway: Doublecortin (DCX), and Sex Determining Region Y box-2 (SOX-2). DCX is a known marker for neurogenesis 44,45 . Its expression starts in type-2b progenitor cells and persists until an immature post-mitotic neuronal cell is produced. SOX-2 is an early stem cell marker expressed in Neuronal Stem Cells (NSCs) and early type 2-a progenitor cells. The expression of DCX and SOX-2 is mutually exclusive 46 . The results depicted in Fig. 10 show that AGS injection caused a significant increase of up to 1.5-fold in the expression of DCX gene (Fig. 10a), while no change in SOX-2 gene has been detected (Fig. 10b).

Discussion
In this study, we examined the effect of increasing TERT gene expression by a pharmaceutical compound on the protection of neurons from the Αβ-induced toxicity. This assumption was based on several studies; it was previously shown that the reduction of TERT expression results in enhanced sensitivity of neurons to the common neurotoxic proteins expressed in AD pathology 16 . In addition, it was suggested that overexpression of telomerase exerted some neuroprotective effects from Aβ in AD models 47 . However, genetic manipulation as a therapeutic strategy possesses several limitations and pharmaceutical treatments are therefore preferred. Indeed, we previously showed that novel tri-aryl compounds designated AGS transiently increased TERT gene and TERT protein expression and telomerase activity both in vivo and in vitro in a time and dose dependent manner 11,17,18 . These compounds can cross the BBB and transiently increase telomerase activity and TERT expression in the brain 11 . Treatment of SOD1 Tg mice with these compounds delayed the onset and progression of ALS and increased the survival of motor neurons in the mouse spinal cord 11 . Here we used a common in vitro model for AD -primary hippocampal cell cultures exposed to oligomeric Aβ. First, we demonstrated that TERT gene expression is transiently increased in these cultures following AGS treatment. The exposure of the primary hippocampal cell culture to cytotoxic concentrations of oligomeric Aβ did not significantly affect TERT gene expression, while treatment with AGS in the presence of Aβ has increased TERT gene expression. These results demonstrate that the AGS compound can increase the expression of TERT in the hippocampal cells also in the presence of Aβ aggregates.
The aggregated exogenic Aβ possesses neurotoxic properties, and it affects the morphology of neurons. Untreated neuronal cultures display normal cellular morphology with: intact axons, connections to neighboring www.nature.com/scientificreports www.nature.com/scientificreports/ cells via neurites, and proper soma size; while treatment of the culture with Aβ, both at 5 μM and 10 μM, caused axonal degradation, synaptic loss and shrinkage of the cell body. Whereas treatment with AGS-499 alone did not affect the neurons in the culture, it significantly conferred partial protection from the cytotoxicity induced by Aβ at both concentrations (5 and 10 μM).
It should be noted that these Aβ concentrations are significantly higher by about 1000 fold than the physiological concentration of the peptide in the brain, which is in the range of pico and nano molars 48,49 .
The neuroprotective effect of AGS 499 is demonstrated here by the morphology of the neurons (normal size of soma body, long axons and neurites). To confirm the neuroprotective effect of AGS by molecular tools we investigated the effect of AGS treatment on the expression of genes that are associated with neuronal soma and neuronal plasticity. Interestingly, we found that treatment of the primary hippocampal cell cultures with telomerase increasing compound alone significantly increased the expression of NeuN and GAP43 genes but not the SYP gene. This data demonstrates a correlation between the increase in the expression of TERT gene and the enhancement in the expression of NeuN and GAP43 genes. It was previously suggested that TERT may act as a www.nature.com/scientificreports www.nature.com/scientificreports/ transcriptional factor for certain genes 3-5 , however it is not yet clear whether this is a direct or indirect effect of TERT on the expression of NeuN and Gap43 genes. Exposure of the primary hippocampal cell culture to cytotoxic aggregates of Aβ slightly but not significantly reduced the expression of NeuN, GAP43, and SYP genes, but when AGS treatment was applied to the Aβ treated cultures, a significant increase in the expression of these genes was observed. The specificity of NeuN expression in neurons of the central nervous system is well established 33 , but its role is not fully understood. The protein is expressed only in post-mitotic neuronal cells and has been implied, among other things, in RNA editing 50 . The increase in NeuN expression in AGS treated hippocampal neurons in the presence or absence of oligomeric Aβ might be due to an increase in the expression levels within existing mature neurons, or is the consequence of maturation of neurons from neuronal precursor cells, or even due to both processes occurring simultaneously. However, our data shows that the number of NeuN positive cells has increased in the hippocampal cell culture by 30% following AGS treatment. Exposure of the culture to Aβ decreased the NeuN positive cells by 40% while treatment with AGS compounds dramatically increased the number of NeuN expressing cells by 183%. In addition, treatment with telomerase increasing compounds gives rise to a morphological new population of neurons; these neurons, while also expressing NeuN, are smaller and round in shape compared to their more common counterparts. The round shape of the cells indicates a lack of a typical neuronal structure known as the axon hillock 51 . These small, NeuN expressing cells that lack structures found in mature neurons, could be a population of young, newly formed, post-mitotic neurons. Therefore, it is possible that treatment of the primary hippocampal cell culture by the telomerase increasing compound induced neurogenesis, a possibility that was previously demonstrated in the brain of telomerase-reactivated mice 52 . GAP43 is highly expressed in neuronal growth cones during development and axonal regeneration 34 . The increase in GAP43 following AGS treatment of either Aβ-treated or untreated hippocampal cell culture may suggest an increase in the growth process of axons. In addition, Gap 43 was identified as a rapidly transported axonal protein that is highly upregulated after sciatic nerve injury. It is localized to growth cones associated with neuropil areas and is highly expressed during nervous system developmental and regenerative axon growth 53 . Therefore, one may assume that the increase of its expression by AGS treatment in damaged axons points toward its involvement in the neuroprotective processes of the telomerase increasing compound.
Although the expression of SYP is known to be limited to synaptic vesicles, its function is not. It is however used as marker to quantify synapses 35 . Synaptic alterations are one of the earliest events in AD and it was found that oligomeric Aβ disrupts synapses 54,55 .
Although AGS-499 treatment alone did not increase SYP gene expression it did however enhance its expression in hippocampal cultures that were exposed to Aβ, thus suggesting that in neurons damaged by Aβ-induced cytotoxicity, treatment with telomerase increasing compound, AGS-499, may restore the synapses formation by increasing the expression of the SYP gene.
All together our data show a significant increase in the expression of NeuN, GAP43 and SYP genes, in the hippocampal cell cultures that were exposed to Aβ and treated with AGS, suggesting that in the in vitro AD model, the increase in their expression is part of the neuroprotective mechanism of the telomerase increasing compound. We injected AGS-499 into adult mice to examine whether the enhancement of the expression of neuronal genes, involved in neuronal plasticity and neuronal growth, is a unique phenomenon observed only in hippocampal cell culture derived from newborn mice, or rather can occur in vivo in adult mice. We previously showed that this compound can cross the BBB and can increase TERT expression in the various regions of the brain of adult mice 11 . Here we found that a single injection of AGS-499 increased TERT gene expression in the hippocampus of both male and female mice. We also showed that a single injection of AGS-499 significantly increased the expression of NeuN, GAP 43 and SYP genes in vivo in the mouse hippocampus. This strongly indicates that the effects of telomerase increasing compound are not only an in vitro phenomenon but also occur in vivo. It should be noted that unlike the in vitro data in which SYP gene expression was not affected by AGS-499 alone, this compound significantly increased SYP gene expression in the mouse hippocampus. The biological effects of the increased expression of NeuN, Gap43 and SYP genes in the mouse brain following AGS treatment are now under investigation in our group.
Since it was suggested that the expression of NeuN, GAP 43 and SYP might be regulated by neurotrophins 36,56,57 , we examined whether treatments with telomerase increasing compound affect the expression of neurotrophins -thus promoting processes such as neuronal development and survival 58 .
Out of the many members in this family, we focused on the main two: BDNF and NGF. In an in vitro primary culture, treatments with AGS-499 significantly increased the expression of BDNF gene but not NGF gene while in vivo the expression of both BDNF and NGF genes and BDNF protein were elevated following AGS treatment. BDNF and NGF play an important role in similar neuronal related pathways such as differentiation, maturation, growth, survival of existing neurons, and neurogenesis [58][59][60] . Since BDNF supports the survival of existing neurons, its increased expression in AGS-499 treated cultures may partially explain the neuroprotective effects of telomerase increasing compounds. In addition to their role in neuronal survival, both BDNF and NGF are important modulators of neuronal development and plasticity and have been shown to increase the expression of both GAP43 and SYP 36,56 . BDNF and NGF play also an important role in the process of formation of new neurons, also known as neurogenesis 58 . This role has led us to hypothesize that the increase in NeuN (gene and protein) expression, which correlates to the increase in both BDNF and NGF following AGS treatments, stems from the formation of new neuronal cells. To further demonstrate the possibility of induced neurogenesis, in AGS treated mice, we used two mutually exclusive markers of the various differentiation stages of the neuronal cell -SOX-2 and DCX. While SOX-2 is expressed only in embryonic stem cells, DCX appears in late neuronal progenitor cells and its expression persists until the early stages of post-mitotic neurons [44][45][46] . Indeed an increase in DCX gene expression was observed in the hippocampus of AGS treated mice suggesting an increase in neuronal differentiation, while the lack of increase in SOX-2 gene expression indicates that no increase in stem cell proliferation was observed. Therefore, it is possible that the increase in the expression of BDNF and NGF by the telomerase increasing compounds result in the enhancement of the expression of neuronal plasticity related genes and induced neurogenesis, and thus contribute to the mode of action of the AGS compound as neuroprotective agent.
We previously showed that AGS compounds exerted their neuroprotective effects in a telomerase dependent manner 11 . To elucidate the mechanism by which increasing TERT expression might affect the expression of neurotrophins we examined the Wnt signaling pathway. Wnt signaling pathway is known to be activated by TERT 29,61 and BDNF and NGF are known to be regulated by Wnt signaling 28,62 . We found that β-catenin expression (gene and protein) is increased in the hippocampus of AGS treated mice. Moreover Cyclin D1 gene that is known to be regulated by the Wnt-beta catenin signaling 43 was also increased in AGS treated mice compared to vehicle treated mice. These data indicates the activation of the Wnt-beta catenin signaling pathway 63 by treatment with the AGS compound.
In conclusion, we propose, as summarized in Fig. 11, that increasing TERT expression in the hippocampus using a pharmaceutical compound partially exerts neuroprotective effect via activation of the Wnt signaling pathway. This activation leads to an increased expression of neurotrophins that in turn enhances the expression of neuronal growth and plasticity genes. This provides damaged neurons, such as those exposed to the cytotoxicity of Aβ, the ability to overcome its cytotoxic effects. In addition, the non-canonical roles of TERT, such as an anti-apoptotic enzyme and its mitochondrial protective function from oxidative stress are also part of the AGS neuroprotective effects as we previously demonstrated 11 . Our data suggest the possibility that the telomerase increasing compound may show some beneficial effects in AD and this is now under investigation using a mouse model for AD. Figure 11. A proposed model for the neuroprotective effects of telomerase increasing compound AGS from the Aβ cytotoxicity in an in-vitro AD model. Increasing TERT expression in hippocampal cells activates the Wnt signaling pathway which upregulates the expression of neurotrophins that increase the expression of neuronal plasticity genes and therefore confer a partial neuroprotective effect from the Aβ induced cytotoxicity.