J Neurosci. 2015 Jan 28;35(4):1659-74. doi: 10.1523/JNEUROSCI.2925-14.2015.

The role of telomerase protein TERT in Alzheimer’s disease and in tau-related pathology in vitro.

 

Spilsbury A1, Miwa S1, Attems J2, Saretzki G3.
  • 1Newcastle University Institute for Ageing, Campus for Ageing and Vitality, Institute for Cell and Molecular Bioscience, and.
  • 2Newcastle University Institute for Ageing, Campus for Ageing and Vitality, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, NE4 5PL, UK.
  • 3Newcastle University Institute for Ageing, Campus for Ageing and Vitality, Institute for Cell and Molecular Bioscience, and gabriele.saretzki@newcastle.ac.uk.

 

Abstract

The telomerase reverse transcriptase protein TERT has recently been demonstrated to have a variety of functions both in vitro and in vivo, which are distinct from its canonical role in telomere extension. In different cellular systems, TERT protein has been shown to be protective through its interaction with mitochondria. TERT has previously been found in rodent neurons, and we hypothesize that it might have a protective function in adult human brain. Here, we investigated the expression of TERT at different stages of Alzheimer’s disease pathology (Braak Stages I-VI) in situ and the ability of TERT to protect against oxidative damage in an in vitro model of tau pathology. Our data reveal that TERT is expressed in vitro in mouse neurons and microglia, and in vivo in the cytoplasm of mature human hippocampal neurons and activated microglia, but is absent from astrocytes. Intriguingly, hippocampal neurons expressing TERT did not contain hyperphosphorylated tau. Vice versa, neurons that expressed high levels of pathological tau did not appear to express TERT protein. TERT protein colocalized with mitochondria in the hippocampus of Alzheimer’s disease brains (Braak Stage VI), as well as in cultured neurons under conditions of oxidative stress. Our in vitro data suggest that the absence of TERT increases ROS generation and oxidative damage in neurons induced by pathological tau. Together, our findings suggest that TERT protein persists in neurons of the adult human brain, where it may have a protective role against tau pathology.

KEYWORDS: Alzheimer’s disease; mitochondria; neuroprotection; oxidative stress; tau; telomerase

PMID: 25632141

 

Alzheimer’s disease (AD) is the leading cause for age-related dementias and affects more than 35 million people worldwide. With an increasing life expectancy this number will rise dramatically in the near future. Many changes occur in the brains of AD patients, but the accumulation of extracellular beta-amyloid and the hyperphosphorylation of the microtubule related protein tau are the most striking hallmarks of the disease.

Telomerase is an enzyme which in its canonical function maintains telomeres, the ends of chromosomes in dividing cells. In humans, most adult somatic cells have no telomerase activity, though there are several cell types such as lymphocytes, endothelial cells and stem cells which continue to express telomerase activity.

Neurons, however, are non-dividing cells in the brain. But they live a long time and understanding the protective mechanisms for guaranteeing a lifelong functioning of these cells is of utmost importance. The protein subunit TERT (Telomerase reverse transcriptase) has been shown to have many non-telomere related functions that have been most comprehensively investigated in cancer cells (for review see Saretzki, 2014).

 

fig 1 Saretzki frontiers

Fig. 1: TERT staining in neurons from human brain.

A-C, TERT is brown using immunohistochemistry , D: Tert is red using immunofluorescence

A: cortex, 10 fold magnification B: hippocampus (CA3 region), 10 fold magnification, C: hippocampus, 40 fold magnification. Blue: haematoxylin staining for nuclei. D: cortex (100x, scale bar = 50 µM).

 

In mice, telomerase activity is high in prenatal neurons, but is quickly down regulated after birth. However, we found that even in adult human brains the telomerase protein TERT can be found in neurons (see figures 1 and 2) as well as in activated microglia cells, which are related to macrophages, but that TERT was not expressed in astrocytes. This seems rather surprising since both cell types, neurons and astrocytes, are generated by the same neural stem cells (NSC). These changes in gene expression may occur later during development.

 

fig 2 Saretzki frontiers

Fig. 2: TERT protein (green) and mitochondria (red) colocalise in neurons from human hippocampus (A) and in Purkinje neurons of mouse cerebellum (B). Blue: DAPI staining for nuclei Scale bar = 50 µM

 

 

We and other have described previously, that the TERT protein can shuttle to mitochondria and has a protective function in the organelles, decreasing oxidative stress, preventing apoptosis and decreasing damage to mitochondrial DNA upon oxidative stress (Ahmed et al., 2008, Haendeler et al., 2009, Singhapol et al., 2013).

Oxidative stress has been demonstrated to play an important role in the progression of neurodegenerative diseases such as AD, although any causal role is still under debate.

The aim of our study was to investigate whether TERT levels in brain tissue decrease during the progression of neurodegeneration from lower to higher Braak stages of tau pathology, and to analyse where the TERT protein localises in neurons. Surprisingly, we did not find any changes in the content of TERT protein in hippocampal tissue from different Braak stages. In contrast, we found an increased localisation of TERT protein within mitochondria in the highest Braak stages, the fully developed AD cases, compared to age-matched healthy controls.

In order to model the disease in an experimental cell system, we cultivated neurons from wild type (wt) and TERT knock-out (k.o.) mice and transduced them with pathological tau. If TERT protein is protective in neurons, those lacking TERT should be preferentially damaged due to the pathological tau than the wild type neurons. And this is precisely what we observed: neurons from TERT k.o. mice produced more oxidative stress in the form of mitochondrial superoxide in their neurites while in their cell bodies more lipid peroxidation occurred in the form of 4-hydroxynonenal. This suggests that TERT protein indeed seems to be protective against the stress induced by pathological hyperphosphorylated tau.

Similarly, when we treat cultivated neurons with other stress-inducing agents such as hydrogen peroxide (H2O2), we also found an increased susceptibility of those neurons without the TERT protein compared to wild type cells harbouring the protein. Most importantly, this treatment also resulted in TERT protein moving into mitochondria (see figure 3).

 

fig 3 Saretzki frontiers

Fig. 3: Under increased oxidative stress (50mM H2O2) TERT protein (green) moves to the mitochondria (red). Blue: nuclear DAPI stain, yellow colour means merged signals of co-localisation. Scale bar = 50 µM

 

We did not find any TERT protein in the nuclei of neurons, corresponding to the fact that these cells do not divide and thus their telomeres are not shortening, although they still can be damaged. This finding corresponds to those from others who had found that TERT forms a complex with other cell components in the cytoplasm that is released when the cell experiences increased stress (Iannilli et al., 2013). However, there are reports that there might also be some nuclear TERT, at least in some neuron types such as Purkinje cells (Eitan et al., 2012).

These findings once again demonstrate how versatile telomerase is, even as an evolutionarily old enzyme, by acquiring new functions in higher eukaryotes. As an example, TERT protein may leave the nucleus and shuttle to mitochondria, in order to reduce oxidative stress and ROS generation from within the organelle. It seems that adult neurons that do not express telomerase activity harness the protective function of the telomerase protein TERT to improve their survival and stress resistance.

We do not yet fully understand whether the localisation of TERT protein in hippocampal neurons of AD brains is the result of increased oxidative stress during disease progression, nor the exact mechanisms by which TERT protects mitochondria against oxidative stress and pathological tau. Intriguingly, we found a mutual exclusion of pathological tau and TERT protein in different areas of the hippocampus (see figure 4). However, we do not know whether neurons without or with very low levels of TERT protein accumulate hyperphosphorylated tau preferentially, or whether accumulation of pathological tau in the form of neuropil threads in the neuronal processes or as neurofibrillary tangles in the cell bodies displaces or leads to down-regulation of TERT protein. More mechanistic experiments are necessary to shed light on the mechanisms by which telomerase may protect neurons in the ageing brain and during neurodegenerative diseases.

 

fig 4 Saretzki Frontiers

Fig. 4: TERT protein (red) and pathological tau (green) are mutually exclusive in the hippocampus of AD brain (right image). Blue: DAPI nuclear stain. Scale bar = 50 µM

 

References:

  1. Ahmed S, Passos JF, Birket MJ, Beckmann T, Brings S, Peters H, Birch-Machin MA, von Zglinicki T, Saretzki G (2008) Telomerase does not counteract telomere shortening but protects mitochondrial function under oxidative stress. Journal of Cell Science 121:1046-1053.
  2. Eitan E, Tichon A, Daniel G, Priel E (2012a) Telomerase expression in adult and old mouse Purkinje neurons. Rejuvenation research 15:206-209.
  3. Haendeler J, Dröse S, Büchner N, Jakob S, Altschmied J, Goy C, Spyridopoulos I, Zeiher AM, Brandt U, Dimmeler S (2009) Mitochondrial Telomerase Reverse Transcriptase Binds to and Protects Mitochondrial DNA and Function From Damage. Arteriosclerosis, Thrombosis, and Vascular Biology 29:929-935.
  4. Iannilli F, Zalfa F, Gartner A, Bagni C, Dotti CG (2013) Cytoplasmic TERT Associates to RNA Granules in Fully Mature Neurons: Role in the Translational Control of the Cell Cycle Inhibitor p15INK4B. PLoS ONE 8:e66602.
  5. Saretzki G. Extra-telomeric Functions of Human Telomerase: Cancer, Mitochondria and Oxidative Stress. Curr Pharm Des. 2014 volume 20 Issue Number 41
  6. Singhapol C, Pal D, Czapiewski R, Porika M, Nelson G, Saretzki GC (2013) Mitochondrial Telomerase Protects Cancer Cells from Nuclear DNA Damage and Apoptosis. PLoS ONE 8:e52989.

 

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