Tamoxifen induces cellular stress in the nervous system by inhibiting cholesterol synthesis
© Denk et al. 2015
Received: 9 October 2015
Accepted: 9 November 2015
Published: 26 November 2015
Tamoxifen (TAM) is an important cancer therapeutic and an experimental tool for effecting genetic recombination using the inducible Cre-Lox technique. Despite its widespread use in the clinic and laboratory, we know little about its effects on the nervous system. This is of significant concern because TAM, via unknown mechanisms, induces cognitive impairment in humans. A hallmark of cellular stress is induction of Activating Transcription Factor 3 (Atf3), and so to determine whether TAM induces cellular stress in the adult nervous system, we generated a knock-in mouse in which Atf3 promoter activity drives transcription of TAM-dependent Cre recombinase (Cre-ERT2); when crossed with tdtomato reporter mice, Atf3 induction results in robust and permanent genetic labeling of cells in which it is up-regulated even transiently.
We found that granular neurons of the olfactory bulb and dentate gyrus, vascular cells and ependymal cells throughout the brain, and peripheral sensory neurons expressed tdtomato in response to TAM treatment. We also show that TAM induced Atf3 up-regulation through inhibition of cholesterol epoxide hydrolase (ChEH): reporter expression was mitigated by delivery in vitamin E-rich wheat germ oil (vitamin E depletes ChEH substrates), and was partially mimicked by a ChEH-specific inhibitor.
This work demonstrates that TAM stresses cells of the adult central and peripheral nervous systems and highlights concerns about clinical and experimental use of TAM. We propose TAM administration in vitamin E-rich vehicles such as wheat germ oil as a simple remedy.
Tamoxifen (TAM), a “selective estrogen receptor modulator” (SERM), is among the most widely-used anti-cancer drugs for women with estrogen receptor-positive breast tumors. Although its side effects have long been considered minor, there is accumulating evidence that a subpopulation of TAM-treated patients experience cognitive disturbances such as confusion, deterioration of verbal memory, and executive function such as decision-making, symptoms often referred to as “TAM brain fog” [1–4]. Changes in human brain structure and metabolism with TAM treatment have also been reported . Yet, there has been surprisingly little work done in animal models to identify TAM-induced changes in structure or function of the nervous system (either central or peripheral) , although in one study it was associated with a loss of glial progenitor cells in the corpus callosum and with reduced cell division in neurogenic regions of the CNS .
The dearth of preclinical work on potential adverse effects of TAM is concerning not only because of its reported cognitive effects in humans, but also because TAM is a widely-used tool for manipulating gene expression in vivo via the inducible Cre-Lox technique. Cre recombinase is a bacteriophage enzyme, which can remove sequences of DNA that lie between engineered loxP sites (referred to as “floxed” sequences). By attaching Cre to a mutated estrogen receptor (ERT2, which binds TAM but not endogenous estrogens; Cre-ERT2), it is possible to control precisely when a DNA sequence can be excised. As an example, mice expressing Cre-ERT2 can be crossed with another transgenic line containing a floxed “stop” signal preceding the sequence for a fluorescent reporter. The resulting mice express Cre-ERT2, which is restricted to the cytoplasm. When it binds TAM, Cre-ERT2 is translocated to the nucleus, where it can excise the stop signal, resulting in reporter expression. This technique has been used in mice to permanently label newly-generated neurons in the hippocampus  and transiently-active neurons following exposure to novel environments .
What remains little-acknowledged in studies using the Cre-ERT2 system is that TAM acts at the endogenous estrogen receptor α (ERα), hence its clinical use in cancer. Furthermore, TAM binds other endogenous molecules, including sigma 1 and 2 receptors on the endoplasmic reticulum , and cholesterol epoxide hydrolase (ChEH) – an enzyme complex involved in cholesterol biosynthesis . In fact, the affinity of TAM for ChEH is second only to that for ERα, which may underlie its counter-intuitive efficacy in ER-negative breast cancers . TAM thus has the potential to alter neurophysiology via any one of these avenues.
We generated a knock-in mouse expressing CreERT2 driven by the native promoter for activating transcription factor 3 (Atf3), one of a number of transcription factors upregulated by cellular stress, such as the unfolded protein response, tyrosine kinase receptor activation, glutamate receptor hyperactivation, and cellular injury . Crossing the ATF3-CreERT2 line with floxed stop ROSA-tdtomato line, when combined with TAM treatment, allows for permanent labelling of cells even transiently stressed by TAM. Here we report TAM-induced recombination in mature granular neurons of the olfactory bulb and dentate gyrus, populations of neurons involved in memory formation and recall. Recombination also occurred in larger-diameter proprioceptive neurons of the dorsal root ganglion, and in vascular endothelial and smooth muscle cells, and ependyma throughout the brain and spinal cord. We also provide evidence that TAM’s induction of Atf3 expression occurs via ChEH binding, and not via ERα or sigma 1/2 receptors.
Material and methods
Generation of mice
We also used a BAC transgenic mouse in which the promoter for advillin, expressed in all dorsal root ganglion (DRG) neurons, drives CreERT2 , and crossed it with the same reporter line as above. For all experiments, mice in treatment and control groups were sex and age-matched.
Compounds and doses used in N number of mice
Dose/ Route of administration
Tamoxifen in sunflower oil
75 mg/kg, i.p. (1 × day or 3 × days)
Tamoxifen in wheat germ oil
75 mg/kg, i.p. (1 × day)
4.47 mg/ml in sunflower oil, i.p.
ICI 182,780 (ICI)
20 mg/kg, gavage
4,4′,4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT)
10 mg/kg, s.c.
5 mg/kg, i.p.
Diethyl-2-[4-(phenylmethyl) phenoxy]ethanamine hydrochloride (DPPE)
50 mg/kg, i.p.
In experiments designed to identify the mechanism of TAM-induced ATF-3 up-regulation, we treated mice with either the anti-estrogen ICI 182,780 (ICI), a “pure” anti-estrogen , 4,4′,4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), a potent ERα agonist , or ditolylguanidine (DTG), a sigma-1 and −2 receptor agonist. ICI 182,780 in SFO (20 μg)  or SFO only was administered to nerve-injured mice by gavage the day before, the day of, and the day following TAM (or oil only) treatment. 4,4′,4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), was dissolved in 50 % DMSO in PBS and given by s.c. injection at 10 mg/kg  following the same schedule as with ICI 182,780. The sigma receptor agonist 1,3-di-o-tolylguanidine (DTG) was prepared as previously described  and delivered by i.p. injection in the same manner as the above drugs. The selective ChEG ligand N,N-Diethyl-2-[4-(phenylmethyl) phenoxy]ethanamine hydrochloride (DPPE, also known as tesmilifene) was administered at a one-time dose of 50 mg/kg i.p. in saline . Control mice received saline-only injections. For treatments with ICI 182,780, PPT, DTG, DPPE, and their controls, mice were killed on the fourth day following treatment.
Tissue processing and analysis
Mice were deeply anesthetized with sodium pentobarbital (Euthanal) and perfused transcardially with phosphate buffered saline followed by 4 % paraformaldehyde in 0.1 M phosphate buffer (PB). Tissues were dissected and transferred to 20 % sucrose in 0.1 M PB. Brains and spinal cords were embedded in gelatin and cut coronally on a vibratome (100 μm), or were frozen and cut at 50 μm on a cryostat (for co-localization studies). Whole dorsal root ganglia or 16 μm-thick cryosections were processed for immunohistochemistry. For DRG whole-mounts and brain sections, quantification was carried out on ~1 μm-thick confocal sections imaged with a Zeiss LSM 710 confocal system. DRG cryosections were imaged with a Zeiss AxioObserver Z.1 equipped with a Yokogawa spinning disk. In the dentate gyrus and olfactory bulb we measured the density of recombined neurons in the granular layers of each structure (3 sections per animal at the level of the median eminence), or counted recombined neurons (for DPPE studies). In the DRG we traced recombined profiles and used recursive translation  to convert profile distributions (in which large profiles are numerically overrepresented and lead to overestimations of the number of small diameter profiles) to cell distributions.
To quantify size-frequency distributions of all DRG neurons, we used cervical DRG whole-mounts from advillin-CreERT2 mice treated for 3 days with 75 mg/kg TAM in SFO, and killed on the fourth day. Cell densities between SFO and WGO groups were compared using Student’s t-test. Proportions of recombined neurons in the DRG were compared with a one-way analysis of variance (ANOVA) followed by a post-hoc Holm-Sidak test for pairwise differences. The Kolmogorov-Smirnov goodness-of-fit test was used to determine whether cell size-frequency distributions differed significantly.
The following antibodies were used: rabbit anti-ATF3 (1:200, Santa Cruz Biotechnology Inc.), mouse anti-neurofilament 200 (NF200; clone N52, 1:500, Sigma), rabbit anti-calcitonin gene related peptide (CGRP; 1:4000, Sigma), rabbit anti-tyrosine hydroxylase (TH; 1:1000, Millipore). To identify non-peptidergic nociceptors, slides were first incubated in isolectin B4 (IB4;1:400, Sigma) followed by an anti-IB4 primary antibody (1:2000, Vector laboratories). To characterize recombined neurons in the brain we used mouse anti-Neuronal Nuclei (NeuN; 1:100, Millipore) and rabbit anti-calbindin d28k (1:1000, Swant). Secondary antibodies included donkey anti-rabbit Alexa-488, donkey anti-goat Alexa-647, donkey anti-mouse Dylight 650 (all at 1:1000, Invitrogen), donkey anti-mouse aminomethylcoumarin (AMCA; 1:100, Jackson labs). Nuclear counterstains were 4′,6-diamidino-2-phenylindole (DAPI) (in ProLong Gold coverslipping medium, Invitrogen) or Hoechst 33342 (1:10,000, Sigma).
Generation and characterization of ATF3-CreERT2:stopfl/fltdtomato mice
A transgenic knock-in strategy was used to replace the endogenous Atf3 locus with an ATF3-CreERT2 allele (Fig. 1a). The resulting mice, even in a homozygous state, which effectively constitutes an Atf3 knock-out, appear phenotypically normal and outwardly indistinguishable from their wild-type littermates. The ATF3-CreERT2 line was then crossed with a ROSA-flox-stop-tdtomato line to obtain a permanent reporter of Atf3 activity.
When examining the resulting mice for tdtomato signal, it became clear that the ATF3-CreERT2 construct displays a small degree of TAM-independent “leakiness”. Under normal circumstances, Atf3 is expressed in very few regions of the adult nervous system . We and others have found that a small number of uninjured sensory neurons express Atf3 , and evidence of rare spontaneous recombination was found in reporter-expressing trigeminal and spinal sensory neurons, and their terminals in the spinal trigeminal nucleus and spinal cord (Fig. 1b, c). Allen Brain Atlas in situ hybridization data  show ATF3 expression only in granular neurons of the olfactory bulb and dentate gyrus, and accordingly, in naïve ATF3-CreERT2:stopfl/fltdtomato mice, occasional reporter-expressing neurons were found in both of these regions (Fig. 1d). We also found occasional clusters (probably clones) of microglia and vascular endothelial cells throughout the brain (Fig. 1e). As would be expected in the case of spontaneous Atf3 activation, recombination events increased with age, ranging from 3 to 5 cells per DRG in 8 week old mice to 10–20 cells in 7 month old mice (data not shown).
TAM-induced Atf3 induction in cortical regions
TAM-induced Atf3 induction in primary afferent neurons
ChEH inhibition accounts for Atf3 induction by TAM
DPPE, a selective ChEH ligand, significantly increased recombination in the DRG, hippocampus and olfactory bulb
12.33 +/− 2.33
26.00 +/− 3.21
Neurons per DRG whole-mount, 2 cervical DRG/ n
1.17 +/− 0.44
8 +/− 0.58
Granule cell neurons per section, 4 sections/ n, anterior dentate gyrus at the level of the median eminence
2 +/− 0.58
12.33 +/− 1.45
Granule cell neurons per 150 × 500 micron field, mid-OB
Since some primary afferent neurons express ERα as well as the sigma-1 receptor [27, 28], we also assessed their putative roles in this context. We treated mice with either ICI 182,780, a “pure” antiestrogen , 4,4′,4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), a potent ERα agonist , or ditolylguanidine (DTG), a sigma-1 and −2 receptor agonist. These drugs were given the day prior, on the day of, and the day following TAM treatment (75 mg/kg). None of these compounds induced recombination if given alone (data not shown), and none significantly altered the proportion of DRG neurons which underwent recombination (Fig. 6b). Drug doses and animal numbers are listed in Table 1.
This is the first study to directly assess the effects of TAM on cellular stress-induced Atf3 expression in the nervous system. We show that granular neurons of the olfactory bulb and dentate gyrus, a subpopulation of sensory neurons, ependymal cells and endothelial and vascular smooth muscle cells upregulate Atf3 in response to a single 75 mg/kg dose of TAM. In addition, we show that TAM does not induce Atf3 via estrogen receptors or sigma 1/2 receptors in these cells. Instead, our results indicate that ChEH is the responsible TAM-binding entity: Atf3 induction is reduced when TAM is delivered in vitamin E rich wheat germ oil and, conversely, direct activation of ChEH using the selective ligand DPPE upregulates Atf3. Whether this is due to accumulation of cholesterol epoxides (ChEH ligands) or reduction in the synthesis of cholestane-3β,5α,6β-triol remains unknown, although the anti-cancer effects of TAM in estrogen-insensitive breast tumors are thought to be attributable to the former [11, 26, 29].
In the cortex, we found TAM to induce recombination in granular neurons of the dentate gyrus of the hippocampus and olfactory bulb, as well as in vascular and ependymal cells. Taking previous findings and our results at face value, they would suggest that recombined neurons in the dentate gyrus occupy relatively superficial positions in the granular cell layer. Both during development and in adulthood, dentate granule neurons are born in an outside-out manner [8, 30], implying that those which are most susceptible to TAM-induced Atf3 upregulation are more mature cells.
A previous investigation of TAM-induced toxicity at therapeutic doses in the brain revealed a loss of oligodendrocyte progenitor cells (OPCs) in the corpus callosum . This was accompanied by a reduction in their proliferation, and that of neural progenitors in the subventricular zone and dentate gyrus, and it was suggested that this may represent a cellular substrate of cognitive dysfunction experienced by breast cancer patients receiving TAM therapy. OPC loss was prevented both in vitro and in vivo by MEK1/2 inhibition, one of a number of intracellular messengers that can activate Atf3 expression . In contrast, we found no evidence of TAM-induced recombination in OPCs or in the deeper neurogenic regions of the dentate gyrus or subventricular zone. It is possible that the reported OPC loss occurs independent of Atf3 induction, and although our study does not rule out effects of TAM on neurogenesis, it does not support a role for Atf3 in this process. Together these data indicate pleiotropic effects of TAM on dentate granule neurons, one involving MEK1/2 activation in immature neural cells (neuronal and glial progenitors), the other reliant upon Atf3 upregulation induced by ChEH inhibition in older adult-born neurons.
One point to take away from our studies in particular is that patients prescribed tamoxifen take the medication on a daily basis. We found that a single tamoxifen dose induced ATF3 expression, albeit transiently, in several populations of neurons. Our data show that ATF3 expression can be maintained in susceptible neurons with repeated daily dosing (Fig. 4), the downstream effects of which are unknown, but likely to be of biological and clinical significance.
TAM also induced Atf3 upregulation in sensory neurons, which was particularly marked in large proprioceptive NF200 positive cells in the trigeminal mesencephalic nucleus and in those projecting to Clarke’s column in the spinal cord, and to a much lesser extent in non-peptidergic IB4 positive neurons. It is tempting to speculate that large fibres have higher metabolic demands and might therefore be more vulnerable to cellular stress. The pattern of induction is certainly evocative of a large fibre neuropathy, which is known to accompany non-hormonal chemotherapy with platinum-based drugs  and has been linked to mitochondrial dysfunction. However, with the exception of rare optic neuropathies [32, 33], we can find no evidence in the literature that TAM treatment results in any sensory disturbances. This could be because Atf3 in the peripheral nervous system is associated with a robust reparative response after stressors such as axotomy. Atf3 is among the earliest genes to be upregulated after axonal injury , and is associated with successful regeneration: it is robustly induced in regeneration-competent sensory neurons by peripheral nerve injury  but not by injury to centrally-projecting dorsal roots . Furthermore, genetic overexpression of Atf3 in sensory neurons improves the regenerative response to peripheral nerve injury [37, 38].
Beyond its therapeutic use in breast cancer, TAM has been exploited experimentally to induce permanent reporter expression in, for example, newly-born neurons in the olfactory bulb and dentate gyrus , in physiologically-active neurons throughout the brain , and in progeny of ependymal cells of the intact and injured murine spinal cord . In each of these examples, the use of TAM to induce recombination may have profound consequences on interpretation of the data.
In the Imayoshi study , very large doses (400 mg/kg) were administered daily for 4 days, a regimen that was repeated twice more in some experiments. What is absent, however, both in anatomical and behavioural experiments assessing the role of newly-generated hippocampal neurons in memory formation/retention, is a TAM-only control in wild-type mice. Based on our results, TAM treatment would (repeatedly) induce cellular stress in older neurons, the effect of which on the neurogenic process remains unknown.
The Guenthner study  used a genetic labeling technique nearly identical to ours, but recombination was driven by promoters for the immediate-early genes Fos and Arc, upregulated as part of the “excitation-transcription” neuronal response to synaptic activity initiated by CREB phosphorylation . TAM-induced recombination in the dentate gyrus and olfactory bulb, particularly when recombination was dependent upon Arc expression, was remarkably similar to what we show here (Figs. 5 and 7), both in terms of number of neurons recombined and dorso-ventral distribution in the granular cell layer of the dentate gyrus. In these regions particularly it is unclear to what extent neuronal activity per se was driving recombination, or whether TAM induced Arc expression via cellular stress as it does expression of Atf3.
Our data show that ependymal cells are particularly prone to TAM-induced Atf3 upregulation. This may have important ramifications for the interpretation of a study by Barnabé-Heider et al. . Here, five daily treatments of TAM (80 mg/kg) were used to induce recombination in ependymal cells in FoxJ1-CreER mice in order to determine whether ependymal cells are multipotential in vitro, and in vivo following a spinal cord injury. The in vitro studies showed that 3 days after cessation of TAM treatment, recombined ependyma could indeed give rise to neurospheres with cells expressing markers for neurons, astrocytes and oligodendrocyte-lineage cells. In vivo, reporter-expressing ependyma self-renewed and produced astrocytes and oligodendrocyte-lineage cells 5 days following the last TAM dose. Again, what remains unknown is the effect that TAM-induced cellular stress has on the multipotentiality of ependymal cells, and thus on the interpretation of the results.
In all three of the above studies, TAM was delivered in corn oil, which has a vitamin E content of ~14.3 mg/100 g, compared to ~40 mg/100 g in SFO and ~150 mg/100 g in WGO. ChEH inhibition, which is complete even at lower (therapeutic) doses of TAM , is thus likely to have contributed significantly to the observed effects.
The ATF3-CreERT2 mice we have generated have allowed the first glimpse into the cellular stress pathway elicited in the central nervous system by TAM. They have revealed that TAM induces Atf3 expression in CNS and PNS neurons, vasculature and ependyma by interfering with cholesterol biosynthesis, an important issue when considering the use of ChEH inhibitors (such as DPPE) in the treatment of cancer. They also give cause for caution when interpreting experiments using the Cre-ERT2 system to manipulate gene expression. Our finding that vitamin E prevents TAM-induced cellular stress represents a solution to this previously unappreciated experimental confound.
L.M.R. was supported by a fellowship from the Canadian Institutes of Health Research. We would like to thank Prof. Fiona Watt at King’s College London for providing the tdtomato line, and Dr. Els Henckaerts and Dr. Nathalie Dutheil for their expert advice on Southern blotting. The Cre-ERT2 construct was kindly provided by Dr. P. Chambon from Université Louis Pasteur.
Compliance with ethical standards
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. Relevant organizations are the United Kingdom Home Office Animals (Scientific Procedures) Act 1986 (project licence 70/0793 to S.B.M), the University of British Columbia (certificate A13-0293 to M.S.R.) and the Canadian Council on Animal Care.
This work was supported by the Wellcome Trust UK (grant number PNWRAQR to S.B.M), and the Natural Science and Engineering Council of Canada (NSERC RGPIN 250355–1 to M.S.R.).
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