Evaluation of the Impact of the Cancer Therapy Everolimus on the Central Nervous System in Mice

<div><p>Cancer and treatments may induce cognitive impairments in cancer patients, and the causal link between chemotherapy and cognitive dysfunctions was recently validated in animal models. New cancer targeted therapies have become widely used, and their impact on brain functions and quality of life needs to be explored. We evaluated the impact of everolimus, an anticancer agent targeting the mTOR pathway, on cognitive functions, cerebral metabolism, and hippocampal cell proliferation/vascular density in mice. Adult mice received everolimus daily for 2 weeks, and behavioral tests were performed from 1 week after the last treatment. Everolimus-treated mice displayed a marked reduction in weight gain from the last day of the treatment period. <i>Ex vivo</i> analysis showed altered cytochrome oxidase activity in selective cerebral regions involved in energy balance, food intake, reward, learning and memory modulation, sleep/wake cycle regulation, and arousal. Like chemotherapy, everolimus did not alter emotional reactivity, learning and memory performances, but in contrast to chemotherapy, did not affect behavioral flexibility or reactivity to novelty. <i>In vivo</i> hippocampal neural cell proliferation and vascular density were also unchanged after everolimus treatments. In conclusion, two weeks daily everolimus treatment at the clinical dose did not evoke alteration of cognitive performances evaluated in hippocampal- and prefrontal cortex-dependent tasks that would persist at one to four weeks after the end of the treatment completion. However, acute everolimus treatment caused selective CO modifications without altering the mTOR effector P70S6 kinase in cerebral regions involved in feeding behavior and/or the sleep/wake cycle, at least in part under control of the solitary nucleus and the parasubthalamic region of the hypothalamus. Thus, this area may represent a key target for everolimus-mediating peripheral modifications, which has been previously associated with symptoms such as weight loss and fatigue.</p></div

Cytochrome oxidase activity in cerebral areas of vehicle and everolimus-treated mice.

<p>Percentage of optical densities of cytochrome oxidase activity staining after everolimus treatment in the telencephalon, the diencephalon, the mesencephalon, the metencephalon, the myelecephalon and the cerebellum.</p><p>Data are means +SEM. (Student <i>t</i> test, *<i>p</i><.05). (a, area; ant, anterior; Diag, diagonal; horiz, horizontal; genic, geniculate; mesenc, mesencephalic; n, nucleus; ret, reticular; vert, vertical).</p><p>Cytochrome oxidase activity in cerebral areas of vehicle and everolimus-treated mice.</p

Effect of everolimus on precursor cell proliferation <i>in vivo</i> and <i>in vitro.</i>

<p>(A) Effect of <i>in vivo</i> administration of everolimus on neural precursor cell proliferation in the hippocampus. Left, BrdU-positive cells (intense green cells, white arrows) were counted in the subgranular zone (SGZ) and the remaining part of the dentate gyrus (outside SGZ). Bregma −2.16 mm; Scale bar, 100 µm. Right, Number of BrdU⁺ cells in the SGZ or outside SGZ (Mann whitney U tests, <i>p</i>>.05). (B and C) Effect of increasing concentrations of vehicle and everolimus on neural stem cell growth in culture. (B) <i>Upper</i>, Phase-contrast image of floating neurospheres at 24 hours after treatment with vehicle and everolimus (10⁻⁸ and 10⁻⁵ M). <i>Below</i>, Neurosphere volume in the absence (control: 0) and presence of vehicle and everolimus after 24 hours and 48 hours of treatment (Kruskal-Wallis ANOVA followed by Dunn's tests ***<i>p<.001 vs</i> 0; Mann Whitney U-tests, ^#<i>p<.05,</i>^##<i>p<.01</i> everolimus <i>vs</i> vehicle. Data represent means +SEM. (C) Proportion of neurospheres according to different categories of volumes. The increase of neurosphere mean volume might be explained by an increased proportion of bigger neurospheres and a decreased proportion of smaller neurospheres associated with everolimus treatment.</p

Spatial learning and memory, and behavioral flexibility of mice treated with vehicle or everolimus in the Morris water maze test.

<p>(A) Motivation and visuo-motor abilities after vehicle or everolimus treatment (Student <i>t</i> test, <i>p</i>>.05). (B and C) During the training and retrieval (R) phases, latency (B) and distance crossed (C) after vehicle or everolimus treatment (ANOVA, Day effect ***<i>p</i><.001). (D) During the probe test, time spent by animals of both groups in the quadrant where the platform was located during the training phase (χ², ***<i>p</i><.001 <i>vs</i> chance). During the transfer phase, escape latency (E), distance crossed (F), and swimming speed (G) in vehicle- or everolimus-treated mice (ANOVA, Treatment effect *<i>p</i><.05, Trial effect *** <i>p</i><.001 followed by LSD post hoc ^##<i>p</i><.01). (H) During the 1st day of the transfer phase, time spent in the previously correct northwest quadrant after vehicle or everolimus treatment (Student <i>t</i> test, <i>p</i>>.05). Data are means +SEM.</p

Impact of everolimus treatment on the mean body weight of mice.

<p>Body weight of vehicle- and everolimus-treated mice was evaluated at long term after the end of the treatment period (gray bar). Mice received vehicle or everolimus once a day during 14 continuous days. Bars represent standard error of the mean. ANOVA, Treatment x Day interaction <i>p</i><.001 followed by LSD post hoc: **<i>p</i><.01, ***<i>p</i><.001.</p

Schematic representation of brain areas presenting cytochrome oxidase activity modifications in mice treated with everolimus.

<p>Everolimus does not induce alteration of the phosphorylated P70S6K in the studied brain areas. It is here hypothesized that everolimus direct action on visceral organs would impact some cerebral areas through the vagus nerve. Among the brain areas with CO activity modifications are the solitary nucleus (Sol) as the principal recipient of visceral information conveyed by the vagus afferents and connected with the autonomic nervous system, motor nuclei of cranial nerves, nuclei in the brainstem and hypothalamus; the parasubthalamic nucleus (PSTh) within the hypothalamus, and motor trigeminal nucleus (MTN) involved in ingestion; the accumbens (Acc) shell important for reward and motivation processes; the preoptic area (LPO) regulating sleep/wake state; and the thalamic reuniens (Re) and rhomboid (Rh) nuclei and cortical areas that integrate arousal, somatosensory and visceral information, and participate to the modulation of learning and memory, and maintenance of arousal and wakefulness. Thus brain areas with modified CO activity are involved in the integration of autonomic and neuroendocrine information important for regulation of food intake, weight, metabolic, motivational processes, and arousal, that could be associated with symptoms suggestive of fatigue and energy homeostasis alterations. Arrows in purple show connections between brain areas metabolically modified. AH, anterior hypothalamic area; BBB, brain blood barrier; DM, dorsomedial hypothalamic nucleus; Ect, ectorhinal cortex; LH, lateral hypothalamic area; PH, posterior hypothalamic nucleus; PrL, prelimbic cortex; PVN, paraventricular hypothalamic nucleus; VM, ventromedial hypothalamic nucleus.</p

Vascular component density in hippocampus and endothelial cell proliferation <i>in vitro.</i>

<p>(A) Vascular density in the dentate gyrus of the hippocampus. <i>Upper</i>, IQGAP1-immunolabeled vascular niches in the dentate gyrus were illustrated from brain slices of vehicle- and everolimus-treated mice (Bregma −1.68 mm). Scale bar, 100 µm. <i>Below</i>, Mean IQGAP1-positive surface labeling and number of branches/nm² (+SEM) in the dentate gyrus (Mann-Whitney U-tests, <i>p</i>>.05). (B) Effects of increasing concentrations of vehicle and everolimus on the bEND.3 endothelial cell growth, as evaluated by endothelial cell number after 24 hours and 48 hours of treatment with vehicle or everolimus. Values are normalized to the mean number of cells in the control condition (white bar). Effect of everolimus, Kruskal-Wallis ANOVA followed by Dunn's tests *<i>p</i><.05, **<i>p</i><.01, ***<i>p</i><.001 <i>vs</i> control; Effect of everolimus compared with vehicle (Mann-Whitney tests, #<i>p</i><.05, ##<i>p</i><.01, ###<i>p</i><.001).</p

Co-localization of UT with γ subunits in neuron and glial components in rat cerebellum.

<p>(A, A′) Double-fluorescence staining for UT (green) and NeuN (red) showing the presence of UT in both mature (arrowhead, merge, A′) and unidentified cells (arrows, merge, A′) in the IGL. (B) Co-staining of UT and the marker of Purkinje cells, calbindin (red), in Purkinje cell soma and dendrites (arrowhead, B′). (C) Staining for UT and the marker of migrating neuroblasts doublecortin DCX (red) depicting a diffuse labeling in the ML. (C′) UT immunopositive fibers contiguous to DCX-expressing migrating granule cells (merge, yellow, arrowhead). (D, D′) Staining for UT and GFAP (red) in glial fibers (merge, yellow, arrowhead) of the ML. (E, F) Distribution of UT and the γ₁ (E) and γ₂ (F) GABA_AR subunits (red), in Purkinje cells (merge, arrowhead) and few extents of glia (merge, arrow) in the ML and IGL. Nuclei (blue) were counterstained with DAPI. Scale bars, 50 µm (A–F); 20 µm (A′–F′). EGL, external granule cell layer; IGL, internal granule cell layer; ML, molecular layer; PCL, Purkinje cell layer. (A′–F′) images of digitally zoomed regions corresponding to the white boxes in A–F.</p

Schematic model depicting the mechanism of UT-mediated GABA_AR down-regulation.

<p>UII efficiently activates the G protein-coupled receptor UT, leading to a fast short-term decrease of the chloride current not sustained by G proteins, calcium, phosphorylation and endocytosis processes. This rapid effect involves the distal 19 C-terminal amino acids of UT and the presence of γ subunits within of the GABA_AR complex (1). During the washout period, a long-term inhibition develops <i>via</i> a dynamin-, calcium- and phosphorylation-dependent endocytic mechanisms, requiring at least in part the 351–370 sequence of UT and GABA_AR γ subunits (2). It is hypothesized that the directional cross-talk between UT and GABA_AR, and the extinction of the latter at the plasma membrane, may relay transition from quiescent to proliferant astrocytes.</p

UII-induced GABA_AR loss from the plasma membrane through the C-terminus fragment of UT in CHO.

<p>The effect of <i>h</i>UII on the proportion of GABA_AR and UT at the cell surface of CHO was assessed by ELISA. (A) CHO transiently transfected with cDNA encoding UT^c-myc and α₂β₃, or α₂β₃γ₂^HA GABA_AR subunits (left), or UT^c-myc, and α₂β₃γ₂^HA GABA_AR subunits cotransfected with the cDNA encoding UT_319–389YFP (right). Background bioluminescence (left) and fluorescence (right) were measured after anti-HA antibody and colorimetric alkaline phophatase substrate incubation, in the absence or presence of 30 min of <i>h</i>UII (10⁻⁸ M, left), or directly on a fluorescent plate reader (right). (B) CHO transiently transfected with cDNA encoding UT^c-myc and α₂β₃γ₂^HA GABA_AR subunits (left), or cotransfected with the cDNA encoding UT_319–389YFP, and immunodetected with anti-HA (left) or anti-c-myc (right) antibodies. Percentage of cell surface γ₂^HA GABA_AR subunit (left) or UT^c-myc (right) are represented as the proportion of receptor at the plasma membrane (non permeabilized cells) to the total expressed receptor (permeabilized cells). One hundred percent correspond to values in the absence of 30 min treatment with <i>h</i>UII (10⁻⁸ M, 37°C). Each bar corresponds to mean ± SEM percent obtained from 5 to 7 independent experiments, in triplicates. ns, non significant; *, <i>P</i><0.05; ***, <i>P</i><0.001.</p