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Adult neural stem cell cycling in vivo requires thyroid hormone and its alpha receptor

UMR CNRS 5166, Evolution des Régulations Endocriniennes, Muséum National d’Histoire Naturelle, Paris, France; and
^* UMR CNRS 5665, Laboratoire de Biologie Moléculaire et Cellulaire, Ecole Normale Supérieure de Lyon, Lyon, France

¹ Correspondence: 7 rue cuvier, Paris 75005, France. E-mail: demeneix{at}mnhn.fr

SPECIFIC AIMS

Neural stem cells (NSC) are found in the subventricular zone (SVZ) of the adult mammalian brain. Understanding the genetic regulation of neural stem cell maintenance, division, and differentiation is a central problem in neural stem cell biology. Few studies have addressed endocrine control of NSC function, particularly in vivo. Thyroid hormones (TH) are essential for brain development in all vertebrates. Given the fragmentary information on whether and how this key endocrine factor affects adult neurogenesis, we analyzed TH effects on mitosis and apoptosis in the SVZ, as well as migration of neuroblasts out of the stem cell niche.

PRINCIPAL FINDINGS

1. Hypothyroidism reduces neural stem cell proliferation
We modified the thyroid status of adult mice and examined DNA synthesis and proliferation in the SVZ. We found that in hypothyroid animals, BrdU labeling, once acquired, is not diluted by successive cell divisions, indicating that cells either are not completing the cell cycle and/or not undergoing further divisions. We therefore examined the actual mitotic state of the cells.

Proliferation was quantified using the active cell cycle marker Ki67, which labels cells in G1, S, G2, and M phases and phosphorylated histone H3 (PhosH3). PhosH3 increases in G2; it is maximal in M phase and rapidly lost as cells enter G1. Hypothyroidism significantly reduced PhosH3-positive cells (Fig. 1 A, P<0.001) and halved the number of Ki67-positive cells in the SVZ (Fig. 1B ).

View larger version (39K): [in this window] [in a new window]   Figure 1. Hypothyroidism reduces the number of mitotic cells in the SVZ of adult mouse brain: in hypothyroid mice, BrdU-positive cells do not re-enter the cell cycle. A) Hypothyroidism reduces numbers of phosH3-positive cells compared with control or hyperthyroid animals (***P<0.001). B) Hypothyroidism halves Ki67-positive cells compared with controls or hyperthyroid animals (***P<0.001). Each experiment was repeated 3 times with similar results. C) Immunohistology of BrdU-positive cells (red), Ki67 (green), and double labeling (merged in yellow) in the ventricular region of untreated (upper panels) or hypothyroid (lower panels) adult mouse brains. D) Hypothyroidism increases the ratio of BrdU-positive/Ki67-negative cells after a 3 day BrdU pulse compared with controls. Cells were counted from 0.7 to 1.2 mm anterior to bregma for each animal using dichroic microscopy.

With the data on proliferation, we infer that the increased BrdU labeling seen in hypothyroid animals is indicative of an increased number of cells that have synthesized DNA but have exited the cell cycle. We performed double labeling between Ki67 (actively cycling cells) and BrdU incorporation after a 3 day BrdU treatment (Fig. 1C ). The proportion of cells that had synthesized DNA but not re-entered the cell cycle increased in hypothyroid mice, indicating that the absence of TH results in accumulation of resting progenitors in the SVZ (Fig. 1D ).

2. Hypothyroidism limits neural stem cell migration
To test whether TH affects the migratory capacity of NSC progeny, we used an in vivo gene transfer protocol that specifically labels NSC by expression of an exogenous reporter gene. This technique exploits polyethylenimine (PEI), which targets plasmid DNA to the SVZ stem cell and neuroprogenitor population in vivo. Stereotaxic delivery of PEI condensed DNA encoding lac-Z to the SVZ resulted in ß-galactosidase expression labeling of cells migrating from the SVZ in the rostral migratory stream toward the olfactory bulbs. Hypothyroidism halved (P<0.05) the numbers of lac-Z-expressing cells migrating in the RMS.

Most progenitors arising from NSC division in the SVZ undergo apoptosis. We followed activated caspase 3 and found that hypothyroidism halved (P<0.05) the number of capsase 3-positive cells in the SVZ. Reduction of apoptosis could also contribute to increased numbers of BrdU-labeled cells accumulating in the SVZ during hypothyroidism.

3. TH regulates of neural stem cell proliferation through the receptor
TH regulates gene transcription through TH receptors (TR), transcription factors belonging to the steroid hormone/retinoic acid receptor superfamily. In vertebrates, two genes encode TRs TR and TRß. Immunocytochemistry with TR-specific antibodies showed TR1, but not TRß, to be expressed in the nestin-expressing cells of the SVZ (Fig. 2 A, B). TR^0/0 and TRß^–/– mice lack all isoforms produced from the TR or TRß loci, respectively. We used ß-galactosidase as a marker for TR-expressing cells in TR mutant mice (Fig. 2C, D ) and homozygous null mice (Fig. 2E, F ). ß-Galactosidase staining reflecting expression from the TR promoter was strong in the SVZ of TR mutant mice (Fig. 2C, E, G ). In contrast, in TRß mutant mice, no ß-galactosidase staining could be detected, suggesting low or undetectable levels of TRß expression in this area (Fig. 2D, F, H ). ß-Galactosidase staining was carried out with nestin immunochemistry to label the SVZ neural stem cells and progenitor cells in homozygous animals. Nestin expression in the TR^0/0 mice was markedly reduced (compare Fig. 2I, J ), differing from the usual pattern recapitulated in TRß^–/– mice, where nestin-positive extensions reach out perpendicularly from the SVZ toward the striatum.

View larger version (77K): [in this window] [in a new window]   Figure 2. TR, but not TRß, is expressed and colocalized with nestin in the SVZ and is required for normal progression of NSC through mitosis. A, B) Deconvolution microscopy and immunocytochemistry on wild-type mice SVZ reveal specifically TR1 (A) and TRß (B). TR (green), nestin (red), nuclei (blue). Bar: 20 µm. C–F) ß-Galactosidase histochemistry (vibratome sections) reveals TR (C, E) or TRß (D, F)-expressing nuclei in mutant heterozygous mice: TR+/0 (C), TRß+/– (D), homozygous mice TR0/0 (E), and TRß–/– (F). Bar: 200 µm. G, H) Semithin sections (1 µm) of the SVZ of TR+/o and TRß+/– mutant mice. Positive ß-galactosidase cells are observed in the subependymal layer of TR heterozygous mice (G); TRß expression in TRß heterozygous mice is not found in the subependymal layer, but more deeply in the striatum (H). Bar: 6 µm. I, J) Nestin immunoreactivity and ß-galactosidase staining in TR null mice. Note that the nestin expression pattern is reduced and limited to the cell bodies in TR0/0 mice, but not in TRß–/–, where typical nestin-positive, long extensions are found (arrows). Bar: 20 µm. K, L) BrdU immunochemistry carried out after a 3 day BrdU treatment on TR0/0 mice shows that the absence of TR (L) increases DNA synthesis compared with wild-type controls (K). Bar: 150 µm. M) Quantification of the number of BrdU labeled cells shows a significant increase in TR0/0 mice (P<0.001). N) PhosH3 immunochemistry shows that absence of TR results in a significant (P>0.001) decrease in the number of mitotic cells in the SVZ.

Given the specific expression of TR in the SVZ, we followed DNA synthesis and mitosis in TR^0/0 mice maintained under euthyroid conditions. BrdU accumulation during a 3 day treatment was significantly increased in TR^0/0 mice vs. wild-type counterparts (Fig. 2 K–M). Inversely, PhosH3-positive cells were halved in TR^0/0 mice (Fig. 2N ). Thus, TR absence inhibits progression of NSC through the cell cycle, demonstrating a role for TR in mediating TH effects on NSC cycling.

4. TH directly regulates gene expression and proliferation in neural stem cells population
Expression of the TR gene in the SVZ strongly argues for a direct effect of TH on transcriptional regulation in these stem cells. To assess this, we used PEI mediated gene delivery to introduce into the stem cell population a construction containing 1.6kb of the mouse c-myc promoter cloned upstream of the luciferase gene. Hypothyroidism doubled transcription from the c-myc promoter whereas transcription was repressed in animals treated with T₃ for 2 wk with expression levels not different from controls. A short T₃ exposure (36 h) experimental paradigm significantly reduced transcription from the c-myc promoter in vivo. This TH effect was direct as it was lost if the TRE in the c-myc promoter was mutated.

These findings suggest a direct action of TH on stem cell activity and should implicate a rapid response to TH. To estimate the delay of action of TH on proliferation, we again used a BrdU incorporation protocol. A 2 h pulse of BrdU, was applied to control or hypothyroid mice injected with saline or T₃ solution for 10 h before sacrifice (8 h before BrdU treatment). With this short pulse of BrdU, which only labels cells in S phase, we observed significantly more positive cells in euthyroid mice than in hypothyroid mice. The rapid treatment with T₃ fully restored the number of cells in S phase to the euthyroid levels, again arguing for a direct effect of TH on proliferation.

CONCLUSIONS

The findings show that TR and TH are required to maintain full proliferative potential of neural stem cells in the SVZ and that the effects of TH on proliferation are direct.

First, we show that lack of TH reduces mitotic figures in the SVZ and that a short pulse of TH revives the process. This rapid effect of TH on mitotic activity within hours in the SVZ suggests that TH is acting directly on the stem cell population.

Second, TR1 is expressed in the subventricular zone. Use of mutant mice lacking the TR receptor showed that the absence of this receptor reproduces the effect of hypothyroidism on mitotic capacity of SVZ neural stem cells. We deduce that TR is necessary for adult neurogenesis. These results corroborate earlier data showing a role for TR in regulating mitosis in chick embryo neuroblasts and can be linked to the finding that an EST similar to TR was found in a genomic expression profile of NSC. Another intriguing finding in the TR^0/0 mice is disruption of nestin expression patterns, observed by Morte and collaborators using another line of mutant mice lacking TR1. Apoptosis, the major fate of neural stem cells’ progeny in the SVZ and probably closely linked to cell cycle to balance proliferation, is affected by hypothyroidism. Taken together, these results indicate that not only proliferation but the entire organization of the stem cell niche is affected by a lack of TH.

A third line of argument for a direct role of TH on neural stem cells biology comes from the finding that TH directly regulates transcription from a c-myc promoter sequence delivered to this population. Given that c-myc is known to be a T₃-dependent gene and that c-myc regulation is related to cell cycle control, it is tempting to speculate that endogenous c-myc may be a key target gene relaying the effects of T₃ on neural progenitor cycle.

These data suggest that, besides the well established multiple roles of TH in early neurogenesis, TH is an essential component of the endocrine environment that activates neural stem cell growth, migration, and apoptosis. Recent work identifies neural stem cells in the adult human brain, which remain relatively quiescent in vivo, suggesting the importance of the cellular environment to activate stem cells. Endocrine factors such as TH could be key factors to reveal regenerative potential of endogenous or grafted stem cells.

These findings could have far-reaching clinical implications particularly in an aging population prone to hypothyroidism. Indeed, hypothyroidism is associated with the loss of a sense of smell in rodents and, more important, in humans with cognitive decline and depression. As active neurogenesis is required in order to see the behavioral effects of anti-depressants, it is tempting to speculate that the link between hypothyroidism and depression could implicate reduced neurogenesis.

View larger version (26K): [in this window] [in a new window]   Figure 3. Schematic diagram of adult neurogenic process. Lack of T3 or its receptor strongly reduces proliferation, apoptosis, and migration of neuronal progenitors, suggesting that thyroid hormone signaling regulates the production of new cells by subventricular NSCs at multiple levels.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2916fje;

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This Article Full Text (PDF) All Versions of this Article: 19/7/863 04-2916fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lemkine, G. F. Articles by Demeneix, B. A. Search for Related Content PubMed PubMed Citation Articles by Lemkine, G. F. Articles by Demeneix, B. A.