Author: José R. Pineda got his Ph.D. from University of Barcelona in 2006. Since 2007 he has worked for Institut Curie and The French Alternative Energies and Atomic Energy Commission. Currently he is a researcher of the UPV/EHU. He investigates the role of stem cells in physiologic and pathologic conditions.
Neurogenesis is a process in which new neurons are generated. It is a process known to happen during the development and after birth in several neurogenic regions in humans including subventricular zone and hippocampus (this last, is a region of the brain key for learning, memory retrieval and spatial memory). However, things were less bright concerning adult and elderly people where an intense debate divided the scientific community between some authors that believed the existence of adult neurogenesis 1 and others that denied it 2 until recent findings demonstrating its existence 34. However, to gain knowledge by which mechanism neurogenesis in elder people could be improved, rodent animal models has been used extensively trying to settle basis for further comprehension and maybe extrapolation to superior mammals. One of the most evident “cause and consequence” of the boost of neurogenesis has been relied to physical exercise 5. However, the molecular mechanism were not completely known.
In a recent publication in the prestigious Cell Metabolism journal, Odette Leiter and collaborators describe one of the molecular mechanisms by which physical exercises increase of the generation of new neurons 6. They observed that exercise-induced systemic release of the antioxidant selenium transport protein called selenoprotein P (SEPP1). However, how did they come to this conclusion? Initially they firstly identified the proteins that were systemically released in response to exercise doing a proteomic screen on the blood plasma and comparing mice housed in standard cages or with a running wheel for 4 days. From 68 proteins with significant running-induced changes, SEPP1 was one of the most significantly upregulated proteins (more than twice the levels of controls). They verified the increase of protein levels using an ELISA-based approach (this technique measure the amount of protein using a specific union of antibodies anti-SEPP1 linked to a colorimetric value) detecting a significant increase of SEPP1 in plasma levels. Interestingly, SEPP1 is known to be the most important protein which function is to maintain selenium levels in the brain. SEPP1 transports selenium in blood and the cells in the blood-brain-barrier express its receptor known as “low-density lipoprotein preceptor-related protein 8 (LRP8)”.
To go straight to the point and decipher if selenium could have a direct impact on neural progenitor cells (NPCs) they decided to perform in vitro studies culturing NPCs in presence of sodium selenite (0.1-0.5M). They observed an increase in cell proliferation and neurosphere generation with increased size. To corroborate the results they decided to repeat the experiments using another form of selenium choosing seleno-L-methionine (an organic form) observing similar results.
In the hippocampus, the subgranular zone is the neurogenic niche where neural precursor cells are generated. However, it is known that in order to assure proper number of NPCs and the future efficient neural connectivity, more NPCs than required are produced. The surplus of cells that do not find their place to integrate suicide by “apoptosis” (programmed cell death) after two waves of cell selection. The first wave of cell selection occurs within the first 4 days, and the second wave occurs at 1-3 weeks after division. Odette and collaborators determined the proportion of death cells using a staining of Annexin V (this compound binds to the phosphatidylserine that flip in the outer leaflet plasma membrane when the cell is dying). She did not find any differences in the proportion of dead cells after selenite treatments, confirming that the compound is not toxic. On the contrary, using a cell viability assay (Resazurin) she found a small but significant increase in the number of viable cells. Furthermore, the induction of differentiation towards neuronal lineage of the NPCs was improved after treating the cells with both forms of selenium.
Next step, after demonstrate the direct effects on selenium in vitro, they moved to determine the effects in vivo. They infused sodium selenite (1 M) directly into the hippocampus for 7 days using micro-osmotic pumps implanted subcutaneously with a tube and microcanula inserted intracranially. To detect proliferating cells, administered BrdU (a thymidine analog that incorporates into the DNA during cell division) 2h prior to sacrifice. They observed how intra-hippocampal selenite administration increased 3-fold the number of proliferating cells in the neurogenic niche without observe an increase of cell death (as they previously observed in ex vivo studies). Then, after observe this huge increase of NPCs, they decided to repeat the experiment infusing selenite during more time (up to 28-days) and labeling the cells using the thymidine analog 5-chloro-20-deoxyuridine (CldU).
They found that selenium treatment resulted in a significant increase in net hippocampal neurogenesis in vivo, with more new neurons, identified by NeuN marker, with double positive staining for CldU+ and NeuN+ surviving 4 weeks after the start of selenium infusion. They corroborated the results administering CIdU prior the 7-day infusion of selenium and at the day 8 they labeled the cells using a secondary thymidine analog, 5-iodo-20-deoxyuridine (IdU). Then, they waited for 3 weeks, allowing that previously CIdU-marked NPCs that were exposed to the selenium treatment and differentiate into neurons. At the outcome of this experimental paradigm, all the CIdU positive neurons observed were the result of the selenium treatment. They found that selenium treatment resulted in approximately 50% more surviving CldU+ cells 3 weeks after the end of a 7-day treatment. Indeed, the number of proliferating NPCs increased by approximately 300%. Thanks to the use of both CIdU and IdU markers, they found that the majority of the newborn cells had differentiated into (NeuN+) neurons. Labeling CIdU NPC population with IdU after 7 days of selenium treatment and waiting the 3-week chase period during which the labeled cells could undergo additional divisions before becoming post-mitotic, they found a 6-fold increase in the number of surviving IdU+ cells. In conclusion, the results indicate that, in addition to its effect on the number of proliferating cells, selenium exerted an additional effect on newborn neuron survival.
One of the additional effects that could explain this increase of neurogenesis could be that physical activity increases the number of proliferating precursors by recruiting quiescent activatable stem cells into proliferation. To prove that, they used a dual thymidine analog (CldU and IdU) labeling paradigm after intraperitoneal injection of selenium. They observed a significant increase in the percentage of CldU-IdU+ cells illustrating that non-dividing cells prior to the selenium treatment (CIdU-) entered proliferation (IdU+) during the 24-h selenium treatment time window, confirming this hypothesis.
Because intracellular reactive oxygen species (ROS) precede the exercise induced activation of quiescent NPCs 7 they further investigated whether selenium could reduce the levels of intracellular ROS in NPCs linking the activation of the neurogenesis due to a selenite-induced ROS fluctuation. Odette and collaborators pretreated NPCs with selenite and measured ROS levels by flow cytometry, observing that cells with high ROS production reduced its levels, becoming the triggering event that lead NPCs activation.
However, one question remained to be resolved: could peripherally-administered selenium reach the brain? To draw this conclusion they used the paradigm of “loss of function” eliminating the expression of SEPP1 receptor known as LRP8. They found that the expression of the receptor was critical for the increase of the hippocampal adult neurogenesis observed after exercise. On the contrary, normal animals expressing SEPP1 were chronically treated with selenium in drinking water observing an increase of hippocampal neural precursor cells proliferation. Interestingly, it is known that selenium levels decrease during ageing 8. They also found dietary supplementation of selenium also could rescue the dramatic decrease in neurogenesis observed in old mice and to reverse cognitive deficits of spatial learning and memory characteristic of aged mice.
Selenium is a cheap, readily available dietary supplement that is found in a number of commonly eaten foods. The findings of how selenium metabolism is involved in mediating the exercise-induced increase in adult hippocampal neurogenesis illustrates the external capacity to regulate neurogenesis and provide new tools to facilitate the discovery of novel therapeutic interventions in both health and disease.
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