Makoto Asashima (AIST Fellow) of the National Institute of Advanced Industrial Science and Technology (AIST: President: Tamotsu Nomakuchi) and Tomoko Kuwabara (Researcher) of Stem Cell Differentiation Research Team, the Research Center for Stem Cell Engineering (Director: Makoto Asashima) of AIST, in collaboration with Hideaki Soya (Professor) and others of the Graduate School of Comprehensive Human Sciences, the University of Tsukuba, have identified, using rats and mice, the mechanism of the aging-related decline in neurogenesis, as well as Wnt3, a key factor in the mechanism, and its role.

Neural stem cells exist in the hippocampus, which is responsible for learning and memory, and new neurons are continuously generated even in the adult brain. The cells composing the bottom of the granule cell layer in the hippocampus are called astrocytes. They produce Wnt3 and have the important role of controlling the mechanism of generating diverse neuronal cells (neurogenesis).

With aging, the number of neural stem cells in the hippocampus decreases significantly, and, at the same time, the ability to generate diverse neuronal populations declines. The present study has shown that astrocytes supporting neural stem cells increase the production of Wnt3 in the aged brain with external stimuli such as physical activity, and as a result promote neurogenesis.

These findings will be published in FASEB Journal, a US scientific journal of experimental biology.

Figure : Wnt3 expression in adult hippocampus

New neurons are continuously generated in the hippocampus of the brain throughout the lifespan (neurogenesis). However, the frequency of neurogenesis decreases with aging, and varies significantly with the environment in which individuals live, including stress and diseases. In neurodegenerative diseases and mental disorders, such as Alzheimer’s disease, dementia, and depression, neurogenesis in the hippocampus declines more significantly. This indicates that neurogenesis in the hippocampus is regulated by a molecular mechanism that can easily vary with external stimuli and the biological environment in which individuals live. Studies have shown that some external stimuli promote the development of new neural networks in the hippocampus (for example, exercise and creating an enriched environment by providing toys) and some cause it to decline (for example, stress, diseases, and aging), and that the amount of gene expressions variously change in response to the stimuli.

The molecular mechanism linking aging to the cellular function of the brain must be understood in order to effectively develop treatments and drugs for neurodegenerative diseases and mental disorders, which concern many people today. To this end, it is important to understand the mechanism regulating neural stem cell differentiation, as well as to analyze the function and role of cells around stem cells. These research activities will lead to the discovery of novel treatments, and the identification of more effective targets for drug development.

AIST conducts research through analysis of neural stem cells in the brain, to help develop treatments and drugs for Alzheimer’s disease and depression. Hippocampal neurogenesis is known to be closely related to neurodegenerative diseases, such as Alzheimer’s disease and depression. However, the relationship between pathogenesis and the combination of molecules and cells is not well known. The Research Center for Stem Cell Engineering has discovered that Wnt3 produced by astrocytes initiates the adult neurogenesis; that is, Wnt3 activates, through signal transduction, NeuroD1 gene required for neural differentiation and retrotransposon genes responsible for the diversification of neurons (a research result announced on October 8, 2009).

In the previous study, the researchers clarified the basic mechanism of the adult neurogenesis. In this study, they investigated in detail how adult neural stem cells and surrounding cells in the brain are involved in phenomena such as diseases and aging in individuals. As part of this study, they conducted an experiment investigating the effect of physical activity on rats, in order to verify which molecule plays a key role in re-activating cells that have lost functionality. Research is underway at the University of Tsukuba to develop a technique for designing exercise that enhances the cognitive function of the brain. The Center analyzed the molecular basis of the enhancement of the cognitive function, using adult neural stem cells.

This study was supported by the Grants-in-Aid for Scientific Research (Young Scientists (B), Basic Research (A)) of the Japan Society for the Promotion of Science.

The researchers extracted and cultured neural stem cells from the hippocampus of old and young rats, and found that in an independent test environment in a test tube (in vitro condition), there is no significant difference in the proliferation potential and gene expression profile of neural stem cells. This is probably due to the effect of the artificial treatment using a proliferation factor in the process of establishing the neural stem cell cultures. This indicates that an externally added factor can bring even aged neural stem cells to the same state as that of young neural stem cells. In other words, it suggests the possibility that the intrinsic ability for neurogenesis of neural stem cells does not decline substantially with aging.

Astrocytes are also extracted and cultured from the hippocampus of old and young rats. In astrocytes extracted from the old rats, the Wnt3 production capacity significantly decreased to about one-thirtieth that of astrocytes extracted from the young rats (Fig. 1).

Figure 1 : Astrocytes extracted and cultured from the hippocampus of young rats (left), and those extracted and cultured from the hippocampus of old rats (right)

GFAP (green: antibody staining), an astrocyte marker, is expressed in both cases. Wnt3 (red: antibody staining) was significantly produced from the hippocampal astrocytes of young rats (left), but Wnt3 secretion from the hippocampal astrocytes of old rats is rarely observed (right).

In addition, when old rats were subjected to moderate exercise (running) for a short period of time, not causing stress, the Wnt3 production capacity of the hippocampal astrocytes increased significantly. It has been shown that as Wnt3 secretion increases, genes required for cell differentiation in Wnt3-receiving neural stem cells are activated, promoting neurogenesis; that is, the number of new neurons generated in the hippocampus increases.

The mechanism causing the diversity (acquisition of specificity) of neurons is involved in the process of generating new neurons in the hippocampus. In diverse neurons in the hippocampus, even identical neuron-specific genes had slightly different expression profiles. Controlling the amount of gene expressions in cells is called chromatin regulation. Chromatin regulation in hippocampal neurons has unique characteristics, different from those of other cells. Retrotransposons contained in the non-coding region of genomes are activated near various genes, resulting in the difference in chromatin regulation and the diversity of neurons. The researchers investigated how the mechanism regulating the expression of various genes changes when stimuli such as exercise (running) are applied to individuals. The investigation has shown that the chromatin-regulated state of retrotransposons in newborn neurons is sensitively dependent on the amount of Wnt3 produced by astrocytes (Fig. 2).

Figure 2 : The factor transmitting information on individuals to neural stem cells (Wnt3: orange)

The regulatory mechanism of the target genes expression itself in neural stem cells responsible for the differentiation for the neuronal diversity remain unchanged, but the Wnt3 productivity of astrocytes is largely dependent on the condition of individuals.

The primary cause of the age-related decrease in the number of newborn neurons produced in the brain has been thought to be the decrease in the number of neural stem cells from which nerves are generated. In this study, the researchers have shown that there is a factor in astrocytes, but not in neural stem cells, which significantly affects neurogenesis, and this factor plays a role leading to the rejuvenation of neural stem cells. They have identified a molecular mechanism that turns on and off a wide range of genomic responses according to changes in external stimuli (such as physical activity) that promote neurogenesis and conditions (such as aging or diseases) that inhibit neurogenesis. They believe that they have acquired knowledge that provides the basis for new approaches to the development of drugs and treatments for neurological and mental disorders.

They will further analyze the role of cells surrounding neural stem cells, which affect aging and various neurological disorders, to search for a new molecular marker detection method useful for diagnosis. They will develop industrial applications of the findings, including the development of drugs and new medical technology that promote the activation of stem cell-supporting cell populations without manipulating stem cells.