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fundamental scientific principle held for the past 100 years is that children
are born with all the neurons they will ever have. New discoveries in
stem cell biology and developmental neuroscience are challenging this
idea. Because stem cells are self-renewing and unspecialized, they can
generate many different cell types. Neuronal stem cells are found within
germinal centers in the adult brain such as the subventricular zone and
the dentate gyrus of the hippocampus. Scientists have discovered how to
harvest these cells from early embryos or the adult CNS for further cultivation
in vitro. A variety of genetic, environmental, and molecular factors can
then be studied to determine whether they influence the types of cells
produced and whether they grow into neurons, heart, muscle, or other cell
types. At present, many of the critical factors that regulate their eventual
fates are poorly understood. However, new evidence suggests that genomic
repair is one of the critical determinants for maintaining stem cells
and mature neurons throughout life. This column reviews studies of the
role of genomic repair in embryonic and stem cell neurogenesis and differentiation.
Although
the interplay between genes and the environment is well established, recent
research has found that physical activity can have a striking effect on
neurogenesis, neuronal survival, and differentiation in the mature brain.
In several studies, voluntary exercise on a running wheel was found to
stimulate proliferation of neuronal stem cells in adult rodent dentate
gyrus. Physical activity may increase the production of growth factors
and other molecules important for neural development. Combinations of
several different growth factors have been used successfully to generate
dopamine- or serotonin-producing neurons from embryonic stem cells in
tissue culture. Once these cells have been propagated and partially differentiated,
they may be transplanted into the brain for further study. This type of
experiment has been successfully used to treat mice with neurodegenerative
disorders of the spinal cord and other brain regions.
Scientists
have also made the startling discovery that stem cells are present in
the adult body that have the capacity to differentiate into a range of
cell types, including blood cells or neurons. Stem cells are not committed
to a specific function, as are most cells of the body such as neurons,
heart cells, and skin cells. The stem cells present in the body have the
unique ability to renew themselves. Beds of neural stem cells reside in
the adult brain and can be stimulated to grow in the subventricular zone
and dentate gyrus. Harvesting these cells and learning how to propagate
them in tissue culture is currently one of the hot test topics of both
basic and clinical research because of its potential application in repairing
brain injury. Propagating adult neurons from neural progenitors or embryonic
stem cells in vitro is now feasible, but much work remains to identify
the exact requirements for producing all of the many neuronal cell types
present in the adult brain.
Studies
are beginning to show that molecules involved in detecting or repairing
DNA or chromosome damage also play a critical role in the process of stem
cell differentiation and survival (Fig.
1). DNA molecules consist of paired strands of ribonucleotides that
are the basis for the genetic code. DNA damage is a serious threat to
the integrity of genetic information within the cell. Nicks or single-strand
DNA breaks occur commonly and are rapidly detected and repaired by nuclear
repair machinery using the intact DNA strand as a template. Cells may
also experience a more severe form of DNA damage involving breakage of
both DNA strands. These double-strand breaks are extremely dangerous for
the cell because they can result in severe chromosomal abnormalities,
including translocations and gene amplifications.
Repairing
this sort of chromosomal damage, called nonhomologous end-joining, tends
to be error-prone because it does not require homologous sequences for
rejoining the broken DNA ends. It is a kind of last-ditch effort to repair
DNA breaks that occasionally occur in all cells of the body and plays
an essential role in suppressing chromosomal aberrations. Apart from this
general role, nonhomologous end-joining serves a specific role during
maturation of immune cells by creating genetic diversity within immunoglobulin
genes. These normal genetic rearrangements are required for the development
of humoral and cellular immunity. Loss of this form of DNA processing
creates severe combined immune deficiency in which an affected child is
unable to produce antibodies required to fight infections.
In
nonhomologous end-joining, DNA breaks are repaired in a step-wise manner.
This process uses several different proteins including DNA ligase 4, X-ray
repair cross-complementation factor 4, and DNA-dependent protein kinase
(DNA-PK). Three subunits called Ku70, Ku80, and DNA-PK catalytic subunit
comprise the DNA-PK enzyme. DNA double-strand breaks are first detected
by Ku70 and Ku80, which bind to the broken DNA and recruit the DNA-PK
catalytic subunit to the damage site. The final step in nonhomologous
end-joining is the ligation of the broken DNA strands involving DNA ligase
4 and X-ray repair cross-complementation factor 4.
Recent
studies in embryonic knockout mice revealed that genes involved in nonhomologous
end-joining are required for proper neurogenesis at early stages of brain
development. Deficits in nonhomologous end-joining proteins resulted in
massive programmed cell death or apoptosis in the embryonic brain, and
in some cases the cellular loss was sufficient to cause lethality. Normally,
newly born neurons migrate away from where they are born in the ventricular
and subventricular zones and begin to extend axons and dendrites. However,
in mice deficient for components of nonhomologous end-joining, many of
the young neurons begin to undergo apoptosis before they differentiate.
The number of apoptotic neurons is extremely high in DNA ligase 4 and
X-ray repair cross-complementation factor 4 knockout embryos, and these
embryos die during gestation. Ku70/80 knockout embryos have improved chances
for survival, but they exhibit significant neurodegeneration at embryonic
and postnatal ages, exhibit dwarfism, and die before reaching adulthood.
The mildest phenotype is associated with mutations or deletions in the
catalytic subunit of DNA-PK, which can result in elevated DNA double-strand
breaks and increased neurodegeneration, but normal survival rates.
Many
questions remain about the triggers for apoptosis in these and other DNA
repair–deficient conditions. Nonhomologous end-joining deficits
appear to cause apoptosis of embryonic neurons because of an inability
to repair DNA breaks. Therefore, what is the source of DNA strand breaks
in embryonic neurons, and do these breaks also appear in differentiating
stem cells? Besides external sources such as radiation, DNA damage can
result from a high rate of cellular metabolism leading to increased levels
of reactive oxygen species that in turn can generate DNA breaks. Such
damage is thought to arise during proliferation or periods of high synaptic
activity. In actively dividing progenitor cells, DNA breaks would passively
accumulate because of suppression of cell cycle checkpoints, which are
the intervals when cells normally perform DNA repairs. Once a neuronal
progenitor leaves the cell cycle and becomes postmitotic, DNA breaks are
detected and repaired continuously. DNA strand breaks in immature neurons
could also originate from genetic rearrangements similar to those known
to occur in developing immune cells during immunoglobulin gene rearrangements,
as this process also requires nonhomologous end-joining proteins. Neuron-specific
multigene families are one of the potential candidates for genetic recombination
in neurons, but attempts to detect any type of rearrangements within these
gene families have failed.
A
growing number of genome caretakers are being found that are essential
for differentiation and survival of both stem cells and immature neurons.
Recently it was shown that telomerase, an enzyme important for protecting
the outer arms of chromosomes, is also critical for embryonic neuronal
survival. Other genomic caretaker genes have been linked to neurodegenerative
disorders of childhood such as xeroderma pigmentosum and trichothiodystrophy.
Furthermore, older patients with amyotrophic lateral sclerosis, Parkinson
disease, and Alzheimer disease show reduced DNA repair and hypersensitivity
to DNA-damaging agents. Taken together, these findings suggest a relationship
between DNA repair deficits and neurodegeneration.
Evidence
that neural progenitors and embryonic neurons sustain high levels of endogenous
DNA breaks has been found in some strains of knockout mice with deficiencies
in DNA repair genes. Our studies of the embryonic brains of these mice
found increased activity of caspases, a family of enzymes that act as
cellular executioners during apoptosis. At birth, the brains of these
mice have fewer differentiated neurons and more neural progenitors compared
with wild-type mice. As young adults, when these mice undergo seizures,
hippocampal neuronal loss is more substantial compared with wild-type
mice of the same strain. Moreover, defective neurogenesis is found in
murine embryos deficient for other forms of DNA repair. Taken together,
the current evidence suggests that many different types of DNA damage
are occurring during neurogenesis and neural differentiation. The range
of DNA defects found in dividing precursors and differentiating neurons
contradicts the notion that neural development requires a specific form
of genetic rearrangement.
What
then causes developing neurons and stem cells to be so sensitive to DNA
repair deficits? One reason may be that during very rapid proliferation,
there is not enough time for a cell to repair its DNA damage. This might
create a higher requirement for genomic surveillance and DNA repair compared
with when the cell ceases dividing. Another important question was recently
addressed: What allows the cell to sense when DNA breaks reach a critical
level? A nuclear DNA damage sensor protein called ATM is responsible for
sending out a signal when DNA breaks occur. ATM signals to a molecule
called p53, which in turn initiates a chain reaction culminating in cell
death in an effort to clear away cells with damaged chromosomes. In mice
with combined knockouts of nonhomologous end-joining proteins and either
ATM or p53, the DNA damage signals to the cell are disrupted, allowing
embryonic neurons to escape apoptosis despite having DNA damage. These
observations have shed light on the pathogenesis of a childhood disease
called ataxia telangiectasia, which is characterized by cancer and neurodegeneration.
Mutations in ATM have been found in children affected with this devastating
disease. Moreover, mutations of p53 are the most common defect present
in many adult human cancers. Once again, mutations in a key gene involved
in monitoring DNA breaks is responsible for disease, as cells without
the ability to undergo apoptosis are able to survive despite their DNA
damage and proceed to proliferate abnormally.
In
light of these findings, it is of great interest to know whether DNA caretakers
regulate long-term survival of stem cells and neural progenitors. Recent
experiments have suggested that ATM-deficient mice also exhibit stem cell
deficits. When the brains of ATM-deficient mice were carefully examined
in regions such as the dentate gyrus of the hippocampus, researchers did
not see the normally observed increase in neurogenesis that is associated
with voluntary running. ATM deficits reduce exercise-dependent survival
and differentiation of newly generated cells in the adult dentate gyrus.
Furthermore, in vitro studies show that ATM-deficient stem cells are unable
to undergo the full range of differentiation into multiple cell types.
Wild-type adult neuronal stem cells located in the subventricular zone
of the cerebral cortex have the capacity to generate neurons, oligodendrocytes,
and astrocytes. By contrast, ATM-deficient neural stem cells generate
only astrocytes. Like ATM, another enzyme called telomerase is expressed
at high levels in dividing neuronal progenitors and immature neurons,
but decreases as neurons differentiate. Telomerase prevents chromosomal
shortening by adding six-base pair repeats to the chromosomal ends called
telomeres. As telomerase levels decrease in both neuronal and non-neuronal
cells, rates of proliferation decline because of telomere shortening.
Low telomerase levels may account for why transplants of human neuronal
precursors cell are often characterized by a progressive decrease in proliferation.
What
happens to other aspects of neural differentiation and maturation when
DNA integrity is compromised by DNA repair deficits? Random alterations
in the genome lead to genetic mutations in important secreted signaling
molecules or cell surface receptors that reduce the ability of a young
neuron to respond to signals in its environment. Delayed or inappropriate
responses to environmental signals would disrupt migration, synapse formation,
or activity-dependent differentiation. Perhaps milder DNA repair deficits
are a contributing factor in the spectrum of human developmental disorders,
including mental retardation or other cognitive disorders.
There
are still many uncertainties about how, when, and where DNA repair serves
a critical role in neuronal survival. It is clear, however, that the stem
cell field will be fertile ground for answering these and other questions.
A better understanding of the requirements for DNA repair in stem cells,
neural progenitors, and immature neurons may also aid development of strategies
for harvesting and transplanting neural progenitors to treat neurological
disorders.
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