Making the Case

By Eric Sauter

In August, Shuji Kishi, an assistant professor in metabolism and aging, published what he called a "mini-review" of the use of zebrafish as a workable model for the study of biological and behavioral gerontology in humans.

Kishi has been working with the zebrafish for almost a decade and this is not his first published research. But it certainly is one of the most clearly articulated, laying out in clear and understandable terms just why the zebrafish may be the next big thing in animal models for aging and why the organism stands a decent chance of, if not replacing, then perhaps unsettling the ubiquitous mouse model.

Kishi, however, tends to see the overview in more practical terms.

"I think that this will help make people more aware of how we are using the zebrafish to study aging," he said. "Many people are using it extensively for developmental studies and some people now think of it as a great disease model, but a number of diseases come with age. So this is a timely review, and maybe it will make people more aware of these aging studies in fish."

Kishi's overview, published in the journal Gerontology ("Zebrafish as a Genetic Model in Biological and Behavioral Gerontology: Where Development Meets Aging in Vertebrates – A Mini-Review" (55:430–44, 2009), incorporates research and observations in the field, including his own research, which was carried out primarily at Harvard Medical School and now continues at Scripps Florida.

"Our recent studies demonstrated that this vertebrate is an excellent model in which to study age-dependent changes in musculoskeletal and eye morphology, endocrine factors, gene expression, circadian clock, sleep, and cognitive functions," Kishi wrote. "Importantly, we have also found that the presence of a senescence-associated biomarker (senescence-associated β-galactosidase) can be documented during early zebrafish development and is predictive of actual aging phenotypes later in adult life."

Cellular senescence plays a role controlling the lifespan of cells, which do not grow forever. When cells enter this later phase, there is increased activity of senescence-associated β-galactosidase (SA-β-gal), a critical biomarker.

Moreover, Kishi explained, the wide availability of mutant genes with identified aging phenotypes (physical characteristics) in zebrafish combined with an existing wealth of information about zebrafish development and genetics, and the existence of multiple strains, should "significantly facilitate the use of this outstanding vertebrate model in deciphering the mechanisms of aging, and in developing preventive and therapeutic strategies to prolong the productive life span in humans."

That sounds like a pretty heavy load to place on the back of a fish that is a member of the carp family, generally grows to a size no longer than your thumb, and lives around three years on average.

On the other hand, models of human aging such as worms, fruit flies, primates, and the ever popular mouse carry with them enough problems that finding an alternative makes the research potential of the humble zebrafish seem outstanding.

A Well Known Fish with Some Well Known Problems

Kishi concluded that one thing that makes the zebrafish a favorite of developmental biologists is that we already know so much about it, but perhaps more important is the fact that aging zebrafish – just like humans – develop problems that sound eerily familiar for anyone over the age of, say, 50.

"With age, they often display spinal curvature that is possibly related to muscle abnormalities," Kishi wrote. "In aging zebrafish, we detected senescence-associated β-galactosidase (SA-β-gal) activity in skin, and oxidized protein accumulation in muscle. Aged zebrafish also demonstrate increased accrual of lipofuscin ("aging pigment") in the liver similar to that reported in mice and humans. Intriguingly, by four years of age, the liver lipofuscin levels become markedly higher than those at one year of age. Aged fish also often develop lipofuscin accumulation and drusen-like lesions in retinal pigment epithelium, similar to that seen in age-related macular degeneration in humans. Moreover, almost all aged zebrafish develop cataracts by four years of age and the majority of old fish show retinal atrophy."

None of which makes the process of aging sound any better for fish than it does for humans. Aging zebrafish also start to look old as well; their tail fins start to crack, plus they have trouble sleeping and they have trouble with their memory. As they age, levels of melatonin begin to decrease relatively early in the zebrafish lifespan and there is a decline in the expression of core circadian clock genes, the genes that manage the rhythm of the lives of both fish and humans.

"For the biomarker (SA-?-gal) we are looking at the skin of the fish and this has also been confirmed in human biomarker of skin senescence," he said. "Even in the early development stage, this biomarker shows up with moderate stress (excessive stress causes cell death and sometimes organismal malformation or acute lethality) or certain specific gene mutations. This is consistent between fish and humans."

In terms of cognitive ability (analogous to lost car keys), zebrafish experience much the same thing, Kishi said. "Our study comparing young and middle-aged wildtype zebrafish and mutants with altered acetylcholinesteraze activity suggested that the cholinergic system is an important player in zebrafish aging and affects their cognitive performance," he wrote.

This is especially important in view of the role the cholinergic system plays in the deficiencies associated with Alzheimer's disease."

The cholinergic system involves the neurotransmitter acetylcholine; lower levels of acetylcholine have been implicated in Alzheimer's disease.

The Biomarkers of Zebrafish

The fact that zebrafish experience gradual aging over an average lifespan of three years (they can live as long as five years) makes them ideal for finding embryonic biomarkers that can predict an aging phenotype later in life – not to mention the fact that you can see right through them, so that any changes that do occur are transparent.

So far, studies have demonstrated that the same environmental challenges that can accelerate aging in adult zebrafish, such as oxidative stress, can also reveal biomarkers of aging during early development.

In fact, if the coupling of stress responses in zebrafish embryos and/or larvae with aging mechanisms in adult fish does hold true, this approach could turn out to be a very useful tool in the search for aging-related genes even in the young.

"Overall, we suggest that the complexity of aging in higher organisms, involving interaction of multiple genes and signaling pathways, warrants a comprehensive search for early predictors of altered aging phenotypes later in life," Kishi concluded. "The zebrafish animal model, with its well-established advantages of forward genetics and improved high-throughput technologies, offers an unparalleled opportunity to identify aging-related genes, and to analyze their function throughout life."

If the human-fish convergence needed anything more, Kishi and his former colleagues at Harvard are also developing an MRI scan for zebrafish.

"We're using a small animal MRI machine," he said. "Obviously they like to be in water, so we're trying several things – like putting the fish in wet paper towels and then putting them into the machine. Of course, you have to be very careful. You don't want them to die."

Send comments to: mikaono[at]scripps.edu

Assistant Professor Shuji Kishi reviews the literature on zebrafish as a model organism in gerontology.