Highlighting Prenatally Damaged Neurons that Remain Vulnerable After Birth

Some 50 years ago, geneticist Ferruccio Ritossa noticed an unusual puffiness in the chromosomes of Drosophila that had been exposed to extreme heat. The phenomenon he described soon became known among scientists as the Heat-Shock Response. But this name, as it turns out, is overly specific: Numerous studies have now identified the Heat-Shock signaling pathway as a protective response not only to heat but to many kinds of physical and chemical stress.

The genetic mechanism for the Heat-Shock Response has been conserved throughout eons of evolution, from bacteria to humans. Now, researchers are taking advantage of the ubiquitous response to gain a closer look at some serious brain illnesses: Heat-Shock Factor 1 (HSF1) serves as the key ingredient in a new technique for detecting neurons in the developing brain that can increase the risk of neuropsychiatric disorders.

“It’s possible that some people are more susceptible to stress than others because of latent damage to their brain cells, and you cannot recognize these individuals in just everyday situations,” says Dana Alliance member Pasko Rakic, professor of both neuroscience and neurology at the Yale School of Medicine. Together with lead author Kazue Hashimoto-Torii, now a neuroscientist at the Children’s National Medical Center, and their co-authors, Rakic was able to identify cells with such risks to brain illness from the early stages through adulthood, which could make it possible to “rescue damaged cells before they become symptomatic.”

The technique has so far been used to study the brains of developing mouse embryos in proof-of-concept testing. The neurons being identified are ones that have suffered some environmental harm or injury during gestation and have survived, but are not restored to full health.

Severe illness or psychological trauma experienced by the mother during pregnancy, such as radiation, smoking, drug or alcohol abuse, or similar stresses, can take a subtle toll on neurons of the embryonic brain without interrupting the pregnancy in any obvious way. The child’s brain, to all appearances, then goes on to develop normally. But it bears traces of impairment that could increase that person’s susceptibility to a serious illness—autism, schizophrenia, or other debilitating condition—two or ten or even twenty years later, under the pressure of some major new stress.

This latent damage could help explain one of the perennial questions about human behavior: why some people break down under circumstances that others seem to bear with equanimity. The circumstances may not be traumatic in themselves—they may even be desirable, as, for instance, when a young person leaves home to attend college—but they present new challenges and may therefore expose an underlying deficiency or vulnerability that had come into existence during gestation and then remained for years without causing any consequences. In the example of new college students, the combination of an unfamiliar social landscape, campus logistics, and perhaps significant changes in sleep patterns may each take their toll; when to these is added the stress of midterm exams or a failed romance, a vulnerability to, say, depression or schizophrenia could emerge as an explicit illness.

Medical science has effective therapies for many a brain illness or mood disorder in children and adults, but the option of treating such a vulnerability at its source—the prenatal harm or injury—has been only a distant goal. In their recent online publication in Proceedings of the National Academy of Science, Hashimoto-Torii and her collaborators describe their technique for finding latently damaged neurons within the embryonic brain, which if it can be translated to humans would represent a big step toward reaching that goal.

They attach a fluorescent “reporter” gene to the HSF1 transcription pathway of mice pups that are then exposed in embryo to some form of physiological or chemical stress. After the mice are born, if they are exposed anew to stress, a fluorescent label is expressed along with heat-shock factor, which can be seen via microscope. This second stress could be something as simple as forcing the mice “to run too much,” says Rakic; nevertheless, it can trigger the stress response, allowing scientists to determine the exact location and level of HSF1 expressed in each mouse’s brain. The fluorescently labeled protein thus serves as a biomarker of latently damaged neurons that could then be targets of further investigation.

The authors first tested their reporter system on dishes of neuronal cultures prepared from embryonic cerebral cortex of mice that had been exposed in utero to ethanol or to heat shock. The results demonstrated the system’s efficacy, and its extreme sensitivity: In one in-vitro experiment, for example, neurons expressed the fluorescent reporter gene after being exposed to ethanol at concentrations as low as 5 mM. Moreover, the percentage of reporter+ neurons (those expressing the fluorescent protein) rose as the cells were exposed to increasingly high concentrations of ethanol. This could explain why offspring of alcoholic mothers react strongly even to small amounts of alcohol.

To test the system in vivo, Hashimoto-Torii and her co-authors exposed embryonic mice (through their mothers) to chemical and physiological stressors, then looked for reporter+ genes using an epifluorescence stereomicroscope (which exhibits the fluorescent protein in stereoscopic vision, to allow depth of view). By this means the researchers were able not only to locate the marked neurons but even, in some cases, to identify the specific nature of the damage, which included abnormal morphology and delays in the cells’ migration through the cerebral cortex. “We were surprised that the reporter expression was quite different [from one assay to another], depending on the type of damage done to the neurons,” says Hashimoto-Torii.

Following this line of investigation, researchers may look for the effects of other prenatal risk factors, such as nicotine. Mitigating or, ultimately, reversing prenatal damage to neurons from common environmental stressors is still a distant goal, but pinpointing both the site and the nature of such damage can offer valuable clues as to the brain functions that could be affected, whether in the realms of cognition, sensory processing, or social behavior.

The applications of cellular probes like the ones developed by Hashimoto-Torii and her colleagues, based on master transcription factors and using fluorescence labeling strategies, may ultimately extend far beyond neuropsychiatry. Mark Mehler, who chairs the department of neurology at Albert Einstein College of Medicine, would like to see similar tactics used against neurodegenerative illnesses as well.

Parkinson’s disease, Alzheimer’s disease, and similar maladies of the adult brain have generally been seen as diseases of aging—but what new insights might be gained by considering them developmental diseases (with a very long latent phase) instead? As with autism, bipolar disorder, or schizophrenia, says Mehler, “Basically we’re talking about a continuum of brain disorders that often occurs many years after the original genetic aberration or pathogenic insult takes place. What [Hashimoto-Torii et al.] have done is essentially allow us to identify the cells that are vulnerable in the embryonic brain and then track their molecular fate throughout life, which makes these tools incredibly valuable”—not only for understanding what causes certain brain diseases but also for coming up with new treatments.