İskemik Hücre Ölümü Pathways Eğitim için seks Katmanlı Nöronal Kültürleri

Mammals are sexually dimorphic with males and females exhibiting different traits and characteristics. Sex differences extend even to the brain and are evident in neuronal susceptibility to injury and disease (reviewed in^1-5). For example, the male brain is more susceptible to ischemic neuronal injury from stroke or cardiac arrest followed by resuscitation (reviewed in⁶). Parkinson's disease (reviewed in⁷) and schizophrenia (reviewed in⁸) are more common in human males than in females, while the female human brain appears more susceptible to Alzheimer's disease (reviewed in⁹), and mood disorders such as depression (reviewed in¹⁰). Despite observable differences between sexes in susceptibility to neuronal injury and disease, most current treatment paradigms fail to consider sex and are similar in both men and women. Greater success might be achieved if sex differences were accounted for and understood.

In vivo animal models of neuronal injury and disease demonstrate sexual dimorphism similar to humans and are critical to enhancing our understanding of sex differences and how those differences contribute to neuronal survival and function. Evidence has demonstrated, for example, that male rodents are more susceptible than females to ischemic neuronal injury^11-15. This difference is thought to be at least partially dependent upon the molecular signaling of testosterone, the primary male sex steroid hormone, and estrogen, the primary female sex steroid hormone. Studies with gonectomized male and female rodents reveals that testosterone and the testosterone metabolite, dihydrotestosterone (DHT) exacerbate and estrogen decreases neuronal injury following cerebral ischemia^12-14,16-18. However, sex differences in cerebral ischemia are present in both neonate and adult animals^19,20. Neonates experience surges in estrogen or testosterone in utero during development, but have relatively low levels of hormone after birth until puberty (reviewed in²¹). Thus, the presence or absence of primary sex hormone is not the only determinant of sex-influenced neuronal susceptibility to injury. Indeed, it is likely that sexual dimorphisms are established during development that result in differences in cell signaling and response to ischemia even when hormone levels are low.

Regardless of whether sex differences are dependent on the presence of sex steroid hormone or whether they are due to dimorphisms established during development, it is becoming clear with further study that there are differences in cell signaling between male and females in the brain. In the Herson Lab at the University of Colorado School of Medicine, we have identified one such difference. We have found that the calcium, sodium, and potassium permeable ion channel, TRPM2 (reviewed in^22,23), induces neuronal cell death following ischemic insult in vivo in the male but not the female mouse²⁴. We demonstrate that pharmacological inhibition or genetic knockdown of TRPM2 is protective in the male but not the female mouse in our in vivo middle carotid arterial occlusion (MCAO) model of stroke²⁴.

Despite their usefulness, in vivo animal models of ischemic neuronal injury may, at times, be limited for molecular studies of neuronal death, due to the difficulty or inability to specifically target neurons for investigation. For this reason, our lab uses primary disassociated cortical or hippocampal neurons in vitro to complement our in vivo work. Primary disassociated neuron cultures present a useful tool to investigate the cell signaling pathways involved in neuronal injury and disease. Neurons in culture can be maintained in a tightly controlled environment, with or without glial input. In the absence of glia, any responses observed in culture are neuronal. Pharmacological agents can be administered to culture without concerns about absorption across the blood brain barrier, allowing for the inhibition or activation of specific components of a given signal transduction pathway. Molecular biology can be used to overexpress or knockdown various proteins of interest, and electrophysiological measurements can be conducted on individual cells, something that would not be possible in an in vivo model system.

The Herson Lab expands the usefulness of primary disassociated neuronal cultures by first sexing the embryos used to prepare the cultures. Male and female sex-stratified neuron cultures demonstrate clearly that male neurons are more susceptible than female neurons to neuronal cell death following oxygen and glucose deprivation²⁵. Oxygen and glucose deprivation is an accepted in vitro model of neuronal ischemia as ischemia reduces oxygen and glucose availability to the brain. Furthermore, we have demonstrated with electrophysiological experiments in disassociated, sex-stratified neurons that TRPM2 is activated in male but not female neurons^24,26. We are currently investigating the molecular sex-specific regulators of TRPM2 activity^24,26. Here, we share and discuss our protocol for establishing sex-stratified primary embryonic disassociated hippocampal neuron cultures and present representative data using this method that suggests that the DNA repair enzyme, PARP-1, contributes to ischemic cell death in a sex specific manner similar to TRPM2. PARP-1 is activated by DNA breakage due to oxidative stress (reviewed in^27,28). When DNA damage is minimal, PARP-1 activity enhances cell survival, but when damage is excessive, increased PARP-1 activity exacerbates cell death. PARP-1 produces poly-(ADP)ribose, a known activator of the TRPM2 channel^28-31, and some evidence suggests that PARP-1 is activated preferentially in the male but not female brain in response to oxidative stress^32,33. Thus, we hypothesize that PARP-1 activates TRPM2 in the male but not female, inducing neuronal cell death in response to oxygen and glucose deprivation.