The collective findings of a new multicenter Japanese study have demonstrated for the first time that expression of the gene Gem via the activity-dependent transcription factor, neuronal PAS domain protein 4 (Npas4), promotes neuroprotection in the injured brain following ischemic stroke.

Importantly, Gem was also shown to be induced in human cerebral organoids cultured under ischemic conditions, revealing Gem to be a new target for stroke drug discovery, the authors reported in the August 2, 2021, online edition of Proceedings of the National Academy of Sciences.

Ischemic stroke is a leading global cause of adult mortality and disability, for which there are no effective pharmacological treatments, "representing a major unmet medical need," said study leader Akio Tsuboi.

"Ischemia triggers pathologic events including excitotoxicity, oxidase stress, and apoptosis, causing cell damage and loss of neurological function," said the professor in the Laboratory for Cellular & Molecular Neurobiology of the Graduate School of Frontier Biosciences at Osaka University.

Given the brain's considerable energy needs, neurons are immediately depleted of energy by any cerebral blood flow impairment, resulting in loss of resting membrane potential and uncontrolled release of the neurotransmitter glutamate.

Lack of blood flow triggers spreading depolarization in neurons within the infarct area, increasing intracellular Ca2+ levels and inflammatory cytokine and growth factor production.

It also initiates transcription of ischemia-induced genes that can activate both neuroprotection and pathogenic cascades, which result in apoptotic or necrotic cell death.

Protection without pathology

Because both protective and pathological cascades are co-activated by ischemia, potentiating protective signaling may block ischemia's cytotoxic effects, but how that neuroprotection can be achieved is unclear.

In the healthy brain, sensory stimulation, notably an enriched environment (EE), induces neuronal Ca2+ transients and gene expression, leading to synaptic plasticity in learning and memory neurons.

Nuclear Ca2+/calmodulin signaling controls neuroprotective gene expression in healthy brain. So far, it is unclear how neuronal activity-regulated genes differ from ischemia-regulated genes in the post-stroke brain.

Moreover, it is uncertain whether inducing healthy brain neuronal activity-regulated genes affects post-stroke neuroprotection.

In their study, the authors profiled gene expression changes common to ischemia in mice, as modeled using middle cerebral artery occlusion (MCAO), and to experience-dependent activation, as reflected by exposure to an EE.

They discovered that Npas4 was upregulated under both MCAO and EE conditions and that transient activation of cortical neurons in healthy brain by the EE decreased cell death after stroke.

"Based on in situ hybridization of mouse cerebral cortex sections, we found that Npas4 was upregulated under both MCAO and EE conditions," Tsuboi told BioWorld Science.

"This revealed that even a short period of 40 minutes of neural activation with EE before stroke induced Npas4 expression, thereby facilitating neuronal protection from ischemic death."

Furthermore, both MCAO in vivo and oxygen-glucose deprivation (ODD) in vitro demonstrated that Npas4 was both necessary and sufficient for neuroprotection.

"Short exposure to EE was shown to be sufficient to decrease infarct volume by approximately 50% compared with controls, so even short exposure to EE induces Npas4 expression, facilitating neuronal protection from ischemic death after stroke," said Tsuboi.

This protection was demonstrated to involve inhibition of L-type voltage-gated Ca2+ channels (VGCCs).

"Based on loss-of-function in Npas4-knockout (KO) mice and the gain-of-function associated with AAV-[adeno-associated virus]-based Npas4 overexpression, we found that Npas4 was necessary and sufficient, respectively, for neuroprotection after stroke," explained Tsuboi.

"We utilized an in vitro model of ischemia in which primary cultured cortical neurons were incubated under ODD conditions," he noted.

"OGD treatment increased both Ca2+ influx and cell death in primary neurons, but these increases were inhibited significantly by either the L-type VGCC antagonist, nifedipine, or Npas4 overexpression before OGD treatment."

A systematic search for Npas4-downstream genes then identified Gem, which encodes a Ras-related small GTPase that mediates the neuroprotective effects of Npas4.

"We showed that Gem suppressed membrane localization of L-type VGCCs to inhibit excess Ca2+ influx in primary cultured neurons, thereby protecting them from excitotoxic death after in vitro ischemia," Tsuboi said.

"In vivo experiments, Gem overexpression using AAV vectors prior to MCAO significantly reduced the infarct volume by approximately one-third compared to controls."

In addition, "we found that expression of the human homologue of Gem (GEM) was increased in human cerebral organoids after OGD, suggesting Gem also plays a role in neuronal protection after ischemia in human brain and we are now assessing the effect of OGD on cell death in these organoids," said Tsuboi.

Together, these findings indicate that Gem expression via Npas4 promotes neuroprotection in injured brain and that Gem is induced in human cerebral organoids grown under ischemic conditions, revealing Gem as a new drug discovery target.

Looking ahead, "we are going to perform high-throughput screening of a chemical library to identify compounds that enhance Gem activity for neuroprotection, using primary cultured cortical neurons with OGD treatment," said Tsuboi.

"Here, we have shown that Gem, a newly identified downstream target of Npas4, protects neurons from ischemic death both in vitro and in vivo, and we now plan to investigate the mechanisms by which Npas4 directly or indirectly induces Gem expression after stroke (Takahashi, H. et al. Proc Natl Acad Sci U S A (PNAS) 2021, 118: e2018850118).