Long-Term Iron Buildup Weakens Brain Cell Defenses Against Stress

Iron is an essential element for brain functions like neuronal development, yet too much of it can be harmful. For decades, scientists studying aging and neurodegenerative diseases have observed iron buildup in certain regions of the brain.¹ Such iron accumulation can trigger ferroptosis, a form of programmed cell death.

While researchers have obtained insights into the mechanisms underlying ferroptosis, these have largely come from studying acute conditions, which do not reflect the chronic stress that neurons encounter in most neurodegenerative diseases.² “If the diseases are progressive that means that if [iron] is accumulating over a period of time, then we need to have a cellular model in the lab that could at least mimic those changes,” said Nawab John Dar, a postdoctoral researcher in biochemist Pamela Maher’s lab at the Salk Institute for Biological Studies.

Now, Dar, Maher, and their team have found that chronic iron overload in nerve cells renders the cells hypersensitive to oxidative stress through a process they named “chronoferroptosis.”³ Their findings, published in Cell Death Discovery, indicate the role of iron accumulation in neurodegenerative diseases and underscore the importance of modeling the progressive nature of such conditions.

“It's a really nice paper,” said Ashley Bush, a neuroscientist who studies metals in neurodegenerative diseases at The Florey Institute of Neuroscience & Mental Health, who was not associated with the study. He added that scientists in the field have noted for many years that while they study iron accumulation in progressive diseases, their experiments cover shorter time frames. “This was a study that was long overdue… We should have been looking at this for a while.”

To compare how short- and long-term iron overload affects neurons, Dar, Maher, and their team exposed a human-derived nerve cell line to ferrous sulfate for either six to eight hours or nine days. Unlike acute exposure, chronic exposure to iron at low concentrations induced a spike in the levels of ferritin, an iron-storing protein, indicating that sustained iron challenge triggers an adaptive response.

Since redox reactions also govern ferroptosis, the researchers investigated how chronic iron stress affects antioxidant enzymes. Sustained iron exposure reduced the levels of glutathione peroxidase (GPX4), a vital antioxidant enzyme, while short-term exposure did not.

This was unexpected, said Dar, since they had initially thought that chronic stress would increase antioxidant levels to protect cells from oxidative damage and cell death. “To see these potentially protective enzyme levels actually significantly decrease was very surprising,” agreed Maher. “[It] suggested how, at least one way, this chronic exposure could be, over the long term, detrimental.”

The researchers then investigated how chronic iron loading influences other molecules and biochemical processes related to GPX4. Immunoblots revealed that chronic, but not acute, iron exposure led to the depletion of glutathione, which maintains mitochondrial redox homeostasis. This, in turn, increased mitochondrial reactive oxygen species (ROS) and lipid peroxidation, wherein ROS interact with cell membrane lipids to degrade them.

Bush was not surprised to see biochemical differences between cells exposed to acute and chronic iron loading. “But we didn't know the specifics about what to expect,” he noted. “They've laid the groundwork for us requiring these sorts of long cell culture experiments to be done,” he said.

Given the changes they observed, Dar and Maher wondered how neurons exposed to iron over a long time would handle stress. Cell survival assays revealed that neither acute nor chronic iron exposure led to cell death, indicating that a chronic ferroptotic state did not lead to ferroptotic death. However, chronic, but not acute iron exposure, in combination with a ferroptosis-triggering molecule significantly increased cell death. Consistent with this, chronic, but not acute, iron exposure sensitized cells to oxidative stress due to hydrogen peroxide. Since chronic iron exposure is what sensitized the cells, the researchers named this process chronoferroptosis.

“These data further support the concept that this pathway [is] more a stress pathway than a death pathway [that] plays a key role in the development of neurodegenerative diseases,” said Maher, adding that interfering with chronoferroptosis could potentially help treat such conditions. She noted that they have already identified some molecules that could inhibit it.

While Bush agreed that these results pave the way for identifying drug targets, he believes that more work needs to be done since the current findings are confined to a single tumor cell line. “[But] you have to start somewhere,” he remarked, adding that the next step would be to conduct experiments with other cell lines and eventually animal models.

Dar agreed that they need to test this pathway in preclinical models next. “But at least for now, we know that the time context is very important, and that plays a major role,” he said.