In a recent study, scientists from MIT’s Picower Institute for Learning and Memory found a direct connection between two issues that appear in Alzheimer’s disease: an accumulation of double-stranded breaks (DSBs) in the DNA of neurons and the inflammatory behavior of microglia, the brain’s immune cells.
The active induction of an inflammatory response to neurons’ genetic damage is a significant new discovery. According to research lead author Gwyneth Welch, a former graduate student in the Department of Brain and Cognitive Sciences at MIT working in the lab of senior author Li-Huei Tsai, neurons had not previously been known to communicate with the brain’s immune system in Alzheimer’s disease.
According to Welch, the notion that neurons may activate inflammatory activity in response to DNA damage is a novel one in neuroscience. In terms of age-associated neuroinflammation, “the overall hypothesis was that neurons have a more passive connection with microglia.”
Instead, Welch, Tsai, and their co-authors describe how neurons dealing with increasing DSBs go through stages of first attempting to heal their shattered DNA and then, when it appears to fail, sending out via chemical signals to microglia, which in response increases their inflammatory state. In trials where the immunological signaling was blocked, the scientists were able to stop the microglia from entering that condition and destroying the synapses that connect brain circuits.
For more than ten years, researchers in Tsai’s group have been examining DSBs in relation to Alzheimer’s. Tsai said that the most recent discoveries help us learn more about their role in Alzheimer’s.
The Picower Professor of Neuroscience and the creator of MIT‘s Aging Brain Initiative, Tsai, stated, “We have a long-standing interest in understanding DNA breaks in neurons. We not only showed that DNA double strand breaks are necessary for activity-regulated gene expression in neurons, but we also found that neurons in the early stages of neurodegeneration have a lot of DNA damage.
According to Tsai, “we now understand that DNA-damaged neurons exhibit senescent characteristics and actively participate in inducing an immunological response from microglia and maybe astrocytes.” The activation of the transcription factor NFkappaB is the mechanism behind this. Additionally, we discovered two cytokines that are released by injured neurons in order to draw in microglia and trigger a response from them. We also show that NFkappaB suppression stopped synaptic loss in neurodegeneration. This shows that the neuroimmune response has an effect on the integrity of synapses and cognitive function.
Welch used the “CK-p25” mouse model of Alzheimer’s developed by the lab for her master’s thesis study. She noticed a chronology in which neurons with DSBs began to emerge within a week, reached their maximal number after two weeks, and then began to decline noticeably six weeks later. These neurons also lost the capacity to express a typical neuronal identity marker. Welch observed that dealing with DSBs appeared to be a multi-stage procedure. At first, neurons have few double-strand breaks (DSBs) and a strong sense of who they are (baseline). Then, they have high DSB levels but don’t lose their identity (stage 1), and finally, they have high DSB levels and lose their identity (stage 2).
Transcripts provide evidence.
Welch and the team employed a variety of “transcriptomics” methods, which monitor variations in gene expression, to comprehend what the cells were doing differently in each stage. Her results showed that the most highly expressed genes at the start were those related to neuronal identity, that the most highly expressed genes during stage 1 were DNA repair genes, and that the most highly expressed genes during stage 2 were immunological signaling genes.
There were immunological signaling genes regulated by NFkappaB, a master transcription regulator. The cytokines Ccl2 and Cxcl10 were among them.
Welch exposed neurons to the DSB-causing drug etoposide in the absence of any induced disease to determine whether these alterations were solely the result of DSBs. The etoposide-treated animals exhibited similar gene expression patterns to those seen in the induced mice. When Welch looked at how genes were expressed in the brains of people with DSBs and Alzheimer’s disease, she also found some interesting overlaps.
She and her co-authors said, “We discovered that stage 1 and stage 2 gene signatures were active in human DSB-bearing neurons.” The study’s authors write that “this neural immune signature was further increased in the context of AD pathology, suggesting that it may have a functional role in disease-associated neuroinflammation increased in the context of AD pathology, suggesting that it may have a functional role in disease-associated neuroinflammation.”
The importance of microglia
Welch and the team next investigated the outcome after proving that DSB-affected neurons use NFkappaB to send immunological signals like Ccl2 and Cxcl10. They made the assumption that neurons might be to blame after the lab in 2017 described a late-stage inflammatory response on the part of microglia in Alzheimer’s.
Welch employed spatial transcriptomics for this investigation. She separated both induced and uninduced mouse brains into various regions, rating each region according to how strong its DSB signal was. She next examined gene transcription in each area and discovered that areas with high DSBs also had a significantly higher number of microglia in an inflammatory state than those with low DSBs. They were also able to see this connection for themselves, which led to the discovery that there were inflammatory microglia near high-DSB neurons, which could be seen by their abnormally big cell bodies.
They interfered with p65, a crucial molecular cog in the machinery that controls NFkappaB-regulated transcription in neurons, to further test the theory. Reduced microglia proliferation and smaller microglia cell bodies were the results of the action. Also, it changed the way microglia’s genes were expressed in a way that was good, making them more like their normal “homeostatic” state.
In previous studies, they discovered that neurons treated with etoposide produced Cxcl10 and Ccl2, but that this expression was decreased when NFkappaB was disrupted. They also saw that when the two chemicals were taken out of the brains, microglia did not start to work.
Looking back at the neurons, they discovered that while inhibiting NFkappaB activity didn’t stop them from dying, it did protect the synapses, or circuit connections, on the neurons that survived. This is important because microglia cut the connections between circuits that help the brain work.
Welch said that knocking it down is probably not a good therapy because NFkappaB is known to help stop cells from dying, which may be why knocking it down didn’t stop neurons from dying.
According to her, it’s more of a proof-of-concept that altering the key switch for inflammation will alter how microglia and neurons interact. “Targeting inflammatory pathways may be more accurately done by focusing on individual signaling molecules.”
Welch, Tsai, Eloi Schmauch, Jose Davila-Velderrain, Matheus Victor, Vishnu Dileep, P. Lorenzo Bozzelli, Qiao Su, Jemmie D. Cheng, Audrey Lee, Noelle S. Leary, Andreas R. Pfenning, and Manolis Kellis are the other authors of the study in addition to them.
The research was funded by the National Institutes of Health, CureAlz, the Glenn Foundation, and the JPB Foundation.
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