Age, sex, and apolipoprotein E isoform alter contextual fear learning, neuronal activation, and baseline DNA damage in the hippocampus; Boutros et al. 2023

“What causes Alzheimer’s disease?” This question lies at the heart of aging research. It has remained elusive since an “unusual disease of the cerebral cortex” was first described by Dr. Alois Alzheimer in 1906.
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 In the last century, progress has been made in understanding causes of and risk factors for Alzheimer’s disease (AD). Notably, increasing age, being biologically female, and carrying a gene called apolipoprotein E4 (apoE4) comprise a “trifecta” that individually and synergistically increase a person’s risk for late-onset AD. The underlying reasons for this increased risk are unclear, though. A recent paper published in Molecular Psychiatry adds some insight into age-, sex-, and apoE-dependent molecular changes that may contribute to the relative increase in AD risk.

Investigating the “trifecta” of risk factors for different neurological diseases using fear conditioning

The researchers conducted their work in mice that express one of the 3 human isoforms of apoE: apoE2 (E2), apoE3 (E3), or apoE4 (E4). Compared to E3, those with E4 are at a relatively increased risk for AD. In contrast, those with E2 are relatively protected from AD. However, other research suggests that E2 increases risk and severity of symptoms in post-traumatic stress disorder (PTSD). These isoform-specific risks for different conditions present an interesting scale: on one side, E2 may contribute to “too much remembering,” reflected in the persistence of traumatic memories, while on the other, E4 may contribute to “too much forgetting,” as seen in memory loss in AD (Figure 1A).


Females are at a higher risk to develop both PTSD and AD compared to males, and increasing age increases risk for AD. This study incorporated these factors: it involved testing young (3 months, about 20-30 years in humans) and middle-aged (12 months, about 50-60 years) male and female apoE mice in a learning task.


The learning task—contextual fear conditioning—is based on Dr. Ivan Pavlov’s observations that dogs will quickly learn that a specific stimulus (such as food) is associated with specific cues (such as a bell) and/or contexts (such as a room). Contextual fear conditioning involves placing mice into an enclosure with a distinct environment, where they receive a mild aversive stimulus. After this, mice should remember that this specific environment is associated with the aversive stimulus and will show a fear response (freezing); mice with impaired memory will freeze less. However, if animals are re-introduced into the environment multiple times and no aversive stimulus is presented, freezing normally decreases, a phenomenon called “extinction.” Fear conditioning is often used to study PTSD-like symptoms in animal models. Animals that do not show extinction mirror one of the primary symptoms of PTSD: the persistence of traumatic memories.  


Previous research has shown that mice with the different apoE genotypes remember the environment differently, with E2 mice displaying persistence of the memory. This contrasts with E3 and E4 mice, which show typical extinction. The recently published study by Boutros & colleagues highlights a new difference: that E4 mice (both males and females) show more fear response during the initial exposure to the aversive stimuli than E2 or E3 mice, and that middle-aged animals express more fear than young animals.


Searching for answers in gene expression and DNA damage

The “why” behind this apoE genotype-dependent behavior has remained elusive. It is known that contextual fear conditioning relies on brain regions involved in spatial learning & memory, including the hippocampus and entorhinal cortex, two regions that are heavily impacted by PTSD and AD. Yet, this task also involves many other brain regions that have been less studied, such as those important for sensation, interpreting the sensations, and expressing the correct behavioral output, which are also known to be affected by PTSD and AD.


The authors used a new technique to look at parts of the brain that were active during the fear conditioning task. Immediate early genes (IEGs), such as a gene called cFos, are only expressed when a specific cell has been “activated,” and thus can tell researchers which cells were “turned on” during a task. A limitation has been the time and effort it takes to look at many brain regions, though. Recent technological advances have led to a method known as whole brain imaging: researchers can take entire brains, turn them transparent, and look at cells expressing IEGs. This technique is similar to magnetic resonance imaging (MRI) in that researchers can look at many brain regions at once as well as their connections to each other. Due to better signal: noise ratio, whole brain imaging provides a clearer picture of active brain regions than MRIs can.


Boutros & colleagues were able to identify differences in cellular activation during contextual fear conditioning between the apoE genotypes in young female mice using whole-brain imaging. E4 mice had less cellular activation in regions that are important for sensation (like the thalamus) and contextual learning (like the entorhinal cortex and hippocampus), whereas E2 mice generally had more cellular activation of these regions. Importantly, the communication between brain regions (which they measured by correlating cFos signal across regions) was altered in young E4 females, which displayed weaker correlations and a communication pattern distinct from those seen in E2 and E3 mice (Figure 1B). These results from the young female mice hint at early, pre-disease differences in neuronal activity and communication specifically in those with E4, which could underlie the risk for late-onset AD. 


Digging even deeper, the question of how IEGs are expressed has been of recent interest. Evidence from others suggest that the fast activation of IEGs occurs as the result of the DNA double-helix breaking, called “double strand breaks” (DSBs). DSBs, as well as other types of DNA damage, increase with age and disease; so, Boutros et al. looked at the amount of DSBs in young and middle aged apoE mice after fear conditioning and in behaviorally naïve mice (a control “baseline” group). They found that DSBs increased in the middle-aged mice at baseline, and that this age-dependency was more pronounced in females than in males. Interestingly, the researchers also found that middle-aged E4 mice had even more DSBs at baseline compared to the other genotypes, and that middle-aged E2 males had more DSBs than young males (Figure 1C). These results contribute to the existing knowledge that there are sex-dependent changes in aging, and that apoE genotype also plays a role in age-related changes.


Lastly, the researchers homed in on the hippocampus, measuring cFos and 53BP1 (a protein that is activated in response to DSBs) in young and middle aged apoE male and female mice (Figure 1D). Young females overall had more hippocampal cFos than young males did after fear conditioning, as well as slightly different patterns based on apoE genotype. The apoE-dependent differences were very apparent in middle-aged mice, though: in both males and females, E2 mice showed high levels of cFos (indicative of active cells), but E3 and E4 mice did not (Figure 1E). This “maintained” cellular activity in middle age could be an underlying mechanism that contributes to the E2-specific protection against AD, and therefore a possible therapeutic target in E4 carriers.


Altogether, these data add insight to the interaction between sex, age, and apoE genotype that lead to risks for different neurological conditions, bringing us closer to an understanding of the underlying, driving mechanisms.


The figure was generated using software.

Figure 1. Summary of the main findings from Boutros et al. 2023. A) The relative risks associated with the 3 aopE genotypes: compared to E3, E2 is protective against Alzheimer’s disease (AD) while E4 is a risk factor for AD. E2 is associated with increased severity of post-traumatic stress disorder (PTSD). B) Whole-brain imaging in young, female apoE mice revealed that E4 was more different than the other 2 genotypes in the amount of inter-region communication and the regions that were communicating. C) Age and E4 increase measures of “baseline” DNA double strand breaks (DSBs) and immediate early genes (IEGs) in both sexes, and age and E2 increased it in only males. D) cFos (green) and 53BP1 (magenta) signal in the hippocampus from mice 1.5 hours after fear conditioning. DAPI (blue) is a counterstain used to identify the nucleus of cells. White arrows indicate cells with cFos, yellow arrows indicate cells with 53BP1. E) Young, female mice had an increase in cFos which was absent in young male mice. Middle-aged E2 female and male mice maintained the increase in cFos, while middle-aged E3 and E4 mice did not. There were no differences detected in 53BP1. Schematics made with

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