Tessa Marzulla

ApoE2 Exaggerates PTSD-Related Behavioral, Cognitive, and Neuroendocrine Alterations

Apolipoprotein E (apoE) is an essential component of lipoprotein particles in both the brain and periphery, and exists in three isoforms in the human population: E2, E3, and E4. ApoE has numerous, well-established roles in neurobiology. Most notably, E4 is associated with earlier onset and increased risk of Alzheimer’s disease (AD). Although possession of E2 is protective in the context of AD, E2 appears to confer an increased incidence and severity of posttraumatic stress disorder (PTSD). However, the biological processes underlying this link remain unclear. In this study, we began to elucidate these associations by examining the effects of apoE on PTSD severity in combat veterans, and on PTSD-like behavior in mice with human apoE. In a group of 92 veterans with PTSD, we observed significantly higher Clinician-Administered PTSD Scale and PTSD Checklist scores in E2+ individuals, as well as alterations in salivary cortisol levels. Furthermore, we measured behavioral and biological outcomes in mice expressing human apoE after a single stressful event as well as following a period of chronic variable stress, a model of combat-related trauma. Mice with E2 showed impairments in fear extinction, and behavioral, cognitive, and neuroendocrine alterations following trauma. To the best of our knowledge, these data constitute the first translational demonstration of PTSD severity in men and PTSD-like symptoms in mice with E2, and point to apoE as a novel biomarker of susceptibility, and potential therapeutic target, for PTSD.

High fat diet-induced diabetes in mice exacerbates cognitive deficit due to chronic hypoperfusion:

Diabetes causes endothelial dysfunction and increases the risk of vascular cognitive impairment. However, it is unknown whether diabetes causes cognitive impairment due to reductions in cerebral blood flow or through independent effects on neuronal function and cognition. We addressed this using right unilateral common carotid artery occlusion to model vascular cognitive impairment and long-term high-fat diet to model type 2 diabetes in mice. Cognition was assessed using novel object recognition task, Morris water maze, and contextual and cued fear conditioning. Cerebral blood flow was assessed using arterial spin labeling magnetic resonance imaging. Vascular cognitive impairment mice showed cognitive deficit in the novel object recognition task, decreased cerebral blood flow in the right hemisphere, and increased glial activation in white matter and hippocampus. Mice fed a high-fat diet displayed deficits in the novel object recognition task, Morris water maze and fear conditioning tasks and neuronal loss, but no impairments in cerebral blood flow. Compared to vascular cognitive impairment mice fed a low fat diet, vascular cognitive impairment mice fed a high-fat diet exhibited reduced cued fear memory, increased deficit in the Morris water maze, neuronal loss, glial activation, and global decrease in cerebral blood flow. We conclude that high-fat diet and chronic hypoperfusion impair cognitive function by different mechanisms, although they share commons features, and that high-fat diet exacerbates vascular cognitive impairment pathology.

28Silicon Irradiation Impairs Contextual Fear Memory in B6D2F1 Mice

The space radiation environment consists of multiple species of charged particles, including 28Si, 48Ti and protons that may impact cognition, but their damaging effects have been poorly defined. In mouse studies, C57Bl6/J homozygous wild-type mice and genetic mutant mice on a C57Bl6/J background have typically been used for assessing effects of space radiation on cognition. In contrast, little is known about the radiation response of mice on a heterozygous background. Therefore, in the current study we tested the effects of 28Si, 48Ti and proton radiation on hippocampus-dependent contextual fear memory and hippocampus-independent cued fear memory in C57Bl6/J × DBA2/J F1 (B6D2F1) mice three months after irradiation. Contextual fear memory was impaired at a 1.6 Gy dose of 28Si radiation, but not cued fear memory. 48Ti or proton irradiation did not affect either type of memory. Based on earlier space radiation cognitive data in C57Bl6/J mice, these data highlight the importance of including different genetic backgrounds in studies aimed at assessing cognitive changes after exposure to space radiation.

Proton irradiation induces persistent and tissue-specific DNA methylation changes in the left ventricle and hippocampus.

BackgroundProton irradiation poses a potential hazard to astronauts during and following a mission, with post-mitotic cells at most risk because they cannot dilute resultant epigenetic changes via cell division. Persistent epigenetic changes that result from environmental exposures include gains or losses of DNA methylation of cytosine, which can impact gene expression. In the present study, we compared the long-term epigenetic effects of whole body proton irradiation in the mouse hippocampus and left ventricle. We used an unbiased genome-wide DNA methylation study, involving ChIP-seq with antibodies to 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) to identify DNA regions in which methylation levels have changed 22 weeks after a single exposure to proton irradiation. We used DIP-Seq to profile changes in genome-wide DNA methylation and hydroxymethylation following proton irradiation. In addition, we used published RNAseq data to assess whether differentially methylated regions were linked to changes in gene expression.ResultsThe DNA methylation data showed tissue-dependent effects of proton irradiation and revealed significant major pathway changes in response to irradiation that are related to known pathophysiologic processes. Many regions affected in the ventricle mapped to genes involved in cardiovascular function pathways, whereas many regions affected in the hippocampus mapped to genes involved in neuronal functions. In the ventricle, increases in 5hmC were associated with decreases in 5mC. We also observed spatial overlap for regions where both epigenetic marks decreased in the ventricle. In hippocampus, increases in 5hmC were most significantly correlated (spatially) with regions that had increased 5mC, suggesting that deposition of hippocampal 5mC and 5hmC may be mechanistically coupled.ConclusionsThe results demonstrate long-term changes in DNA methylation patterns following a single proton irradiation, that these changes are tissue specific, and that they map to pathways consistent with tissue specific responses to proton irradiation. Further, the results suggest novel relationships between changes in 5mC and 5hmC.

Amelioration of Metabolic Syndrome-Associated Cognitive Impairments in Mice via a Reduction in Dietary Fat Content or Infusion of Non-Diabetic Plasma

Obesity, metabolic syndrome (MetS) and type 2 diabetes (T2D) are associated with decreased cognitive function. While weight loss and T2D remission result in improvements in metabolism and vascular function, it is less clear if these benefits extend to cognitive performance. Here, we highlight the malleable nature of MetS-associated cognitive dysfunction using a mouse model of high fat diet (HFD)-induced MetS. While learning and memory was generally unaffected in mice with type 1 diabetes (T1D), multiple cognitive impairments were associated with MetS, including deficits in novel object recognition, cued fear memory, and spatial learning and memory. However, a brief reduction in dietary fat content in chronic HFD-fed mice led to a complete rescue of cognitive function. Cerebral blood volume (CBV), a measure of vascular perfusion, was decreased during MetS, was associated with long term memory, and recovered following the intervention. Finally, repeated infusion of plasma collected from age-matched, low fat diet-fed mice improved memory in HFD mice, and was associated with a distinct metabolic profile. Thus, the cognitive dysfunction accompanying MetS appears to be amenable to treatment, related to cerebrovascular function, and mitigated by systemic factors.

Impairment in Extinction of Contextual and Cued Fear Following Post-Training Whole-Body Irradiation

Because of the use of radiation in cancer therapy, the risk of nuclear contamination from power plants, military conflicts, and terrorism, there is a compelling scientific and public health interest in the effects of environmental radiation exposure on brain function, in particular hippocampal function and learning and memory. Previous studies have emphasized changes in learning and memory following radiation exposure. These approaches have ignored the question of how radiation exposure might impact recently acquired memories, which might be acquired under traumatic circumstances (cancer treatment, nuclear disaster, etc.). To address the question of how radiation exposure might affect the processing and recall of recently acquired memories, we employed a fear conditioning paradigm wherein animals were trained, and subsequently irradiated (whole-body X-ray irradiation) 24 h later. Animals were given 2 weeks to recover, and were tested for retention and extinction of hippocampus-dependent contextual fear conditioning or hippocampus-independent cued fear conditioning. Exposure to irradiation following training was associated with reduced daily increases in body weights over the 22-days of the study and resulted in greater freezing levels and aberrant extinction 2 weeks later. This was also observed when the intensity of the training protocol was increased. Cued freezing levels and measures of anxiety 2 weeks after training were also higher in irradiated than sham-irradiated mice. In contrast to contextual freezing levels, cued freezing levels were even higher in irradiated mice receiving 5 shocks during training than sham-irradiated mice receiving 10 shocks during training. In addition, the effects of radiation on extinction of contextual fear were more profound than those on the extinction of cued fear. Thus, whole-body irradiation elevates contextual and cued fear memory recall.

16)Oxygen irradiation enhances cued fear memory in B6D2F1 mice.

The space radiation environment includes energetic charged particles that may impact cognitive performance. We assessed the effects of (16)O ion irradiation on cognitive performance of C57BL/6J × DBA/2J F1 (B6D2F1) mice at OHSU (Portland, OR) one month following irradiation at Brookhaven National Laboratory (BNL, Upton, NY). Hippocampus-dependent contextual fear memory and hippocampus-independent cued fear memory of B6D2F1 mice were tested. (16)O ion exposure enhanced cued fear memory. This effect showed a bell-shaped dose response curve. Cued fear memory was significantly stronger in mice irradiated with (16)O ions at a dose of 0.4 or 0.8 Gy than in sham-irradiated mice or following irradiation at 1.6 Gy. In contrast to cued fear memory, contextual fear memory was not affected following (16)O ion irradiation at the doses used in this study. These data indicate that the amygdala might be particularly susceptible to effects of (16)O ion exposure.

An Optimized Protocol for High-Throughput In Situ Hybridization of Zebra Finch Brain

In situ hybridization (ISH) is a sensitive technique for documenting the tissue distribution of mRNAs. Advanced nonradioactive ISH methods that are based on the use of digoxigenin (DIG)-labeled probes and chromogenic detection have better spatial resolution than emulsion autoradiography techniques and, when paired with high-resolution digital imaging, allow for large-scale profiling of gene expression at cellular resolution within a histological context. However, technical challenges restrict the number of genes that can be investigated in a small laboratory setting. This protocol describes an optimized, low-cost, small-footprint, high-throughput ISH procedure to detect gene expression patterns in 10-µm brain sections from zebra finches. It uses DIG-labeled riboprobes synthesized from cDNA templates available through the Songbird Neurogenomics Consortium. The method is compatible with high-resolution digital imaging; it produces images with low background and a resolution approaching that of immunohistochemical methods. Approximately 180 slides can be processed each week using this protocol, but it can be scaled to accommodate a broad range of tissues from which cryosections can be obtained.

Short- and long-term effects of 56 Fe irradiation on cognition and hippocampal DNA methylation and gene expression

BackgroundAstronauts are exposed to 56Fe ions that may pose a significant health hazard during and following prolonged missions in deep space. We showed previously that object recognition requiring the hippocampus, a structure critical for cognitive function, is affected in 2-month-old mice irradiated with 56Fe ions. Here we examined object recognition in 6-month-old mice irradiated with 56Fe ions, a biological age more relevant to the typical ages of astronauts. Moreover, because the mechanisms mediating the detrimental effects of 56Fe ions on hippocampal function are unclear, we examined changes in hippocampal networks involved in synaptic plasticity and memory, gene expression, and epigenetic changes in cytosine methylation (5mC) and hydroxymethylation (5hmC) that could accompany changes in gene expression. We assessed the effects of whole body 56Fe ion irradiation at early (2 weeks) and late (20 weeks) time points on hippocampus-dependent memory and hippocampal network stability, and whether these effects are associated with epigenetic changes in hippocampal DNA methylation (both 5mC and 5hmC) and gene expression.ResultsAt the two-week time point, object recognition and network stability were impaired following irradiation at the 0.1 and 0.4 Gy dose, but not following irradiation at the 0.2 Gy dose. No impairments in object recognition or network stability were seen at the 20-week time point at any irradiation dose used. Consistent with this pattern, the significance of pathways for gene categories for 5hmC was lower, though not eliminated, at the 20-week time point compared to the 2-week time point. Similarly, significant changes were observed for 5mC gene pathways at the 2-week time point, but no significant gene categories were observed at the 20-week time point. Only the 5hmC changes tracked with gene expression changes.ConclusionsDose- and time-dependent epigenomic remodeling in the hippocampus following 56Fe ion exposure correlates with behavioral changes.

Bi-directional and shared epigenomic signatures following proton and 56 Fe irradiation

The brain’s response to radiation exposure is an important concern for patients undergoing cancer therapy and astronauts on long missions in deep space. We assessed whether this response is specific and prolonged and is linked to epigenetic mechanisms. We focused on the response of the hippocampus at early (2-weeks) and late (20-week) time points following whole body proton irradiation. We examined two forms of DNA methylation, cytosine methylation (5mC) and hydroxymethylation (5hmC). Impairments in object recognition, spatial memory retention, and network stability following proton irradiation were observed at the two-week time point and correlated with altered gene expression and 5hmC profiles that mapped to specific gene ontology pathways. Significant overlap was observed between DNA methylation changes at the 2 and 20-week time points demonstrating specificity and retention of changes in response to radiation. Moreover, a novel class of DNA methylation change was observed following an environmental challenge (i.e. space irradiation), characterized by both increased and decreased 5hmC levels along the entire gene body. These changes were mapped to genes encoding neuronal functions including postsynaptic gene ontology categories. Thus, the brain’s response to proton irradiation is both specific and prolonged and involves novel remodeling of non-random regions of the epigenome.