Brain Cell-Type Specific Enhancer-Promoter Interactions and Disease Risk

Post by Stephanie Williams

What's the science?

The relationship between common genetic variation, regulatory elements in different types of brain cells, and disease risk is poorly characterized. Regulatory elements, such as promoters and enhancers, which control the expression of genes, vary across cell types. It is possible that particular common genetic variants (i.e. single nucleotide polymorphisms) that confer disease risk, may also interact with cell-type-specific regulatory elements to affect the risk of psychiatric and neurological diseases. This week in Science, Nott, and colleagues analyzed the regulatory elements of four major cell types in the brain to establish cell-type specific enhancer-promoter maps and to understand the relationship between regulatory elements and disease risk across different cells. 

How did they do it?                             

First, the authors performed a series of analyses to characterize the regulatory regions for four major cell types in the human brain. They extracted their samples from post-mortem cortical tissue from six individuals. They group cells into four major types, including neuronal cells and three types of non-neuronal cells, including microglia, oligodendrocytes, and astrocytes. The authors identified active promoters and enhancers in each cell type in order to establish enhancer-promoter maps. The authors then used statistics from previous genome-wide association studies (GWAS) that characterized different risk alleles (common genetic variants conferring risk) for neurological disorders, psychiatric disorders, and neurobehavioral traits to investigate how risk variants were related to the identified regulatory elements. They used the results from these analyses to further probe the relationship between particular disease risk variants (eg. Alzheimer’s risk variants) and regulatory elements within specific sets of cells (eg. microglia). The authors selected a subset of previously-defined Alzheimer’s risk variants and analyzed whether those variants overlapped with cell-type-specific regulatory elements that they had identified. They performed a series of steps to confirm that those genomic regions containing the risk variants interacted with active promoters. These steps resulted in 41 genes that spanned all 4 cell types, which were linked to the variants. Of the 41 genes, 25 were identified in microglia, and the remaining were not identified in the other types of cells that the authors looked at. They then repeated this analysis with other Alzheimer’s disease genome-wide association studies and found a greater number of risk genes. They focus on one particular enhancer that contains an Alzheimer’s Disease risk variant with the second highest Alzheimer’s Disease risk score. They performed analyses to verify the functionality of the enhancer and to investigate the cell-type specificity of the enhancer.

What did they find?

When the authors analyzed which regulatory elements were active in the nuclei of their different cell types, they found that their data clustered according to cell type and showed cell-type-specific patterns. They found that the promoters associated with cell-type-specific signatures exhibited specific H3K27ac (a type of histone modification) profiles. From their analysis on the relationship between promoters and enhancers, the authors found that there was a large amount of overlap in the active promoters across different cell types, while there was very little overlap in the active enhancers across cell types. They suggest that the cell-type specificity is therefore captured by the group of active enhancers. From their analysis on the relationship between the regulatory regions of their four classes of cells and complex traits and diseases, the authors found that 1) psychiatric disorders and behavioral trait heritability was most highly enriched for enhancers and promoters in neuronal cells, and 2) Alzheimer’s heritability was most highly enriched in microglia-specific regulatory regions. In particular, the authors showed that Alzheimer’s heritability was enhanced in microglial enhancers. Methodologically, results from their analysis on promotors and distal regulatory regions with a technique called proximity ligation-assisted ChIP seq (PLAC), showed that PLAC could be used to identify cell-type-specific promoter-enhancer interactions.

They identified several thousand clusters of multiple enhancers, called super-enhancers, which are thought to be important in driving the expression of cell identity genes. Of the super-enhancers that they identified, many contained GWAS disease-risk variants and were related to cell-type-specific genes. These results suggest that some GWAS variants may act on these clusters of enhancers to affect gene expression. From their series of analyses on Alzheimer’s Disease-risk variants, the authors identified several interesting characteristics of Alzheimer’s Disease variants. They found that the risk variants were usually linked to active promoters that were far away, and not to the closest gene promoter. They also found that although some Alzheimer’s Disease risk variants were expressed in different cell types, there were microglia-specific enhancers that contained the risk variants. The authors focused on the BIN1 (a gene involved in Alzheimer’s risk) enhancer, which contained a risk variant with the second highest AD-risk score after APOE, and confirmed that the risk allele associated with BIN1 was contained in the microglia-specific enhancers.

What's the impact?

The authors collected evidence that advances our understanding of promoter-enhancer interactions in specific cell types and characterized associations between cell-type-specific regulatory elements and particular disease risk variants. They show that risk variants for psychiatric disorders are associated with regulatory elements within neurons, while a particular risk variant for sporadic Alzheimer’s disease is regulated by a microglia-specific enhancer. 

Nott et al. Brain cell type-specific enhancer-promoter interactome maps and disease-risk association. Science. (2019). Access the original scientific publication here.

Exercise Improves Brain Tissue Oxygenation in a Mouse Model of Alzheimer’s Disease

Post by Elisa Guma

What's the science?

Alzheimer’s disease (AD) is a progressive neurodegenerative disease associated with loss of memory, as well as neuronal cell death due to the aggregation of tangles (formed by misfolded tau protein in neurons), and plaques (formed by a protein amyloid-beta). Further, the vascular system has been shown to be compromised in AD, but it is unclear what role it plays in disease progression and whether it could be a potential treatment target. Voluntary exercise has been identified as an effective means for preserving brain function and preventing cognitive decline in both rodent and human research. However, the oxygenation mechanism through which voluntary exercise affects AD remains unknown. This week in the Neurobiology of Aging, Lu, and colleagues use a novel approach to image brain oxygenation and blood flow in awake mice to investigate whether brain oxygenation is compromised in a mouse model of AD and if voluntary exercise can reverse impairments. 

How did they do it?

The authors used a transgenic AD mouse model, which expresses Amyloid Precursor Protein Presenilin-1 gene mutations that have been identified in humans with AD, and develops cognitive impairments and memory loss at 4.5 months. Tissue oxygenation was measured in AD and wild-type (normal) mice at 3 and 6 months of age, as well as in a group of AD mice who were given a running wheel at 3 months and assessed at 6 months.

The authors used a novel two-photon microscopy system with a phosphorescent lifetime arm, which measures the lifetime of a fluorescent signal as opposed to just identifying its presence, in order to measure tissue oxygenation and cerebral blood flow at a sub-capillary resolution around cortical arterioles, capillaries, and venules in awake mice. Tissue oxygenation was measured using oxygen partial pressure in brain tissue which is affected by variations in blood flow and metabolic demand in different regions. To be able to view the blood vessels in the brain, the authors drilled a hole in the mouse skull (under anesthesia) above the barrel cortex, while keeping the dura mater (a layer of tissue surrounding the brain and spinal cord) intact, and mounted a glass cover over it, keeping a small silicone-covered hole on the side to act as a biocompatible port. 

An oxygen-sensitive dye was injected into the brain tissue via the biocompatible port, and tissue oxygenation was measured using the two-photon phosphorescence lifetime microscopy. This allowed the authors to see how quickly the oxygen-sensitive dye was decaying (an advantage of the phosphorescence lifetime microscopy), to assess how oxygenated the tissue was at a given time. The authors used another kind of imaging technique, Doppler Optical Coherence Tomography, to acquire cerebral blood flow estimates in the blood vessels. During the experiment, the animals could move on a treadmill wheel which allowed free movement of the limbs. Following the awake imaging, the authors collected the brains of their mice and stained them for amyloid plaques, neuronal cell density, and the LRP1 receptor in the cortex, which has been associated with improving amyloid clearance, to determine if exercise was able to reverse AD pathology.

What did they find?

The authors found a decrease in tissue oxygenation over time in the AD mice. Further, there was more heterogeneity of tissue oxygenation indicative of vascular dysfunction both with increased age and due to AD. They also observed more regions of hypoxia or near-hypoxia (low oxygenation) and decreased cerebral blood flow in the AD mice. An increase in amyloid-beta and a decrease in neuronal cell density in the AD mice were also found.

Voluntary exercise for 3 months seemed to reverse all of the observed impairments in vascular function in the AD mice. First, the authors found increased tissue oxygenation and decreased heterogeneity of oxygenation in the AD mice. Exercise also decreased the number of near-hypoxic areas in AD mice and improved cerebral blood flow. At a cellular level, the authors found that exercise decreased the amount of amyloid-beta plaque and reversed the decrease in neuronal cell density. Interestingly, exercise increased the levels of a receptor called LRP1 in the cortex, suggesting that perhaps it may have improved amyloid clearance. Not all mice ran the same amount over the 3 months, so the authors were able to investigate whether the distance ran correlated with the improvement in oxygenation and blood flow; in fact, they found that it correlated with the amount of brain oxygenation and blood flow observed, suggesting that there is a dose-response between the two. 

What's the impact?

This study is the first to measure brain tissue oxygenation and cerebral blood flow in a live awake mouse using a cutting-edge microscopy technique. They show that blood oxygenation and cerebral blood flow are compromised in a mouse model of AD and that voluntary exercise is able to reverse many of these impairments. This suggests that exercise may be an interesting treatment intervention for individuals with dementia. Unfortunately, this work was only performed in male mice; future work should extend this work to female mice as well, as AD is more prevalent in females. 

Xuecogn Lu et al. Voluntary exercise increases brain tissue oxygenation and spatially homogenized oxygen delivery in a mouse model of Alzheimer’s Disease. Neurobiology of Aging (2019). Access the original scientific publication here.

The Role of Gut Microbes in Neurological Disorders

Post by Amanda McFarlan

What's the science?

Research investigating the relationship between the gut microbiota (the ensemble of microorganisms in the gut) and overall health has gained increasing popularity in recent years. Studies have shown that the gut microbiota is important for regulating many major systems of the body, including the central nervous system. This week in The Lancet Neurology, Cryan and colleagues summarized past and present research that highlights the important role of gut microbiota in regulating the central nervous system.

What do we already know?

Much of our understanding of how the gut microbiota affects the central nervous system comes from studies using germ-free mice (i.e. lacking any gut microbiota). Researchers have shown that without the gut microbiota, germ-free mice have deficits in many neural processes that are critical for development and aging, including the maturation and myelination of neurons, neurogenesis and microglia activation. Additionally, germ-free mice had higher levels of neuroinflammation and an increase in the permeability of the blood-brain barrier, which are hallmarks of the immune-related neurological disease, multiple sclerosis. These studies with germ-free mice have provided researchers with important insights into how the gut microbiome is implicated in neurodevelopment, aging, and neurodegenerative disease. Researchers have continued to use animal models as well as clinical studies to further our understanding of the interaction between the gut and the brain. 

What’s new?

In recent studies, researchers have focused on how gut microorganisms can be targeted for therapeutic purposes in neurological diseases. In the case of treating multiple sclerosis, for example, researchers found that a multispecies probiotic, administered two times a day for two months, had an anti-inflammatory effect and reversed changes in gut microbiota. Moreover, a clinical trial for children with autism spectrum disorder revealed that children who received a microbiota transfer had significantly reduced gastrointestinal problems (commonly found to be co-morbid with autism spectrum disorder) as well as behavioral improvements. These findings suggest that some gut microbiota may be a potential target for the treatment of neurological disorders. Additionally, researchers have started to look at how gut microbiota may regulate the physiology and behavior of neurological disorders like Huntington’s disease, amyotrophic lateral sclerosis (ALS) and epilepsy. For example, researchers found that the gut microbiota is involved in synaptic changes that occur in brain regions that are associated with epilepsy. They also found that the therapeutic effects of a ketogenic diet on epilepsy are dependent on gut microbiota. Finally, recent studies have shown that many non-antibiotic drugs influence gut microbiota, highlighting the importance of investigating the relationship between the gut microbiome and medications (especially those that are prescribed for treating a neurological disorder).

What's the bottom line?

The importance of gut microbiota on the development and overall health of the brain is becoming more and more evident. Studies with animal models and clinical trials have provided strong evidence for the role of the gut microbiota in neurological diseases like multiple sclerosis and autism spectrum disorder, with a growing body of research showing implications of the gut microbiota in many other diseases. Still, there is much more to be done in this field to elucidate the mechanisms by which gut microorganisms influence the brain. A better understanding of the gut-brain axis will provide insight into new therapies that may help treat or prevent a wide range of neurological disorders. 

Cryan et al. The gut microbiome in neurological disorders. The Lancet Neurology (2019). Access the original scientific publication here.