Advanced Gene Sequencing Study Recognized by AJP Editors

The American Journal of Psychiatry has long been a place where readers could find the top research on the pharmacology, neurobiology, genetics, and epidemiology of mental illness. In recent years, AJP has made a concerted effort to also be a hub for cutting-edge neuroscience that will guide the future of psychiatric care. An AJP report showcasing a technique known as single-nucleus RNA sequencing was named by the journal’s editorial board as one of AJP’s “2023 Articles of Import and Impact.”

Single-nucleus RNA sequencing (snRNAseq) allows researchers to study the gene expression of individual cells in complex tissue, such as in the human brain, where neurons and supporting cells are tightly intertwined. Rather than trying to separate brain cells, snRNAseq pops them open and collects the individual nuclei that house the genetic material and express RNA.

In the AJP article “Single-Nucleus Transcriptome Profiling of Dorsolateral Prefrontal Cortex: Mechanistic Roles for Neuronal Gene Expression, Including the 17q21.31 Locus, in PTSD Stress Response,” Nikolaos Daskalakis, M.D., Ph.D., the director of the Neurogenomics and Translational Bioinformatics Laboratory at McLean Hospital and colleagues, used this technique to analyze postmortem brain tissue of people with posttraumatic stress disorder (PTSD). The findings offered new details into how the stress response is altered in people with PTSD.

The analysis by Daskalakis and colleagues focused on 32 postmortem samples of the prefrontal cortex from 11 individuals who had PTSD, 10 who had major depressive disorder, and 11 who had neither disorder. To complete snRNAseq, individual cell nuclei were extracted from the tissue and individually encased in tiny gel droplets containing barcodes. These barcoded droplets were then fed into a machine that reads the barcode and sequences the RNA inside each nucleus. (RNA is the template used to make proteins and thus is a measure of gene expression.)

In total, about 415,000 nuclei were analyzed, with a median of 2,700 genes being sequenced from these nuclei. Daskalakis said this makes for an immense amount of genetic data, but by looking for the presence of genes that are known to be conserved across the dataset (called “anchor genes”), the gene expression data can be easily sorted. In the AJP study, his team sorted the data into eight different cell types and 22 cell subtypes.

The analysis by Daskalakis and colleagues revealed some notable differences in gene expression in PTSD and depression samples in multiple types of neurons, particularly genes related to the activity of stress-related steroid hormones known as glucocorticoids (for example, cortisol). A follow-up sequencing of 15 different brain samples (five each from people with PTSD, depression, or control), produced similar results.

To further support their snRNAseq data, Daskalakis’ team created neurons in lab dishes (from the stem cells of people who did not have PTSD or depression) and stressed them with the corticosteroid dexamethasone. They found that dexamethasone altered the expression of several glucocorticoid receptors in a similar pattern as was seen in the cells from people with PTSD, and in a different pattern as seen in depression.

“This paper gives a taste of the power of single cell resolution analyzes,” wrote AJP Assistant Editor Elisabeth Binder, M.D., Ph.D., who selected this study as her favorite of 2023. “While previous large-scale RNA sequencing studies in postmortem brain have indicated the relevance of inhibitory neurons in the biology of PTSD, this manuscript can now pinpoint that excitatory and inhibitory neurons show most changes in gene expression, as well as differentiate which transcripts and pathways are regulated in which cell type.” Binder is the director of the Department of Genes and Environment at the Max Planck Institute of Psychiatry in Munich.

“What really amazed us was that in our small sample of 30 brains we could also see signs of how genetic risk influences PTSD biology,” Daskalakis said. A previously conducted genome-wide analysis involving hundreds of thousands of DNA samples had suggested that a region on chromosome 17 was associated with PTSD risk. In this study, Daskalakis’ team found altered gene expression in four genes that are in the chromosome 17 region.

“It’s wonderful that the editors recognized our efforts. Our team put all our heart and resources into this study,” Daskalakis said. He expressed enthusiasm about next steps in this research, including studies to compare the changes in gene expression in tissue from people with PTSD who were exposed to different kinds of trauma.

Additionally, Daskalakis noted the importance of analyzing gene expression from other brain regions, such as the amygdala and hippocampus.

“I hope projects like this will help our field develop a larger appetite for complex studies needed to address the complexity of brain disorders,” Daskalakis said. “We have to be more lateral thinkers to fast-track progress; when we come to a scientific problem, we should ask ourselves ‘How many different ways can we attack this?’ ”

Childhood Trauma, OCD Among Other Studies of Import and Impact

Another study that stood out to the journal editors in 2023 included “A Comprehensive Multilevel Analysis of the Bucharest Early Intervention Project: Causal Effects on Recovery From Early Severe Deprivation.”

The landmark Bucharest Early Intervention Project (BEIP, initiated in 2001) took advantage of Romania’s dramatic societal changes following the fall of communism to examine the developmental differences in children receiving institutional care versus foster care.

While numerous studies using BEIP data have shown the benefits of quality foster care on measures such as IQ or social behavior, the AJP study combined over 7,000 clinical observations of 136 children, taken from infancy to late adolescence. The results showed that children placed in foster families had better physical, behavioral, and cognitive outcomes than those who continued to receive institutional care that continued across the children’s lifespan.

“This study is remarkable,” AJP Editor in Chief Ned Kalin, M.D., wrote in his summary of the study. “It is likely the first scientific demonstration of an intervention that promotes healthy physical and brain development in neglected children and the data from this study were used to support public policy changes [in Romania] related to the care of orphaned children. This is a great demonstration of how effective science can promote societal change.”

Deputy Editor Carolyn Rodriguez, M.D., Ph.D., selected an article reflecting how brain imaging can help inform clinical data: “Resting-State Connectivity and Response to Psychotherapy Treatment in Adolescents and Adults With OCD: A Randomized Clinical Trial.”

In this trial, 54 adolescents and 62 adults with obsessive-compulsive disorder (OCD) received an MRI scan prior to 12 weeks of either exposure and response prevention (ERP) or stress management therapy to see if there were any patterns of brain connectivity that might predict treatment response. The researchers found baseline brain connections between regions in the frontal cortex and deeper areas of the brain that signaled a general tendency to respond to psychotherapy, but also uncovered a couple of connections that were specific to people who improved with ERP. These ERP-specific connectivity patterns also differed with age.

“Taken together, the findings indicate that it may be possible to improve the effectiveness of ERP via novel cognitive training or neuromodulation interventions targeting baseline connectivity between cognitive control and subcortical regions,” Rodriguez wrote.

Daskalakis’ study and the OCD study were supported by grants from the National Institute of Mental Health; the BEIP study was supported by the National Institutes of Health, the John D. and Catherine T. MacArthur Foundation, the Palix Foundation, and the Jacobs Foundation. ■