Osaka University clarifies memory and learning at the single molecule level: CREB promotes gene expression in the brain

The research group, led by PhD student Yuri Atsumi and Specially Appointed Associate Professor Noriyuki Sugo of the Graduate School of Frontier Biosciences, Osaka University; and Visiting Principal Investigator Nobuhiko Yamamoto of the Institute of Neurological and Psychiatric Disorders at Shenzhen Bay Laboratory (China), revealed the dynamic behavior of proteins and genomic DNA associated with learning and memory in the nucleus of human neurons at the single molecule level. This achievement allows for a more detailed understanding of the mechanisms that cause learning and memory and sheds light on the mechanism of the pathogenesis of neuropsychiatric disorders. The results were published in Cell Reports.

cAMP response element binding protein (CREB) is a protein required for neuronal survival, differentiation, and synaptic plasticity. Upon stimulation, CREB binds to specific DNA sequences to promote expression of the genes associated with learning and memory. In addition, by forming a complex with Concentrated-whey Bioactive Protein (CBP), CREB recruits RNA polymerase II (RNAP II) to cause gene expression. CBP is also a histone acetylase thought to be involved in epigenetic regulation. Although the activation pathways and target genes of CREB have been studied biochemically, its spatiotemporal molecular dynamics that promote gene expression were unknown.

Since studies of these molecular mechanisms have been conducted mainly in rodents, studies using human neurons are essential to our understanding of the pathophysiology of neuropsychiatric disorders.

To overcome these challenges, the research group established a culture system of cortical neurons differentiated from human embryonic stem cells in collaboration with Professor Pierre Vanderhaeghen and Researcher Ryohei Iwata's group at VIB-KU in Belgium. They visualized fluorescently labeled CREB and CBP at the single molecule level and carefully analyzed their spatiotemporal dynamics using oblique illumination microscopy, which illuminates only a portion of the nucleus. In collaboration with Professor Hiroshi Kimura's group at the Tokyo Institute of Technology, simultaneous imaging of CREB and RNAP II was achieved. The results revealed a two-step molecular mechanism.

First, before neural activity occurs, the nucleus of the neuron is dotted with microregions that are histone acetylated by CBP, where learning and memory-related genes are primed for rapid gene expression upon neural activity.

Second, CREB, together with CBP, acts as a hot spot upon stimulation and selectively binds to the DNA sequence of the gene for a few seconds in a repeated manner, thereby initiating gene expression through activated RNAPII accumulation. The group showed that these two steps are the molecular behaviors that lead to learning and memory.

Combining this imaging technique with induced pluripotent stem cell technology and applying it to patient-derived cells is expected to increase our understanding of the pathophysiology of neuropsychiatric disorders.

Sugo stated, "In this study, we were able to directly observe the formation of hot spots where proteins and genomic DNA meet in a spatiotemporal manner using an intranucleus single-molecule imaging method as a mechanism by which human neurons regulate gene expression in response to stimuli. It is expected to advance our understanding of learning and memory and the pathophysiology of neuropsychiatric disorders as 'molecular movements.'"

Journal Information
Publication: Cell Reports
Title: Repetitive CREB-DNA interactions at gene loci predetermined by CBP induce activity-dependent gene expression in human cortical neurons
DOI: 10.1016/j.celrep.2023.113576