In this study, social deficits were recovered through chemogenetic inhibition of Emx1-expressing cells in the vDG of Emx1-Cre;IRSp53 flox/flox mice, and protein and transcriptome experiments were conducted to investigate the mechanism of social deficits and their recovery. Behavioral experiments confirmed that chemogenetic activation of Emx1-expressing vDG cells in Emx1-Cre mice reduced social behavior, and chemical inhibition of Emx1-expressing vDG cells in Emx1-Cre;IRSp53 flox/flox mice improved social deficits. At the protein level, increased protein expression of CRHR1 and SOX2 was observed in the hippocampus of Emx1-Cre;IRSp53 flox/flox mice compared to control mice, and a decrease in CRHR1 was observed after chemogenetic inhibition of Emx1-Cre;IRSp53 flox/flox mice. Through bulk RNA sequencing of the hippocampus, changes in the regulation of multicellular organism processes and vitamin binding were revealed in Emx1-Cre;IRSp53 flox/flox mice, and differences in Htd2, Ccn1, and Atp6ap1l were identified. Single-cell RNA sequencing of the hippocampus showed that Eya1 expression was decreased in astrocytes, and Ecrg4 expression was increased in the microglia of Emx1-Cre;IRSp53 flox/flox mice.
In this study, we demonstrated the rescue of social deficits by chemogenetic inhibition of the vDG in Emx1-Cre;IRSp53 flox/flox mice, and we suggest that chemogenetic inhibition of Emx1-expressing neural stem cells, neurons, and glial cells is related to the recovery of sociability. A difference from previous studies showing social deficits through chemogenetic inhibition in Nestin-Cre WT mice is that nestin expression was restricted to early-stage neural stem and progenitor cells 28. Since Emx1 expression is present in the neural stem cells of newly born post-mitotic neurons, Cre-selective chemogenetic modulation may have affected Emx1-expressing cells with a wider cell growth cycle 10, 11, 14, 25, 29. Because we used an AAV vector from the hSyn promoter, the effects of neuron-specific chemical modulation may differ 27, 30–32. In particular, according to previous IRSp53 studies 6, 7, it can be inferred that the social deficits due to the increased excitability of newborn neurons in the vDG of IRSp53 knockout mice are corrected by chemogenetic inhibition. In addition, we found that increased neuronal excitability caused by chemogenetic activation induced social deficits in Emx1-Cre mice.
Stress-vulnerable individuals exhibit stronger activation of the HPA axis, including CRHR1, and fail to adapt to stress caused by relatively weak external environmental changes. Genome-wide association studies have shown that genes associated with stress vulnerability are also associated with HPA axis activation 33–37. Furthermore, stress vulnerability is studied in clinical areas such as PTSD and mood disorders 38–40. Psychiatric risk genes appear not only in one diagnostic category of mental disorders, such as schizophrenia, mood disorders, and autism spectrum disorders but also in vulnerable factors across multiple diagnostic categories. Genes known to be susceptible to stress include serotonin transporter, brain-derived neurotrophic factor, bromodomain 1, Cacna1a, Catechol-O-methyltransferase, Disrupted-in-schizophrenia-1, Estrogen receptor alpha, Fkbp5, Glutamate decarboxylase1, and Neuregulin1 41–46. In this study, we observed that the deletion of IRSp53 can lead to stress vulnerability phenomena associated with HPA axis activation, including increased CRHR1 and social deficits. We attempted to restore CRHR1 and social deficits through chemogenetic modulation of vDG.
CRHR activation is expected to be affected by environmental changes such as stress, but genetic factors also affect vulnerability 47, 48. CRHR activation in the hippocampus, especially in the dentate gyrus, improves synaptic function and structure when it acts for a short period; however, deteriorating effects, such as decreased numbers of synapses, appear when it acts for a long time 49. In particular, CRHR1 activation has been reported to increase the excitability of hippocampal CA1 glutamatergic neurons 50. The increased excitability of hippocampal neurons has been actively studied for its role in stress in epileptic disorders 51–55. Furthermore, the deletion of CRHR1 in telencephalon glutamatergic neurons results in decreased cognitive function, including memory impairment 56, 57. CRHR1 overexpression is associated with vulnerability to stress and increased alcohol consumption during stressful situations 58, 59. The mutant mice in this study were expected to have genetic susceptibilities due to IRSp53 deletion in telencephalic glutamatergic neurons without prominent stress triggers. We attempted to normalize changes in CRHR1 and social behavior using chemogenetic modulation in the vDG. Therefore, this study suggests that social abnormalities can be restored by correcting the increased excitability of the vDG caused by genetic or environmental problems.
Among the results of bulk RNA sequencing analyses, Htd2 (Hydroxyacyl-Thioester Dehydratase Type 2) is known to exist in adipocytes as a key enzyme in mitochondrial fatty acid synthesis (mtFAS) 60–63. Previous reports have suggested that abnormalities in mtFAS can impact CNS function and are associated with neurodegeneration 64. The extracellular matrix protein Ccn1 (a cystine-rich inducer of angiogenesis 61) is involved in repair, fibroblast senescence, and DNA damage response pathways 65. Ccn1 mutant mice showed exacerbated fibrosis, which was reversed by topical application of CCN1. In addition, increased CCN1 secretion in glioblastoma cells results in macrophage infiltration into the tumor tissue 66. Studies of psychedelics and stimulants that induced Ccn1 expression in the rodent neocortex suggest that Ccn1 is associated with the age of onset of schizophrenia 67. Atp6apl1 (ATPase proton transporting accessory protein 1 like) was found to be decreased in the PTSD patient group compared to the control group, and ATP6AP1l, a paralog of the vesicular ATPase whose function has not yet been identified, was considered one of the stress-related factors in a previous study 68–70. A comprehensive interpretation of the results of the bulk RNA sequencing study showed that, although no direct association with CRHR1 was found, factors that could have a wide range of effects on brain function were identified, particularly those clinically associated with schizophrenia, neurodegeneration, and stress-related disorders, such as PTSD.
According to single-cell RNA sequencing results, Eya1 plays an important role in cranial placode neurogenesis by acting as a cofactor for the homeobox transcription factor Six1 71, 72. Deletion of Eya1 (eye defects) causes BOR syndrome (branchio-oto-renal syndrome) and branchio-oto syndrome 73. Cases of autism spectrum disorder with BOR syndrome have been reported 74, and Eya1 is widely expressed throughout the brain, as well as in neurogenesis-related regions of the central nervous system related to vision and hearing 75. Ecrg4 (esophageal cancer-associated gene 4), also known as C2orf40, is a tumor suppressor gene identified in esophageal, breast, and colorectal carcinomas and gliomas 76, 77. It is also highly expressed in leukocytes, and ECRG4 knockout mice showed abnormal wound healing 76. Ecrg4 is expressed in the choroid plexus to produce augurin and functions in fetal brain development, cerebrospinal fluid homeostasis, and neural progenitor cell response to CNS injury 78. A single-nucleotide variant in rs34487851 upstream of Ecrg4 has been reported in Alzheimer's disease 79, 80. Studies using oligodendrocyte progenitor cells have shown that this gene is associated with neural aging 81.
Further studies are needed to determine the mechanism by which the deletion of IRSp53 and CRHR1 increases, the interaction between genetic susceptibility and different levels of environmental stress, and the key steps in treating social deficits during neural stem cell development, even in adults. One limitation of this study is that chemogenetic modulation was restricted to Emx1-expressing cells in the vDG. We focused on the expression of restricted proteins such as CRHR1. It is difficult to explain and connect directly the results from protein expression, such as CRHR1 and transcriptomics. Additionally, owing to the fragility of adult neurons, many neuronal losses are considered to occur during hippocampal dissociation for single-cell RNA sequencing. Thus, more meticulous protocols and sufficient experience are needed for successful hippocampal dissociation 82–85.
Chemogenetic inhibition of vDG significantly affected the recovery of social deficits in Emx1-Cre;IRSp53 flox/flox mice, and the restoration of CRHR1 reduction in the hippocampus was observed as a mechanism. Further studies are needed to determine whether regulating excitability in Emx1-expressing cells of the vDG helps mice with genetic susceptibility to stress or prominent environmental stress to recover from social abnormalities. If this approach helps adult mice with stress vulnerability and social deficits recover, it may provide clues for the treatment of psychiatric disorders in the future.