Biology subjects

Oguro-Ando, A.

Publications and source records attributed to Oguro-Ando, A..

5 recordsLinked to original sources

bioRxiv2025

CYFIP1 overexpression amplifies IL-6/STAT3 and IFN-γ/STAT1 signaling: potential implications for neuroinflammation and autism spectrum disorder

Autism spectrum disorder (ASD) encompasses a group of neurodevelopmental disorders characterised by impaired social interaction, delayed language development, and repetitive or restrictive behaviours. While both genetic and environmental factors contribute to ASD, the specific molecular mechanisms underlying these interactions remain unclear. In a recent preprint, we showed that cytokine signaling may be dysregulated in chromosome 15q-duplication syndrome (Dup(15q)), one of the most common syndromic forms of ASD. Specifically, Dup(15q) induced pluripotent stem cells (iPSC)-derived neurons exhibit an amplified Signal Transducer and Activator of Transcription-3 (STAT3) response to Interleukin-6 (IL-6), a proinflammatory cytokine often upregulated in ASD. To identify which gene in the 15q region may be responsible for modifying cytokine signalling responses in Dup(15q), we focus on the Cytoplasmic FMRP-Interacting Protein 1 (CYFIP1) gene, as increased CYFIP1 dosage in Dup(15q) is associated with increased severity of neurobehavioral symptoms in individuals with Dup(15q) and CYFIP1 dysregulation has been linked to altered expression of various genes involved in immunoregulatory signalling pathways. Here, we explore the eeects of CYFIP1 overexpression (CYFIP1-OE) on cytokine signaling, demonstrating that CYFIP1-OE in HEK293 cells modifies the expression and activity of several cytokine signaling-related transcription factors, including STAT3 and STAT1. Additionally, we use SH-SY5Y neuroblastoma cells to assess neuron-related phenotypes, showing that CYFIP1-OE alters IL-6-induced neurite outgrowth. These findings provide novel insights into how CYFIP1 may contribute to dysregulated cytokine responses in ASD, advancing our understanding of the molecular mechanisms that underlie neuroinflammatory processes in this disorder.

molecular biology↗

bioRxiv2025

Sleeping ORANGE: A CRISPR-Transposase Hybrid Approach to Boost Endogenous Protein Tagging Efficiency

BackgroundInvestigating the subcellular distribution of proteins is crucial for understanding complex cell behaviours and disease mechanisms, and fluorescence microscopy has become a key tool for visualising protein localisation. Endogenous protein tagging, where the sequence for a tag (typically a peptide or fluorescent protein) is integrated into the native genetic sequence encoding a protein of interest, enables proteins to be visualised without the need for antibodies against the target protein. ORANGE (Open Resource for the Application of Neuronal Genome Editing) is a CRISPR-Cas9-based endogenous protein tagging technique which relies on homology-independent targeted integration (HITI)-mediated gene editing. Utilising HITI as the DNA repair pathway of choice gives ORANGE the advantage of being more efficient than classical homology-directed repair (HDR)-based endogenous protein tagging techniques and additionally, means it can be used in post-mitotic cells. ResultsWe applied the ORANGE system to tag three proteins, CYFIP1, JAKMIP1, and STAT3, and confirmed that the expressed fusion proteins demonstrate expected subcellular localisations through fluorescence microscopy. Unexpectedly, the efficiency of ORANGE editing was less than 1% in HEK293 cells, despite high transfection efficiency. To improve the editing efficiency associated with ORANGE, we combined the ORANGE method with an established Sleeping Beauty transposase/CRISPR-Cas9 fusion technique, which has been shown to enhance HITI-mediated gene editing. Using this new method, which we term Sleeping ORANGE, we successfully tagged CYFIP1 with the fluorescent protein mNeonGreen. Importantly, quantitative analysis by fluorescence microscopy and flow cytometry demonstrated an increase in editing efficiency using Sleeping ORANGE, with an approximately 12.85-fold increase in the percentage of mNeonGreen-expressing cells at 72 hours post-transfection relative to populations of cells edited with the ORANGE method. ConclusionsWe have incorporated the DNA-binding domain of the Sleeping Beauty transposase to create a new system that improves the gene-editing efficiency of the ORANGE technique. With further developments to optimise CRISPR gRNA design and reduce off-target effects, the Sleeping ORANGE technique may form a valuable tool for researchers to better understand subcellular localisation and dynamics.

molecular biology↗

bioRxiv2025

Exploring the functions of JAKMIP1 in neuronal IL-6/STAT3 signaling and its relevance to chromosome 15q-duplication syndrome

Growing evidence supports neuroinflammation as a risk factor for neurodevelopmental and psychiatric disorders. Interleukin 6 (IL-6), a classical pro-inflammatory cytokine, has been associated with autism spectrum disorder (ASD)-related phenotypes. To better understand molecular factors that modify neuronal cytokine responses in ASD, we investigated potential roles for JAKMIP1, a gene linked to chromosome 15q-duplication syndrome (Dup15q; a form of syndromic ASD), in regulating IL-6/STAT3 signaling. We observe that JAKMIP1 deficiency impairs IL-6/STAT3 signaling and IL-6-induced neuritogenesis in SH-SY5Y cells; and discover that JAKMIP1 may regulate STAT3 expression via its C-terminus, which exhibits nucleoplasmic localization. Additionally, we find that IL-6/STAT3 signaling is altered in Dup15q hiPSCs-derived cortical neurons, which display heightened responsiveness to IL-6; though it is unclear whether and how JAKMIP1 contributes to this. Overall, our findings identify JAKMIP1 as a modulator of neuronal IL-6/STAT3 signaling and support that ASD-linked genetic variants can alter the inflammatory landscape of ASD.

neuroscience↗

bioRxiv2023

Regulation of astrocyte metabolism by mitochondrial translocator protein 18kDa

The mitochondrial translocator protein 18kDa (TSPO) has been linked to a variety of functions from steroidogenesis to regulation of cellular metabolism and is an attractive therapeutic target for chronic CNS inflammation. Studies in the periphery using Leydig cells and hepatocytes, as well as work in microglia, indicate that the function of TSPO may vary between cells depending on their specialised roles. Astrocytes are critical for providing trophic and metabolic support in the brain as part of their role in maintaining brain homeostasis. Recent work has highlighted that TSPO expression increases in astrocytes under inflamed conditions and may drive astrocyte reactivity. However, relatively little is known about the role TSPO plays in regulating astrocyte metabolism and whether this protein is involved in immunometabolic processes in these cells. Using TSPO-deficient (TSPO-/-) mouse primary astrocytes in vitro (MPAs) and a human astrocytoma cell line (U373 cells), we performed metabolic flux analyses. We found that loss of TSPO reduced basal astrocyte respiration and increased the bioenergetic response to glucose reintroduction following glucopenia, while increasing fatty acid oxidation (FAO). Lactate production was significantly reduced in TSPO-/- astrocytes. Co-immunoprecipitation studies in U373 cells revealed that TSPO forms a complex with carnitine palmitoyltransferase 1a, which presents a mechanism wherein TSPO may regulate FAO in astrocytes. Compared to TSPO+/+ cells, inflammation induced by 3h lipopolysaccharide (LPS) stimulation of TSPO-/- MPAs revealed attenuated tumour necrosis factor release, which was enhanced in TSPO-/- MPAs at 24h LPS stimulation. Together these data suggest that while TSPO acts as a regulator of metabolic flexibility in astrocytes, loss of TSPO does not appear to modulate the metabolic response of astrocytes to inflammation, at least in response to the stimulus/time course used in this study.

neuroscience↗

bioRxiv2023

CNTN4 modulates neural elongation through interplay with APP

The neuronal cell adhesion molecule contactin-4 (CNTN4) has been genetically linked to autism spectrum disorders (ASD) and other psychiatric disorders. The Cntn4-deficient mouse model has previously shown that CNTN4 has important roles in axon guidance and synaptic plasticity in the hippocampus. However, the pathogenesis and functional role of CNTN4 in the cortex have not yet been investigated. Using Nissl staining, immunohistochemistry and Golgi staining the motor cortex of Cntn4-/- mice was analysed for abnormalities. Interacting partners of CNTN4 were identified by immunoprecipitation and mass spectrometry. Further analysis of the interaction between CNTN4 and APP utilised knockout human cells generated via CRISPR-Cas9 gene editing. Our study newly identified reduced cortical thickness in the motor cortex of Cntn4-/- mice, but cortical cell migration and differentiation were unaffected. Significant morphological changes were observed in neurons in the M1 region of the motor cortex, indicating that CNTN4 is also involved in the morphology and spine density of neurons in the motor cortex. Furthermore, mass spectrometry analysis identified an interaction partner for CNTN4, and we confirmed an interaction between CNTN4 and APP. Knockout human cells of CNTN4 and/or APP revealed a relationship between CNTN4 and APP. This study demonstrates that CNTN4 contributes to cortical development, and that its binding and interplay with APP controls neural elongation. This is an important finding for understanding the function of APP, a target protein for Alzheimers disease. The binding between Cntn4 and APP, which is involved in neurodevelopment, is essential for healthy nerve outgrowth.

neuroscience↗