Biology subjects

Imamura Kawasawa, Y.

Publications and source records attributed to Imamura Kawasawa, Y..

8 recordsLinked to original sources

bioRxiv2021

Neurexins in serotonergic neurons regulate serotonin transmission and complex mouse behaviors

Extensive serotonin (5-HT) innervation throughout the brain corroborates 5-HTs modulatory role in numerous cognitive activities. Volume transmission is the major mode for 5-HT transmission but mechanisms underlying 5-HT signaling are still largely unknown. Abnormal brain 5-HT levels and function have been implicated in autism spectrum disorder (ASD). Neurexin (Nrxn) genes encode presynaptic cell adhesion molecules important for the regulation of synaptic neurotransmitter release, notably glutamatergic and GABAergic transmission. Mutations in Nrxn genes are associated with neurodevelopmental disorders including ASD. However, the role of Nrxn genes in the 5-HT system is poorly understood. Here, we generated a mouse model with all three Nrxn genes disrupted specifically in 5-HT neurons to study how Nrxns affect 5-HT transmission. Loss of Nrxns in 5-HT neurons reduced the number of serotonin neurons in the early postnatal stage, impaired 5-HT release, and decreased 5-HT release sites and serotonin transporter expression. Furthermore, 5-HT neuron-specific Nrxn knockout reduced sociability and increased depressive-like behavior. Our results highlight functional roles for Nrxns in 5-HT neurotransmission, 5-HT neuron survival, and the execution of complex behaviors.

neuroscience↗

bioRxiv2020

DOT1L modulates the senescence-associated secretory phenotype through epigenetic regulation of IL1A

Cellular senescence is characterized as a stable cell cycle arrest that can occur as a stress response associated with oncogenic activation, termed oncogene-induced senescence (OIS). Cells undergoing OIS acquire a unique microenvironment termed the senescence-associated secretory phenotype (SASP), which can be both beneficial and detrimental in a context-dependent manner. Additionally, senescent cells are characterized by robust changes in their epigenome. Here, we globally assessed the histone landscape of cells induced to senesce by oncogenic RAS and discovered a novel epigenetic regulatory mechanism of the key SASP regulator IL1A. OIS cells displayed increased di- and tri-methylation of histone H3 lysine 79 (H3K79me2/3), two active histone marks. Depletion of the H3K79 methyltransferase disruptor of telomeric silencing 1-like (DOT1L) during OIS resulted in decreased H3K79me2/3 occupancy at the IL1A gene locus, which corresponded to decreased IL1A mRNA and cell surface expression. Decreased expression and secretion of downstream cytokines without a change in senescence markers were also observed upon DOT1L depletion. Overexpression of DOT1L increased H3K79me2/3 occupancy at the IL1A locus and upregulated the SASP, indicating that DOT1L is both necessary and sufficient for SASP gene expression. Mechanistically, we found that STING, an essential mediator of SASP transcription, is upstream of DOT1L in the epigenetic regulation of the SASP. Together, our studies establish DOT1L as an epigenetic regulator of the SASP whose expression is uncoupled from the senescence-associated cell cycle arrest, providing a potential strategy to inhibit the negative side effects of senescence while maintaining the beneficial inhibition of proliferation.

cell biology↗

bioRxiv2020

Neuroligin3 Splice Isoforms Shape Mouse Hippocampal Inhibitory Synaptic Function

Synapse formation is a dynamic process essential for neuronal circuit development and maturation. At the synaptic cleft, trans-synaptic protein-protein interactions constitute major biological determinants of proper synapse efficacy. The balance of excitatory and inhibitory synaptic transmission (E-I balance) stabilizes synaptic activity. Dysregulation of the E-I balance has been implicated in neurodevelopmental disorders including autism spectrum disorders. However, the molecular mechanisms underlying E-I balance remain to be elucidated. Here, we investigate Neuroligin (Nlgn) genes that encode a family of postsynaptic adhesion molecules known to shape excitatory and inhibitory synaptic function. We demonstrate that Nlgn3 protein differentially regulates inhibitory synaptic transmission in a splice isoform-dependent manner at hippocampal CA1 synapses. Distinct subcellular localization patterns of Nlgn3 isoforms contribute to the functional differences observed among splice variants. Finally, single-cell sequencing analysis reveals that Nlgn1 and Nlgn3 are the major Nlgn genes and that expression of Nlgn splice isoforms are highly diverse in CA1 pyramidal neurons.

neuroscience↗

bioRxiv2019

Identification of regulators of poly-ADP-ribose polymerase (PARP) inhibitor response through complementary CRISPR knockout and activation screens

Inhibitors of poly-ADP-ribose polymerase 1 (PARPi) are highly effective in killing cells deficient in the homologous recombination (HR) DNA repair pathway, such as those lacking BRCA2. In light of this, PARPi have been utilized in recent years to treat BRCA2-mutant tumors, with many patients deriving impressive clinical benefit. However, positive response to PARPi is not universal, even among patients with HR-deficient tumors. Here, we present the results of three genome-wide CRISPR knockout and activation screens which provide an unbiased look at genetic determinants of PARPi response in wildtype or BRCA2-knockout cells. Strikingly, we reveal that depletion of the histone acetyltransferase TIP60, a top hit from our screens, robustly reverses the PARPi sensitivity caused by BRCA2 deficiency. Mechanistically, we show that TIP60 depletion rewires double strand break repair in BRCA2-deficient cells by promoting 53BP1 binding to double strand breaks to suppress end resection. Our work provides a comprehensive set of putative biomarkers that serve to better understand and predict PARPi response, and identifies a novel pathway of PARPi resistance in BRCA2-deficient cells.

molecular biology↗

bioRxiv2019

Placental neurosteroids shape cerebellar development and social behaviour

Compromised placental function or premature loss has been linked to diverse neurodevelopmental disorders 1,2. The placenta is the first functional foetal endocrine organ, but the direct impact of placental hormone loss on foetal brain in late gestation has not been empirically tested. Allopregnanolone (ALLO) is a non-glucocorticoid, progesterone derivative that acts as a positive modulator of GABA-A receptor activity3 with the potential to alter critical GABA-mediated developmental processes 4,5. To directly test the role of placental ALLO, we generated a novel mouse model in which the gene encoding the synthetic enzyme for ALLO (Akr1c14) is specifically deleted in trophoblasts using a tissue-specific Cre-Lox strategy. ALLO concentrations are significantly decreased in late gestation in placenta and brain when placental Akr1c14 is removed, indicating placenta as the primary gestational ALLO source. We now demonstrate that targeted placental ALLO loss leads to permanent changes in brain development in a sex- and regionally-specific manner. Placental ALLO insufficiency led to male-specific cerebellar white matter (WM) abnormalities characterized by excess myelination with increased myelin protein expression, similar to changes reported in boys with autism spectrum disorders (ASD)6,7. Behavioural testing of these mice revealed increased repetitive behaviour and sociability deficits, two hallmarks of ASD, only in male offspring with placental ALLO insufficiency. Notably, a strong positive correlation was seen between the cerebellar contents of myelin basic protein (MBP) and the severity of ASD-like behaviours. A single injection of ALLO during gestation was sufficient to rescue both cerebellar MBP levels and aberrant behaviours. This study reveals a new role for a placental hormone in shaping specific brain structures and behaviours, and suggests that identifying placental hormone insufficiency or preterm loss may offer novel therapeutic opportunities to prevent later neurobehavioural disorders.

neuroscience↗

bioRxiv2019

Loss of microbial diversity and body site heterogeneity in individuals with Hidradenitis Suppurativa

Hidradenitis Suppurativa (HS) is a chronic, scarring, inflammatory skin disease affecting hair follicles in axillae, inguinal, and anogenital regions. Dysbiosis in HS patients compared to healthy subjects is documented. However, whether dysbiosis is specific to particular body sites or skin niches is unknown. We investigated the follicular and skin surface microbiome of the axilla and groin of HS patients (n=11) and healthy individuals (n=10) using 16S rRNA gene sequencing (V3-V4). We sampled non-lesional (HSN) and lesional skin (HSL) of HS patients. {beta}-diversity was significantly decreased (p<0.05) in HSN and HSL skin compared to normal skin with loss of body site and skin niche heterogeneity in HS samples. The relative bacterial abundance of specific microbes was also significantly different between normal and HSN (15 genera) or HSL (21 genera) skin. Smoking and alcohol use influenced the {beta}-diversity (p<0.08) in HS skin. We investigated metabolic profiles of bacterial communities in HS and normal skin using a computational approach. Metabolism, Genetic Information Processing, and Environmental Information Processing were significantly different between normal and HS samples. Altered metabolic pathways associated with dysbiosis of HS skin suggest mechanisms underlying the disease pathology and information about treatment with drugs targeting those pathways.

microbiology↗

bioRxiv2018

Repression of Transcription Factor AP-2 Alpha by Peroxisome Proliferator Activated Receptor Gamma Reveals a Novel Transcriptional Circuit in basal-squamous Bladder Cancer

The discovery of bladder cancer transcriptional subtypes provides an opportunity to identify high risk patients, and tailor disease management. Recent studies suggest tumor heterogeneity contributes to \"plasticity\" of molecular subtype during progression and following treatment. Nonetheless, the transcriptional drivers of the aggressive basal-squamous subtype remain unidentified. As PPAR{gamma} has been repeatedly implicated in the luminal subtype of bladder cancer, we hypothesized inactivation of this transcriptional master regulator during progression results in increased expression of basal-squamous specific transcription factors (TFs) which act to drive aggressive behavior. We initiated a pharmacologic and RNA-seq-based screen to identify PPAR{gamma}-repressed, basal-squamous specific TFs. Hierarchical clustering of RNA-seq data following treatment of a panel of human bladder cancer cell lines with a PPAR{gamma} agonist identified a number of TFs regulated by PPAR{gamma} activation, several of which are implicated in urothelial and squamous differentiation. One PPAR{gamma}-repressed TF implicated in squamous differentiation identified is Transcription Factor Activating Protein 2 alpha (TFAP2A). We show TFAP2A and its paralog TFAP2C are overexpressed in basal-squamous bladder cancer and in squamous areas of cystectomy samples, and that overexpression is associated with increased lymph node metastasis and distant recurrence, respectively. Biochemical analysis confirmed the ability of PPAR{gamma} activation to repress TFAP2A, while PPAR{gamma} antagonist studies indicate the requirement of a functional receptor. In vivo tissue recombination studies show TFAP2A and TFAP2C promote tumor growth in line with the aggressive nature of basal-squamous bladder cancer. Our findings suggest PPAR{gamma} inactivation, as well as TFAP2A and TFAP2C overexpression cooperate with other TFs to promote the basal-squamous transition.

cancer biology↗

bioRxiv2018

Effects of duplicated mapped read PCR artifacts on RNA-seq differential expression analysis based on qRNA-seq

Best practices to handling duplicated mapped reads in RNA-seq analyses has long been discussed but a gold standard method has yet to be established, as such duplicates could originate from valid biological transcripts or they could be PCR-related artifacts. Here we used the NEXTflex qRNA-SeqTM (aka Molecular Indexing) technology to identify PCR duplicates via the random attachment of unique molecular labels to each cDNA molecule prior to PCR amplification. We found that up to 64.3% of the single end and 19.3% of the mouse paired end duplicates originated from valid biological transcripts rather than PCR artifacts. For single end reads, either removing or retaining all duplicates resulted in a substantial number of false positives (up to 47.0%) and false negatives (up to 12.1%) in the sets of significantly differentially expressed genes. For paired end reads, only the alignment retaining all duplicates resulted in a substantial number of false positives. This is the first effort to evaluate the performance of qRNA-seq using real-world biomedical samples, and we found that PCR duplicate identification provided minor benefits for paired end reads but greatly improved the sensitivity and specificity in the determination of the significantly differentially expressed genes for single end reads.

genomics↗