Hepatic Expression of the Na+-Taurocholate Cotransporting Polypeptide Is Independent from Genetic Variation.

The hepatic Na+-taurocholate cotransporting polypeptide NTCP/SLC10A1 is important for the uptake of bile salts and selected drugs. Its inhibition results in increased systemic bile salt concentrations. NTCP is also the entry receptor for the hepatitis B/D virus. We investigated interindividual hepatic SLC10A1/NTCP expression using various omics technologies. SLC10A1/NTCP mRNA expression/protein abundance was quantified in well-characterized 143 human livers by real-time PCR and LC-MS/MS-based targeted proteomics. Genome-wide SNP arrays and SLC10A1 next-generation sequencing were used for genomic analyses. SLC10A1 DNA methylation was assessed through MALDI-TOF MS. Transcriptomics and untargeted metabolomics (UHPLC-Q-TOF-MS) were correlated to identify NTCP-related metabolic pathways. SLC10A1 mRNA and NTCP protein levels varied 44-fold and 10.4-fold, respectively. Non-genetic factors (e.g., smoking, alcohol consumption) influenced significantly NTCP expression. Genetic variants in SLC10A1 or other genes do not explain expression variability which was validated in livers (n = 50) from The Cancer Genome Atlas. The identified two missense SLC10A1 variants did not impair transport function in transfectants. Specific CpG sites in SLC10A1 as well as single metabolic alterations and pathways (e.g., peroxisomal and bile acid synthesis) were significantly associated with expression. Inter-individual variability of NTCP expression is multifactorial with the contribution of clinical factors, DNA methylation, transcriptional regulation as well as hepatic metabolism, but not genetic variation.

Developmental dysregulation of excitatory-to-inhibitory GABA-polarity switch may underlie schizophrenia pathology: A monozygotic-twin discordant case analysis in human iPS cell-derived neurons.

Schizophrenia is a major psychiatric disorder, but the molecular mechanisms leading to its initiation or progression remain unclear. To elucidate the pathophysiology of schizophrenia, we used an in vitro neuronal cell culture model involving human induced pluripotent stem cells (hiPSCs) derived from a monozygotic-twin discordant schizophrenia pair. The cultured neurons differentiated from hiPSCs were composed of a mixture of glutamatergic excitatory neurons and gamma aminobutyric acid (GABA)ergic inhibitory neurons. In the electrophysiological analysis, a different pattern of spontaneous neuronal activity was observed under the condition without any stimulants. The frequency of spontaneous excitatory post-synaptic currents (sEPSCs) was significantly higher in the hiPSC-derived neurons of the patient with schizophrenia than in the control sibling at day-in-vitro 30. However, the synaptic formation was not different between the patient with schizophrenia and the control sibling during the same culture period. To explain underlying mechanisms of higher excitability of presynaptic cells, we focused on the potassium-chloride co-transporter KCC2, which contributes to excitatory-to-inhibitory GABA polarity switch in developing neurons. We also revealed the altered expression pattern of KCC2 in hiPSC-derived neurons from the patient with schizophrenia, which could contribute to understanding the pathology of schizophrenia in the developing nervous system.

LIFU Alleviates Neuropathic Pain by Improving the KCC2 Expression and Inhibiting the CaMKIV-KCC2 Pathway in the L4-L5 Section of the Spinal Cord.

Effective treatment remains lacking for neuropathic pain (NP), a type of intractable pain. Low-intensity focused ultrasound (LIFU), a noninvasive, cutting-edge neuromodulation technique, can effectively enhance inhibition of the central nervous system (CNS) and reduce neuronal excitability. We investigated the effect of LIFU on NP and on the expression of potassium chloride cotransporter 2 (KCC2) in the spinal cords of rats with peripheral nerve injury (PNI) in the lumbar 4-lumbar 5 (L4-L5) section. In this study, rats received PNI surgery on their right lower legs followed by LIFU stimulation of the L4-L5 section of the spinal cord for 4 weeks, starting 3 days after surgery. We used the 50% paw withdraw threshold (PWT50) to evaluate mechanical allodynia. Western blotting (WB) and immunofluorescence (IF) were used to calculate the expression of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), calcium/calmodulin-dependent protein kinase type IV (CaMKIV), phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB), and KCC2 in the L4-L5 portion of the spinal cord after the last behavioral tests. We found that PWT50 decreased (P < 0.05) 3 days post-PNI surgery in the LIFU- and LIFU+ groups and increased (P < 0.05) after 4 weeks of LIFU stimulation. The expression of p-CREB and CaMKIV decreased (P < 0.05) and that of KCC2 increased (P < 0.05) after 4 weeks of LIFU stimulation, but that of p-ERK1/2 (P > 0.05) was unaffected. Our study showed that LIFU could effectively alleviate NP behavior in rats with PNI by increasing the expression of KCC2 on spinal dorsal corner neurons. A possible explanation is that LIFU could inhibit the activation of the CaMKIV-KCC2 pathway.

S-acylation status of bile acid transporter hASBT regulates its function, metabolic stability, membrane expression, and phosphorylation state.

The human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) is the rate-limiting step of intestinal bile acid absorption in the enterohepatic circulation system of bile acids. Therefore, the regulation and stability of hASBT is vital in maintaining bile acid and cholesterol homeostasis and may serve as a potential target for cholesterol-related disorders. We hypothesized that post-translational mechanisms that govern hASBT function and regulation will provide novel insight on intestinal bile acid transport and homeostasis. In this study, we confirm the S-acylation status of hASBT via acyl biotin exchange in COS-1 cells and its impact on hASBT expression, function, kinetics, and protein stability. Using the acylation inhibitor, 2-bromopalmitate, we show that S-acylation is an important modification which modulates the function, surface expression, and maximal transporter flux (Jmax) of hASBT. By means of proteasome inhibitors, S-acylated hASBT was found to be cleared via the proteasome whereas a reduction in the palmitoylation status of hASBT resulted in rapid proteolytic degradation compared to the unmodified transporter. Screening of cysteine mutants in and or near transmembrane domains, some of which are exposed to the cytosol, confirmed Cys314 to be the predominate S-acylated residue. Lastly, we show that S-acylation was reduced in a mutant form of hASBT devoid of cytosolic facing tyrosine residues, suggestive of crosstalk between acylation and phosphorylation post-translational modification mechanisms.

Preventing neuronal edema increases network excitability after traumatic brain injury.

Edema is an important target for clinical intervention after traumatic brain injury (TBI). We used in vivo cellular resolution imaging and electrophysiological recording to examine the ionic mechanisms underlying neuronal edema and their effects on neuronal and network excitability after controlled cortical impact (CCI) in mice. Unexpectedly, we found that neuronal edema 48 hours after CCI was associated with reduced cellular and network excitability, concurrent with an increase in the expression ratio of the cation-chloride cotransporters (CCCs) NKCC1 and KCC2. Treatment with the CCC blocker bumetanide prevented neuronal swelling via a reversal in the NKCC1/KCC2 expression ratio, identifying altered chloride flux as the mechanism of neuronal edema. Importantly, bumetanide treatment was associated with increased neuronal and network excitability after injury, including increased susceptibility to spreading depolarizations (SDs) and seizures, known agents of clinical worsening after TBI. Treatment with mannitol, a first-line edema treatment in clinical practice, was also associated with increased susceptibility to SDs and seizures after CCI, showing that neuronal volume reduction, regardless of mechanism, was associated with an excitability increase. Finally, we observed an increase in excitability when neuronal edema normalized by 1 week after CCI. We conclude that neuronal swelling may exert protective effects against damaging excitability in the aftermath of TBI and that treatment of edema has the potential to reverse these effects.

Iodide Transporters in the Endometrium: A Potential Diagnostic Marker for Women with Recurrent Pregnancy Failures.

OBJECTIVE: The element iodine is an essential nutrient utilized by the thyroid glands, and deficiency of this element has been linked to reproductive failures. Iodide transporters are also present in reproductive tissues and cells of embryonic origin such as the endometrium and trophoblasts, respectively. The aim of this study is to understand if levels of iodide transporters are linked to pregnancy outcomes. SUBJECTS AND METHODS: RNA derived from endometrial biopsies from controls or women with recurrent reproductive failures was analyzed utilizing RT-PCR and targeted RNASeq. RESULTS: When compared to controls, women with 2 or more reproductive failures had a significant increase (>5 fold) in mRNA levels of the iodine transporters NIS and PENDRIN, but not thyroglobulin when probed vis RT-PCR. Targeted RNASeq analysis confirmed these findings when another group of patients were analyzed. CONCLUSION: These findings suggest possible abnormal iodine metabolism and a deficiency of iodine in endometrial tissues from some of the women with reproductive failures. We hypothesize from these findings that inorganic iodide and/or iodine is required for optimal cellular function in reproductive tissues, and that iodide transporters may potentially be used as a marker for infertility or for probing potential localized iodine deficiency that may not present in a typical thyroid panel analysis.

Spatiotemporal Changes of Cerebral Monocarboxylate Transporter 8 Expression.

Background: Mutations of monocarboxylate transporter 8 (MCT8), a thyroid hormone (TH)-specific transmembrane transporter, cause a severe neurodevelopmental disorder, the Allan-Herndon-Dudley syndrome. In MCT8 deficiency, TH is not able to reach those areas of the brain where TH uptake depends on MCT8. Currently, therapeutic options for MCT8-deficient patients are missing, as TH treatment is not successful in improving neurological deficits. Available data on MCT8 protein and transcript levels indicate complex expression patterns in neural tissue depending on species, brain region, sex, and age. However, information on human MCT8 expression is still scattered and additional efforts are needed to map sites of MCT8 expression in neurovascular units and neural tissue. This is of importance because new therapeutic strategies for this disease are urgently needed. Methods: To identify regions and time windows of MCT8 expression, we used highly specific antibodies against MCT8 to perform immunofluorescence labeling of postnatal murine brains, adult human brain tissue, and human cerebral organoids. Results: Qualitative and quantitative analyses of murine brain samples revealed stable levels of MCT8 protein expression in endothelial cells of the blood-brain barrier (BBB), choroid plexus epithelial cells, and tanycytes during postnatal development. Conversely, the neuronal MCT8 protein expression that was robustly detectable in specific brain regions of young mice strongly declined with age. Similarly, MCT8 immunoreactivity in adult human brain tissue was largely confined to endothelial cells of the BBB. Recently, cerebral organoids emerged as promising models of human neural development and our first analyses of forebrain-like organoids revealed MCT8 expression in early neuronal progenitor cell populations. Conclusions: With respect to MCT8-deficient conditions, our analyses not only strongly support the contention that the BBB presents a lifelong barrier to TH uptake but also highlight the need to decipher the TH transport role of MCT8 in early neuronal cell populations in more detail. Improving the understanding of the spatiotemporal expression in latter barriers will be critical for therapeutic strategies addressing MCT8 deficiency in the future.

Novel Chimeric Immuno-Oncolytic Virus CF33-hNIS-antiPDL1 for the Treatment of Pancreatic Cancer.

BACKGROUND: Peritoneal carcinomatosis (PC) from pancreatic ductal adenocarcinoma (PDAC) is fatal. Our preclinical study presents an effective treatment against PDAC PC using a novel oncolytic viral agent, CF33-hNIS-antiPDL1. STUDY DESIGN: CF33-hNIS-antiPDL1 is a genetically engineered chimeric orthopoxvirus, CF33, armed with the human Sodium Iodide Symporter (hNIS) and anti-PD-L1 antibody (anti-PD-L1). The in vitro cytotoxic ability of this virus against 5 PDAC cell lines was tested at various doses (multiplicity of infection [MOI] = 0.01, 0.1, 1, 10). Production and blockade function of virus-encoded anti-PD-L1 antibody were verified using immunoblot, immunoprecipitation, and PD-1/PD-L1 bioassay. In vivo mouse models of PC, with or without subcutaneous (SC) tumors, created by injecting AsPC-1-ffluc cells into nude mice, were treated with PBS or a single dose (1×105 plaque-forming units) of either intraperitoneal (IP) or IV injection of CF33-hNIS-antiPDL1. Mice with PC tumors were treated on days 0, 2, or 14 after tumor implantation. RESULTS: CF33-hNIS-antiPDL1 killed PDAC cells in a dose-dependent manner, achieving >90% cell killing by day 8. Cells infected with CF33-hNIS-antiPDL1 produced bioactive anti-PD-L1 antibody, which blocked PD-1/PD-L1 interaction. In vivo, a single dose of virus reduced tumor burden and prolonged survival of treated mice. It was observed that IP administration of CF33-hNIS-antiPDL1 was more effective than IV administration. CONCLUSIONS: CF33-hNIS-antiPDL1 virus is effective in infecting and killing human PDACs and producing functional anti-PD-L1 antibody. Intraperitoneal delivery of CF33-hNIS-antiPDL1 effectively reduces peritoneal tumor burden and improves survival after only 1 dose and is superior to IV delivery.

Expression of the organic cation/carnitine transporter family (Octn1,-2 and-3) in mdx muscle and heart: Implications for early carnitine therapy in Duchenne muscular dystrophy to improve cellular carnitine homeostasis.

INTRODUCTION: Carnitine is essential for long-chain fatty acid oxidation in muscle and heart. Tissue stores are regulated by organic cation/Cn transporter plasmalemmal Octn2. We previously demonstrated low carnitine in quadriceps/gluteus and heart of adult mdx mice. METHODS: We studied protein and mRNA expression of Octn2, mitochondrial Octn1 and peroxisomal Octn3 in adult male C57BL/10ScSn-DMD mdx/J quadriceps, heart, and diaphragm compared to C57BL/10SnJ mice. RESULTS: We demonstrated reduction in mOctn2 expression on Western blot and similar expression of mOctn1 and mOctn3 in mdx quadriceps, heart and diaphragm. There was a significant upregulation of mOctn1 and mOctn2 mRNA by qRT-PCR in mdx quadriceps and of mOctn2 and mOctn3 mRNA in mdx heart. We showed upregulation of mdx mOctn1 and mOctn3 mRNA but no increase in protein expression. DISCUSSION: Dystrophin deficiency likely disrupts Octn2 expression decreasing muscle carnitine uptake thus contributing to membranotoxic long-chain acyl-CoAs with sarcolemmal and organellar membrane oxidative injury providing a treatment rationale for early L-carnitine in DMD.

CAIX is a predictor of pathological complete response and is associated with higher survival in locally advanced breast cancer submitted to neoadjuvant chemotherapy.

BACKGROUND: Locally advanced breast cancer often undergoes neoadjuvant chemotherapy (NAC), which allows in vivo evaluation of the therapeutic response. The determination of the pathological complete response (pCR) is one way to evaluate the response to neoadjuvant chemotherapy. However, the rate of pCR differs significantly between molecular subtypes and the cause is not yet determined. Recently, the metabolic reprogramming of cancer cells and its implications for tumor growth and dissemination has gained increasing prominence and could contribute to a better understanding of NAC. Thus, this study proposed to evaluate the expression of metabolism-related proteins and its association with pCR and survival rates. METHODS: The expression of monocarboxylate transporters 1 and 4 (MCT1 and MCT4, respectively), cluster of differentiation 147 (CD147), glucose transporter-1 (GLUT1) and carbonic anhydrase IX (CAIX) was analyzed in 196 locally advanced breast cancer samples prior to NAC. The results were associated with clinical-pathological characteristics, occurrence of pCR, disease-free survival (DFS), disease-specific survival (DSS) and overall survival (OS). RESULTS: The occurrence of pCR was higher in the group of patients whith tumors expressing GLUT1 and CAIX than in the group without expression (27.8% versus 13.1%, p = 0.030 and 46.2% versus 13.5%, p = 0.007, respectively). Together with regional lymph nodes staging and mitotic staging, CAIX expression was considered an independent predictor of pCR. In addition, CAIX expression was associated with DFS and DSS (p = 0.005 and p = 0.012, respectively). CONCLUSIONS: CAIX expression was a predictor of pCR and was associated with higher DFS and DSS in locally advanced breast cancer patients subjected to NAC.

KCC2 expression levels are reduced in post mortem brain tissue of Rett syndrome patients.

Rett Syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the Methyl CpG binding protein 2 (MECP2) gene. Deficient K+-Cl-co-transporter 2 (KCC2) expression is suggested to play a key role in the neurodevelopmental delay in RTT patients' neuronal networks. KCC2 is a major player in neuronal maturation by supporting the GABAergic switch, through the regulation of neuronal chlorine homeostasis. Previous studies suggest that MeCP2 mutations lead to changed KCC2 expression levels, thereby causing a disturbance in excitation/inhibition (E/I) balance. To investigate this, we performed protein and RNA expression analysis on post mortem brain tissue from RTT patients and healthy controls. We showed that KCC2 expression, in particular the KCC2a isoform, is relatively decreased in RTT patients. The expression of Na+-K+-Cl- co-transporter 1 (NKCC1), responsible for the inward transport of chlorine, is not affected, leading to a reduced KCC2/NKCC1 ratio in RTT brains. Our report confirms KCC2 expression alterations in RTT patients in human brain tissue, which is in line with other studies, suggesting affected E/I balance could underlie neurodevelopmental defects in RTT patients.

Overexpression of SLC12A5 is associated with tumor progression and poor survival in ovarian carcinoma.

INTRODUCTION: The solute carrier family 12 member 5 (SLC12A5) gene is playing a putative oncogenic role in colorectal carcinoma. However, the status of SLC12A5 amplification and expression in ovarian carcinoma and its potential clinical and/or prognostic significance has not yet been investigated. METHODS: In the present study, semi-quantitative staining and fluorescence in situ hybridization were used to investigate SLC12A5 protein expression and gene amplification levels. Samples were obtained from archival, formalin-fixed, paraffin-embedded pathological specimens consisting of 30 normal ovaries, 30 ovarian cystadenomas, 30 borderline ovarian tumors, and 147 invasive ovarian carcinomas. SLC12A5 immunohistochemical staining results, pathological parameters, and patient prognosis were then evaluated using various statistical models. Patient survival rate was also assessed using receiver-operator curve analysis. RESULTS: Our results revealed no SLC12A5 protein overexpression in normal ovaries. However, 7% of cystadenomas had SLC12A5 protein overexpression along with 17% of borderline tumors and 37% of ovarian carcinomas (P<0.01). Amplification of SLC12A5 was detected in 10.3% of ovarian carcinomas. Further correlational analyses showed that SLC12A5 protein overexpression in ovarian carcinomas was significantly associated with ascending histological grade, pT/pN/pM status, as well as FIGO stage (P<0.05). A subsequent univariate survival analysis of our ovarian carcinoma cohorts resulted in a significant association between SLC12A5 protein overexpression and decreased patient survival (44.3 and 85.9 months for high and low SLC12A5 protein expression, respectively; P<0.001). Importantly, additional multivariate analysis revealed that SLC12A5 protein expression was a significant, independent prognostic factor for overall survival in ovarian carcinoma patients (P=0.003). CONCLUSIONS: Collectively, these findings support the conclusion that SLC12A5 protein overexpression could indicate an invasive and/or aggressive phenotype of ovarian carcinoma. Future work will need to investigate whether SLC12A5 protein can serve as an independent prognostic molecular marker in patients with ovarian carcinoma.

Expression of Monocarboxylate Transporter 1 Is Associated With Better Prognosis and Reduced Nodal Metastasis in Pancreatic Ductal Adenocarcinoma.

OBJECTIVES: Because lactate is believed to support tumor growth, monocarboxylate transporters (MCTs), which transport lactate, have been investigated in multiple tumors. However, the significance of MCTs in pancreatic cancer is unclear. METHODS: A retrospective survey was conducted on 240 patients who underwent surgical resection for pancreatic ductal adenocarcinoma without preoperative treatment. The expression of MCT1, MCT2, MCT3, MCT4, and the glucose transporter 1 (GLUT1) was assessed in tumor cells and cancer-associated fibroblasts (CAFs) by tissue microarrays and immunohistochemistry. The impact of their expression on patient outcome and clinicopathological characteristics was also analyzed. RESULTS: In tumor cells, MCT1, MCT2, MCT3, MCT4, and GLUT1 were detected in 52 (22%), 31 (13%), 149 (62%), 204 (85%), and 235 (98%) cases, respectively. In CAFs, MCT2, MCT4, and GLUT1 were detected in 9 (3.8%), 178 (74%), and 36 (15%) cases, respectively. In tumor cells, MCT1 expression was associated with extended overall and progression-free survival and decreased nodal metastasis. Conversely, MCT4 expression in CAFs was associated with shortened survival. CONCLUSIONS: In tumor cells, MCT1 expression is associated with better prognosis and reduced nodal metastasis in pancreatic cancer, contrary to findings of past in vitro studies. Conversely, MCT4 expression in CAFs is indicative of worse prognosis.

Decreased expression of SLC16A12 mRNA predicts poor prognosis of patients with clear cell renal cell carcinoma.

Solute carrier family 16, member 12 (SLC16A12) is a highly -expressed protein in the kidney and has been reported to participate in the transport of creatine. However, the clinical values of SLC16A12 in clear cell renal cell carcinoma (ccRCC) have not been explored.SLC16A12 RNA-seq data and clinical information were downloaded from the Cancer Genome Atlas (TCGA) database. We compared its expression in ccRCC and paracancerous tissues, then the result was further validated with our cohort. The impact on the clinical significance of SLC16A12 in ccRCC was also assessed.Compared with paracancerous tissue, SLC16A12 was significantly downregulated in the tumor tissues both in mRNA and protein level. In TCGA cohort, SLC16A12 mRNA expression was associated with several clinicopathological parameters, including T stages (P < .001), M stages (P = .009), TNM stages (P < .001), and differentiated grades (P = .001). Kaplan-Meier analysis showed that the overall survival of patients with low expression of SLC16A12 mRNA was significantly worse than that of patients with high expression (P < .001). Furthermore, both univariate (HR = 0.371, 95%CI: 0.269-0.513, P < .001) and multivariate (HR = 0.485, 95%CI: 0.297-0.793, P = .004) Cox regression analyses suggested that low expression of SLC16A12 mRNA was an independent prognostic factor for patients with ccRCC.Overall, we uncovered that decreased expression of SLC16A12 is a poor prognostic factor for patients with ccRCC. SLC16A12 might be a potential biomarker and therapeutic target in ccRCC.

Regulation of renal NaDC1 expression and citrate excretion by NBCe1-A.

Citrate is critical for acid-base homeostasis and to prevent calcium nephrolithiasis. Both metabolic acidosis and hypokalemia decrease citrate excretion and increase expression of Na+-dicarboxylate cotransporter 1 (NaDC1; SLC13A2), the primary protein involved in citrate reabsorption. However, the mechanisms transducing extracellular signals and mediating these responses are incompletely understood. The purpose of the present study was to determine the role of the Na+-coupled electrogenic bicarbonate cotransporter (NBCe1) A variant (NBCe1-A) in citrate metabolism under basal conditions and in response to acid loading and hypokalemia. NBCe1-A deletion increased citrate excretion and decreased NaDC1 expression in the proximal convoluted tubules (PCT) and proximal straight tubules (PST) in the medullary ray (PST-MR) but not in the PST in the outer medulla (PST-OM). Acid loading wild-type (WT) mice decreased citrate excretion. NaDC1 expression increased only in the PCT and PST-MR and not in the PST-MR. In NBCe1-A knockout (KO) mice, the acid loading change in citrate excretion was unaffected, changes in PCT NaDC1 expression were blocked, and there was an adaptive increase in PST-MR. Hypokalemia in WT mice decreased citrate excretion; NaDC1 expression increased only in the PCT and PST-MR. NBCe1-A KO blocked both the citrate and NaDC1 changes. We conclude that 1) adaptive changes in NaDC1 expression in response to metabolic acidosis and hypokalemia occur specifically in the PCT and PST-MR, i.e., in cortical proximal tubule segments; 2) NBCe1-A is necessary for normal basal, metabolic acidosis and hypokalemia-stimulated citrate metabolism and does so by regulating NaDC1 expression in cortical proximal tubule segments; and 3) adaptive increases in PST-OM NaDC1 expression occur in NBCe1-A KO mice in response to acid loading that do not occur in WT mice.

Retrotransposon elements among initial sites of hepatitis B virus integration into human genome in the HepG2-NTCP cell infection model.

Integration of hepatitis B virus (HBV) DNA into host's genome is evident in all stages and models of HBV infection. Investigations of the initial virus-host junctions have been just recently initiated since their nature may promote liver oncogenesis immediately following infection. We examined the time-frame and host sites at which HBV integrates in HepG2 cells overexpressing sodium taurocholate co-transporting polypeptide (NTCP) receptor mediating HBV entry. HepG2-NTCP cells were analyzed from 15 min to 13 days post-infection (p.i.). The results showed that except for 15 min p.i., HBV-host integrations were detected at all time points thereafter. At 30 min p.i., virus junctions with retrotransposon SINE and with neuroblastoma breakpoint family member 1 gene were detected. At one-hour p.i., HBV integration with retrotransposon THE-1B-LTR was identified, while virus insertions into proline-rich protein and protein kinase cGMP-dependent type 1 encoding genes were found at 3 h p.i. Fusion with runt-related transcription factor 1 was detected at 24 h p.i. and merges with 9 different genes at 13 day p.i. The data showed that retrotransposon elements are frequent among first-hit sites of HBV insertion. This may suggest a mechanism by which HBV DNA may spread across host's genome from earliest stages of infection.

A triheptanoin-supplemented diet rescues hippocampal hyperexcitability and seizure susceptibility in FoxG1+/- mice.

The Forkhead Box G1 (FOXG1) gene encodes a transcription factor with an essential role in mammalian telencephalon development. FOXG1-related disorders, caused by deletions, intragenic mutations or duplications, are usually associated with severe intellectual disability, autistic features, and, in 87% of subjects, epileptiform manifestations. In a subset of patients with FoxG1 mutations, seizures remain intractable, prompting the need for novel therapeutic options. To address this issue, we took advantage of a haploinsufficient animal model, the FoxG1+/- mouse. In vivo electrophysiological analyses of FoxG1+/- mice detected hippocampal hyperexcitability, which turned into overt seizures upon delivery of the proconvulsant kainic acid, as confirmed by behavioral observations. These alterations were associated with decreased expression of the chloride transporter KCC2. Next, we tested whether a triheptanoin-based anaplerotic diet could have an impact on the pathological phenotype of FoxG1+/- mice. This manipulation abated altered neural activity and normalized enhanced susceptibility to proconvulsant-induced seizures, in addition to rescuing altered expression of KCC2 and increasing the levels of the GABA transporter vGAT. In conclusion, our data show that FoxG1 haploinsufficiency causes dysfunction of hippocampal circuits and increases the susceptibility to a proconvulsant insult, and that these alterations are rescued by triheptanoin dietary treatment.

Role of epigenetic mechanisms in transmitting the effects of neonatal sevoflurane exposure to the next generation of male, but not female, rats.

BACKGROUND: Clinical studies report learning disabilities and attention-deficit/hyperactivity disorders in those exposed to general anaesthesia early in life. Rats, primarily males, exposed to GABAergic anaesthetics as neonates exhibit behavioural abnormalities, exacerbated responses to stress, and reduced expression of hypothalamic K+-2Cl- Cl- exporter (Kcc2). The latter is implicated in development of psychiatric disorders, including male predominant autism spectrum disorders. We tested whether parental early life exposure to sevoflurane, the most frequently used anaesthetic in paediatrics, affects the next generation of unexposed rats. METHODS: Offspring (F1) of unexposed or exposed to sevoflurane on postnatal day 5 Sprague-Dawley rats (F0) were subjected to behavioural and brain gene expression evaluations. RESULTS: Male, but not female, progeny of sevoflurane-exposed parents exhibited abnormalities in behavioural testing and Kcc2 expression. Male F1 rats of both exposed parents exhibited impaired spatial memory and expression of hippocampal and hypothalamic Kcc2. Offspring of only exposed sires had abnormalities in elevated plus maze and prepulse inhibition of startle, but normal spatial memory and impaired expression of hypothalamic, but not hippocampal, Kcc2. In contrast to exposed F0, their progeny exhibited normal corticosterone responses to stress. Bisulphite sequencing revealed increased CpG site methylation in the Kcc2 promoter in F0 sperm and F1 male hippocampus and hypothalamus that was in concordance with the changes in Kcc2 expression in specific F1 groups. CONCLUSIONS: Neonatal exposure to sevoflurane can affect the next generation of males through epigenetic modification of Kcc2 expression, while F1 females are at diminished risk.

Postnatal Changes in K+/Cl- Cotransporter-2 Expression in the Forebrain of Mice Bearing a Mutant Nicotinic Subunit Linked to Sleep-Related Epilepsy.

The Na+/K+/Cl- cotransporter-1 (NKCC1) and the K+/Cl- cotransporter-2 (KCC2) set the transmembrane Cl- gradient in the brain, and are implicated in epileptogenesis. We studied the postnatal distribution of NKCC1 and KCC2 in wild-type (WT) mice, and in a mouse model of sleep-related epilepsy, carrying the mutant ß2-V287L subunit of the nicotinic acetylcholine receptor (nAChR). In WT neocortex, immunohistochemistry showed a wide distribution of NKCC1 in neurons and astrocytes. At birth, KCC2 was localized in neuronal somata, whereas at subsequent stages it was mainly found in the somatodendritic compartment. The cotransporters' expression was quantified by densitometry in the transgenic strain. KCC2 expression increased during the first postnatal weeks, while the NKCC1 amount remained stable, after birth. In mice expressing ß2-V287L, the KCC2 amount in layer V of prefrontal cortex (PFC) was lower than in the control littermates at postnatal day 8 (P8), with no concomitant change in NKCC1. Consistently, the GABAergic excitatory to inhibitory switch was delayed in PFC layer V of mice carrying ß2-V287L. At P60, the amount of KCC2 was instead higher in mice bearing the transgene. Irrespective of genotype, NKCC1 and KCC2 were abundantly expressed in the neuropil of most thalamic nuclei since birth. However, KCC2 expression decreased by P60 in the reticular nucleus, and more so in mice expressing ß2-V287L. Therefore, a complex regulatory interplay occurs between heteromeric nAChRs and KCC2 in postnatal forebrain. The pathogenetic effect of ß2-V287L may depend on altered KCC2 amounts in PFC during synaptogenesis, as well as in mature thalamocortical circuits.

Changes in the expression and localization of signaling molecules in mouse facial motor neurons during regeneration of facial nerves.

After injury, peripheral axons usually re-extend toward their target, and neuronal functions recover. Previous studies have reported that expression of various molecules are transiently altered in motor neurons after nerve injury, but the time course of these changes and their relationship with functional recovery have not been clearly demonstrated. We used the mouse facial nerve transection and suturing model, and examined the changes in expression of five molecules, choline acetyl transferase (ChAT), galanin, calcitonin gene-related protein (CGRP), gephyrin, and potassium chloride co-transporter 2 (KCC2) in the facial motor neurons after surgery until recovery. Number of ChAT-positive neurons was markedly decreased at days 3 and 7, and recovered to the normal level by day 60, when facial motor functions recovered. Localization of two neuropeptides, CGRP and galanin, was increased in the perikarya and axons during regeneration, and returned to the normal levels by days 60 and 28, respectively. Expression of two postsynaptic elements of γ-amino butyric acid synapses, gephyrin and KCC2, was decreased at days 3 and 7, and recovered by day 60. These results suggest that ChAT, CGRP, and KCC2 may be objective indicators of regeneration, and altering their expression may be related to the functional recovery and axonal re-extension.