Brain Isoform Research Articles

Phosphorylation of STAT3 by Human Serotonin 2C Receptor

Serotonin (5‐HT) is an indolamine neurotransmitter involved in a variety of functions including regulation of mood, appetite, sleep, learning and memory, and neuronal excitability. 5‐HT elicits its effects by binding to at least 14 different receptor subtypes that mediate both excitatory and inhibitory neurotransmission. The many classes of serotonin receptors have been subjected to significant studies in order to discern the functionality and produce effective therapeutic targets. One of the receptors, the 5‐HT2C receptor, has been studied and used as a target for antipsychotic medication. 5‐HT2C subtype receptor exists as a 7‐transmembrane spanning G protein‐coupled receptor (GPCR) found in the central nervous system, and is the only GPCR known to undergo RNA editing, creating 14 known isoforms in the human brain which affect its ability to couple with G proteins and other signaling proteins. The structurally similar 5‐HT2A receptor has been shown to activate the G protein independent JAK/STAT pathway. The purpose of this study was to determine if the human 5‐HT2C receptor also activates the JAK/STAT pathway. Human Embryonic Kidney (HEK) 293 cells were transfected with human 5‐HT2C receptors to produce stable cell lines. Cells were then treated with vehicle (control), 5‐HT, olanzapine (antagonist), and SB206553 (inverse agonist) for 30 minutes or 1 hour. Treated cells were lysed, proteins were separated by SDS‐PAGE, and levels of phosphorylated JAK2 and STAT3 were analyzed using western blots. In cells treated for 30 minutes with vehicle, phosphorylation of STAT3 was observed in cells transfected with the 5‐HT2C receptor but not in untransfected cells indicating constitutive phosphorylation of STAT3 by the 5‐HT2C receptor. Upon treatment with serotonin, phosphorylated levels of STAT3 were increased in cells expressing the 5‐HT2C receptor while STAT3 phosphorylation was completely blocked with cells treated with olanzapine or SB206553. Phosphorylated levels of JAK2 were unaffected across all treatments. Phosphorylated STAT3 levels showed the same patterns in cells expressing the 5‐HT2C receptor when treated for 1 hour. These findings indicate that 2C receptors are activating STAT3 independently of JAK2 and may be involved in cell growth, differentiation, transcription of other STAT genes, and immune response. Future studies will examine different edited isoforms of the 5‐HT2C receptor and whether STAT3 activation is dependent or independent of G‐protein activation to elucidate the phosphorylation mechanism of STAT3 by the human 5‐HT2C receptors.Support or Funding InformationSupported by NSF Grant HRD‐1238723 to HMFThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structure and Functions of Microtubule Associated Proteins Tau and MAP2c: Similarities and Differences

The stability and dynamics of cytoskeleton in brain nerve cells are regulated by microtubule associated proteins (MAPs), tau and MAP2. Both proteins are intrinsically disordered and involved in multiple molecular interactions important for normal physiology and pathology of chronic neurodegenerative diseases. Nuclear magnetic resonance and cryo-electron microscopy recently revealed propensities of MAPs to form transient local structures and long-range contacts in the free state, and conformations adopted in complexes with microtubules and filamentous actin, as well as in pathological aggregates. In this paper, we compare the longest, 441-residue brain isoform of tau (tau40), and a 467-residue isoform of MAP2, known as MAP2c. For both molecules, we present transient structural motifs revealed by conformational analysis of experimental data obtained for free soluble forms of the proteins. We show that many of the short sequence motifs that exhibit transient structural features are linked to functional properties, manifested by specific interactions. The transient structural motifs can be therefore classified as molecular recognition elements of tau40 and MAP2c. Their interactions are further regulated by post-translational modifications, in particular phosphorylation. The structure-function analysis also explains differences between biological activities of tau40 and MAP2c.

Abstract P2-02-01: Inhibition of creatine kinase metabolism represses invasion and sensitizes estrogen-receptor negative breast cancer cells to standard chemotherapies

Abstract Dysregulated tumor cell metabolism is a hallmark of cancer progression and therapeutic resistance. In a screen for Hypoxia-Inducible Factor (HIF)-dependent genes regulating metabolism, we identified creatine kinase, brain isoform (CKB) as down-regulated in HIF-1 knockout mammary tumor cells. CKB is a member of a family of cytosolic and mitochondrial creatine kinases (CKs) that reversibly catalyze the transfer of a high-energy phosphoryl group from ATP to creatine, generating local stores of phosphocreatine in the forward reaction, and re-generating ATP in the reverse reaction. Creatine kinases are up-regulated in a variety of solid tumors, including breast cancers. CK activity is inhibited by cyclocreatine (cCr), a creatine kinase substrate that represses the CK-dependent generation of ATP from phosphocreatine >170-fold. Knockdown of CKB in tumor cells derived from the polyoma middle T (PyMT) transgenic model of metastatic breast cancer did not impair cell proliferation or wound healing, but potently suppressed invasion in vitro, suggesting utility for treating stage IV disease. When female FVB/Nj mice were injected with wild type PyMT cells in a tail vein assay and then treated with cCr, lung metastasis was repressed to the same extent as CKB gene knockdown. These data were validated using a variety of human estrogen receptor (ER)-negative breast cancer cell lines. CKB loss- and gain-of-function models were created and effects on cell proliferation, survival, wound healing and ATP production were compared to cCr treatment. Overall, whereas deletion of CKB had no effect on cell proliferation or survival in either adherent or suspension conditions, either deletion of CKB or cCr therapy potently reduced intracellular ATP levels, which was associated with reduced invasive potential. In contrast, over-expression of CKB enhanced chemotaxis to EGF and invasion towards serum. Whereas cCr monotherapy is generally cytostatic, combination with either doxorubicin or paclitaxel is synergistic to kill tumor cells. Together, these data suggest that inhibition of CK activity may be effective in treating stage IV breast cancer patients with established metastatic disease. We are currently exploring the cell signaling and cell survival pathways that are altered by CKB gene deletion or cCr treatment to understand how the creatine kinase arm of metabolism promotes tumor progression, metastasis and response to conventional chemotherapies. Citation Format: Barch HP, Krutilina R, Brooks DL, Parke D, Seagroves TN. Inhibition of creatine kinase metabolism represses invasion and sensitizes estrogen-receptor negative breast cancer cells to standard chemotherapies [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-02-01.

Effects of Long Form of CAPON Overexpression on Glioma Cell Proliferation are Dependent on AKT/mTOR/P53 Signaling.

Background: CAPON has two isoforms in human brain: long form of CAPON (CAPON-L) and short form of CAPON (CAPON-S). Recent studies have indicated the involvement of CAPON in tumor cell growth. We aimed to reveal the role of the two CAPON isoforms in the proliferation of glioma cells in this study.Materials and Methods: Lentivirus-mediated stable cell lines with CAPON-L or CAPON-S overexpression were established in U87 and U251 glioma cells. Cell counting kit-8 and colony formation assays were used to evaluate cell proliferation. Western blot analysis of cell cycle-related proteins and flow cytometry were performed to analyze cell cycle progression. Some important molecules of the AKT/mTOR pathway and P53 were also measured by Western blot analysis.Results: Overexpression of CAPON-L showed a significantly inhibitory role in U251 cells, while it exhibited a promoting role in U87 cells. Consistently, overexpressing CAPON-L impeded the cell cycle progression and down-regulated the expression levels of Cyclin D1, CDK4 and CDK6 in U251 cells, whereas it up-regulated the CDK6 level in U87 cells. The overexpression of CAPON-L significantly decreased the phosphorylation and/or total levels of AKT, mTOR and S6 in U251 cells, while it did not affect these signaling molecules in U87 cells, except for a significant increase in the phosphorylation of AKT at Thr-308 site. Transfecting constitutively active AKT (myr-AKT) partially reversed the decreased phosphorylation of AKT and S6 in the CAPON-L-overexpressing U251 cells. In addition, we found a significant decrease in the wild-type P53 level in the CAPON-L-overexpressing U87 cells. The overexpression of CAPON-S also inhibited cell proliferation, blocked cell cycle progression, and decreased the AKT/mTOR pathway activity in U251 cells.Conclusion: The effects of CAPON-L overexpression on glioma cell proliferation are dependent on the AKT/mTOR/P53 activity. The overexpression of CAPON inhibits U251 cell proliferation through the AKT/mTOR signaling pathway, while overexpressing CAPON-L promoted U87 cell proliferation, possibly through down-regulating the P53 level.

Calcineurin Binds to a Unique C-Terminal Region of NBCe1-C, the Brain Isoform of NBCe1 and Enhances its Surface Expression

Calcineurin Binds to a Unique C-Terminal Region of NBCe1-C, the Brain Isoform of NBCe1 and Enhances its Surface Expression

The Structure and the Regulation of Glycogen Phosphorylases in Brain.

Glycogen constitutes the main store of glucose in animal cells. Being present at much lower concentrations in the brain than in liver and muscles, brain glycogen has long been considered as an emergency source of glucose, mobilized under stress conditions (including hypoglyceamia). Nevertheless, over the past decade, multiple studies have shed a new light on the roles of brain glycogen, being notably an energy supply critical for high-cognitive processes such as learning and memory consolidation. Glycogen phosphorylase (GP) is the key enzyme regulating the mobilization of glycogen in cells. It is found in humans as three isozymes: muscle (mGP), liver (lGP) and brain GP (bGP). In the brain, astrocytes express both mGP and bGP while neurons only express the brain isoform. Although GP isozymes are very similar, their distinct regulatory features confer them distinct metabolic functions that are strongly related to the roles of glycogen in different tissues. Here, we provide an overview of the functions, the regulations and the structures of GPs in the brain and their relation to the specific roles of glycogen in astrocytes and neurons. We also discuss novel findings concerning the specific regulations of bGP by oxidative stress, and the potential of these enzymes as therapeutic targets in the brain.

Deep proteomic network analysis of Alzheimer\u2019s disease brain reveals alterations in RNA binding proteins and RNA splicing associated with disease

BackgroundThe complicated cellular and biochemical changes that occur in brain during Alzheimer’s disease are poorly understood. In a previous study we used an unbiased label-free quantitative mass spectrometry-based proteomic approach to analyze these changes at a systems level in post-mortem cortical tissue from patients with Alzheimer’s disease (AD), asymptomatic Alzheimer’s disease (AsymAD), and controls. We found modules of co-expressed proteins that correlated with AD phenotypes, some of which were enriched in proteins identified as risk factors for AD by genetic studies.MethodsThe amount of information that can be obtained from such systems-level proteomic analyses is critically dependent upon the number of proteins that can be quantified across a cohort. We report here a new proteomic systems-level analysis of AD brain based on 6,533 proteins measured across AD, AsymAD, and controls using an analysis pipeline consisting of isobaric tandem mass tag (TMT) mass spectrometry and offline prefractionation.ResultsOur new TMT pipeline allowed us to more than double the depth of brain proteome coverage. This increased depth of coverage greatly expanded the brain protein network to reveal new protein modules that correlated with disease and were unrelated to those identified in our previous network. Differential protein abundance analysis identified 350 proteins that had altered levels between AsymAD and AD not caused by changes in specific cell type abundance, potentially reflecting biochemical changes that are associated with cognitive decline in AD. RNA binding proteins emerged as a class of proteins altered between AsymAD and AD, and were enriched in network modules that correlated with AD pathology. We developed a proteogenomic approach to investigate RNA splicing events that may be altered by RNA binding protein changes in AD. The increased proteome depth afforded by our TMT pipeline allowed us to identify and quantify a large number of alternatively spliced protein isoforms in brain, including AD risk factors such as BIN1, PICALM, PTK2B, and FERMT2. Many of the new AD protein network modules were enriched in alternatively spliced proteins and correlated with molecular markers of AD pathology and cognition.ConclusionsFurther analysis of the AD brain proteome will continue to yield new insights into the biological basis of AD.

Biological predictors shared by dementia and bullous pemphigoid patients point out a cross-antigenicity between BP180/BP230 brain and skin isoforms.

Bullous pemphigoid (BP) following dementia diagnosis has been reported in the elderly. Skin and brain tissues express BP180 and BP230 isoforms. Dementia has been associated with rs6265 (Val66Met) polymorphism of the brain-derived neurotrophic factor (BDNF) gene and low serum BDNF. Here we investigated a possible cross-antigenicity between BP180/BP230 brain and skin isoforms. We assessed antibodies against BP180/BP230 and BDNF levels by ELISA and BDNF Val66Met SNP by PCR in three groups: 50 BP patients, 50 patients with dementia, and 50 elderly controls. Heatmap hierarchical clustering and data mining decision tree were used to analyze the patients' demographic and laboratorial data as predictors of dementia-BP association. Sixteen percent of BP patients with the lowest serological BDNF presented dementia-BP clinical association. Anti-BP180/230 positivity was unexpected observed among dementia patients (10%, 10%) and controls (14%, 1%). Indirect immunofluorescence using healthy human skin showed a BP pattern in two of 10 samples containing antibodies against BP180/BP230 obtained from dementia group but not in the control samples. Neither allelic nor genotypic BDNF Val66Met SNP was associated with dementia or with BP (associated or not with clinical manifestation of dementia). Heatmap analysis was able to differentiate the three studied groups and confirmed the ELISA results. The comprehensive data mining analysis revealed that BP patients and dementia patients shared biological predictors that justified the dementia-BP association. Autoantibodies against the BP180/BP230 brain isoforms produced by dementia patients could cross-react with the BP180/BP230 skin isoforms, which could justify cases of dementia preceding the BP disease.

Novel antibodies reveal presynaptic localization of C9orf72 protein and reduced protein levels in C9orf72 mutation carriers

Hexanucleotide repeat expansion in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis, but the pathogenic mechanism of this mutation remains unresolved. Haploinsufficiency has been proposed as one potential mechanism. However, insights if and how reduced C9orf72 proteins levels might contribute to disease pathogenesis are still limited because C9orf72 expression, localization and functions in the central nervous system (CNS) are uncertain, in part due to the poor specificity of currently available C9orf72 antibodies.Here, we generated and characterized novel knock-out validated monoclonal rat and mouse antibodies against C9orf72. We found that C9orf72 is a low abundant, cytoplasmic, highly soluble protein with the long 481 amino acid isoform being the predominant, if not exclusively, expressed protein isoform in mouse tissues and human brain. As consequence of the C9orf72 repeat expansion, C9orf72 protein levels in the cerebellum were reduced to 80% in our series of C9orf72 mutation carriers (n = 17) compared to controls (n = 26). However, no associations between cerebellar protein levels and clinical phenotypes were seen. Finally, by utilizing complementary immunohistochemical and biochemical approaches including analysis of human iPSC derived motor neurons, we identified C9orf72, in addition to its association to lysosomes, to be localized to the presynapses and able to interact with all members of the RAB3 protein family, suggestive of a role for C9orf72 in regulating synaptic vesicle functions by potentially acting as guanine nucleotide exchange factor for RAB3 proteins.In conclusion, our findings provide further evidence for haploinsufficiency as potential mechanism in C9orf72 pathogenesis by demonstrating reduced protein levels in C9orf72 mutation carriers and important novel insights into the physiological role of C9orf72 in the CNS. Moreover, the described novel monoclonal C9orf72 antibodies will be useful tools to further dissect the cellular and molecular functions of C9orf72.

Genetically Engineered iPSC-Derived FTDP-17 MAPT Neurons Display Mutation-Specific Neurodegenerative and Neurodevelopmental Phenotypes.

SummaryTauopathies such as frontotemporal dementia (FTD) remain incurable to date, partially due to the lack of translational in vitro disease models. The MAPT gene, encoding the microtubule-associated protein tau, has been shown to play an important role in FTD pathogenesis. Therefore, we used zinc finger nucleases to introduce two MAPT mutations into healthy donor induced pluripotent stem cells (iPSCs). The IVS10+16 mutation increases the expression of 4R tau, while the P301S mutation is pro-aggregant. Whole-transcriptome analysis of MAPT IVS10+16 neurons reveals neuronal subtype differences, reduced neural progenitor proliferation potential, and aberrant WNT/SHH signaling. Notably, these neurodevelopmental phenotypes could be recapitulated in neurons from patients carrying the MAPT IVS10+16 mutation. Moreover, the additional pro-aggregant P301S mutation revealed additional phenotypes, such as an increased calcium burst frequency, reduced lysosomal acidity, tau oligomerization, and neurodegeneration. This series of iPSCs could serve as a platform to unravel a potential link between pathogenic 4R tau and FTD.

Subchronic administration of creatine produces antidepressant-like effect by modulating hippocampal signaling pathway mediated by FNDC5/BDNF/Akt in mice

Creatine has been shown to play a significant role in the pathophysiology and treatment of major depressive disorder (MDD) in preclinical and clinical studies. However, the biological mechanisms underlying its antidepressant effect is still not fully elucidated. This study investigated the effect of creatine (p.o.) administered for 21 days in the behavior of mice submitted to tail suspension test (TST), a predictive test of antidepressant activity. Creatine reduced the immobility time in the TST (1–10 mg/kg), without affecting locomotor activity, a finding consistent with an antidepressant profile. Creatine administration increased the ubiquitous creatine kinase (uCK) and creatine kinase brain isoform (CK-B) mRNA in the hippocampus of mice. Taking into account that PGC-1α induces FNDC5/irisin expression mediating BDNF-dependent neuroplasticity, the effect of creatine administration (1 mg/kg, p. o.) on the hippocampal PGC-1α, FNDC5 and BDNF gene expression was investigated. Creatine treatment increased PGC-1α, FNDC5 and BDNF mRNA in the hippocampus as well as BDNF immunocontent. The involvement of BDNF downstream intracellular signaling pathway mediated by Akt, proapoptotic proteins BAX and BAD and antiapoptotic proteins Bcl2 and Bcl-xL was also investigated following creatine treatment. Creatine increased Akt phosphorylation (Ser 473), and Bcl2 mRNA and protein levels, and Bcl-xL mRNA, whereas BAD mRNA was decreased following creatine administration in the hippocampus. Altogether these results indicate that creatine antidepressant-like effect may be dependent on Akt activation and increased expression of the neuroprotective proteins in the hippocampus of mice. The obtained data reinforce the antidepressant property of creatine and highlight the role of these molecular targets in the pathophysiology of MDD.

Structure of a human synaptic GABAA receptor.

Fast inhibitory neurotransmission in the brain is principally mediated by the neurotransmitter γ-aminobutyric acid (GABA) and its synaptic target, the GABA-A receptor. Dysfunction of this receptor results in neurological disorders and mental illnesses including epilepsy, anxiety and insomnia. The GABA-A receptor is also a prolific target for therapeutic, illicit, and recreational drugs, including benzodiazepines, barbiturates, anesthetics and ethanol. We present high resolution cryo-electron microscopy structures of the human α1β2γ2 GABA-A receptor, the predominant isoform in the adult brain. The receptor is bound to GABA and the benzodiazepine site antagonist flumazenil, the first-line clinical treatment for benzodiazepine overdose. The receptor architecture reveals unique heteromeric interactions for this important class of inhibitory neurotransmitter receptors. This work provides a template for understanding receptor modulation by GABA and benzodiazepines, and will assist rational approaches to therapeutic targeting of this receptor for neurological disorders and mental illness.

Evolutionary analysis of the carnitine- and choline acyltransferases suggests distinct evolution of CPT2 versus CPT1 and related variants

Carnitine/choline acyltransferases play diverse roles in energy metabolism and neuronal signalling. Our knowledge of their evolutionary relationships, important for functional understanding, is incomplete. Therefore, we aimed to determine the evolutionary relationships of these eukaryotic transferases. We performed extensive phylogenetic and intron position analyses. We found that mammalian intramitochondrial CPT2 is most closely related to cytosolic yeast carnitine transferases (Sc-YAT1 and 2), whereas the other members of the family are related to intraorganellar yeast Sc-CAT2. Therefore, the cytosolically active CPT1 more closely resembles intramitochondrial ancestors than CPT2. The choline acetyltransferase is closely related to carnitine acetyltransferase and shows lower evolutionary rates than long chain acyltransferases. In the CPT1 family several duplications occurred during animal radiation, leading to the isoforms CPT1A, CPT1B and CPT1C. In addition, we found five CPT1-like genes in Caenorhabditis elegans that strongly group to the CPT1 family. The long branch leading to mammalian brain isoform CPT1C suggests that either strong positive or relaxed evolution has taken place on this node. The presented evolutionary delineation of carnitine/choline acyltransferases adds to current knowledge on their functions and provides tangible leads for further experimental research.

Alternative Splicing at N Terminus and Domain I Modulates CaV1.2 Inactivation and Surface Expression

The CaV1.2 L-type calcium channel is a key conduit for Ca2+ influx to initiate excitation-contraction coupling for contraction of the heart and vasoconstriction of the arteries and for altering membrane excitability in neurons. Its α1C pore-forming subunit is known to undergo extensive alternative splicing to produce many CaV1.2 isoforms that differ in their electrophysiological and pharmacological properties. Here, we examined the structure-function relationship of human CaV1.2 with respect to the inclusion or exclusion of mutually exclusive exons of the N-terminus exons 1/1a and IS6 segment exons 8/8a. These exons showed tissue selectivity in their expression patterns: heart variant 1a/8a, one smooth-muscle variant 1/8, and a brain isoform 1/8a. Overall, the 1/8a, when coexpressed with CaVβ2a, displayed a significant and distinct shift in voltage-dependent activation and inactivation and inactivation kinetics as compared to the other three splice variants. Further analysis showed a clear additive effect of the hyperpolarization shift in V1/2inact of CaV1.2 channels containing exon 1 in combination with 8a. However, this additive effect was less distinct for V1/2act. However, the measured effects were β-subunit-dependent when comparing CaVβ2a with CaVβ3 coexpression. Notably, calcium-dependent inactivation mediated by local Ca2+-sensing via the N-lobe of calmodulin was significantly enhanced in exon-1-containing CaV1.2 as compared to exon-1a-containing CaV1.2 channels. At the cellular level, the current densities of the 1/8a or 1/8 variants were significantly larger than the 1a/8a and 1a/8 variants when coexpressed either with CaVβ2a or CaVβ3 subunit. This finding correlated well with a higher channel surface expression for the exon 1-CaV1.2 isoform that we quantified by protein surface-expression levels or by gating currents. Our data also provided a deeper molecular understanding of the altered biophysical properties of alternatively spliced human CaV1.2 channels by directly comparing unitary single-channel events with macroscopic whole-cell currents.

Structural principles of distinct assemblies of the human α4β2 nicotinic receptor.

SummaryFast chemical communication in the nervous system is mediated by neurotransmitter-gated ion channels. The prototypical member of this class of cell surface receptors is the cation-selective nicotinic acetylcholine receptor. As with most ligand-gated ion channels, nicotinic receptors assemble as oligomers of subunits, usually as hetero-oligomers and often with variable stoichiometries1. This intrinsic heterogeneity in protein composition provides the fine tunability in channel properties essential to brain function but frustrates structural and biophysical characterization. The α4β2 subtype of the nicotinic acetylcholine receptor is the most abundant isoform in the human brain and is the principal target in nicotine addiction. This pentameric ligand-gated ion channel assembles in two stoichiometries of α and β subunits, 2α:3β and 3α:2β. Both assemblies are functional, have distinct biophysical properties, and a misbalance in the ratio of assemblies is tied to both nicotine addiction2,3 and congenital epilepsy4,5. Here we leverage cryo-electron microscopy (cryo-EM) to obtain structures of both receptor assemblies from a single sample. Antibody fragments specific to β2 were used to ‘break’ symmetry during particle alignment and obtain high resolution reconstructions of both stoichiometries, in complex with nicotine. The results reveal principles of subunit assembly and the structural basis of the distinctive biophysical and pharmacological properties of the two different stoichiometries of this receptor.

Germline deletion of CIN85 in humans with X chromosome-linked antibody deficiency.

Ubiquitously expressed Cbl-interacting protein of 85 kD (CIN85) is a multifunctional adapter molecule supposed to regulate numerous cellular processes that are critical for housekeeping as well as cell type-specific functions. However, limited information exists about the in vivo roles of CIN85, because only conditional mouse mutants with cell type-specific ablation of distinct CIN85 isoforms in brain and B lymphocytes have been generated so far. No information is available about the roles of CIN85 in humans. Here, we report on primary antibody deficiency in patients harboring a germline deletion within the CIN85 gene on the X chromosome. In the absence of CIN85, all immune cell compartments developed normally, but B lymphocytes showed intrinsic defects in distinct effector pathways of the B cell antigen receptor, most notably NF-κB activation and up-regulation of CD86 expression on the cell surface. These results reveal nonredundant functions of CIN85 for humoral immune responses.

The Role of the Creatine Kinase Energy System in Intestinal Inflammation

Inflammatory bowel disease (IBD) is a chronic inflammation of the intestine affecting more than 1 million people in the United States. Intestinal epithelia facilitate nutrient transport as well as protect against luminal bacteria and disruption of the intestinal epithelial barrier is a hallmark of IBD. The regulation of intestinal epithelial barrier is made possible by junctional complexes including tight junctions and adherens junctions that are supported by the actin cytoskeleton. Maintenance of the actin cytoskeleton is an energy demanding process and intestinal epithelial cells shuttle energy to the locations of the cellular junctions through the creatine energy circuit. Within the cell, creatine and its associated enzyme creatine kinase (CK) facilitate the shuttling of high energy phosphates in the form of phosphocreatine between sites of ATP generation. Previous work in our lab has shown that CK contributes to apical junction assembly in vitro and that supplementation of creatine in murine colitis models has a protective effect. Further studies have shown that mice lacking the brain and mitochondrial isoforms of CK have worse outcomes in a DSS model of colitis. In addition to impaired barrier function, this may be due in part to exacerbated intestinal bleeding observed in these mice. To that end, we measured platelet counts and aggregation and found that platelet counts are lower in CK double knockout mice compared to wild‐type mice. The platelets from the knockout mice also showed a hyporesponsive phenotype in a collagen adhesion assay. Together, these results provide evidence that the CK energy circuit is essential in maintaining intestinal epithelial barrier function as well as supporting proper platelet function.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

S186. KCNH2 POLYMORPHISM ASSOCIATED TO ALTERED EEG FUNCTIONAL NETWORK MODULATION IN SCHIZOPHRENIA

BackgroundThe rs3800779 polymorphism at KCNH2 gene, which encodes for a Voltage-Gated Potassium Channel Subunit, has been associated with the risk for schizophrenia (SZ) and with changes in the expression of a brain isoform with specific electrophysiological characteristics (Huffaker et al., 2009). It has been hypothesized that the KCNH2 gene variability could be involved in SZ by means of modulating the neuronal excitability. Graph-theory parameters applied to electroencephalographic (EEG) activity are useful to assess functional connectivity in the brain and have shown altered patterns of global connectivity in schizophrenia. We aimed to investigate whether KCNH2 contributes to functional connectivity alterations replicated in SZ patients.MethodsEEG data were acquired during the performance of an odd-ball task in 50 schizophrenia patients and 101 matched healthy controls. The rs3800779 at KCNH2 was genotyped. From the EEG activity, the Small World index (SW, a measure of network efficiency) was calculated as the coefficient between clustering coefficient (CLC, a measure of network segregation) and path length (PL, a measure of network integration). SW was calculated in two temporal windows with respect to the target tone (pre-stimulus and response). Functional SW modulation (SWm) was calculated as the difference in SW between pre-stimulus and response windows. Finally, the association between KCNH2 polymorphism and functional connectivity modulation was assessed.ResultsPatients carrying the A allele (AA or AC, n=25) showed smaller SW modulation in comparison with patients with the CC genotype (n=25) (t=-2.84, df=48, p=0.007). Moreover, patients carrying the A allele showed smaller SW modulation than healthy controls with the A allele (n=45) (t=-3.41, df=68, p=0.001) or without the A allele (n=56) (t=-3.87, df=79 p<0.001). There were no significant SW modulation differences between healthy controls carrying or not the A allele. Patients with the AA/AC genotype showed an inverse SW modulation pattern (decreased SW at response) in comparison with patients without the A allele and controls (increased SW at response).DiscussionOur data indicate that, within SZ patients, the A allele is associated with smaller SW modulation and lower SW values at response, which might be interpreted as an altered ability to coordinate the activity of neural assemblies during cognitive tasks (Basar et al 2016). Although replication analyses are needed, our findings suggest that genetic variation at KCNH2 might contribute to the efficiency of brain functional networks in schizophrenia patients.

Identification of new PDE9A isoforms and how their expression and subcellular compartmentalization in the brain change across the life span

3′,5′-Cyclic nucleotide phosphodiesterases (PDEs) degrade 3′,5′ cyclic adenonosine monophosphate (cAMP) and 3′,5′ cyclic guanosine monophosphate (cGMP), with PDE9A having the highest affinity for cGMP. We show PDE9A6 and 3 novel PDE9 isoforms (PDE9X-100, PDE9X-120, and PDE9X-175) are reliably detected in the brain and lung of mice, whereas PDE9A2 and other isoforms are found elsewhere. PDE9A localizes to the membrane in all organs except the bladder, where it is cytosolic. Brain additionally shows PDE9 in the nuclear fraction. PDE9A mRNA expression/localization dramatically changes across neurodevelopment in a manner that is strikingly consistent between mice and humans (i.e., decreased expression in the hippocampus and cortex and inverted-U in the cerebellum). Study of the 4 PDE9 isoforms in the mouse brain from postnatal day 7 through 24 months similarly identifies dramatic effects of age on expression and subcellular compartmentalization that are isoform specific and brain region specific. Finally, PDE9A mRNA is elevated in the aged human hippocampus with dementia when there is a history of traumatic brain injury. Thus, brain PDE9 is localized to preferentially regulate nuclear- and membrane-proximal pools of cGMP, and its function likely changes across the life span.

Fetal-type Glycogen Phosphorylase Expression in Intestinal Metaplasia as a Predictor of the Development of Gastric Cancer

Glycogen phosphorylase (GP; EC 2.4.1.1) plays a central role in the mobilization of carbohydrate reserves in a wide variety of organs and tissues. There are three major isoforms of mammalian GP; i.e., the muscle, liver, and brain isoforms. The physiological roles of muscle and liver GP are to provide fuel for energy production (to produce the energy required for muscle contraction) and to ensure a constant supply of glucose to extra hepatic tissues, respectively. However, the physiological role of brain GP (BGP) is poorly understood. It has been demonstrated that BGP is the major isoform of GP found in fetal tissue and tumor tissue, and BGP is identical to fetal-type GF (FGP). We have demonstrated that the significant enzymatic activity of FGP in the gastric carcinoma and proliferating cells of particular Intestinal Metaplasia (IM). We studied 136 specimens with gastric carcinoma and the adjacent IM using specific anti-FGP antibody. FGP was expressed in 80% of the intestinal type and 19% of the diffuse type of carcinoma and in 88% and 42% in the generative zone of IM adjacent to each type of cancer foci, respectively. The proportion of the positivity of FGP expression in the cancer and IM was significantly greater in intestinal type carcinoma than in diffuse type. In addition, according to the proliferating cell nuclear antigen labeling index analysis, IM with FGP expression (FGP-IM) were significantly higher in a proliferating state than in IM without FGP, and some of them were co-expressed accumulated p53 in the generative cells. These data indicate that FGP could be one of the fetal biomarkers and FGP-IM could be a premalignant lesion of intestinal type adenocarcinoma. We conducted another study to investigate the incidence of FGP-IM in gastric biopsy specimens and to identify FGP-IM as a predictor of the coexistence of accessory carcinoma and/or metachronous carcinoma. Eight endoscopic biopsy specimens of methylene blue-positive mucosa of the stomach were obtained from the patients with multiple gastric carcinomas (n=14), a single carcinoma (n=25) and atrophic gastritis (n=20), examined the incidence of FGP-IM. FGP positivity was 93.3% in the multiple carcinomas and 80.0% in the single carcinomas. The incidences of FGP-IM in the stomachs with multiple carcinomas, single carcinoma and atrophic gastritis were 83.2 ± 22.8%, 36.5 ± 41.3% and 7.1 ± 18.0%, respectively. The incidence of FGP-IM was significantly higher in the stomachs with multiple carcinomas than in those with a single carcinoma or atrophic gastritis. It is suggested that the frequent appearance of FGP-IM reflects the high potential of carcinogenesis of intestinal type gastric carcinoma and FGP-IM could be a predictive indicator of metachronous gastric carcinoma. Recently, there are increasing opportunities where endoscopic and laparoscopic local treatments are applied for the early gastric carcinoma, therefore, it is significantly important to identify the high-risk group of metachronous recurrence of gastric carcinoma. FGP could serve as a potential predictor of the risk of the development of multiple and/or metachronous carcinomas. It might be possible to follow-up new lesions using this method, and follow-up studies would provide better information on whether FGP-IM positivity is a good predictor of metachronous recurrence after local treatment for gastric cancer.