kDa Isoform Research Articles

Dysbindin-1 contributes to prefrontal cortical dendritic arbor pathology in schizophrenia

Deep layer III pyramidal cells in the dorsolateral prefrontal cortex (DLPFC) from subjects with schizophrenia and bipolar disorder previously were shown to exhibit dendritic arbor pathology. This study sought to determine whether MARCKS, its regulatory protein dysbindin-1, and two proteins, identified using microarray data, CDC42BPA and ARHGEF6, were associated with dendritic arbor pathology in the DLPFC from schizophrenia and bipolar disorder subjects. Using western blotting, relative protein expression was assessed in the DLPFC (BA 46) grey matter from subjects with schizophrenia (n = 19), bipolar disorder (n = 17) and unaffected control subjects (n = 19). Protein expression data were then correlated with dendritic parameter data obtained previously. MARCKS and dysbindin-1a expression levels did not differ among the three groups. Dysbindin-1b expression was 26% higher in schizophrenia subjects (p = 0.01) and correlated inversely with basilar dendrite length (r = −0.31, p = 0.048) and the number of spines per basilar dendrite (r = −0.31, p = 0.048), but not with dendritic spine density (r = −0.16, p = 0.32). The protein expression of CDC42BPA was 33% higher in schizophrenia subjects (p = 0.03) but, did not correlate with any dendritic parameter (p > 0.05). ARHGEF6 87 kDa isoform expression did not differ among the groups. CDC42BPA expression was not altered in frontal cortex from rats chronically administered haloperidol or clozapine. Dysbindin-1b appears to play a role in dendritic arbor pathology observed previously in the DLPFC in schizophrenia.

Expression of the onconeural protein CDR1 in cerebellum and ovarian cancer.

Cerebellar degeneration related protein 1 (CDR1) is expressed in the cerebellum, and CDR1 antibodies have been associated with paraneoplastic cerebellar degeneration (PCD). In this study, we examined CDR1 expression in cerebellum and in ovarian and breast tumors, as well as the intracellular localization of CDR1 in cancer cells in culture. CDR1 was strongly expressed in the cytosol and dendrites of Purkinje cells and in interneurons of the molecular layer in cerebellum. CDR1 was also present in ovarian and breast tumors, as well as in ovarian and breast cancer cell lines, but was not present in normal breast or ovarian tissue. In cells overexpressing CDR1, CDR1 localized close to the plasma membrane in a polarized pattern at one edge. CDR1 was strongly expressed on the outer surface, apparently in filopodias or lamellipodias, in cells endogenously expressing CDR1. Overexpression of CDR1 showed a 37 and a 45 kDa band in western blot. The 37-kDa isoform was present in 16 ovarian cancer lysates, while the 45-kDa isoform was only found in three ovarian cancer patients. The presence of CDR1 in ovarian cancer was not associated with PCD. CDR1 antibodies were only found in serum from one patient with PCD and ovarian tumor with metastases. Therefore, CDR1 is probably not a marker for PCD. However, CDR1 may be associated with cell migration and differentiation.

Regulators of glucocorticoid receptor function in an animal model of depression and obesity.

Obesity is a disease that often co-occurs with depression, and some evidence indicates that chronic stress in the perinatal period, in association with overactive glucocorticoids, can cause permanent changes that increase the risk of the development of both depression and obesity later in life. However, the mechanism responsible for the overly potent action of glucocorticoids in both depression and obesity is not known. The aim of the present study was to determine the expression of glucocorticoid receptors (GRs) and mineralocorticoid receptors (MRs) and the factors that affect GR function (FKBP51, Bag-1 and HSP70) in a prenatal stress animal model of depression, a model of obesity and a model of both depression and obesity. Prenatal stress but not high-fat diet (HFD) was found to decrease the GR concentration in the frontal cortex. The level of the Bag-1M (46 kDa) isoform was also decreased in this structure but only in prenatal-stressed animals that did not show depression-like behaviour in the Porsolt test and were fed the standard diet (STD). In the model of depression employed here, decreases in MR expression and GR co-chaperone (FKBP51) levels in the hippocampus were also observed, and HFD intensified the prenatal stress-induced changes in MR expression. The obtained results indicated that prenatal stress affected the expression of GRs, MRs and their co-chaperones in the brain, but its effects were different in the frontal cortex and hippocampus. The decrease in MR density in the hippocampus and increased plasma insulin level seemed to be the most significant changes observed in the model of the co-occurrence of depression and obesity, which could limit the neuroprotective effects associated with the activation of MR and be a marker of peripheral insulin resistance, respectively. This article is protected by copyright. All rights reserved.

Identification of Placental Aspartic Proteinase in the Eurasian Beaver (Castor fiber L.).

Aspartic proteinases (AP) form a multigenic group widely distributed in various organisms and includes pepsins (pep), cathepsins D and E, pregnancy associated glycoproteins (PAGs) as well as plant, fungal, and retroviral proteinases. This study describes the transcript identification and expression localization of the AP within the discoid placenta of the Castor fiber. We identified 1257 bp of the AP cDNA sequence, encoding 391 amino acids (aa) of the polypeptide precursor composed of 16 aa signal peptide, 46 aa pro-piece, and 329 aa of the mature protein. Within the AP precursor, one site of potential N-glycosylation (NPS119–121) and two Asp residues (D) specific for the catalytic cleft of AP were identified (VLFDTGSSNLWV91–102 and GIVDTGTSLLTV277–288). The highest homology of the identified placental AP nucleotide and aa sequence was to mouse pepsinogen C (75.8% and 70.1%, respectively). Identified AP also shared high homology with other superfamily members: PAGs, cathepsins, and napsins. The AP identified in this study was named as pepsinogen/PAG-Like (pep/PAG-L). Diversified pep/PAG-L protein profiles with a dominant 58 kDa isoform were identified. Immune reactive signals of the pep/PAG-L were localized within the trophectodermal cells of the beaver placenta. This is the first report describing the placental AP (pep/PAG-L) in the C. fiber.

Circulating histocompatibility antigen (HLA) gene products may help differentiate benign from malignant indeterminate pulmonary lesions

BackgroundThis study explores the potential diagnostic utility of soluble Human Leukocyte Antigen (sHLA) molecules differentially released by lung adenocarcinoma and benign lung lesions. MethodsConditioned media from the NSCLC cell lines H358 and H1703 were immunoblotted for soluble isoforms of major histocompatibility complex (MHC) class I (ABC) and II (DRB1, DMB, and DQ) antigens. Sera from 25 patients with benign and 25 patients with malignant lesions were similarly evaluated to appraise the potential diagnostic value. ResultsHigher concentrations of soluble HLA class I molecules were observed in conditioned medium for the highly-invasive H1703 cell line, relative to the more indolent H358 cells. Evaluation of these markers against a cohort of 50 cases demonstrated that patients with malignant lesions possess higher levels of HLA class I and II molecules relative to those with benign lesions (p < 0.05), with exception to the primary isoform, DQA1, which was suppressed in malignancies. An analysis of biomarker performance via ROC analysis revealed promising performance (AUC > 0.75) for DMB and the 26 kDa isoform of DQ in distinguishing lesion pathology. ConclusionsSoluble HLA molecules may have diagnostic value for early-stage NSCLC. Validation studies are currently underway using sera from a lung cancer screening cohort.

Expression Profile of G Protein‐Coupled Receptor 37L1 in mouse

The orphan G protein‐coupled receptor GPR37L1, also known as ETBR‐LP2, has strong homology with the endothelin‐B receptor (ETBR). However, GPR37L1 does not interact with endothelins. GPR37L1 is implicated to participate in brain development and the regulation of arterial blood pressure. GPR37L1 has been shown to be expressed in several regions in the brain, however its expression profile and the function in peripheral organs is poorly understood. We studied the expression profile of GPR37L1 in the brain, heart, kidney, liver, lung, spleen, stomach, pancreas, skeletal muscle, testis, and ovaries by RT‐PCR and immunoblot analyses and further used immunohistochemical analyses on tissue sections to identify the location of GPR37L1 expression within the tissue. Immunoblot analyses revealed the predicted 50 kDa and 40 kDa isoforms in most organs, and a ~35 kDa isoform mainly in the stomach and to a lesser extent in the brain, and a diffuse band of 45 kDa isoform mainly in the testis. The expression of GPR37L1 was highest in the brain, heart, stomach, pancreas, and skeletal muscle while GPR37L1 protein expression was lower in the kidney, liver, lung, spleen, testis, and ovary. The brain and pancreas expressed predominantly a 50 kDa isoform, while heart and skeletal muscle expressed mainly a 40 kDa isoform of GPR37L1. The kidney, liver, lung and ovary expressed almost equivalent levels of 50 kDa and 40 kDa isoforms of GPR37L1. The spleen expressed mainly a 40 kDa isoform of GPR37L1. RT‐PCR analyses confirmed the relative levels of GPR37L1 expression in the organs analyzed. Immunohistochemical analyses revealed the expression of GPR37L1, in the granule cell layer of the cerebellum, apical membrane of proximal tubule cells in the kidney, and airway epithelial cells in the lung. Further, RT‐PCR of proximal tubule and collecting duct cells obtained by laser capture micro‐dissection of mouse kidney sections, confirmed that the GPR37L1 is expressed in renal the proximal tubule but not in the collecting duct. These data show that GPR37L1 is expressed in the specific cell types within the organs and may play a key role in the physiology of the respective organs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Prospective Study To Investigate The Prevalence Of The 90 kDa Isoform Of Angiotensin Converting Enzyme In Vitória ‐ Brazil

Hypertension is considered a worldwide public health problem and identified as a risk factor for cardiovascular morbidity and mortality. The Renin Angiotensin System (RAS) has been a focus of interest of researchers in the area of hypertension both for the identification of its etiopathogenesis and for its treatment. In order to identify the components involved in the onset of hypertension and the participation of Angiotensin I Converting Enzyme (ACE) in this problem, the RAS has been the object of many studies. ACE has a great physiological importance for converting angiotensin I (Ang I) to angiotensin II (Ang II), a potent vasoconstrictor. Some papers suggest the association of ACE isoforms, especially the 90 kDa isoform, with hypertension. The aim of this study was to investigate the association between ACE isoforms and the presence of arterial hypertension in the second phase of the prospective study (MONICA Project) in the population of Vitória ‐ ES, Brazil.MethodsThe demographic, clinical and biochemical parameters of 220 individuals were evaluated. Urine samples were concentrated and ACE isoforms were identified by the Western Blotting technique.ResultsThe groups were classified as Group 1 (presence of isoforms with 65, 90 and 190 KDa); Group 2 (presence of 65 and 90 kDa isoforms) and Group 3 (presence of 65 and 190 kDa isoforms). The results showed a high prevalence of the 90 kDa isoform with a higher incidence of hypertension (higher in group 2) after five years of segment. The groups containing 90 kDa isoform showed higher systolic and diastolic pressure profiles in the second phase and the frequency of normotensives expressing the isoforms with 65 and 190 kDa was higher than that the hypertensive ones. Loss of isoform expression at 190 kDa increases the chances of developing hypertension as well as family history of hypertension.ConclusionThe results suggest the 90‐kDa N‐domain isoform as a possible biological marker of hypertension, which confirms the results obtained in the first phase of the study.Support or Funding InformationCNPq and FAPESPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interplay between gut microbiota and p66Shc affects obesity-associated insulin resistance.

The 66 kDa isoform of the mammalian Shc gene promotes adipogenesis, and p66Shc-/- mice accumulate less body weight than wild-type (WT) mice. As the metabolic consequences of the leaner phenotype of p66Shc-/- mice is debated, we hypothesized that gut microbiota may be involved. We confirmed that p66Shc-/- mice gained less weight than WT mice when on a high-fat diet (HFD), but they were not protected from insulin resistance and glucose intolerance. p66Shc deletion significantly modified the composition of gut microbiota and their modification after an HFD. This was associated with changes in gene expression of Il-1b and regenerating islet-derived protein 3 γ ( Reg3g) in the gut and in systemic trimethylamine N-oxide and branched chain amino acid levels, despite there being no difference in intestinal structure and permeability. Depleting gut microbiota at the end of HFD rendered both strains more glucose tolerant but improved insulin sensitivity only in p66Shc-/- mice. Microbiota-depleted WT mice cohoused with microbiota-competent p66Shc-/- mice became significantly more insulin resistant than WT mice cohoused with WT mice, despite no difference in weight gain. These findings reconcile previous inconsistent observations on the metabolic phenotype of p66Shc-/- mice and illustrate the complex microbiome-host-genotype interplay under metabolic stress.-Ciciliot, S., Albiero, M., Campanaro, S., Poncina, N., Tedesco, S., Scattolini, V., Dalla Costa, F., Cignarella, A., Vettore, M., Di Gangi, I. M., Bogialli, S., Avogaro, A., Fadini, G. P. Interplay between gut microbiota and p66Shc affects obesity-associated insulin resistance.

Six-month cultured cerebral organoids from human ES cells contain matured neural cells

Recently, researchers have developed protocols for human cerebral organoids using human pluripotent stem cells, which mimic the structure of the developing human brain. Existing research demonstrated that human cerebral organoids which undergo short cultivation periods, contain astrocytes, neurons, and neural stem cells, but lacked mature oligodendrocytes, and mature, fully functional neurons. In this study, we analyzed organoids induced from H9 human embryonic stem (ES) cells that were cultivated for as long as six months. We observed mature oligodendrocytes, positive for MBP (myelin-basic protein), and mature GAD67 (glutamate decarboxylase 67 kDa isoform)-positive inhibitory neurons and VGLUT1 (vesicular glutamate transporter 1)-positive excitatory neurons via immunohistochemical analysis. These observations suggest that long-term cultivation of cerebral organoids can lead to the maturation of human cerebral organoids, which can be used as a tool to study the development of human brains.

Change in distribution of cytoskeleton-associated proteins, lasp-1 and palladin, during uterine receptivity in the rat endometrium.

The epithelium of the uterine lumen is the first point of contact with the blastocyst before implantation. To facilitate pregnancy, these uterine epithelial cells (UECs) undergo morphological changes specific to the receptive uterus. These changes include basal, lateral and apical alterations in the plasma membrane of UECs. This study looked at the cytoskeletal and focal adhesion-associated proteins, lasp-1 and palladin, in the uterus during early pregnancy in the rat. Two palladin isoforms, 140 kDa and 90 kDa, were analysed, with the migration-associated 140-kDa isoform increasing significantly at the time of implantation when compared with the time of fertilisation. Lasp-1 was similarly increased at this time, whilst also being located predominantly apically and laterally in the UECs, suggesting a role in the initial contact between the UECs and the blastocyst. This is the first study to investigate palladin and lasp-1 in the uterine luminal epithelium and suggests an importance for these cytoskeletal proteins in the morphological changes the UECs undergo for pregnancy to occur.

Distinct Roles of Different Presynaptic and Postsynaptic NCAM Isoforms in Early Motoneuron-Myotube Interactions Required for Functional Synapse Formation.

The neural cell adhesion molecule (NCAM) is expressed both presynaptically and postsynaptically during neuromuscular junction formation. Genetic deletion in mice of all three isoforms (180, 140, and 120 kDa), or just the 180 isoform, suggested that different isoforms played distinct roles in synaptic maturation. Here we characterized in mice of either sex the earliest adhesive contacts between the growth cones of motoneurons and myotubes and their subsequent maturation into functional synapses in cocultures of motoneurons and myotubes, which expressed their normal complement of NCAM isoforms, or were lacking all isoforms either presynaptically or postsynaptically. Growth cone contact with +/+ mouse myotubes resulted in immediate adhesive contacts and the rapid downregulation of growth cone motility. When contacting NCAM-/- myotubes, growth cones touched and retracted/collapsed multiple times and failed to form stable contacts, even after 10 h. Exogenous expression in myotubes of either the 180 or 140 isoform, but not the 120 kDa isoform, rescued the rapid formation of stable contacts, the accumulation of presynaptic and postsynaptic molecules, and functional transmission. When NCAM was absent only in motoneurons, growth cones did not retract upon myotube contact, but, since their motility was not downregulated, they grew off the ends of the myotubes, failing to form synapses. The agrin receptor Lrp4 was strongly downregulated in NCAM-negative myotubes, and motoneuron growth cones did not make stable contacts with Lrp4-negative myotubes. These studies have identified novel roles for presynaptic and postsynaptic NCAM in mediating early cell-cell interactions required for synapse formation.SIGNIFICANCE STATEMENT Although many molecular signals needed to form the functionally effective neuromuscular synapses required for normal movement have been described, the earliest signals that let motoneuron growth cones make stable adhesive contacts with myotubes and cease motility are not well understood. Using dynamic imaging of motoneuron-myotube cocultures, we show that NCAM is required on both the growth cone and myotube and that different NCAM isoforms mediate initial adhesion and the downregulation of growth cone motility. The agrin receptor Lrp4 was also essential for initial adhesive contacts and was downregulated on NCAM-/- myotubes. Our identification of novel roles for NCAM and Lrp4 and possible interactions between them in transforming motile growth cones into stable contacts opens interesting new avenues for exploration.

Bazedoxifene and raloxifene protect neocortical neurons undergoing hypoxia via targeting ERα and PPAR-γ

Selective estrogen receptor modulators (SERMs) such as bazedoxifene and raloxifene are recognized to mainly act via estrogen receptors (ERs), but there is no study examining the involvement of PPAR-γ in their actions, especially in neurons undergoing hypoxia. Little is also known about age-dependent actions of the SERMs on neuronal tissue challenged with hypoxia. In this study, bazedoxifene and raloxifene protected neocortical cells against hypoxia at early and later developmental stages. Both SERMs evoked caspase-3-independent neuroprotection and increased protein levels of ERα (66 and 46 kDa isoforms) and PPAR-γ. In addition, bazedoxifene enhanced expression of ERα-regulated Cyp19a1 mRNA. Using double siRNA silencing, for the first time we demonstrated a key role of ERα and PPAR-γ in the neuroprotective action of the SERMs in neocortical neurons undergoing hypoxia. This study provides prospects for the development of a new therapeutic strategies against hypoxic brain injury that selectively target ERα and/or PPAR-γ.

GSK3\u03b2 and ERK regulate the expression of 78\u2009kDa SG2NA and ectopic modulation of its level affects phases of cell cycle

Striatin and SG2NA are essential constituents of the multi-protein STRIPAK assembly harbouring protein phosphatase PP2A and several kinases. SG2NA has several isoforms generated by mRNA splicing and editing. While the expression of striatin is largely restricted to the striatum in brain, that of SG2NAs is ubiquitous. In NIH3T3 cells, only the 78 kDa isoform is expressed. When cells enter into the S phase, the level of SG2NA increases; reaches maximum at the G2/M phase and declines thereafter. Downregulation of SG2NA extends G1 phase and its overexpression extends G2. Ectopic expression of the 35 kDa has no effects on the cell cycle. Relative abundance of phospho-SG2NA is high in the microsome and cytosol and the nucleus but low in the mitochondria. Okadoic acid, an inhibitor of PP2A, increases the level of SG2NA which is further enhanced upon inhibition of proteasomal activity. Phospho-SG2NA is thus more stable than the dephosphorylated form. Inhibition of GSK3β by LiCl reduces its level, but the inhibition of ERK by PD98059 increases it. Thus, ERK decreases the level of phospho-SG2NA by inhibiting GSK3β. In cells depleted from SG2NA by shRNA, the levels of pGSK3β and pERK are reduced, suggesting that these kinases and SG2NA regulate each other’s expression.

Positive transcription elongation factor b (P-TEFb) is a therapeutic target in human multiple myeloma.

The role of the positive RNA Pol II regulator, P-TEFb (positive transcription elongation factor b), in maintenance of the anti-apoptotic protein Mcl-1 and bortezomib (btz) resistance was investigated in human multiple myeloma (MM) cells. Mcl-1 was up-regulated in all MM lines tested, including bortezomib-resistant lines, human MM xenograft mouse models, and primary CD138+ MM cells. Mcl-1 over-expression significantly reduced bortezomib lethality, indicating a functional role for Mcl-1 in bortezomib resistance. MM cell lines, primary MM specimens, and murine xenografts exhibited constitutive P-TEFb activation, manifested by high CTD (carboxy-terminal domain) S2 phosphorylation, associated with a) P-TEFb subunit up-regulation i.e., CDK9 (42 and 55 kDa isoforms) and cyclin T1; and b) marked CDK9 (42 kDa) T186 phosphorylation. In marked contrast, normal hematopoietic cells failed to exhibit up-regulation of p-CTD, CDK9, cyclin T1, or Mcl-1. CDK9 or cyclin T1 shRNA knock-down dramatically inhibited CTD S2 phosphorylation and down-regulated Mcl-1. Moreover, CRISPR-Cas CDK9 knock-out triggered apoptosis in MM cells and dramatically diminished cell growth. Pan-CDK e.g., dinaciclib or alvocidib and selective CDK9 inhibitors (CDK9i) recapitulated the effects of genetic P-TEFb disruption. CDK9 shRNA or CDK9 inhibitors significantly potentiated the susceptibility of MM cells, including bortezomib-resistant cells, to proteasome inhibitors. Analogously, CDK9 or cyclin T1 knock-down or CDK9 inhibitors markedly increased BH3-mimetic lethality in bortezomib-resistant cells. Finally, pan-CDK inhibition reduced human drug-naïve or bortezomib-resistant CD138+ cells and restored bone marrow architecture in vivo. Collectively, these findings implicate constitutive P-TEFb activation in high Mcl-1 maintenance in MM, and validate targeting the P-TEFb complex to circumvent bortezomib-resistance.

Acetylated Signal Transducer and Activator of Transcription 3 Functions as Molecular Adaptor Independent of Transcriptional Activity During Human Cardiogenesis.

Activation of signal transducer and activator of transcription 3 (STAT3) is imperative for mammalian development, specifically cardiogenesis. STAT3 phosphorylation and acetylation are key post-translational modifications that regulate its transcriptional activity. Significance of such modifications during human cardiogenesis remains elusive. Using human pluripotent stem cells to recapitulate cardiogenesis, two independently modified STAT3α (92 kDa) isoforms (phosphorylated and acetylated), which perform divergent functions were identified during cardiomyocyte (CM) formation. Phosphorylated STAT3α functioned as the canonical transcriptional activator, while acetylated STAT3α underwent caspase-3-mediated cleavage to generate a novel STAT3ζ fragment (∼45 kDa), which acted as a molecular adaptor integral to the ErbB4-p38γ signaling cascade in driving CM formation. While STAT3α knockdown perturbed cardiogenesis by eliminating both post-translationally modified STAT3α isoforms, caspase-3 knockdown specifically abrogates the function of acetylated STAT3α, resulting in limited STAT3ζ formation thereby preventing nuclear translocation of key cardiac transcription factor Nkx2-5 that disrupted CM formation. Our findings show the coexistence of two post-translationally modified STAT3α isoforms with distinct functions and define a new role for STAT3 as a molecular adaptor that functions independently of its canonical transcriptional activity during human cardiogenesis. Stem Cells 2017;35:2129-2137.

Distinct Localization of SNAP47 Protein in GABAergic and Glutamatergic Neurons in the Mouse and the Rat Hippocampus.

Synaptosomal-associated protein of 47 kDa (SNAP47) isoform is an atypical member of the SNAP family, which does not contribute directly to exocytosis and synaptic vesicle (SV) recycling. Initial characterization of SNAP47 revealed a widespread expression in nervous tissue, but little is known about its cellular and subcellular localization in hippocampal neurons. Therefore, in the present study we applied multiple-immunofluorescence labeling, immuno-electron microscopy and in situ hybridization (ISH) and analyzed the localization of SNAP47 in pre- and postsynaptic compartments of glutamatergic and GABAergic neurons in the mouse and rat hippocampus. While the immunofluorescence signal for SNAP47 showed a widespread distribution in both mouse and rat, the labeling pattern was complementary in the two species: in the mouse the immunolabeling was higher over the CA3 stratum radiatum, oriens and cell body layer. In contrast, in the rat the labeling was stronger over the CA1 neuropil and in the CA3 stratum lucidum. Furthermore, in the mouse high somatic labeling for SNAP47 was observed in GABAergic interneurons (INs). On the contrary, in the rat, while most INs were positive, they blended in with the high neuropil labeling. ISH confirmed the high expression of SNAP47 RNA in INs in the mouse. Co-staining for SNAP47 and pre- and postsynaptic markers in the rat revealed a strong co-localization postsynaptically with PSD95 in dendritic spines of pyramidal cells and, to a lesser extent, presynaptically, with ZnT3 and vesicular glutamate transporter 1 (VGLUT1) in glutamatergic terminals such as mossy fiber (MF) boutons. Ultrastructural analysis confirmed the pre- and postsynaptic localization at glutamatergic synapses. Furthermore, in the mouse hippocampus SNAP47 was found to be localized at low levels to dendritic shafts and axon terminals of putative INs forming symmetric synapses, indicating that this protein could be trafficked to both post- and presynaptic sites in both major cell types. These results reveal divergent localization of SNAP47 protein in mouse and rat hippocampus indicating species- and cell type-specific differences. SNAP47 is likely to be involved in unique fusion machinery which is distinct from the one involved in presynaptic neurotransmitter release. Nonetheless, our data suggest that SNAP47 may be involved not only postsynaptic, but also in presynaptic function.

Abstract 3181: A novel topoisomerase IIα 90 kDa isoform in etoposide resistant human leukemia K562 cells produced as a result of alternative RNA processing

Abstract DNA topoisomerase IIα (TOP2α) is a prominent target for anticancer drugs whose clinical efficacy is often limited by chemoresistance. We previously characterized acquired resistance to etoposide (VP-16) in a cloned human K562 leukemia cell line, K/VP.5, containing reduced TOP2α. In the present study, using an antibody specific for the amino-terminus of TOP2α, immunoassays indicated the existence of two TOP2α isoforms, 170 and 90 kDa, present in K562 leukemia cells and in the etoposide resistant K/VP.5 cells. TOP2α/90 expression was dramatically increased in etoposide-resistant K/VP.5 compared to parental K562 cells. We hypothesized that TOP2α/90 was the translation product of novel alternatively processed pre-mRNA, confirmed by 3’-RACE, PCR, and sequencing. TOP2α/90 mRNA includes retained intron 19 which harbors an in-frame stop codon, and two consensus poly(A) sites. The processed transcript is polyadenylated. TOP2α/90 mRNA encodes a 90,076 Da translation product missing the carboxyl-terminal 770 amino acids of TOP2α/170, replaced by 25 unique amino acids through translation of the exon 19/intron 19 ‘readthrough’. Immunoassays, utilizing antisera raised against these unique amino acids, confirmed that TOP2α/90 is expressed in both cell types, with overexpression in K/VP.5 cells. Immunodetection of Complex of Enzyme-to-DNA (ICE) and single cell gel electrophoresis (Comet) assays demonstrated that K562 cells transfected with a TOP2α/90 expression plasmid, exhibited reduced etoposide-mediated TOP2α-DNA covalent complexes and decreased etoposide-induced DNA damage, respectively, compared to similarly treated K562 cells transfected with empty vector. Since TOP2α/90 lacks the active site tyrosine (Tyr805) of full length TOP2α, these results strongly suggest that TOP2α/90 exhibits dominant-negative properties. . In separate studies the TOP2α/90 mRNA splice variant was found to be expressed in most human tissues suggesting that this novel protein isoform may play a role in both intrinsic chemosensitivity as well as in acquired resistance. Further studies are underway to characterize the mechanism(s) by which TOP2α/90 plays a role in acquired resistance to etoposide and other TOP2α targeting agents. In addition, future studies will be directed to examine the RNA processing mechanism(s) operational that suppress intron 19 splicing in TOP2α pre-mRNA. Citation Format: Ragu Kanagasabai, Lucas Serdar, Soumendrakrishna Karmahapatra, Corey A. Kientz, Mary K. Ritke, Terry S. Elton, Jack C. Yalowich. A novel topoisomerase IIα 90 kDa isoform in etoposide resistant human leukemia K562 cells produced as a result of alternative RNA processing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3181. doi:10.1158/1538-7445.AM2017-3181

Regulation of sub-compartmental targeting and folding properties of the Prion-like protein Shadoo

Shadoo (Sho), a member of prion protein family, has been shown to prevent embryonic lethality in Prnp0/0 mice and to be reduced in the brains of rodents with terminal prion diseases. Sho can also affect PrP structural dynamics and can increase the prion conversion into its misfolded isoform (PrPSc), which is amyloidogenic and strictly related to expression, intracellular localization and association of PrPC to lipid rafts. We reasoned that if Sho possesses a natural tendency to convert to amyloid-like forms in vitro, it should be able to exhibit “prion-like” properties, such as PK-resistance and aggregation state, also in live cells. We tested this hypothesis, by different approaches in neuronal cells, finding that Sho shows folding properties partially dependent on lipid rafts integrity whose alteration, as well as proteasomal block, regulated generation of intermediate Sho isoforms and exacerbated its misfolding. Moreover, a 18 kDa isoform of Sho, likely bearing the signal peptide, was targeted to mitochondria by interacting with the molecular chaperone TRAP1 which, in turn controlled Sho dual targeting to ER or mitochondria. Our studies contribute to understand the role of molecular chaperones and of PrP-related folding intermediates in “prion-like” conversion.

VGLUT1 synapses and P-boutons on regenerating motoneurons after nerve crush.

Stretch-sensitive Ia afferent monosynaptic connections with motoneurons form the stretch reflex circuit. After nerve transection, Ia afferent synapses and stretch reflexes are permanently lost, even after regeneration and reinnervation of muscle by motor and sensory afferents is completed in the periphery. This loss greatly affects full recovery of motor function. However, after nerve crush, reflex muscle forces during stretch do recover after muscle reinnervation and reportedly exceed 140% baseline values. This difference might be explained by structural preservation after crush of Ia afferent synapses on regenerating motoneurons and decreased presynaptic inhibitory control. We tested these possibilities in rats after crushing the tibial nerve (TN), and using Vesicular GLUtamate Transporter 1 (VGLUT1) and the 65 kDa isoform of glutamic acid-decarboxylase (GAD65) as markers of, respectively, Ia afferent synapses and presynaptic inhibition (P-boutons) on retrogradely labeled motoneurons. We analyzed motoneurons during regeneration (21 days post crush) and after they reinnervate muscle (3 months). The results demonstrate a significant loss of VGLUT1 terminals on dendrites and cell bodies at both 21 days and 3 months post-crush. However, in both cellular compartments, the reductions were small compared to those observed after TN full transection. In addition, we found a significant decrease in the number of GAD65 P-boutons per VGLUT1 terminal and their coverage of VGLUT1 boutons. The results support the hypothesis that better preservation of Ia afferent synapses and a change in presynaptic inhibition could contribute to maintain or even increase the stretch reflex after nerve crush and by difference to nerve transection.

Identification of Novel Placentally Expressed Aspartic Proteinase in Humans.

This study presents pioneering data concerning the human pregnancy-associated glycoprotein-Like family, identified in the genome, of the term placental transcriptome and proteome. RNA-seq allowed the identification of 1364 bp hPAG-L/pep cDNA with at least 56.5% homology with other aspartic proteinases (APs). In silico analyses revealed 388 amino acids (aa) of full-length hPAG-L polypeptide precursor, with 15 aa-signal peptide, 47 aa-blocking peptide and 326 aa-mature protein, and two Asp residues (D), specific for a catalytic cleft of the APs (VVFDTGSSNLWV91-102 and AIVDTGTSLLTG274-285). Capillary sequencing identified 9330 bp of the hPAG-L gene (Gen Bank Acc. No. KX533473), composed of nine exons and eight introns. Heterologous Western blotting revealed the presence of one dominant 60 kDa isoform of the hPAG-L amongst cellular placental proteins. Detection with anti-pPAG-P and anti-Rec pPAG2 polyclonals allowed identification of the hPAG-L proteins located within regions of chorionic villi, especially within the syncytiotrophoblast of term singleton placentas. Our novel data extend the present knowledge about the human genome, as well as placental transcriptome and proteome during term pregnancy. Presumably, this may contribute to establishing a new diagnostic tool for examination of some disturbances during human pregnancy, as well as growing interest from both scientific and clinical perspectives.