Inhibition of p25/Cdk5 Attenuates Tauopathy in Mouse and iPSC Models of Frontotemporal Dementia.

Increased p25, a proteolytic fragment of the regulatory subunit p35, is known to induce aberrant activity of cyclin-dependent kinase 5 (Cdk5), which is associated with neurodegenerative disorders, including Alzheimer's disease. Previously, we showed that replacing endogenous p35 with the noncleavable mutant p35 (Δp35) attenuated amyloidosis and improved cognitive function in a familial Alzheimer's disease mouse model. Here, to address the role of p25/Cdk5 in tauopathy, we generated double-transgenic mice by crossing mice overexpressing mutant human tau (P301S) with Δp35KI mice. We observed significant reduction of phosphorylated tau and its seeding activity in the brain of double transgenic mice compared with the P301S mice. Furthermore, synaptic loss and impaired LTP at hippocampal CA3 region of P301S mice were attenuated by blocking p25 generation. To further validate the role of p25/Cdk5 in tauopathy, we used frontotemporal dementia patient-derived induced pluripotent stem cells (iPSCs) carrying the Tau P301L mutation and generated P301L:Δp35KI isogenic iPSC lines using CRISPR/Cas9 genome editing. We created cerebral organoids from the isogenic iPSCs and found that blockade of p25 generation reduced levels of phosphorylated tau and increased expression of synaptophysin. Together, these data demonstrate a crucial role for p25/Cdk5 in mediating tau-associated pathology and suggest that inhibition of this kinase can remedy neurodegenerative processes in the presence of pathogenic tau mutation.SIGNIFICANCE STATEMENT Accumulation of p25 results in aberrant Cdk5 activation and induction of numerous pathological phenotypes, such as neuroinflammation, synaptic loss, Aß accumulation, and tau hyperphosphorylation. However, it was not clear whether p25/Cdk5 activity is necessary for the progression of these pathological changes. We recently developed the Δp35KI transgenic mouse that is deficient in p25 generation and Cdk5 hyperactivation. In this study, we used this mouse model to elucidate the role of p25/Cdk5 in FTD mutant tau-mediated pathology. We also used a frontotemporal dementia patient-derived induced pluripotent stem cell carrying the Tau P301L mutation and generated isogenic lines in which p35 is replaced with noncleavable mutant Δp35. Our data suggest that p25/Cdk5 plays an important role in tauopathy in both mouse and human model systems.

Enhanced cryoprecipitate for skin graft and donor site wound healing in pigs.

Due to similarities in skin characteristics, the authors hypothesise that a pig model would most accurately show the ability of autologous, enhanced cryoprecipitate (eCryo) to improve the wound healing of split-thickness skin grafts (STSGs) and corresponding donor sites. Fifty-two STSGs (5 × 5 cm) were fashioned and treated according to a randomised protocol with an autologous eCryo-treated and a control group. Macroscopic assessment, histological evaluation and cellular composition were completed at days 7, 14, 21 and 28. Thirty-two donor sites were also created and assessed in a similar manner. Histologic analysis showed enhancement of healing over all time points for eCryo-treated donor sites. All other results showed no statistically significant improvement with the use of eCryo. Autologous cryoprecipitate appears to be a safe, inexpensive and easy-to-use alternative to fibrin glue, which carries risks and is, in many cases, prohibitively expensive. Further studies are necessary to evaluate the full potential of eCryo. Interestingly, eCryo application may improve donor site aesthetic appearance. We believe that a pig model most reliably simulates eCryo's behaviour in humans to accurately reflect its future clinical applicability.

Receptor-binding and oncogenic properties of polyoma viruses isolated from feral mice.

Laboratory strains of the mouse polyoma virus differ markedly in their abilities to replicate and induce tumors in newborn mice. Major determinants of pathogenicity lie in the sialic binding pocket of the major capsid protein Vp1 and dictate receptor-binding properties of the virus. Substitutions at two sites in Vp1 define three prototype strains, which vary greatly in pathogenicity. These strains replicate in a limited fashion and induce few or no tumors, cause a disseminated infection leading to the development of multiple solid tumors, or replicate and spread acutely causing early death. This investigation was undertaken to determine the Vp1 type(s) of new virus isolates from naturally infected mice. Compared with laboratory strains, truly wild-type viruses are constrained with respect to their selectivity and avidity of binding to cell receptors. Fifteen of 15 new isolates carried the Vp1 type identical to that of highly tumorigenic laboratory strains. Upon injection into newborn laboratory mice, the new isolates induced a broad spectrum of tumors, including ones of epithelial as well as mesenchymal origin. Though invariant in their Vp1 coding sequences, these isolates showed considerable variation in their regulatory sequences. The common Vp1 type has two essential features: 1) failure to recognize "pseudoreceptors" with branched chain sialic acids binding to which would attenuate virus spread, and 2) maintenance of a hydrophobic contact with true receptors bearing a single sialic acid, which retards virus spread and avoids acute and potentially lethal infection of the host. Conservation of these receptor-binding properties under natural selection preserves the oncogenic potential of the virus. These findings emphasize the importance of immune protection of neonates under conditions of natural transmission.

APOE4 Causes Widespread Molecular and Cellular Alterations Associated with Alzheimer's Disease Phenotypes in Human iPSC-Derived Brain Cell Types.

The apolipoprotein E4 (APOE4) variant is the single greatest genetic risk factor for sporadic Alzheimer's disease (sAD). However, the cell-type-specific functions of APOE4 in relation to AD pathology remain understudied. Here, we utilize CRISPR/Cas9 and induced pluripotent stem cells (iPSCs) to examine APOE4 effects on human brain cell types. Transcriptional profiling identified hundreds of differentially expressed genes in each cell type, with the most affected involving synaptic function (neurons), lipid metabolism (astrocytes), and immune response (microglia-like cells). APOE4 neurons exhibited increased synapse number and elevated Aß42 secretion relative to isogenic APOE3 cells while APOE4 astrocytes displayed impaired Aß uptake and cholesterol accumulation. Notably, APOE4 microglia-like cells exhibited altered morphologies, which correlated with reduced Aß phagocytosis. Consistently, converting APOE4 to APOE3 in brain cell types from sAD iPSCs was sufficient to attenuate multiple AD-related pathologies. Our study establishes a reference for human cell-type-specific changes associated with the APOE4 variant. VIDEO ABSTRACT.

Modulation of liver-intestine cadherin (Cadherin 17) expression, ERK phosphorylation and WNT signaling in EPHB6 receptor-expressing MDA-MB-231 cells.

Aberrant expression of erythropoietin-producing hepatocellular carcinoma cell (EPH) receptors has been reported in a variety of human cancer types. In addition to modulating cell proliferation and migration, EPH receptors are also involved in tumor progression. The transcriptional activation and silencing of EPH receptors are also associated with tumorigenesis. However, the mechanisms underlying the involvement of EPH receptors in tumorigenesis have not been completely deciphered. We have investigated and described the role of EPHB6, a kinase-deficient receptor, in modulating the abundance of cadherin 17 and activation of other intracellular signaling proteins. We previously showed that EPHB6 alters the tumor phenotype of breast carcinoma cells. However, the mechanisms underlying these phenotypic changes had not previously been investigated. Herein we demonstrated the downstream effects of EPHB6 expression on the abundance of cadherin 17, mitogen-activated protein kinase (MEK2), extracellular signal-regulated kinase (ERK), phospho-ERK, ß-catenin, phospho- glycogen synthase kinase 3 beta (GSK3ß) (ser21/9), cell morphology and actin cytoskeleton. These comparisons were made between EPHB6-deficient MDA-MB-231 cells transfected with an empty pcDNA3 vector and cells stably transfected with an expression construct of EPHB6. The results indicate elevated levels of MEK2 and phospho-ERK. While there was no change in the amount of ERK, the abundance of cadherin 17, ß-catenin and phospho-GSK3ß was significantly reduced in EPHB6-transfected cells. These studies clearly demonstrate an inverse relationship between the levels of phospho-ERK and the abundance of cadherin 17, ß-catenin and phospho-GSK3ß in EPHB6-expressing MDA-MB-231 cells. From these data we conclude that EPHB6-mediated alterations arise due to changes in abundance and localization of cadherin 17 and activation of WNT signaling pathway. Transcriptional silencing of EPHB6 in native MDA-MB-231 cells and consequent effects on cadherin 17 and WNT pathway may, thus, be responsible for the invasive behavior of these cells.

Cisplatin causes over-expression of tachykinin NK(1) receptors and increases ERK1/2- and PKA- phosphorylation during peak immediate- and delayed-phase emesis in the least shrew (Cryptotis parva) brainstem.

Scant information is available regarding the effects of cisplatin on the expression profile of tachykinin NK(1) receptors and downstream signaling during cisplatin-induced emesis. Cisplatin causes peak early- and delayed-phase emesis in the least shrew at 1-2 and 33 h post-injection. To investigate the expression profile of NK(1) receptor during both emetic phases, we cloned the cDNA corresponding to a ~700 base pairs of mRNA flanked by two stretches of nucleotides conserved among different species and demonstrated that the shrew NK(1) receptor nucleotide sequence shares ~90% sequence identity with the human NK(1) receptor. Of the 12 time-points tested, significant increases in expression levels of NK(1) receptor mRNA in the shrew brainstem occurred at 2 and 28 h post-cisplatin injection, whereas intestinal NK(1) receptor mRNA was increased at 28 h. Shrew brainstem and intestinal substance P mRNA levels also tended to increase during the two phases. Furthermore, expression levels of NK(1) receptor protein were significantly increased in the brainstem at 2, 8, and 33 h post-cisplatin. No change in brainstem 5-HT(3) receptor protein expression was observed. The temporal enhancements in NK(1) receptor protein expression were mirrored by significant increases in the phosphorylation status of the brainstem ERK1/2 at 2, 8, and 33 h post-cisplatin. Phosphorylation of PKA significantly increased at 33rd and 40th hour. Our results indicate associations between cisplatin's peak immediate- and delayed-phase vomiting frequency with increased: (1) expression levels of NK(1) receptor mRNA and its protein level, and (2) downstream NK(1) receptor-mediated phosphorylation of ERK1/2 and PKA signaling.

The tumor suppressor CDKN3 controls mitosis.

Mitosis is controlled by a network of kinases and phosphatases. We screened a library of small interfering RNAs against a genome-wide set of phosphatases to comprehensively evaluate the role of human phosphatases in mitosis. We found four candidate spindle checkpoint phosphatases, including the tumor suppressor CDKN3. We show that CDKN3 is essential for normal mitosis and G1/S transition. We demonstrate that subcellular localization of CDKN3 changes throughout the cell cycle. We show that CDKN3 dephosphorylates threonine-161 of CDC2 during mitotic exit and we visualize CDC2(pThr-161) at kinetochores and centrosomes in early mitosis. We performed a phosphokinome-wide mass spectrometry screen to find effectors of the CDKN3-CDC2 signaling axis. We found that one of the identified downstream phosphotargets, CKß phosphorylated at serine 209, localizes to mitotic centrosomes and controls the spindle checkpoint. Finally, we show that CDKN3 protein is down-regulated in brain tumors. Our findings indicate that CDKN3 controls mitosis through the CDC2 signaling axis. These results have implications for targeted anticancer therapeutics.

Generation of Induced Pluripotent Stem (iPS) Cells by Nuclear Reprogramming.

During embryonic development pluripotency is progressively lost irreversibly by cell division, differentiation, migration and organ formation. Terminally differentiated cells do not generate other kinds of cells. Pluripotent stem cells are a great source of varying cell types that are used for tissue regeneration or repair of damaged tissue. The pluripotent stem cells can be derived from inner cell mass of blastocyte but its application is limited due to ethical concerns. The recent discovery of iPS with defined reprogramming factors has initiated a flurry of works on stem cell in various laboratories. The pluripotent cells can be derived from various differentiated adult cells as well as from adult stem cells by nuclear reprogramming, somatic cell nuclear transfer etc. In this review article, different aspects of nuclear reprogramming are discussed.

Differential temporal changes in brain and gut substance P mRNA expression throughout the time-course of cisplatin-induced vomiting in the least shrew (Cryptotis parva).

Cisplatin and related chemotherapeutics are potent emetogens in humans and least shrews, a small animal emesis model which also vomits in response to substance P (SP). The SP-producing preprotachykinin-1 (PPT1) mRNA is transcribed from the Tac1 gene, which has been sequenced from several animal species and humans and is highly conserved. Despite its prominent role in chemotherapy-induced vomiting, the tachykininergic system is not well-characterized in emesis-competent species. This study was undertaken to further establish Cryptotis parva as an emesis model, by sequencing and characterizing SP mRNA, and then comparing the least shrew tachykininergic system to other mammalian species (vomiting and non-vomiting). The cDNA for least shrew beta-PPT1 was successfully cloned and partially sequenced, and found to be 90% homologous to the human sequence, with the SP-producing portion identical to humans. Initial in situ hybridization results demonstrated induction of beta-PPT1 mRNA in the gut following cisplatin administration. These were followed up with mRNA quantification (via QPCR) at multiple time points following cisplatin injection. PPT1 mRNA levels in the brain spiked at 4 h (19-fold increase) and 24 h (20-fold increase) in correlation with cisplatin-induced emesis. PPT1 mRNA in the gut spiked at 28 h (approximately 6.5-fold increase), correlated with the later phase of vomiting. These results validate the least shrew as a tachykinin model at the molecular level.

Zebrafish-encoded 3-O-sulfotransferase-3 isoform mediates herpes simplex virus type 1 entry and spread.

Heparan sulfate proteoglycans modified by human glucosaminyl 3-O-sulfotransferase-3 (3-OST-3) isoform generates the cellular receptor for herpes simplex virus type 1 (HSV-1). Interestingly, the ability of zebrafish (ZF)-encoded 3-OST-3 isoform to modify heparan sulfate to mediate HSV-1 entry and cell-cell fusion has not been determined although it is predominantly expressed in ZF, a popular model organism to study viral infections. Here, we demonstrate that expression of ZF-encoded 3-OST-3 isoform renders the resistant Chinese hamster ovary (CHO-K1) cells to become susceptible for HSV-1 entry. The following lines of evidence support the important role of ZF-encoded 3-OST-3 isoform as the mediator of HSV-1 entry into CHO-K1 cells: (1) ZF 3-OST-3-expressing CHO-K1 cells were able to preferentially bind HSV-1 glycoprotein D, and (2) CHO-K1 cells expressing ZF-encoded 3-OST-3 acquire the ability to fuse with cells expressing HSV-1 glycoproteins. Finally, knocking down 3-OST-3 receptor by siRNA in ZF fibroblasts cells significantly reduced HSV-1 entry and glycoprotein D binding to cells. Taken together, our results provide novel insight into the significance of ZF 3-OST-3 isoform as an HSV-1 entry and fusion receptor and its potential involvement in the HSV-1 disease model of ZF.

Induction and bypass of p53 during productive infection by polyomavirus.

Lytic infection by polyomavirus leads to elevated levels of p53 and induction of p53 target genes p21Cip1/WAF1 (p21) and BAX. This is seen both in polyomavirus-infected primary mouse cell cultures and in kidney tissue of infected mice. Stabilization of p53 and induction of a p53 response are accompanied by phosphorylation of p53 on serine 18, mimicking a DNA damage response. Stabilization of p53 does not depend on p19Arf interaction with mdm2. Cells infected by a mutant virus defective in binding pRb and in inducing G(1)-to-S progression show a greatly diminished p53 response. However, cells infected by wild-type virus and blocked from entering S phase by addition of mimosine still show a p53 response. These results suggest a role of E2F target genes in inducing a p53 response. Polyomavirus large T antigen coprecipitates with p53 phosphorylated on serine 18 and also with p21Cip1/WAF1. Implications of these and other findings on possible mechanisms of induction and override of p53 functions during productive infection by polyomavirus are discussed.

6-(2-Hydroxybenzoyl)-5-(pyrrol-2-yl)-3H-pyrrolizine.

The title compound, 2-hydroxyphenyl 5-(pyrrol-2-yl)-3H-pyrrolizin-6-yl ketone, C(18)H(14)N(2)O(2), was isolated from the base-catalyzed 1:2 condensation of 2-hydroxyacetophenone with pyrrole-2-carbaldehyde. The pyrrole N-H and hydroxybenzoyl O-H groups are hydrogen bonded to the benzoyl O atom. The allylic C=C double bond of the 3H-pyrrolizine system is located between ring positions 1 and 2, the C atom at position 3 (adjacent to the N atom) being single bonded.

[5-Bromo-N-(2-carboxylatophenyl)salicylideniminato]dimethyltin(IV).

The structure of the title dimethyltin(IV) complex, [2-(5-bromo-2-oxidobenzylideneamino)benzoato-kappa3O,N,O']dimethyltin(IV), [Sn(CH(3))(2)(C(14)H(8)BrNO(3))], features centrosymmetric dimers disposed about a central Sn(2)O(2) core. Each Sn centre has seven-coordinate pentagonal-bipyramidal geometry, taking into account two moderately long Sn-O contacts about an inversion centre [2.679 (4) and 2.981 (4) A]. The methyl groups are in an axial orientation.