Opposing roles of transient and prolonged expression of p25 in synaptic plasticity and hippocampus-dependent memory

While deregulation of cyclin-dependent kinase 5 (Cdk5) has been implicated in neurodegenerative diseases, its precise role in synaptic plasticity and memory remains elusive. Proteolytic cleavage of p35, a regulatory subunit of Cdk5, by calpain results in the generation of the truncated p25 protein, which causes hyperactivation of Cdk5. Using region-specific and inducible transgenic mice, we show that transiently increased p25 expression in the hippocampus enhanced long-term potentiation (LTP) and facilitated hippocampus-dependent memory. Moreover, p25 expression increased the number of dendritic spines and synapses. Importantly, enhanced memory achieved by a transient expression of p25 followed by its repression did not cause neurodegeneration. In contrast, prolonged p25 production caused severe cognitive deficits, which were accompanied by synaptic and neuronal loss and impaired LTP. Our data suggest a role for p25 in synaptic plasticity, synaptogenesis, learning, and memory and provide a model whereby deregulation of a plasticity factor can contribute to neurodegeneration.     

The yin and yang of neurotrophin action

Neurotrophins have diverse functions in the CNS. Initially synthesized as precursors (proneurotrophins), they are cleaved to produce mature proteins, which promote neuronal survival and enhance synaptic plasticity by activating Trk receptor tyrosine kinases. Recent studies indicate that proneurotrophins serve as signalling molecules by interacting with the p75 neurotrophin receptor (p75NTR). Interestingly, proneurotrophins often have biological effects that oppose those of mature neurotrophins. Therefore, the proteolytic cleavage of proneurotrophins represents a mechanism that controls the direction of action of neurotrophins. New insights into the 'yin and yang' of neurotrophin activity have profound implications for our understanding of the role of neurotrophins in a wide range of cellular processes.     

Identification of amino acids in the transmembrane and juxtamembrane domains of the platelet-derived growth factor receptor required for productive interaction with the bovine papillomavirus E5 protein.

The bovine papillomavirus E5 protein forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in receptor activation and cell transformation. Amino acids in both the putative transmembrane domain and extracytoplasmic carboxyl-terminal domain of the E5 protein appear important for PDGF receptor binding and activation. Previous analysis indicated that the transmembrane domain of the receptor was also required for complex formation and receptor activation. Here we analyzed receptor chimeras and point mutants to identify specific amino acids in the PDGF beta receptor required for productive interaction with the E5 protein. These receptor mutants were analyzed in murine Ba/F3 cells, which do not express endogenous receptor. Our results confirmed the importance of the transmembrane domain of the receptor for complex formation, receptor tyrosine phosphorylation, and mitogenic signaling in response to the E5 protein and established that the threonine residue in this domain is required for these activities. In addition, a positive charge in the extracellular juxtamembrane domain of the receptor was required for E5 interaction and signaling, whereas replacement of the wild-type lysine with either a neutral or acidic amino acid inhibited E5-induced receptor activation and transformation. All of the receptor mutants defective for activation by the E5 protein responded to acute treatment with PDGF and to stable expression of v-Sis, a form of PDGF. The required juxtamembrane lysine and transmembrane threonine are predicted to align precisely on the same face of an alpha helix packed in a left-handed coiled-coil geometry. These results establish that the E5 protein and v-Sis recognize distinct binding sites on the PDGF beta receptor and further clarify the nature of the interaction between the viral transforming protein and its cellular target.     

Transformation of mortal human fibroblasts and activation of a growth inhibitory pathway by the bovine papillomavirus E5 oncoprotein.

The 44-amino acid bovine papillomavirus E5 protein induces tumorigenic transformation of immortal rodent fibroblasts by binding to and activating the platelet-derived growth factor beta receptor (PDGFbetaR). Here E5 was expressed in mortal human diploid fibroblasts (HDFs), which lack the accumulated genetic changes that are present in immortal rodent cells. E5 induced focus formation and morphological transformation of HDFs without inducing anchorage independence or immortalization. Similar effects were observed with the v-sis and neu* oncogenes. E5-PDGFbetaR complexes were observed in the E5-expressing HDFs, as was constitutive PDGFbetaR activation, which was required for the transforming activity of E5. The E5 HDFs attained a higher saturation density than the control cells, expressing increased levels of hyperphosphorylated retinoblastoma protein at subconfluent densities. However, when these cells reached confluence, growth inhibition accompanied by dramatic down-regulation of the PDGFbetaR, and retinoblastoma protein was induced apparently by a factor secreted into the medium. This may represent a novel negative feedback mechanism controlling PDGFbetaR-induced proliferation and thereby protecting against complete transformation.     

Specific interaction between the bovine papillomavirus E5 transforming protein and the beta receptor for platelet-derived growth factor in stably transformed and acutely transfected cells.

The E5 protein of bovine papillomavirus is a 44-amino-acid membrane protein which induces morphologic and tumorigenic transformation of fibroblasts. We previously showed that the E5 protein activates and forms a complex with the endogenous beta receptor for platelet-derived growth factor (PDGF) in transformed rodent fibroblasts and that the PDGF beta receptor can mediate tumorigenic transformation by the E5 protein in a heterologous cell system. Other workers have identified the receptor for epidermal growth factor (EGF) as a potential target of the E5 protein in NIH 3T3 cells. Here, we investigate the specificity of the interaction of the E5 protein with various growth factor receptors, with particular emphasis on the PDGF beta receptor and the EGF receptor. Under conditions where both the PDGF beta receptor and the EGF receptor are stably expressed in E5-transformed mouse and bovine fibroblasts and in E5-transformed epithelial cells, the E5 protein specifically forms a complex with and activates the PDGF receptor and not the EGF receptor. Under conditions of transient overexpression in COS cells, the E5 protein has the potential to associate with several growth factor receptors, including the EGF receptor. However, upon coexpression of PDGF beta receptors and EGF receptors in COS cells, the E5 protein preferentially forms a complex with the PDGF receptor. Therefore, we conclude that the PDGF beta receptor is the primary target for the E5 protein in a variety of cell types, including bovine fibroblasts.     

Immobilization of anaerobic sludge using chitosan crosslinked with lignosulfonate

A new method for immobilization of anaerobic sludge in chitosan is described. It is based on the reaction of basic NH₂ groups of chitosan with the acidic sulfonic-groups of lignosulfonate to form sulfonilamide linkages. The new procedure features simplicity, low-cost and mild immobilization conditions. Batch tests of acetate consumption along with a continuous reactor operation confirmed the effectiveness of the immobilization technique for maintenance of long-term stability of the polymer. Received 27 March 1997/ Accepted in revised form 01 October 1997     

Identity and ranking of colonic mesenchymal stromal cells

Although ongoing clinical trials utilize systemic administration of bone-marrow mesenchymal stromal cells (BM-MSCs) in Crohn's disease (CD), nothing is known about the presence and the function of mesenchymal stromal cells (MSCs) in the normal human bowel. MSCs are bone marrow (BM) multipotent cells supporting hematopoiesis with the potential to differentiate into multiple skeletal phenotypes. A recently identified new marker, CD146, allowing to prospectively isolate MSCs from BM, renders also possible their identification in different tissues. In order to elucidate the presence and functional role of MSCs in human bowel we analyzed normal adult colon sections and isolated MSCs from them. In colon (C) sections, resident MSCs form a net enveloping crypts in lamina propria, coinciding with structural myofibroblasts or interstitial stromal cells. Nine sub-clonal CD146(+) MSC lines were derived and characterized from colon biopsies, in addition to MSC lines from five other human tissues. In spite of a phenotype qualitative identity between the BM- and C-MSC populations, they were discriminated and categorized. Similarities between C-MSC and BM-MSCs are represented by: Osteogenic differentiation, hematopoietic supporting activity, immune-modulation, and surface-antigen qualitative expression. The differences between these populations are: C-MSCs mean intensity expression is lower for CD13, CD29, and CD49c surface-antigens, proliferative rate faster, life-span shorter, chondrogenic differentiation rare, and adipogenic differentiation completely blocked. Briefly, BM-MSCs, deserve the rank of progenitors, whereas C-MSCs belong to the restricted precursor hierarchy. The presence and functional role of MSCs in human colon provide a rationale for BM-MSC replacement therapy in CD, where resident bowel MSCs might be exhausted or diverted from their physiological functions.     

Tailoring structure-function and pharmacokinetic properties of single-chain Fv proteins by site-specific PEGylation

The utility of single-chain Fv proteins as therapeutic agents would be realized if the circulating lives of these minimal antigen-binding polypeptides could be both prolonged and adjustable. We have developed a general strategy for creating tailored monoPEGylated single-chain antibodies. Free cysteine residues were engineered in an anti-TNF-alpha scFv at the C-terminus or within the linker segments of both scFv orientations, V(L)-linker-V(H) and V(H)-linker-V(L). High-level expression of 10 designed variant scFv proteins in Pichia pastoris allowed rapid purification. Optimization of site-specific conjugate preparation with 5, 20 and 40 kDa maleimide-PEG polymers was achieved and a comparison of the structural and functional properties of the scFv proteins and their PEGylated counterparts was performed. Peptide mapping and MALDI-TOF mass spectrometric analysis confirmed the unique attachment site for each PEG polymer. Independent biochemical and bioactivity analyses, including binding affinities and kinetics, antigenicity, flow cytometric profiling and cell cytotoxicity rescue, demonstrated that the functional activities of the 10 designed scFv conjugates are maintained, while scFv activity variations between these alternative assays can be correlated with conjugate and analytical designs. Pharmacokinetic studies of the PEGylated scFv in mice demonstrated up to 100-fold prolongation of circulating lives, in a range comparable to clinical antibodies.     

Molecular examination of the transmembrane requirements of the platelet-derived growth factor beta receptor for a productive interaction with the bovine papillomavirus E5 oncoprotein

The small transmembrane E5 protein of bovine papillomavirus (BPV) transforms cells by forming a stable complex with and activating the platelet-derived growth factor beta receptor (PDGFbetaR). The E5/PDGFbetaR interaction is thought to involve specific physical contacts between the transmembrane domains of the two proteins. Lys(499) at the extracellular juxtamembrane position and Thr(513) within the transmembrane domain of the PDGFbetaR are required for the interaction and are predicted to contact analogously positioned residues in the E5 protein. Here, mutagenic analysis of the transmembrane region of the PDGFbetaR was performed to further characterize the nature of the E5/PDGFbetaR interaction. We show that the receptor transmembrane domain, with minimal extracellular and intracellular sequence, is sufficient for the interaction. In addition, we provide evidence that the polar nature of Thr(513) as well as its positioning along the transmembrane alpha-helix is important for the interaction. We also identify the receptor transmembrane amino acids Ile(506) and Leu(520) as additional requirements for the interaction. Because Lys(499), Thr(513), Ile(506), and Leu(520) all align along the same face of the predicted PDGFbetaR transmembrane alpha-helix, our data support the model that the PDGFbetaR contacts the E5 protein via multiple amino acids along a single alpha-helical interface.