Niels A. Jensen

Human and mouse proteomic databases: novel resources in the protein universe

Proteomics 1 is an emerging area of research of the post‐genomic era that deals with the global analysis of gene expression using a plethora of techniques to resolve (high resolution two‐dimensional polyacrylamide gel electrophoresis, 2D PAGE), identify (peptide sequencing by Edman degradation, mass spectrometry, Western immunoblotting, etc.), quantitate and characterize proteins, as well as to store (comprehensive 2D PAGE databases), communicate and interlink protein and DNA sequence and mapping information from genome projects. Here we review the current status as well as applications of human and mouse proteomic 2D PAGE databases that are being systematically constructed for the global analysis of gene expression in both health and disease (http://biobase.dk/cgi‐bin/celis). Furthermore, we discuss the problems one faces when using powerful proteomic technology to study heterogeneous tissue and tumor biopsies, and emphasize the importance of building comprehensive databases that contain a critical mass of information for both known and novel proteins in normal and disease conditions.

GABAA Increases Calcium in Subventricular Zone Astrocyte-Like Cells Through L- and T-Type Voltage-Gated Calcium Channels

In the adult neurogenic subventricular zone (SVZ), the behavior of astrocyte-like cells and some of their functions depend on changes in intracellular Ca2+ levels and tonic GABAA receptor activation. However, it is unknown whether, and if so how, GABAA receptor activity regulates intracellular Ca2+ dynamics in SVZ astrocytes. To monitor Ca2+ activity selectively in astrocyte-like cells, we used two lines of transgenic mice expressing either GFP fused to a Gq-coupled receptor or DsRed under the human glial fibrillary acidic protein (hGFAP) promoter. GABAA receptor activation induced Ca2+ increases in 40–50% of SVZ astrocytes. GABAA-induced Ca2+ increases were prevented with nifedipine and mibefradil, blockers of L- and T-type voltage-gated calcium channels (VGCC). The L-type Ca2+ channel activator BayK 8644 increased the percentage of GABAA-responding astrocyte-like cells to 75%, suggesting that the majority of SVZ astrocytes express functional VGCCs. SVZ astrocytes also displayed spontaneous Ca2+ activity, the frequency of which was regulated by tonic GABAA receptor activation. These data support a role for ambient GABA in tonically regulating intracellular Ca2+ dynamics through GABAA receptors and VGCC in a subpopulation of astrocyte-like cells in the postnatal SVZ.

Hippocampus-like corticoneurogenesis induced by two isoforms of the BTB-zinc finger gene Zbtb20 in mice

Hippocampus-associated genes that orchestrate the formation of the compact stratum pyramidale are largely unknown. The BTB (broad complex, tramtrack, bric-a-brac)-zinc finger gene Zbtb20 (also known as HOF, Znf288, Zfp288) encodes two protein isoforms, designated Zbtb20S and Zbtb20L, which are expressed in newborn pyramidal neurons of the presumptive hippocampus in mice. Here, we have generated transgenic mice with ectopic expression of Zbtb20S and Zbtb20L in immature pyramidal neurons differentiated from multipotent non-hippocampal precursors. The subiculum and posterior retrosplenial areas in these mice were transformed into a three-layered hippocampus-like cortex with a compact homogenous pyramidal cell layer. Severe malformations of lamination occur in neocortical areas, which coincide with a deficiency in expression of cortical lamination markers. The alterations in cortical cytoarchitecture result in behavioral abnormalities suggestive of a deficient processing of visual and spatial memory cues in the cerebral cortex of adult Zbtb20 transgenic mice. Overall, our in vivo data suggest that Zbtb20 functions as a molecular switch for a pathway that induces invariant pyramidal neuron morphogenesis and suppression of cell fate transitions in newborn neurons.

High expression of microRNA-625-3p is associated with poor response to first-line oxaliplatin based treatment of metastatic colorectal cancer

The backbone of current cytotoxic treatment of metastatic colorectal cancer (mCRC) consists of a fluoropyrimidine together with either oxaliplatin (XELOX/FOLFOX) or irinotecan (XELIRI/FOLFIRI). With an overall objective response rate of approximately 50% for either treatment combination, a major unsolved problem is that no predictors of response to these treatments are available. To address this issue, we profiled 742 microRNAs in laser‐capture microdissected cancer cells from responding and non‐responding patients receiving XELOX/FOLFOX as first‐line treatment for mCRC, and identified, among others, high expression of miR‐625‐3p, miR‐181b and miR‐27b to be associated with poor clinical response. In a validation cohort of 94 mCRC patients treated first‐line with XELOX, high expression of miR‐625‐3p was confirmed to be associated with poor response (OR = 6.25, 95%CI [1.8; 21.0]). Independent analyses showed that miR‐625‐3p was not dysregulated between normal and cancer samples, nor was its expression associated with recurrence of stage II or III disease, indicating that miR‐625‐3p solely is a response marker. Finally, we also found that these miRNAs were up‐regulated in oxaliplatin resistant HCT116/oxPt (miR‐625‐3p, miR‐181b and miR‐27b) and LoVo/oxPt (miR‐181b) colon cancer cell lines as compared with their isogenic parental cells. Altogether, our results suggest an association between miR‐625‐3p and response to first‐line oxaliplatin based chemotherapy of mCRC.

muFKBP38: A novel murine immunophilin homolog differentially expressed in Schwannoma cells and central nervous system neurons in vivo

To better understand the process of multistage carcinogenesis in Schwann cells, we have attempted to isolate novel candidate genes involved in neoplastic progression of mouse malignant Schwannoma cells. The semi‐differentiated Schwannoma cell line 56‐24 and the less differentiated Schwannoma cell line 64‐39 were established from peripheral nerve sheath tumors arising in transgenic mice of the MBP/SV40 large T strain Tg29. By using the chemical cross‐linking subtraction technique, we have cloned a novel murine cDNA that detects pronounced expression in 56‐24 cells but not in 64‐39 cells. The longest open reading frame of the cDNA predicts a peptide showing 95% amino acid sequence homology to the recorded sequence of the human immunophilin homolog huFKBPr38, one of a family of proteins that are thought to interface with a wide range of intracellular signal transduction systems. The predicted open reading frame of the corresponding gene, named muFKBP38, encodes a 38 kDa protein that harbors an FK‐binding protein (FKBP) domain that is 36% identical to that of muFKBP52, a three‐unit tetratricopeptide repeat and a consensus leucine‐zipper repeat. Although muFKBP38 mRNA was detected in both neurons and glial cells, pronounced expression of the immunophilin homolog appeared in various classes of neurons associated with the hippocampal formation, as shown by in situ hybridization analysis of adult mouse brains. Taken together, these data indicate that muFKBP38 is (i) a novel potential marker for semi‐differentiated Schwannomas, (ii) may form homomultimers and/or interact with other proteins, and (iii) may have a role in neurons associated with memory function.

Expression of a novel murine phospholipase D homolog coincides with late neuronal development in the forebrain.

Members of the phospholipase D (PLD) superfamily are defined by the conserved HXKXXXXD motif, which is essential for the catalytic function of mammalian PLD. PLD enzymes are thought to play roles in signal transduction and membrane vesicular trafficking in mammalian cells. Here we describe a 54-kDa novel murine polypeptide (designated SAM-9) that is predicted to be a membrane-associated member of the PLD superfamily. SAM-9 shares 40, 30, and 29% amino acid identity with potential orthologs, in vaccinia virus, Caenorhabditis elegans, and Dictyostelium discoideum, respectively, and belongs to a subclass of PLD homologs in which the second HXKXXXXD motif is imperfect and harbors a conserved Asp to Glu substitution. Thesam-9 gene has more than eight exons, and the two HXKXXXXD motifs are encoded by two highly conserved exons. The expression of the sam-9 gene is greater in the brain than in non-nervous tissue and appears to be predominantly of neuronal origin. sam-9 expression is pronounced in mature neurons of the forebrain and appears to be turned on at late stages of neurogenesis as revealed by in situhybridization analysis of sam-9 expression during postnatal development of the hippocampal formation and the primary somatosensory cortex.

Zbtb20-Induced CA1 Pyramidal Neuron Development and Area Enlargement in the Cerebral Midline Cortex of Mice

Expression of the transcriptional repressor Zbtb20 is confined to the hippocampal primordium of the developing dorsal midline cortex in mice. Here, we show that misexpression of Zbtb20 converts projection neurons of the subiculum and postsubiculum (dorsal presubiculum) to CA1 pyramidal neurons that are innervated by Schaffer collateral projections in ectopic strata oriens and radiatum. The Zbtb20-transformed neurons express Bcl11B, Satb2, and Calbindin-D28k, which are markers of adult CA1 pyramidal neurons. Downregulation of Zbtb20 expression by RNA interference impairs the normal maturation of CA1 pyramidal neurons resulting in deficiencies in Calbindin-D28k expression and in reduced apical dendritic arborizations in stratum lacunosum moleculare. Overall, the results show that Zbtb20 is required for various aspects of CA1 pyramidal neuron development such as the postnatal extension of apical dendritic arbors in the distal target zone and the subtype differentiation of Calbindin-D28k-positive subsets. They further suggest that Zbtb20 plays a role in arealization of the midline cortex.

Altered splicing in exon 8 of the DNA replication factor CIZ1 affects subnuclear distribution and is associated with Alzheimer's disease.

In order to understand the gene-mediated processes underlying sporadic Alzheimers disease (AD), we carried out a subtractive cloning screen for novel AD candidate genes. We identified the gene encoding the DNA replication factor CIZ1 (CDKN1A interacting zinc finger protein 1) as being more highly expressed in Alzheimer tissue than in healthy brains. We show here that an isoform of CIZ1 which lacks a glutamine-rich region, due to alternative splicing in exon 8, is upregulated in AD brains relative to the full-length CIZ1 protein. We demonstrate for the first time that a minimal 28 amino acid sequence within this region is required for CIZ1 to associate with the nuclear matrix and to form nuclear foci.

Zbtb20 Defines a Hippocampal Neuronal Identity Through Direct Repression of Genes That Control Projection Neuron Development in the Isocortex

Hippocampal pyramidal neurons are important for encoding and retrieval of spatial maps and episodic memories. While previous work has shown that Zbtb20 is a cell fate determinant for CA1 pyramidal neurons, the regulatory mechanisms governing this process are not known. In this study, we demonstrate that Zbtb20 binds to genes that control neuronal subtype specification in the developing isocortex, including Cux1, Cux2, Fezf2, Foxp2, Mef2c, Rorb, Satb2, Sox5, Tbr1, Tle4, and Zfpm2. We show that Zbtb20 represses these genes during ectopic CA1 pyramidal neuron development in transgenic mice. These data reveal a novel regulatory mechanism by which Zbtb20 suppresses the acquisition of an isocortical fate during archicortical neurogenesis to ensure commitment to a CA1 pyramidal neuron fate. We further show that the expression pattern of Zbtb20 is evolutionary conserved in the fetal human hippocampus, where it is complementary to the expression pattern of the Zbtb20 target gene Tbr1. Therefore, the disclosed Zbtb20-mediated transcriptional repressor mechanism may be involved in development of the human archicortex.

Failure of central nervous system myelination in MBP/c-myc transgenic mice: evidence for c-myc cytotoxicity.

c-myc is a member of the helix–loop–helix/leucine zipper family of proteins that modulate the transcriptional activity of specific target genes. Although aberrant c-myc expression has been reported to play a role in multistage carcinogenesis in astrocytic gliomas, little is known about the effects of the expression of c-myc on oligodendrocytes. Using transgenic animals expressing a human c-myc oncogene under transcriptional control of the myelin basic protein gene, we investigated the effect of overexpression of this oncogene in oligodendrocytes. The MBP/c-myc transgenic mice developed severe neurological disturbances characterized by action tremors and recurrent seizures, and premature death during postnatal weeks three to five. Affected transgenic mice of various strains had severely hypomyelinated central nervous systems and expressed low levels of c-myc, myelin basic protein (MBP) and proteolipid protein (PLP) mRNAs in the brain. These c-myc transgenic mice also exhibited an increased number of TUNEL positive nuclei, which in most cases were located in cells that expressed c-myc, as judged by double immunohistochemistry. There was no evidence of brain tumors in the c-myc transgenic mice, including heterozygous mice from two strains that had normal lifespans. These observations indicate that the myelin deficiency observed in the MBP/c-myc transgenic animals results from a cytotoxic effect of the c-myc transgene.