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Markus U Ehrengruber

Publications and source records attributed to Markus U Ehrengruber.

At least 19 recordsLinked to original sources
PubMed2006

Synaptic scaffolding protein Homer1a protects against chronic inflammatory pain.

Glutamatergic signaling and intracellular calcium mobilization in the spinal cord are crucial for the development of nociceptive plasticity, which is associated with chronic pathological pain. Long-form Homer proteins anchor glutamatergic receptors to sources of calcium influx and release at synapses, which is antagonized by the short, activity-dependent splice variant Homer1a. We show here that Homer1a operates in a negative feedback loop to regulate the excitability of the pain pathway in an activity-dependent manner. Homer1a is rapidly and selectively upregulated in spinal cord neurons after peripheral inflammation in an NMDA receptor-dependent manner. Homer1a strongly attenuates calcium mobilization as well as MAP kinase activation induced by glutamate receptors and reduces synaptic contacts on spinal cord neurons that process pain inputs. Preventing activity-induced upregulation of Homer1a using shRNAs in mice in vivo exacerbates inflammatory pain. Thus, activity-dependent uncoupling of glutamate receptors from intracellular signaling mediators is a novel, endogenous physiological mechanism for counteracting sensitization at the first, crucial synapse in the pain pathway. Furthermore, we observed that targeted gene transfer of Homer1a to specific spinal segments in vivo reduces inflammatory hyperalgesia. Thus, Homer1 function is crucially involved in pain plasticity and constitutes a promising therapeutic target for the treatment of chronic inflammatory pain.

Animals↗
PubMed2005

HGF induces fibronectin matrix synthesis in melanoma cells through MAP kinase-dependent signaling pathway and induction of Egr-1.

The matrix fibronectin protein is a multifunctional adhesive molecule that promotes migration and invasiveness of many tumors including melanomas. Increased fibronectin synthesis has been associated with the metastatic potential of melanoma cells; however, the molecular mechanisms underlying fibronectin overexpression during melanoma development are poorly understood. We report that hepatocyte growth factor/scatter factor (HGF) induces fibronectin expression and its extracellular assembly on the surface of melanoma cells through activation of mitogen-activated protein (MAP) kinase pathway, and induction and transcriptional activation of Early growth response-1 (Egr-1). Inhibition of B-RAF/MAP kinase pathway by dominant-negative mutants and by U0126-abrogated HGF-induced Egr-1, and chromatin immunoprecipitation showed that Egr-1 is bound to the fibronectin promoter in response to HGF. Exogenously expressed Egr-1 increased fibronectin levels, while blockage of Egr-1 activation by expression of the Egr-1 corepressor NAB2 interfered with the upregulation of fibronectin synthesis induced by HGF, indicating that Egr-1 exerts a significant role in fibronectin expression in response to HGF. Finally, analysis of the expression pattern of fibronectin in melanoma cells demonstrated that fibronectin levels are correlated with constitutive MAP kinase signaling. Our data define a novel mechanism that might have important implications in regulation of melanoma progression by autocrine HGF signaling or by constitutive activation of MAP kinase pathway.

Autocrine Communication↗
PubMed2004

Alphaviral cytotoxicity and its implication in vector development.

A great variety of viruses have been engineered to serve as expression vectors. Among them, the alphaviruses Semliki Forest virus and Sindbis virus represent promising tools for heterologous gene expression in a wide variety of host cells. Several applications have already been described in neurobiological studies, in gene therapy, for vaccine development and in cancer therapy. Both viruses trigger stress pathways in the cells they infect, sometimes culminating in the death of the host. This inherent property is either an advantage or a drawback, depending on the type of application. This review covers the development and applications of alphavirus vectors and, as our work has been mainly with Semliki Forest virus, we have focused on this virus with special emphasis on how the understanding of Semliki Forest virus cytotoxicity enables it to be manipulated and used.

Alphavirus↗
PubMed2004

The protooncogene Ski controls Schwann cell proliferation and myelination.

Schwann cell proliferation and subsequent differentiation to nonmyelinating and myelinating cells are closely linked processes. Elucidating the molecular mechanisms that control these events is key to the understanding of nerve development, regeneration, nerve-sheath tumors, and neuropathies. We define the protooncogene Ski, an inhibitor of TGF-beta signaling, as an essential component of the machinery that controls Schwann cell proliferation and myelination. Functional Ski overexpression inhibits TGF-beta-mediated proliferation and prevents growth-arrested Schwann cells from reentering the cell cycle. Consistent with these findings, myelinating Schwann cells upregulate Ski during development and remyelination after injury. Myelination is blocked in myelin-competent cultures derived from Ski-deficient animals, and genes encoding myelin components are downregulated in Ski-deficient nerves. Conversely, overexpression of Ski in Schwann cells causes an upregulation of myelin-related genes. The myelination-regulating transcription factor Oct6 is involved in a complex modulatory relationship with Ski. We conclude that Ski is a crucial signal in Schwann cell development and myelination.

Animals↗
PubMed2004

Homer/Vesl proteins and their roles in CNS neurons.

Since their initial discovery in 1997, Homer/Vesl proteins have become increasingly investigated as putative regulators of receptor and ion-channel function in the central nervous system. Within a relatively brief period, numerous research reports have described manifold effects of Homer proteins, including the modulation of the trafficking of type I metabotropic glutamate receptors (mGluRs), axonal pathfinding, mGluR coupling to calcium and potassium channels, agonist-independent mGluR activity, ryanodine receptor regulation, locomotor activity, and behavioral plasticity. This review summarizes our current knowledge on the induction, expression, and structure of the various forms of Homer proteins, as well as their roles in neuronal function. In addition, we provide an outlook on novel developments with regard to the involvement of Homer-1a in hippocampal synaptic function.

Animals↗
PubMed2003

Epileptiform activity in rat hippocampus strengthens excitatory synapses.

Although epileptic seizures are characterized by excessive excitation, the role of excitatory synaptic transmission in the induction and expression of epilepsy remains unclear. Here, we show that epileptiform activity strengthens excitatory hippocampal synapses by increasing the number of functional (RS)-alpha-amino-3hydroxy-5methyl-4-isoxadepropionate (AMPA)-type glutamate receptors in CA3-CA1 synapses. This form of synaptic strengthening occludes long-term potentiation (LTP) and enhances long-term depression (LTD), processes involved in learning and memory. These changes in synaptic transmission and plasticity, which are fully blocked with N-methyl-D-aspartate (NMDA) receptor antagonists, may underlie epilepsy induction and seizure-associated memory deficits.

Animals↗
PubMed2003

Egr-1 upregulates the Alzheimer's disease presenilin-2 gene in neuronal cells.

Regulation of the Alzheimer's disease (AD)-related gene, presenilin-2 (PSEN2), was analyzed in neuronal (SK-N-SH) and non-neuronal (human embryonic kidney 293, HEK293) cells. We show that the PSEN2 regulatory region includes two separate promoter elements, each located upstream of multiple transcription start sites in the first and second exons. The stronger upstream promoter, P1, has housekeeping characteristics: it resides in a CpG island, is TATA-less, and up to 83% of PSEN2-P1 activity depends on a stimulating protein 1 (Sp1) site at the most 5' initiation site. However, the downstream promoter P2 includes neuronal-specific elements and two sites for early growth response gene-1 (Egr-1), a transcription factor upregulated in learning paradigms and implicated in neuronal plasticity, in response to injury. We show that Egr-1 binds to PSEN-P2, and that PSEN-P2 activity is increased threefold by overexpression of Egr-1, and by 12-O-tetradecanoylphorbol-13-acetate (TPA), which induces physiological Egr-1 levels. Egr-1 represses PSEN2-P1 activity by 50% in neuronal cells, suggesting it partially shifts promoter usage from PSEN2-P1 to PSEN2-P2. This could lead to a relative increase in shorter exon 2 transcripts, which may be more efficiently translated than exon 1 transcripts. Identification of PSEN2 as an Egr-1 target suggests a link between PSEN2 expression and Egr-1-related processes, which may impact on understanding PSEN-2's physiological function and its role in Alzheimer's disease.

Base Sequence↗
PubMed2003

Egr-1 target genes in human endothelial cells identified by microarray analysis.

Early growth response factor 1 (Egr-1) is a key transcriptional factor to mediate gene expression after vascular injury. To better understand the role of Egr-1 in vasculature, we globally profiled Egr-1 target genes in human endothelial cells using adenoviral gene transfer and Affymetrix oligonucleotide-based microarray technology. More than 300 genes regulated by >/=3-fold with Egr-1 overexpression were identified and, partially, confirmed by Northern and Western blotting, including genes for transcriptional regulators, signaling proteins, cell cycle regulatory proteins, growth factors, and cytokines. Among them, thymus-expressed chemokine (TECK) and IP-30 were dramatically induced by Egr-1, but TNFalpha-related apoptosis inducing ligand (TRAIL) was significantly repressed by Egr-1, suggesting that Egr-1 is a key mediator of inflammation and apoptosis in vascular cells. These data provide novel Egr-1 target genes and contribute to the understanding of the role of Egr-1 in vasculature.

Adenoviridae↗
PubMed2003

K+-dependent cerebellar granule neuron apoptosis. Role of task leak K+ channels.

Rat mature cerebellar granule, unlike hippocampal neurons, die by apoptosis when cultured in a medium containing a physiological concentration of K+ but survive under high external K+ concentrations. Cell death in physiological K+ parallels the developmental expression of the TASK-1 and TASK-3 subunits that encode the pH-sensitive standing outward K+ current IKso. Genetic transfer of the TASK subunits in hippocampal neurons, lacking IKso, induces cell death, while their genetic inactivation protects cerebellar granule neurons. Neuronal death of cultured rat granule neurons is also prevented by conditions that specifically reduce K+ efflux through the TASK-3 channels such as extracellular acidosis and ruthenium red. TASK leak K+ channels thus play an important role in K+-dependent apoptosis of cerebellar granule neurons in culture.

Animals↗
PubMed2003

Early stimulation and late inhibition of peroxisome proliferator-activated receptor gamma (PPAR gamma) gene expression by transforming growth factor beta in human aortic smooth muscle cells: role of early growth-response factor-1 (Egr-1), activator protein 1 (AP1) and Smads.

Transforming growth factor beta (TGF beta) and peroxisome proliferator-activated receptor gamma (PPAR gamma) play major roles in the development of vascular diseases. It has been documented that PPAR gamma activation inhibits the TGF beta signal pathway in vascular smooth muscle cells (VSMC). Here we examined whether TGF beta can regulate PPAR gamma expression. Northern blot analyses revealed that both TGF beta 1 and 2 exert a biphasic effect (early stimulation and late repression) on PPAR gamma gene expression in VSMC. TGF beta rapidly and transiently induced early growth-response factor-1 (Egr-1) expression through the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK1)/ERK-mediated pathway. Inhibition of MEK1/ERK by PD98059 not only abrogated the induction of Egr-1 but also abolished the rapid and transient induction of PPAR gamma by TGF beta. Furthermore, overexpression of NAB2, a repressor of Egr-1 activation, also blocked the induction of PPAR gamma by TGF beta in VSMC, suggesting that Egr-1 mediates the rapid and transient induction of PPAR gamma by TGF beta. With regard to the TGF beta repression of PPAR gamma expression, activator protein 1 (AP1) and Smad3/4 dramatically inhibited the PPAR gamma promoter activity in transient-transfection studies. In contrast, adenovirus-mediated overexpression of a dominant-negative form of c-Jun partially rescued the TGF beta-induced PPAR gamma repression in VSMC. Taken together, our data demonstrate that Egr-1, AP1 and Smad are part components of the TGF beta signal transduction pathway that regulates PPAR gamma expression.

Aorta↗
PubMed2003

Novel Semliki Forest virus vectors with reduced cytotoxicity and temperature sensitivity for long-term enhancement of transgene expression.

Alphaviral vectors inhibit host cell protein synthesis and are cytotoxic. To overcome these limitations, we modified the nonstructural protein-2 (nsP2) gene in the Semliki Forest virus (SFV) vector, pSFV1. Packaging of SFV replicons with two point mutations in nsP2 resulted in high-titer recombinant SFV(PD) particles. SFV(PD) led to more efficient host cell protein synthesis, exhibited reduced cytotoxicity and improved cell survival, and allowed greater and prolonged transgene expression than the original vector, SFV. In dissociated hippocampal neurons and organotypic rat hippocampal slices, SFV(PD) infection preserved neuronal morphology and synaptic function more efficiently than SFV. Combination of the two point mutations with a replication-persistent mutation in nsP2 resulted in a highly temperature-sensitive vector, SFV(PD713P), which efficiently transduced neurons in hippocampal slice cultures. At 31 degrees C, SFV(PD713P) allowed continuous transgene expression in BHK cells, at amounts comparable to SFV(PD). These new SFV mutants are expected to substantially broaden the application of alphaviral vectors in neurons and other mammalian cells.

Animals↗
PubMed2003

Homer-1a/Vesl-1S enhances hippocampal synaptic transmission.

Homer/Vesl proteins are involved in regulating metabotropic glutamate receptors, synaptogenesis, dendritic spine development and axonal pathfinding. We investigated the potential modulation of glutamatergic synaptic transmission by the immediate early gene product Homer-1a/Vesl-1S and by the constitutively expressed long-form Homer-1c/Vesl-1L in CA1 pyramidal cells from cultured rat hippocampal slices. Semliki Forest virus vector-mediated overexpression of Homer-1a enhanced alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor function, but did not detectably affect N-methyl-d-aspartate (NMDA) receptor function and presynaptic glutamate release. Overexpression of Homer-1c, by contrast, did not alter synaptic transmission. To corroborate our electrophysiological results obtained in slice cultures, we performed quantitative immunocytochemistry in cultures of dissociated hippocampal neurons. Homer-1a also increased synaptic clustering of AMPA but not NMDA receptors, whereas Homer-1c had no detectable effect. Our results show that Homer-1a potentiates synaptic AMPA receptor function, supporting a critical role for Homer-1a in hippocampal synaptic plasticity.

Animals↗
PubMed2003

Semliki Forest virus A7(74) transduces hippocampal neurons and glial cells in a temperature-dependent dual manner.

In central nervous system (CNS) tissue preparations, wild-type Semliki Forest virus (SFV) mainly infects neurons, and in vivo it causes lethal encephalitis in neonatal and adult rodents. The SFV strain A7(74), by contrast, is avirulent in adult rodents, triggering only limited CNS infection. To examine A7(74) infection in hippocampal tissue, the authors constructed a replicon, termed SFV(A774nsP)-GFP, expressing green fluorescent protein. The results were compared to replication-proficient recombinant A7(74) encoding GFP, named VA7-EGFP. As nonstructural gene mutations can confer temperature sensitivity, the authors also tested whether infection was temperature-dependent. Indeed, at 31 degrees C both viral recombinants transduced significantly more baby hamster kidney cells than at 37 degrees C. When rat hippocampal slices and dissociated cells were incubated at 37 degrees C, SFV(A774nsP)-GFP transduced glial cells but virtually no neurons-the opposite of conventional SFV. For VA7-EGFP at 37 degrees C, the preferred GFP-positive cells in hippocampal slices were also non-neuronal cells. At 31 degrees C, however, a more wild-type phenotype was found, with 33% and 94% of the GFP-positive cells being neurons for SFV(A774nsP)-GFP in slices and dissociated cells, respectively, and 94% neurons for VA7-EGFP in slices. Immunochemical and electrophysiological analyses confirmed that at 37 degrees C virtually all cells transduced by SFV(A774nsP)-GFP in slices were astrocytes, while at 31 degrees C they also contained neurons. These results show that in addition to the developmental age, the temperature determines which cell type becomes infected by A7(74). Our data suggest that A7(74) is avirulent in adult animals because it does not readily replicate in mature neurons at body temperature, whereas it still does so at lower temperatures.

Animals↗
PubMed2003

A novel neurotropic expression vector based on the avirulent A7(74) strain of Semliki Forest virus.

Semliki Forest virus (SFV), an enveloped alphavirus of the family Togaviridae, infects a wide range of mammalian host cells. Most strains are neurotropic but differ in virulence. The authors took advantage of the nonpathogenic properties of SFV strain A7(74), cloned recently in their laboratory, and constructed a replication-proficient expression vector to target the central nervous system (CNS) for heterologous gene expression. The vector, termed VA7, was engineered to drive expression of foreign inserts through a second subgenomic promoter inserted in the viral 3' nontranslated region (NTR). Infectious virus was obtained by in vitro transcription and transfection into BHK cells, and was shown to direct synthesis of heterologous proteins in several mammalian cell lines. Although novel expression vehicle is not applicable for targeting specific cell populations within the CNS in its present form, in cultured rat hippocampal slices, VA7 encoding enhanced green fluorescent protein (EGFP) efficiently transduced pyramidal cells, interneurons, and glial cells. With prolonged time post infection, the number of EGFP-expressing neurons in hippocampal slices increased. Mice infected intraperitoneally with the recombinant virus remained completely asymptomatic but showed CNS expression of EGFP as evidenced by immunohistochemistry. SFV A7(74) is a nonintegrating virus, which gives rise to a randomly distributed, patchy infection of the adult CNS that is cleared within 10 days. With the advantage of noninvasive administration, the expression vector described in this work is thus applicable for short-term gene expression in the CNS.

Animals↗
PubMed2002

Advanced modular self-inactivating lentiviral expression vectors for multigene interventions in mammalian cells and in vivo transduction.

In recent years, lentiviral expression systems have gained an unmatched reputation among the gene therapy community for their ability to deliver therapeutic transgenes into a wide variety of difficult-to-transfect/transduce target tissues (brain, hematopoietic system, liver, lung, retina) without eliciting significant humoral immune responses. We have cloned a construction kit-like self-inactivating lentiviral expression vector family which is compatible to state-of-the-art packaging and pseudotyping technologies and contains, besides essential cis-acting lentiviral sequences, (i) unparalleled polylinkers with up to 29 unique sites for restriction endonucleases, many of which recognize 8 bp motifs, (ii) strong promoters derived from the human cytomegalovirus immediate-early promoter (P(hCMV)) or the human elongation factor 1alpha (P(hEF1)(alpha)), (iii) P(hCMV-) or P(PGK-) (phosphoglycerate kinase promoter) driven G418 resistance markers or fluorescent protein-based expression tracers and (iv) tricistronic expression cassettes for coordinated expression of up to three transgenes. In addition, we have designed a size-optimized series of highly modular lentiviral expression vectors (pLenti Module) which contain, besides the extensive central polylinker, unique restriction sites flanking any of the 5'U3, R-U5-psi+-SD, cPPT-RRE-SA and 3'LTR(DeltaU3) modules or placed within the 5'U3 (-78 bp) and 3'LTR(DeltaU3) (8666 bp). pLentiModule enables straightforward cassette-type module swapping between lentiviral expression vector family members and facilitates the design of Tat-independent (replacement of 5'LTR by heterologous promoter elements), regulated and self-excisable proviruses (insertion of responsive operators or LoxP in the 3'LTR(DeltaU3) element). We have validated our lentiviral expression vectors by transduction of a variety of insect, chicken, murine and human cell lines as well as adult rat cardiomyocytes, rat hippocampal slices and chicken embryos. The novel multi-purpose construction kit-like vector series described here is compatible with itself as well as many other (non-viral) mammalian expression vectors for straightforward exchange of key components (e.g. promoters, LTRs, resistance genes) and will assist the gene therapy and tissue engineering communities in developing lentiviral expression vectors tailored for optimal treatment of prominent human diseases.

Animals↗
PubMed2002

Alphaviral gene transfer in neurobiology.

Semliki Forest virus (SFV), Sindbis virus (SIN), and Venezuelan equine encephalitis virus are simple, enveloped plus-strand RNA viruses belonging to the Alphavirus genus of the Togaviridae family. They have been developed into expression vectors that infect a wide host cell range and cause rapid and high-level transgene expression. Their easy and fast generation, classification into biosafety levels 1 and 2, and preferential transduction of neurons in cell and tissue cultures makes them an increasingly used gene transfer system. This review summarizes the alphaviral replication and expression, the replicon system, and its application in neurobiology. Alphaviral vectors can introduce multiple transgenes into host cells, and mutants with low or absent cytotoxicity and increased or decreased transgene expression levels are available. Temperature-dependent mutants permit to control the host cell specificity as well as the on- and offset of gene expression. These features, together with the transduction characteristics revealed in a direct comparison of alphaviral and other viral vectors in hippocampal slice cultures, make SFV and SIN vectors a powerful tool for neurobiological studies.

Alphavirus↗
PubMed2002

Knockout of ERK1 MAP kinase enhances synaptic plasticity in the striatum and facilitates striatal-mediated learning and memory.

Extracellular signal-regulated kinases (ERK1 and 2) are synaptic signaling components necessary for several forms of learning. In mice lacking ERK1, we observe a dramatic enhancement of striatum-dependent long-term memory, which correlates with a facilitation of long-term potentiation in the nucleus accumbens. At the cellular level, we find that ablation of ERK1 results in a stimulus-dependent increase of ERK2 signaling, likely due to its enhanced interaction with the upstream kinase MEK. Consistently, such activity change is responsible for the hypersensitivity of ERK1 mutant mice to the rewarding properties of morphine. Our results reveal an unexpected complexity of ERK-dependent signaling in the brain and a critical regulatory role for ERK1 in the long-term adaptive changes underlying striatum-dependent behavioral plasticity and drug addiction.

Amygdala↗