Jürgen Engele

Regulation of glial glutamate transporter expression by growth factors

Injuries to the brain result in the decline of glial glutamate transporter expression within hours and a recovery after several days. One consequence of this disturbed expression seems to consist in the temporary accumulation of toxic extracellular glutamate levels followed by secondary neuronal cell death. Whereas evidence exists that the decline in glutamate transporter expression results from a loss of neuronal PACAP influences on astroglia, the mechanism(s) inducing the reexpression of glial glutamate transporters is presently unknown. We now demonstrate that the injury-induced growth factors EGF, TGFalpha, FGF-2, and PDGF all promote the expression of the glutamate transporters GLT-1 and/or GLAST in cultured cortical astroglia. In contrast, similar stimulatory influences were absent with GDNF and BDNF, growth factors not affected by brain injuries. The effects of EGF, TGFalpha, FGF-2, and PDGF on glial glutamate transport were only partly redundant and involved distinctly different signaling pathways. Unlike EGF, TGFalpha, and FGF-2, PDGF promoted GLT-1, but not GLAST expression and further failed to increase the maximal velocity of sodium-dependent glutamate uptake. Moreover, FGF-2 only affected glial glutamate transport when the RAF-MEK-ERK signaling pathway was concomitantly inhibited with PD98059. Depending on the extracellular growth factor and glutamate transporter subtype, the observed stimulatory effects required the activation of PKA, PKC, and/or AKT. We suggest that after brain injury, reactive processes may limit secondary neuronal cell death by promoting glial glutamate transport. The detailed knowledge of these compensatory mechanisms will eventually allow us to therapeutically interfere with glutamate-associated neuronal cell death in the brain.

CXCR7 is an active component of SDF-1 signalling in astrocytes and Schwann cells

The alternative SDF-1 (stromal cell derived factor-1) receptor, CXCR7, has been suggested to act as either a scavenger of extracellular SDF-1 or a modulator of the primary SDF-1 receptor, CXCR4. CXCR7, however, also directly affects the function of various tumor-cell types. Here, we demonstrate that CXCR7 is an active component of SDF-1 signalling in astrocytes and Schwann cells. Cultured cortical astrocytes and peripheral nerve Schwann cells exhibit comparable total and cell-surface levels of expression of both SDF-1 receptors. Stimulation of astrocytes with SDF-1 resulted in the temporary activation of Erk1/2, Akt and PKCζ/λ, but not p38 and PKCα/β. Schwann cells showed SDF-1-induced activation of Erk1/2, Akt and p38, but not PKCα/β and PKCζ/λ. The respective signalling pattern remained fully inducible in astrocytes from CXCR4-deficient mice, but was abrogated following depletion of astrocytic CXCR7 by RNAi. In Schwann cells, RNAi-mediated depletion of either CXCR4 or CXCR7 silenced SDF-1 signalling. The findings of the astrocytic receptor-depletion experiments were reproduced by CXCR7 antagonist CCX754, but not by CXCR4 antagonist AMD3100, both of which abolished astrocytic SDF-1 signalling. Further underlining the functional importance of CXCR7 signalling in glial cells, we show that the mitogenic effects of SDF-1 on both glial cell types are impaired upon depleting CXCR7.

CNS region-specific regulation of glial glutamate transporter expression

The neuronal cell death associated with certain neurodegenerative disorders as well as acute brain injuries is in part due to the reduced expression of glial glutamate transporters and the subsequent accumulation of toxic extracellular glutamate concentrations. Extracellular factors previously found to potently stimulate the expression of the glial glutamate transporters, GLT‐1/EAAT2 and GLAST/EAAT1, in astroglial cultures of rat cerebral hemispheres are PACAP, TGFα, and EGF. In the present study, we sought to determine whether similar stimulatory influences apply for astroglia from other areas of the central nervous system (CNS). Immunoblot and real‐time RT‐PCR analysis of striatal astroglial cultures maintained for 72 h with PACAP, TGFα, or EGF revealed a prominent increase in GLT‐1 and GLAST expression. In apparent contrast, all factors completely failed to affect GLT‐1 and GLAST expression in astroglial cultures from the cerebellum, mesencephalon, and spinal cord between 36 h and 7 days. This failure was not due to the absence of functional recognition or transduction machineries for the extracellular factors as suggested by the additional observations that cerebellar, mesencephalic and spinal cord glia were capable of responding to stimulation with PACAP, TGFα, or EGF for 10 min with activation of CREB. Moreover, dibutyryl cyclic AMP (dbcAMP) potently promoted GLT‐1 and/or GLAST expression in mesencephalic, cerebellar and spinal cord glia, further indicating that extracellular factors regulate glial glutamate transporter expression throughout the CNS. Together these findings identify PACAP, TGFα and EGF as potent regulators of glutamate transporter expression in striatal glia. In addition, these findings provide evidence for a CNS region‐specific regulation of glial glutamate transport.

Interleukin-6 and cAMP Induce Stromal Cell-derived Factor-1 Chemotaxis in Astroglia by Up-regulating CXCR4 Cell Surface Expression IMPLICATIONS FOR BRAIN INFLAMMATION

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 control the migration of neurons and microglial cells in the central nervous system. Although functional CXCR4 is also expressed by astroglia, recent studies have failed to observe a chemotactic response of these cells to SDF-1. Here, we demonstrate that SDF-1-dependent chemotaxis can be induced by treating cultured cortical astroglia with either dibutyryl cyclic AMP (dbcAMP; 10−4 m) or interleukin-6 (IL-6; 10 ng/ml). Flow cytometric analysis revealed that both the dbcAMP- and IL-6-induced onset of SDF-1-dependent chemotaxis of astroglia are due to the increased cell surface expression of CXCR4. In addition, dbcAMP and IL-6 also increased CXCR4 transcript levels, further suggesting that both treatments primarily affect CXCR4 surface expression in astroglia by stimulation of gene expression. Moreover, unlike the case with IL-6 and dbcAMP, which allowed for an optimal chemotactic response to SDF-1 only after 48 h, a similar chemotactic response, associated with an increase in CXCR4 cell surface expression, already occurred after 24 h when astroglial cultures were maintained with medium conditioned by IL-6- or dbcAMP-pretreated astrocytes, indicating that the stimulatory effects of IL-6 and cAMP on CXCR4 cell surface expression involve a secondary mechanism. The findings that elevated extracellular levels of IL-6 or factors positively coupled to cAMP result in increased CXCR4 cell surface expression levels and subsequent SDF-1-dependent chemotaxis in central nervous system astrocytes point to a crucial role of this chemokine during reactive gliosis and human immunodeficiency virus-mediated dementia.

Estrogen stimulates brain-derived neurotrophic factor expression in embryonic mouse midbrain neurons through a membrane-mediated and calcium-dependent mechanism

We have provided evidence that 17β‐estradiol (E) synthesized in the midbrain promotes the differentiation of midbrain dopamine neurons through nonclassical steroid action. Because these developmental effects resemble those reported for brain‐derived neurotrophic factor (BDNF), we hypothesized that E influences dopaminergic cell differentiation through a BDNF‐dependent mechanism. Competitive RT‐PCR and ELISA techniques were employed to study first the developmental pattern of BDNF and trkB expression in the mouse midbrain. BDNF protein/mRNA levels peaked postnatally, whereas trkB did not fluctuate perinatally. To prove the hypothesis that E regulates BDNF expression in vivo, fetuses and newborns were treated with the aromatase antagonist CGS 16949A. CGS 16949A exposure reduced midbrain BDNF mRNA/protein levels. The coapplication of CGS 16949A and E abolished this effect. Midbrain cultures from mouse fetuses were used to investigate intracellular signaling mechanisms involved in transmitting E effects. Estrogen increased expression of BDNF but not of other neurotrophins. As concerns the related signaling mechanism, these effects were antagonized by interrupting intracellular Ca2+ signaling with BAPTA and thapsigargin but not by the estrogen receptor antagonist ICI 182,780. Insofar as E effects on BDNF mRNA expression were inhibited by cycloheximide, it appears likely that other, not yet characterized intermediate proteins take part in the estrogenic regulation of BDNF expression. We conclude that E exerts its stimulatory effect on the differentiation of dopaminergic neurons by coordinating BDNF expression. This particular E effect appears to be transmitted through Ca2+‐dependent signaling cascades upon activation of putative membrane estrogen receptors. J. Neurosci. Res. 66:221–230, 2001.

Activation of CXCR7 receptor promotes oligodendroglial cell maturation.

Differentiation of oligodendroglial precursor cells is crucial for central nervous system (re)myelination and is influenced by multiple extrinsic and intrinsic factors. Chemokines, a group of small proteins, are highly conserved among mammals and have been implicated in a variety of biological processes during development, tissue homeostasis, and repair. We investigated whether the chemokine CXCL12 influences oligodendrocytes and what cellular differentiation/maturation processes are controlled by this molecule.

Effects of glial cell line-derived neurotrophic factor (GDNF) on dopaminergic neurons require concurrent activation of cAMP-dependent signaling pathways

Abstract.Glial cell line-derived neurotrophic factor (GDNF) is a highly selective neurotrophic factor for midbrain dopaminergic neurons and might thus be of potential use in the therapy of Parkinson’s disease. In this study, we present evidence that the survival-promoting action of GDNF on dopaminergic neurons requires the concurrent activation of cAMP-dependent signaling pathways. In serum-free low density cultures of the dissociated embryonic day 15 mesencephalon, dopaminergic neurons undergo constant cell death as evidenced by a 90% reduction in tyrosine hydroxylase-immunoreactive (TH-IR) cell numbers between days 1 and 9 of cultivation. This decline was not affected by GDNF (5 ng/ml) within the initial 3 days of cultivation, but was in part attenuated with prolonged treatment. In contrast, stimulation of 3-day-old mesencephalic cultures with GDNF induced c-fos expression in 73% of all TH-IR neurons, indicative for the early presence of efficient signal-transduction coupling in these neurons. Combined treatment of mesencephalic cultures with dibutyryl cyclic AMP (dbcAMP; 100 μM) and GDNF accelerated the onset of the survival effects of GDNF on dopaminergic neurons, resulting in a 1.5-fold increase in the number of surviving TH-IR neurons at 3 days in vitro. In addition, activation of cAMP-dependent signal pathways significantly potentiated the survival-promoting effects of GDNF on dopaminergic neurons in older cultures. dbcAMP alone had no effect on dopaminergic cell survival. Taken together, our findings suggest that the action of GDNF on midbrain dopaminergic neurons is modulated by other extracellular signals.

Adipocyte-derived angiopoietin-1 supports neurite outgrowth and synaptogenesis of sensory neurons

Sensory and sympathetic innervation of the white fat tissue (WAT) contributes to lipolysis. In addition, both fiber types adapt in density to weight gain and loss. Because these findings are indicative for a tight control of nerve fiber plasticity by adipokines, we tested whether adipocytes control neurite growth of sensory neurons through angiopoietin‐1 (Ang‐1). We further considered initial hints that Ang‐1‐induced neuritogenesis involves transactivation of the high‐affinity nerve growth factor (NGF) receptor trkA. Coculturing dorsal root ganglion (DRG) cells with 3T3‐L1 adipocytes supported neurite outgrowth. These neurotrophic effects were associated with the increased expression of Ang‐1 (presumably in adipocytes) as well as of trkA. The effects were abolished upon inactivating Ang‐1 in culture with selective antibodies. Likewise, neurite outgrowth was impaired in the presence of inactivating NGF antibodies as well as upon inhibition of the NGF high‐affinity trkA receptor with the antagonist K252a, indicating a tight cooperation of Ang‐1 and NGF in the control of neuritogenesis. DRG‐adipipocyte cocultures were further used to establish whether sensory neurons would form synaptic contacts with adipocytes. Electron microscopy demonstrated that cultured sensory neurons develop predominantly neuroneuronal synapses but seem to affect adipocytes by synapses en passant. Comparably to the case for neuritogenesis, expression of the presynaptic protein synaptophysin as well of the postsynaptic protein PSD‐95 correlated with Ang‐1 levels in culture. It is concluded that adipocyte‐secreted Ang‐1 supports neurite outgrowth, which is involved in synaptogenesis. The novel function of Ang‐1 appears to play a physiological role in WAT plasticity.

High extracellular glutamate modulates expression of glutamate transporters and glutamine synthetase in cultured astrocytes.

Astroglial cells clear extracellular glutamate through the glutamate transporters, GLT-1 and GLAST, and subsequently convert the incorporated glutamate into glutamine by the enzyme, glutamine synthetase (GS). Several forms of acute brain injury are associated with the increased expression of GS and the decreased expression of GLT-1 and/or GLAST, eventually leading to the accumulation of excitotoxic extracellular glutamate concentrations. Although of clinical interest, the actual trigger of these injury-related changes of glial glutamate turnover remains unknown. Our present studies provide evidence that increases in extracellular glutamate, as present in many brain injuries, are sufficient to modulate the expression of glutamate transporters and GS. Subjecting cultured cortical astrocytes to glutamate concentrations of 0.5-20 mM resulted in a 25% loss of GLT-1 and GLAST protein levels after 24 h; GLT-1 and GLAST levels maximally decreased by 40% and 75%, respectively, after 72 h. This decline was not due to astroglial cell death, since glutamate up to 50 mM did not affect the survival of cultured astrocytes within 72 h. Major astrocytic cell death, however, occurred in cultures maintained under severe (4% O(2)), but not mild (9% O(2)), hypoxia, as well as in the presence of aspartate (>or=20 mM). Glutamate at >or=1 mM induced a prolonged increase of GS expression in contrast to glutamate transporters. Neither the decline of glutamate transporter expression nor the increase in GS expression induced by high extracellular glutamate was further modulated by mild hypoxia. Whereas the stimulatory influences of glutamate on GS expression were prevented by the non-competitive NMDA receptor antagonist, MK801, the inhibitory influences on glutamate transporter expression were neither sensitive to MK801, the non-competitive mGluR5 antagonist, MTEP, nor the non-competitive AMPA receptor antagonist, GYKI52466, implying that glutamate controls glial glutamate transport by a glutamate receptor-independent mechanism.

The chemokine SDF1 controls multiple steps of myogenesis through atypical PKCζ

Mice deficient in the SDF1-chemokine-receptor CXCR4, exhibit severe defects of secondary limb myogenesis. To further elucidate the role of SDF1 in muscle development, we have now analyzed putative effects of this chemokine on proliferation, migration and myogenic differentiation of mouse C2C12 myogenic progenitor/myoblast cells. In addition, we have characterized the signaling pathways employed by SDF1-CXCR4 to control myogenesis. We found that SDF1 stimulates proliferation and induces migration of C2C12 cells with a potency similar to that of FGF2 and HGF, which both represent prototypical extracellular regulators of myogenesis. In addition, SDF1 inhibits myogenic differentiation in both C2C12 cells and primary myoblasts, as assessed by MyoD, myosin heavy chain and/or myogenin expression. Regarding signaling pathways, C2C12 cells responded to SDF1 with activation (phosphorylation) of Erk and PKCζ, whereas even after prolonged SDF1 treatment for up to 120 minutes, levels of activated Akt, p38 and PKCα or PKCβ remained unaffected. Preventing activation of the classic MAP kinase cascade with the Erk inhibitor UO126 abolished SDF1-induced proliferation and migration of C2C12 cells but not the inhibitory action of SDF1 on myogenic differentiation. Moreover, the effects of SDF1 on proliferation, migration and differentiation of C2C12 cells were all abrogated in the presence of myristoylated PKCζ peptide pseudosubstrate and/or upon cellular depletion of PKCζ by RNA interference. In conclusion, our findings unravel a previously unknown role of CXCR4-PKCζ signaling in myogenesis. The potent inhibitory effects of SDF1 on myogenic differentiation point to a major function of CXCR4-PKCζ signaling in the control of secondary muscle growth.