Deletion of the BDNF truncated receptor TrkB.T1 delays disease onset in a mouse model of amyotrophic lateral sclerosis

Brain Derived Neurotrophic Factor (BDNF) exerts strong pro-survival effects on developing and injured motoneurons. However, in clinical trials, BDNF has failed to benefit patients with amyotrophic lateral sclerosis (ALS). To date, the cause of this failure remains unclear. Motoneurons express the TrkB kinase receptor but also high levels of the truncated TrkB.T1 receptor isoform. Thus, we investigated whether the presence of this receptor may affect the response of diseased motoneurons to endogenous BDNF. We deleted TrkB.T1 in the hSOD1(G93A) ALS mouse model and evaluated the impact of this mutation on motoneuron death, muscle weakness and disease progression. We found that TrkB.T1 deletion significantly slowed the onset of motor neuron degeneration. Moreover, it delayed the development of muscle weakness by 33 days. Although the life span of the animals was not affected we observed an overall improvement in the neurological score at the late stage of the disease. To investigate the effectiveness of strategies aimed at bypassing the TrkB.T1 limit to BDNF signaling we treated SOD1 mutant mice with the adenosine A2A receptor agonist CGS21680, which can activate motoneuron TrkB receptor signaling independent of neurotrophins. We found that CGS21680 treatment slowed the onset of motor neuron degeneration and muscle weakness similarly to TrkB.T1 removal. Together, our data provide evidence that endogenous TrkB.T1 limits motoneuron responsiveness to BDNF in vivo and suggest that new strategies such as Trk receptor transactivation may be used for therapeutic intervention in ALS or other neurodegenerative disorders.     

TTIP is a novel protein that interacts with the truncated T1 TrkB neurotrophin receptor

Alternative splicing of the TrkB gene produces a full length tyrosine kinase receptor as well as two truncated isoforms that contain extracellular and transmembrane domains but lack the kinase domain and have unique C terminal tails. The function of the truncated TrkB isoforms is unclear and to gain insights into their function, we have isolated a protein from 15N neuroblastoma cells that specifically binds the TrkB.T1 isoform. Pulldown experiments using a GST fusion protein containing the TrkB.T1 intracellular domain identified a 61 kDa protein from radiolabeled 15N lysates. Coimmunoprecipitation experiments showed that the 61 kDa protein interacted with epitope-tagged TrkB.T1 overexpressed in 15N cells as well as with TrkB.T1 which was endogenously expressed. Peptide competition experiments revealed that the protein, designated TTIP (for Truncated TrkB Interacting Protein), showed specific binding to the TrkB.T1 tail. MALDI MS and MS/MS analysis has revealed that TTIP is a novel protein not yet listed in the current databases.     

Antidepressant-like effect of guanosine involves activation of AMPA receptor and BDNF/TrkB signaling

Purinergic Signalling - Guanosine is a purine nucleoside that has been shown to exhibit antidepressant effects, but the mechanisms underlying its effect are not well established. We investigated if...     

BDNF heightens the sensitivity of motor neurons to excitotoxic insults through activation of TrkB

The survival promoting and neuroprotective actions of brain-derived neurotrophic factor (BDNF) are well known but under certain circumstances this growth factor can also exacerbate excitotoxic insults to neurons. Prior exploration of the receptor through which BDNF exerts this action on motor neurons deflects attention away from p75. Here we investigated the possibility that BDNF acts through the receptor tyrosine kinase, TrkB, to confer on motor neurons sensitivity to excitotoxic challenge. We blocked BDNF activation of TrkB using a dominant negative TrkB mutant or a TrkB function blocking antibody, and found that this protected motor neurons against excitotoxic insult in cultures of mixed spinal cord neurons. Addition of a function blocking antibody to BDNF to mixed spinal cord neuron cultures is also neuroprotective indicating that endogenously produced BDNF participates in vulnerability to excitotoxicity. We next examined the intracellular signaling cascades that are engaged upon TrkB activation. Previously we found that inhibition of the phosphatidylinositide-3'-kinase (PI3'K) pathway blocks BDNF-induced excitotoxic sensitivity. Here we show that expression of a constitutively active catalytic subunit of PI3'K, p110, confers excitotoxic sensitivity (ES) upon motor neurons not incubated with BDNF. Parallel studies with purified motor neurons confirm that these events are likely to be occuring specifically within motor neurons. The abrogation of BDNF's capacity to accentuate excitotoxic insults may make it a more attractive neuroprotective agent.     

DRD1 signaling modulates TrkB turnover and BDNF sensitivity in direct pathway striatal medium spiny neurons

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In vivo restoration of physiological levels of truncated TrkB.T1 receptor rescues neuronal cell death in a trisomic mouse model

Imbalances in neurotrophins or their high-affinity Trk receptors have long been reported in neurodegenerative diseases. However, a molecular link between these gene products and neuronal cell death has not been established. In the trisomy 16 (Ts16) mouse there is increased apoptosis in the cortex, and hippocampal neurons undergo accelerated cell death that cannot be rescued by administration of brain-derived neurotrophic factor (BDNF). Ts16 neurons have normal levels of the TrkB tyrosine kinase receptor but an upregulation of the TrkB.T1 truncated receptor isoform. Here we show that restoration of the physiological level of the TrkB.T1 receptor by gene targeting rescues Ts16 cortical cell and hippocampal neuronal death. Moreover, it corrects resting Ca2+ levels and restores BDNF-induced intracellular signaling mediated by full-length TrkB in Ts16 hippocampal neurons. These data provide a direct link between neuronal cell death and abnormalities in Trk neurotrophin receptor levels.     

Estrogen rescues heart failure through estrogen receptor Beta activation

Background

Recently, we showed that exogenous treatment with estrogen (E2) rescues pre-existing advanced heart failure (HF) in mice. Since most of the biological actions of E2 are mediated through the classical estrogen receptors alpha (ERα) and/or beta (ERβ), and both these receptors are present in the heart, we examined the role of ERα and ERβ in the rescue action of E2 against HF.

Methods

Severe HF was induced in male mice by transverse aortic constriction-induced pressure overload. Once the ejection fraction (EF) reached ~?35%, mice were treated with selective agonists for ERα (PPT, 850 μg/kg/day), ERβ (DPN, 850 μg/kg/day), or E2 (30 μg/kg/day) together with an ERβ-antagonist (PHTPP, 850 μg/kg/day) for 10 days.

Results

EF of HF mice was significantly improved to 45.3?±?2.1% with diarylpropionitrile (DPN) treatment, but not with PPT (31.1?±?2.3%). E2 failed to rescue HF in the presence of PHTPP, as there was no significant improvement in the EF at the end of the 10-day treatment (32.5?±?5.2%). The improvement of heart function in HF mice treated with ERβ agonist DPN was also associated with reduced cardiac fibrosis and increased cardiac angiogenesis, while the ERα agonist PPT had no significant effect on either cardiac fibrosis or angiogenesis. Furthermore, DPN improved hemodynamic parameters in HF mice, whereas PPT had no significant effect.

Conclusions

E2 treatment rescues pre-existing severe HF mainly through ERβ. Rescue of HF by ERβ activation is also associated with stimulation of cardiac angiogenesis, suppression of fibrosis, and restoration of hemodynamic parameters.

     

Mouse resistin modulates adipogenesis and glucose uptake in 3T3-L1 preadipocytes through the ROR1 receptor

Mouse resistin, a cysteine-rich protein primarily secreted from mature adipocytes, is involved in insulin resistance and type 2 diabetes. Human resistin, however, is mainly secreted by immune mononuclear cells, and it competes with lipopolysaccharide for the binding to Toll-like receptor 4, which could mediate some of the well-known proinflammatory effects of resistin in humans. In addition, resistin has been involved in the regulation of many cell differentiation and proliferation processes, suggesting that different receptors could be involved in mediating its numerous effects. Thus, a recent work identifies an isoform of Decorin (Δ Decorin) as a functional resistin receptor in adipocyte progenitors that may regulate white adipose tissue expansion. Our work shows that the mouse receptor tyrosine kinase-like orphan receptor (ROR)1 could mediate some of the described functions of resistin in 3T3-L1 adipogenesis and glucose uptake. We have demonstrated an interaction of mouse resistin with specific domains of the extracellular region of the ROR1 receptor. This interaction results in the inhibition of ROR1 phosphorylation, modulates ERK1/2 phosphorylation, and regulates suppressor of cytokine signaling 3, glucose transporter 4, and glucose transporter 1 expression. Moreover, mouse resistin modulates glucose uptake and promotes adipogenesis of 3T3-L1 cells through ROR1. In summary, our results identify mouse resistin as a potential inhibitory ligand for the receptor ROR1 and demonstrate, for the first time, that ROR1 plays an important role in adipogenesis and glucose homeostasis in 3T3-L1 cells. These data open a new line of research that could explain important questions about the resistin mechanism of action in adipogenesis and in the development of insulin resistance.     

The EphB6 receptor inhibits JNK activation in T lymphocytes and modulates T cell receptor-mediated responses

EphB6 is the most recently identified member of the Eph receptor tyrosine kinase family. EphB6 is primarily expressed in thymocytes and a subpopulation of T cells, suggesting that it may be involved in regulation of T lymphocyte differentiation and functions. We show here that overexpression of EphB6 in Jurkat T cells and stimulation with the EphB6 ligand, ephrin-B1, results in the selective inhibition of TCR-mediated activation of JNK but not the MAPK pathway. EphB6 appears to suppress the JNK pathway by preventing T cell receptor (TCR)-induced activation of the small GTPase Rac1, a critical event in initiating the JNK cascade. Furthermore, EphB6 blocked anti-CD3-induced secretion of IL-2 and CD25 expression in a ligand-dependent manner. Dominant negative EphB6 suppressed the inhibitory activity of the endogenous receptor and enhanced anti-CD3-induced JNK activation, CD25 expression, and IL-2 secretion, confirming the requirement for EphB6-specific signaling. Activation of the JNK pathway and the establishment of an IL-2/IL-2R autocrine loop have been shown to play a role in the negative selection of CD4(+)CD8(+) self-reacting thymocytes. In agreement, stimulation of murine thymocytes with ephrin-B1 not only blocked anti-CD3-induced CD25 up-regulation and IL-2 production, but also inhibited TCR-mediated apoptosis. Thus, EphB6 may play an important role in regulating thymocyte differentiation and modulating responses of mature T cells.     

Wip1 phosphatase modulates both long-term potentiation and long-term depression through the dephosphorylation of CaMKII

Synaptic plasticity is an important mechanism that underlies learning and cognition. Protein phosphorylation by kinases and dephosphorylation by phosphatases play critical roles in the activity-dependent alteration of synaptic plasticity. In this study, we report that Wip1, a protein phosphatase, is essential for long-term potentiation (LTP) and long-term depression (LTD) processes. Wip1-deletion suppresses LTP and enhances LTD in the hippocampus CA1 area. Wip1 deficiency-induced aberrant elevation of CaMKII T286/287 and T305 phosphorylation underlies these dysfunctions. Moreover, we showed that Wip1 modulates CaMKII dephosphorylation. Wip1^?/? mice exhibit abnormal GluR1 membrane expression, which could be reversed by the application of a CaMKII inhibitor, indicating that Wip1/CaMKII signaling is crucial for synaptic plasticity. Together, our results demonstrate that Wip1 phosphatase plays a vital role in regulating hippocampal synaptic plasticity by modulating the phosphorylation of CaMKII.     

LAR protein tyrosine phosphatase receptor associates with TrkB and modulates neurotrophic signaling pathways

The identities of receptor protein tyrosine phosphatases (PTPs) that associate with Trk protein tyrosine kinase (PTK) receptors and modulate neurotrophic signaling are unknown. The leukocyte common antigen-related (LAR) receptor PTP is present in neurons expressing TrkB, and like TrkB is associated with caveolae and regulates survival and neurite outgrowth. We tested the hypothesis that LAR associates with TrkB and regulates neurotrophic signaling in embryonic hippocampal neurons. Coimmunoprecipitation and coimmunostaining demonstrated LAR interaction with TrkB that is increased by BDNF exposure. BDNF neurotrophic activity was reduced in LAR-/- and LAR siRNA-treated LAR+/+ neurons and was augmented in LAR-transfected neurons. In LAR-/- neurons, BDNF-induced activation of TrkB, Shc, AKT, ERK, and CREB was significantly decreased; while in LAR-transfected neurons, BDNF-induced CREB activation was augmented. Similarly, LAR+/+ neurons treated with LAR siRNA demonstrated decreased activation of Trk and AKT. LAR is known to activate the Src PTK by dephosphorylation of its negative regulatory domain and Src transactivates Trk. In LAR-/- neurons, or neurons treated with LAR siRNA, phosphorylation of the Src regulatory domain was increased (indicating Src inactivation), consistent with a role for Src in mediating LAR's ability to up-regulate neurotrophic signaling. Interactions between LAR, TrkB, and Src were further confirmed by the findings that Src coimmunoprecipitated with LAR, that the Src inhibitor PP2 blocked the ability of LAR to augment TrkB signaling, and that siRNA-induced depletion of Src decreased LAR interaction with TrkB. These studies demonstrate that receptor PTPs can associate with Trk complexes and promote neurotrophic signaling and point to receptor PTP-based strategies as a novel approach for modulating neurotrophin function.     

MAPK scaffold IQGAP1 binds the EGF receptor and modulates its activation

Cellular responses produced by EGF are mediated through the receptor (EGFR) and by various enzymes and scaffolds. Recent studies document IQGAP1 as a scaffold for the MAPK cascade, binding directly to B-Raf, MEK, and ERK and regulating their activation in response to EGF. We previously showed that EGF is unable to activate B-Raf in cells lacking IQGAP1. However, the mechanism by which IQGAP1 links B-Raf to EGFR was unknown. Here we report that endogenous EGFR and IQGAP1 co-localize and co-immunoprecipitate in cells. EGF has no effect on the association, but Ca(2+) attenuates binding. In vitro analysis demonstrated a direct association mediated through the IQ and kinase domains of IQGAP1 and EGFR, respectively. Calmodulin disrupts this interaction. Using a mass spectrometry-based assay, we show that EGF induces phosphorylation of IQGAP1 Ser(1443), a residue known to be phosphorylated by PKC. This phosphorylation is eliminated by pharmacological inhibition of either EGFR or PKC and transfection with small interfering RNA directed against the PKCα isoform. In IQGAP1-null cells, EGF-stimulated tyrosine phosphorylation of EGFR is severely attenuated. Normal levels of autophosphorylation are restored by reconstituting wild type IQGAP1 and enhanced by an IQGAP1 S1443D mutant. Collectively, these data demonstrate a functional interaction between IQGAP1 and EGFR and suggest that IQGAP1 modulates EGFR activation.     

The a2b adenosine receptor modulates glucose homeostasis and obesity

Background

High fat diet and its induced changes in glucose homeostasis, inflammation and obesity continue to be an epidemic in developed countries. The A2b adenosine receptor (A2bAR) is known to regulate inflammation. We used a diet-induced obesity murine knockout model to investigate the role of this receptor in mediating metabolic homeostasis, and correlated our findings in obese patient samples.

Methodology/Principal Findings

Administration of high fat, high cholesterol diet (HFD) for sixteen weeks vastly upregulated the expression of the A2bAR in control mice, while A2bAR knockout (KO) mice under this diet developed greater obesity and hallmarks of type 2 diabetes (T2D), assessed by delayed glucose clearance and augmented insulin levels compared to matching control mice. We identified a novel link between the expression of A2bAR, insulin receptor substrate 2 (IRS-2), and insulin signaling, determined by Western blotting for IRS-2 and tissue Akt phosphorylation. The latter is impaired in tissues of A2bAR KO mice, along with a greater inflammatory state. Additional mechanisms involved include A2bAR regulation of SREBP-1 expression, a repressor of IRS-2. Importantly, pharmacological activation of the A2bAR by injection of the A2bAR ligand BAY 60-6583 for four weeks post HFD restores IRS-2 levels, and ameliorates T2D. Finally, in obese human subjects A2bAR expression correlates strongly with IRS-2 expression.

Conclusions/Significance

Our study identified the A2bAR as a significant regulator of HFD-induced hallmarks of T2D, thereby pointing to its therapeutic potential.     

Expression of BDNF and TrkB receptor subtypes in the postnatal developing Purkinje cells of monkey cerebellum

In the previous study, we have shown the complementary expression of TrkB subtypes (TK+ and T1) in the adult monkey cerebellar cortex. In this study, to clarify when that expression pattern appeared, we examined the expressions of TrkB subtypes and its ligand brain-derived neurotrophic factor (BDNF) by immunohistochemistry and Western blot analysis. At the newborn stage, both TK+ and T1 were expressed uniformly in the cerebellar cortex. At postnatal month 3.5, the uneven expression of TrkB subtypes was observed, while the BDNF immunoreactivity was strongly detected in all regions of the cerebellar cortex. The expression patterns of TrkB subtypes and BDNF at both postnatal month 6 and year 7 were the same as those at postnatal month 3.5. Western blot analysis demonstrated that TK+ and T1 were expressed at high levels in the synaptic membrane from newborn to adult stages. Furthermore, the dimerization of TrkB subtypes changed at postnatal month 3, which was similar to the adult pattern: at the newborn stage, the TK+ and TK- homodimers; after postnatal month 3.5, the TK+ and TK- homodimers, and the TK+/TK- heterodimer. These findings suggest that the localization of TrkB subtypes in each Purkinje would be changed at postnatal month 3.5, resulting in the uneven expression of TrkB subtypes and the change of TrkB dimerization.     

T cell leukemia-1 modulates TCR signal strength and IFN-gamma levels through phosphatidylinositol 3-kinase and protein kinase C pathway activation

A signaling role for T cell leukemia-1 (TCL1) during T cell development or in premalignant T cell expansions and mature T cell tumors is unknown. In this study, TCL1 is shown to regulate the growth and survival of peripheral T cells but not precursor thymocytes. Proliferation is increased by TCL1-induced lowering of the TCR threshold for CD4(+) and CD8(+) T cell activation through both PI3K-Akt and protein kinase C-MAPK-ERK signaling pathways. This effect is submaximal as CD28 costimulation coupled to TCL1 expression additively accelerates dose-dependent T cell growth. In addition to its role in T cell proliferation, TCL1 also increases IFN-gamma levels from Th1-differentiated T cells, an effect that may provide a survival advantage during premalignant T cell expansions and in clonal T cell tumors. Combined, these data indicate a role for TCL1 control of growth and effector T cell functions, paralleling features provided by TCR-CD28 costimulation. These results also provide a more detailed mechanism for TCL1-augmented signaling and help explain the delayed occurrence of mature T cell expansions and leukemias despite tumorigenic TCL1 dysregulation that begins in early thymocytes.     

A discrete domain of the human TrkB receptor defines the binding sites for BDNF and NT-4

TrkB is a member of the Trk family of tyrosine kinase receptors. In vivo, the extracellular region of TrkB is known to bind, with high affinity, the neurotrophin protein brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). We describe the expression and purification of the second Ig-like domain of human TrkB (TrkBIg(2)) and show, using surface plasmon resonance, that this domain is sufficient to bind BDNF and NT-4 with subnanomolar affinity. BDNF and NT-4 may have therapeutic implications for a variety of neurodegenerative diseases. The specificity of binding of the neurotrophins to their receptor TrkB is therefore of interest. We examine the specificity of TrkBIg(2) for all the neurotrophins, and use our molecular model of the BDNF-TrkBIg(2) complex to examine the residues involved in binding. It is hoped that the understanding of specific interactions will allow design of small molecule neurotrophin mimetics.     

Angiotensin II type 1 receptor activation modulates L- and T-type calcium channel activity through distinct mechanisms in bovine adrenal glomerulosa cells

In adrenal zona glomerulosa cells, calcium entry is crucial for aldosterone production and secretion. This influx is stimulated by increases of extracellular potassium in the physiological range of concentrations and by angiotensin II (Ang II). The high threshold voltage-activated (L-type) calcium channels have been shown to be the major mediators for the rise in cytosolic free calcium concentration, [Ca2+]c, observed in response to a depolarisation by physiological potassium concentrations. Paradoxically, both T- and L-type calcium channels have been shown to be negatively modulated by Ang II after activation by a sustained depolarisation. While the modulation of T-type channels involves protein kinase C (PKC) activation, L-type channel inhibition requires a pertussis toxin-sensitive G protein. In order to investigate the possibility of additional modulatory mechanisms elicited by Ang II on L-type channels, we have studied the effect of PKC activation or tyrosine kinase inhibition. Neither genistein or MDHC, two strong inhibitors of tyrosine kinases, nor the phorbol ester PMA, a specific activator of PKC, affected the Ang II effect on the [Ca2+]c response and on the Ba2+ currents elicited by cell depolarisation with the patch-clamp method. We propose a model describing the mechanisms of the [Ca2+]c modulation by Ang II and potassium in bovine adrenal glomerulosa cells.     

A cell surface ADP-ribosyltransferase modulates T cell receptor association and signaling.

ART-1, a cell surface ADP-ribosyltransferase, is imbedded in the membrane by a glycosylphosphatidylinositol anchor. Function of this enzyme in mouse T lymphocytes is to transfer ADP-ribose groups from NAD to arginine residues, exposed on the extracellular domain of cell surface molecules. As a consequence, T cell responses are modulated. To explore the precise action of the enzyme, the T cell lymphoma EL-4 was transfected with the ART-1 gene, and its effects were examined. It is shown that ART-1 ADP-ribosylates distinct cell surface molecules, causing inhibition of T cell receptor signaling, concomitant to suppression of p56(lck) kinase activation. These effects are explained by failure of T cell receptors and co-receptors to associate into a contiguous and functional receptor cluster.