Abdul H. Fauq

The nervous system and innate immunity: the neuropeptide connection

Notch receptors are processed by γ-secretase acting in synergy with T cell receptor signaling to sustain peripheral T cell activation. Activated CD4+ T cells differentiate into T helper type 1 (TH1) or TH2 subsets. Molecular cues directing TH1 differentiation include expression of the TH1-specific transcription factor T-bet, encoded by Tbx21. However, the regulation of Tbx21 remains incompletely defined. Here we report that Notch1 can directly regulate Tbx21 through complexes formed on the Tbx21 promoter. In vitro, γ-secretase inhibitors extinguished expression of Notch, interferon-γ and Tbx21 in TH1-polarized CD4+ cells, whereas ectopic expression of activated Notch1 restored Tbx21 transcription. In vivo, administration of γ-secretase inhibitors substantially impeded TH1-mediated disease progression in the mouse experimental autoimmune encephalomyelitis model of multiple sclerosis. Thus, using γ-secretase inhibitors to modulate Notch signaling may prove beneficial in treating TH1-mediated autoimmunity.

Substrate-targeting γ-secretase modulators

Selective lowering of Aβ42 levels (the 42-residue isoform of the amyloid-β peptide) with small-molecule γ-secretase modulators (GSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer’s disease. To identify the target of these agents we developed biotinylated photoactivatable GSMs. GSM photoprobes did not label the core proteins of the γ-secretase complex, but instead labelled the β-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-β peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP γ-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28–36 of amyloid-β, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-β act as GSMs, and some GSMs alter the production of cell-derived amyloid-β oligomers. Furthermore, mutation of the GSM binding site in the APP alters the sensitivity of the substrate to GSMs. These findings indicate that substrate targeting by GSMs mechanistically links two therapeutic actions: alteration in Aβ42 production and inhibition of amyloid-β aggregation, which may synergistically reduce amyloid-β deposition in Alzheimer’s disease. These data also demonstrate the existence and feasibility of ‘substrate targeting’ by small-molecule effectors of proteolytic enzymes, which if generally applicable may significantly broaden the current notion of ‘druggable’ targets.

Diverse compounds mimic Alzheimer disease–causing mutations by augmenting Aβ42 production

Increased Aβ42 production has been linked to the development of Alzheimer disease. We now identify a number of compounds that raise Aβ42. Among the more potent Aβ42-raising agents identified are fenofibrate, an antilipidemic agent, and celecoxib, a COX-2–selective NSAID. Many COX-2–selective NSAIDs tested raised Aβ42, including multiple COX-2–selective derivatives of two Aβ42-lowering NSAIDs. Compounds devoid of COX activity and the endogenous isoprenoids FPP and GGPP also raised Aβ42. These compounds seem to target the γ-secretase complex, increasing γ-secretase–catalyzed production of Aβ42 in vitro. Short-term in vivo studies show that two Aβ42-raising compounds increase Aβ42 levels in the brains of mice. The elevations in Aβ42 by these compounds are comparable to the increases in Aβ42 induced by Alzheimer disease–causing mutations in the genes encoding amyloid β protein precursor and presenilins, raising the possibility that exogenous compounds or naturally occurring isoprenoids might increase Aβ42 production in humans.

Notch1 augments NF-κB activity by facilitating its nuclear retention

Notch1 specifically upregulates expression of the cytokine interferon‐γ in peripheral T cells through activation of NF‐κB. However, how Notch mediates NF‐κB activation remains unclear. Here, we examined the temporal relationship between Notch signaling and NF‐κB induction during T‐cell activation. NF‐κB activation occurs within minutes of T‐cell receptor (TCR) engagement and this activation is sustained for at least 48 h following TCR signaling. We used γ‐secretase inhibitor (GSI) to prevent the cleavage and subsequent activation of Notch family members. We demonstrate that GSI blocked the later, sustained NF‐κB activation, but did not affect the initial activation of NF‐κB. Using biochemical approaches, as well as confocal microscopy, we show that the intracellular domain of Notch1 (N1IC) directly interacts with NF‐κB and competes with IκBα, leading to retention of NF‐κB in the nucleus. Additionally, we show that N1IC can directly regulate IFN‐γ expression through complexes formed on the IFN‐γ promoter. Taken together, these data suggest that there are two ‘waves’ of NF‐κB activation: an initial, Notch‐independent phase, and a later, sustained activation of NF‐κB, which is Notch dependent.

Notch1 and TGFβ1 cooperatively regulate Foxp3 expression and the maintenance of peripheral regulatory T cells

Notch and its ligands have been implicated in the regulation and differentiation of various CD4(+) T-helper cells. Regulatory T cells (T(regs)), which express the transcription factor Foxp3, suppress aberrant immune responses that are typically associated with autoimmunity or excessive inflammation. Previous studies have shown that transforming growth factor beta (TGFbeta1) induces Foxp3 expression and a regulatory phenotype in peripheral T cells. Here, we show that pharmacologic inhibition of Notch signaling using gamma-secretase inhibitor (GSI) treatment blocks (1) TGFbeta1-induced Foxp3 expression, (2) the up-regulation of Foxp3-target genes, and (3) the ability to suppress naive T-cell proliferation. In addition, the binding of Notch1, CSL, and Smad to conserved binding sites in the foxp3 promoter can be inhibited by treatment with GSI. Finally, in vivo administration of GSI results in reduced Foxp3 expression and development of symptoms consistent with autoimmune hepatitis, a disease previously found to result from dysregulation of TGFbeta signaling and regulatory T cells. Together, these findings indicate that the Notch and TGFbeta signaling pathways cooperatively regulate Foxp3 expression and regulatory T-cell maintenance both in vitro and in vivo.

Phase III Double-Blind, Randomized, Placebo-Controlled Crossover Trial of Black Cohosh in the Management of Hot Flashes: NCCTG Trial N01CC1

PURPOSE Hot flashes can cause significant morbidity in postmenopausal women undergoing or finished with breast cancer treatment. Black cohosh has been used to treat hot flashes, but definitive clinical data about efficacy have been equivocal. METHODS A double-blind, randomized, cross-over clinical trial with two 4-week periods, was used to study the efficacy of black cohosh (1 capsule, Cimicifuga racemosa 20 mg BID) for the treatment of hot flashes in women. Participants kept a daily hot flash diary during a baseline week and then during two 4-week crossover treatment periods. Hot flash scores were measured by assigning points (1 to 4 for mild to very severe) to each hot flash based on severity and then adding the points for a given time period. RESULTS Between October 31, 2003, to March 4, 2004, 132 patients were randomly assigned. Toxicity was minimal and not different by treatment group. Patients receiving black cohosh reported a mean decrease in hot flash score of 20% (comparing the fourth treatment week to the baseline week) compared with a 27% decrease for patients on placebo (P = .53). Mean hot flash frequency was reduced 17% on black cohosh and 26% on placebo (P = .36). Patient treatment preferences were measured after completion of both treatment periods by ascertaining which treatment period, if any, the patient preferred. Thirty-four percent of patients preferred the black cohosh treatment, 38% preferred the placebo, and 28% did not prefer either treatment. CONCLUSION This trial failed to provide any evidence that black cohosh reduced hot flashes more than the placebo.

Regulation of Steady-state β-Amyloid Levels in the Brain by Neprilysin and Endothelin-converting Enzyme but Not Angiotensin-converting Enzyme

The deposition of β-amyloid in the brain is a pathological hallmark of Alzheimer disease (AD). Normally, the accumulation of β-amyloid is prevented in part by the activities of several degradative enzymes, including the endothelin-converting enzymes, neprilysin, insulin-degrading enzyme, and plasmin. Recent reports indicate that another metalloprotease, angiotensin-converting enzyme (ACE), can degrade β-amyloid in vitro and in cellular overexpression experiments. In addition, ACE gene variants are linked to AD risk in several populations. Angiotensin-converting enzyme, neprilysin and endothelin-converting enzyme function as vasopeptidases and are the targets of drugs designed to treat cardiovascular disorders, and ACE inhibitors are commonly prescribed. We investigated the potential physiological role of ACE in regulating endogenous brain β-amyloid levels for two reasons: first, to determine whether β-amyloid degradation might be the mechanism by which ACE is associated with AD, and second, to determine whether ACE inhibitor drugs might block β-amyloid degradation in the brain and potentially increase the risk for AD. We analyzed β-amyloid accumulation in brains from ACE-deficient mice and in mice treated with ACE inhibitors and found that ACE deficiency did not alter steady-state β-amyloid concentration. In contrast, β-amyloid levels are significantly elevated in endothelin-converting enzyme and neprilysin knock-out mice, and inhibitors of these enzymes cause a rapid increase in β-amyloid concentration in the brain. The results of these studies do not support a physiological role for ACE in the degradation of β-amyloid in the brain but confirm roles for endothelin-converting enzyme and neprilysin and indicate that reductions in these enzymes result in additive increases in brain amyloid β-peptide levels.

Notch signaling is activated by TLR stimulation and regulates macrophage functions

Notch signaling is a well‐conserved pathway involved in cell fate decisions, proliferation and apoptosis. We report on the involvement of Notch signaling in regulating gene expression in activated macrophages. Toll‐like receptors (TLR) agonists such as bacterial lipopeptide, polyI:C, lipopolysaccharide and unmethylated CpG DNA all induced up‐regulation of Notch1 in primary and macrophage‐like cell lines. Notch1 up‐regulation was dependent on the MyD88 pathway when stimulated through TLR2, but not TLR4. Activated Notch1 and expression of the Notch target genes, Hes1 and Deltex, were detected in activated macrophages, suggesting that Notch signaling was activated upon stimulation. Inhibiting processing of Notch receptor by γ‐secretase using a γ‐secretase inhibitor (GSI), the expression of Notch1 was down‐regulated to basal levels. This treatment significantly modulated expression of TNF‐α, IL‐6, and IL‐10. In addition, the amount of nitric oxide produced was significantly lower and the expression of MHC class II was up‐regulated in GSI‐treated cells. Treatment with GSI or silencing Notch1 resulted in decreased translocation of NF‐κBp50 into nucleus upon stimulation. Taken together, stimulation of macrophages through the TLR signaling cascade triggered activation of Notch signaling, which in turn regulated gene expression patterns involved in pro‐inflammatory responses, through activation of NF‐κB.

Notch signaling mediates G1/S cell-cycle progression in T cells via cyclin D3 and its dependent kinases

Notch signaling plays a role in normal lymphocyte development and function. Activating Notch1-mutations, leading to aberrant downstream signaling, have been identified in human T-cell acute lymphoblastic leukemia (T-ALL). While this highlights the contribution of Notch signaling to T-ALL pathogenesis, the mechanisms by which Notch regulates proliferation and survival in normal and leukemic T cells are not fully understood. Our findings identify a role for Notch signaling in G(1)-S progression of cell cycle in T cells. Here we show that expression of the G(1) proteins, cyclin D3, CDK4, and CDK6, is Notch-dependent both in vitro and in vivo, and we outline a possible mechanism for the regulated expression of cyclin D3 in activated T cells via CSL (CBF-1, mammals; suppressor of hairless, Drosophila melanogaster; Lag-1, Caenorhabditis elegans), as well as a noncanonical Notch signaling pathway. While cyclin D3 expression contributes to cell-cycle progression in Notch-dependent human T-ALL cell lines, ectopic expression of CDK4 or CDK6 together with cyclin D3 shows partial rescue from gamma-secretase inhibitor (GSI)-induced G(1) arrest in these cell lines. Importantly, cyclin D3 and CDK4 are highly overexpressed in Notch-dependent T-cell lymphomas, justifying the combined use of cell-cycle inhibitors and GSI in treating human T-cell malignancies.

Liver X Receptor Agonist Treatment Ameliorates Amyloid Pathology and Memory Deficits Caused by High-Fat Diet in APP23 Mice

High-fat diet and certain dietary patterns are associated with higher incidence of sporadic Alzheimers disease (AD) and cognitive decline. However, no specific therapy has been suggested to ameliorate the negative effects of high fat/high cholesterol levels on cognition and amyloid pathology. Here we show that in 9-month-old APP23 mice, a high-fat/high-cholesterol (HF) diet provided for 4 months exacerbates the AD phenotype evaluated by behavioral, morphological, and biochemical assays. To examine the therapeutic potential of liver X receptor (LXR) ligands, APP23 mice were fed HF diet supplemented with synthetic LXR agonist T0901317 (T0). Our results demonstrate that LXR ligand treatment causes a significant reduction of memory deficits observed during both acquisition and retention phases of the Morris water maze. Moreover, the effects of T0 on cognition correlate with AD-like morphological and biochemical parameters. We found a significant decrease in amyloid plaque load, insoluble Aβ and soluble Aβ oligomers. In vitro experiments with primary glia demonstrate that Abca1 is essential for the proper lipidation of ApoE and mediates the effects of T0 on Aβ degradation by microglia. Microdialysis experiments performed on awake freely moving mice showed that T0 decreased Aβ levels in the interstitial fluid of the hippocampus, supporting the conclusion that this treatment increases Aβ clearance. The data presented conclusively shows that LXR activation in the context of a metabolic challenge has critical effects on AD phenotype progression by attenuating Aβ deposition and facilitating its clearance.