Using Plants as a Source of Potential Therapeutics for the Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common form of dementia with the numbers expected to increase dramatically as our society ages. There are no treatments to cure, prevent, or slow down the progression of the disease. Age is the single greatest risk factor for AD. However, to date, AD drug discovery efforts have generally not taken this fact into consideration. Multiple changes associated with brain aging, including neuroinflammation and oxidative stress, are important contributors to disease development and progression. Thus, due to the multifactorial nature of AD, the one target strategy to fight the disease needs to be replaced by a more general approach using pleiotropic compounds to deal with the complexity of the disease. In this perspectives piece, our alternative approach to AD drug development based on the biology of aging is described. Starting with plants or plant-derived natural products, we have used a battery of cell-based screening assays that reflect multiple, age-associated toxicity pathways to identify compounds that can target the aspects of aging that contribute to AD pathology. We have found that this combination of assays provides a replicable, cost- and time-effective screening approach that has to date yielded one compound in clinical trials for AD (NCT03838185) and several others that show significant promise.

Back to the future with phenotypic screening.

There are no disease-modifying drugs for any old age associated neurodegenerative disease or stroke. This is at least in part due to the failure of drug developers to recognize that the vast majority of neurodegenerative diseases arise from a confluence of multiple toxic insults that accumulate during normal aging and interact with genetic and environmental risk factors. Thus, it is unlikely that the current single target approach based upon rare dominant mutations or even a few preselected targets is going to yield useful drugs for these conditions. Therefore, the identification of drug candidates for neurodegeneration should be based upon their efficacy in phenotypic screening assays that reflect the biology of the aging brain, not a single, preselected target. It is argued here that this approach to drug discovery is the most likely to produce safe and effective drugs for neurodegenerative diseases.

Modulation of 5-lipoxygenase in proteotoxicity and Alzheimer's disease.

The accumulation of intracellular ß amyloid (Aß) may be one of the factors leading to neuronal cell death in Alzheimer's disease (AD). Using a pyrazole called CNB-001, which was selected for its ability to reduce intracellular Aß, we show that the activation of the eIF2α/ATF4 arm of the unfolded protein response is sufficient to degrade aggregated intracellular Aß. CNB-001 is a potent inhibitor of 5-lipoxygenase (5-LOX), decreases 5-LOX expression, and increases proteasome activity. 5-LOX inhibition induces eIF2α and PERK (protein kinase R-like extracellular signal-regulated kinase) phosphorylation, and HSP90 and ATF4 levels. When fed to AD transgenic mice, CNB-001 also increases eIF2α phosphorylation and HSP90 and ATF4 levels, and limits the accumulation of soluble Aß and ubiquitinated aggregated proteins. Finally, CNB-001 maintains the expression of synapse-associated proteins and improves memory. Therefore, 5-LOX metabolism is a key element in the promotion of endoplasmic reticulum dysfunction, and its inhibition under conditions of stress is sufficient to reduce proteotoxicity both in vivo and in vitro.

De-Risking of Stilbazulenyl Nitrone (STAZN), a Lipophilic Nitrone to Treat Stroke Using a Unique Panel of In Vitro Assays.

In the present study, we used a comprehensive panel of in vitro assays to evaluate the efficacy and safety of stilbazulenyl nitrone (STAZN) as a lead compound to treat acute ischemic stroke. First, we measured neuroprotection in vitro using two different HT22 hippocampal nerve cell assays. Secondly, to de-risk drug development, we used CeeTox analysis with the H4IIE rat hepatoma cell line to determine the acute toxicity profile of STAZN. Third, STAZN was tested in microsomes from four species for measures of metabolic stability. Last, we determined the Ames test genotoxicity profile of STAZN using Salmonella typhimurium TA989 and TA100. In vitro, STAZN was neuroprotective against toxicity induced by iodoacetic acid, and oxytosis-induced glutathione depletion was initiated by glutamate, with an EC(50) value of 1-5 µM. Secondly, using CeeTox analysis, the estimated C(Tox) value (i.e., sustained concentration expected to produce toxicity in a rat 14-day repeat dose study) for STAZN was calculated to be 260 µM. Third, the half-life of STAZN in humans, dogs, and rats was 60-78 min. Last, the genotoxicity profile showed that STAZN did not induce bacterial colony growth under any conditions tested, indicating the lack of mutagenicity with this compound. STAZN appears to be a multi-target neuroprotective compound that has an excellent safety profile in both the CeeTox and Ames mutagenicity assays. STAZN may have significant potential as a novel neuroprotective agent to treat stroke and should be pursued in clinically relevant embolic stroke models.

Delayed treatment with a novel neurotrophic compound reduces behavioral deficits in rabbit ischemic stroke.

Acute ischemic stroke is a major risk for morbidity and mortality in our aging population. Currently only one drug, the thrombolytic tissue plasminogen activator, is approved by the US Food and Drug Administration to treat stroke. Therefore, there is a need to develop new drugs that promote neuronal survival following stroke. We have synthesized a novel neuroprotective molecule called CNB-001 (a pyrazole derivative of curcumin) that has neurotrophic activity, enhances memory, and blocks cell death in multiple toxicity assays related to ischemic stroke. In this study, we tested the efficacy of CNB-001 in a rigorous rabbit ischemic stroke model and determined the molecular basis of its in vivo activity. CNB-001 has substantial beneficial properties in an in vitro ischemia assay and improves the behavioral outcome of rabbit ischemic stroke even when administered 1 h after the insult, a therapeutic window in this model comparable to tissue plasminogen activator. In addition, we elucidated the protein kinase pathways involved in neuroprotection. CNB-001 maintains the calcium-calmodulin-dependent kinase signaling pathways associated with neurotrophic growth factors that are critical for the maintenance of neuronal function. On the basis of its in vivo efficacy and novel mode of action, we conclude that CNB-001 has a great potential for the treatment of ischemic stroke as well as other CNS pathologies.

Metabolic links between diabetes and Alzheimer's disease.

There is a cluster of risk factors for Type 2 diabetes and vascular disease that include high blood glucose, obesity, high blood pressure, increased blood triacylglycerols and insulin resistance. All of these factors, both individually and collectively, increase the risk of Alzheimer's disease (AD) and vascular dementia. Alterations in insulin signaling, glucose and fatty acid metabolism, as well as the accumulation of oxidatively modified and glycated proteins, are associated with both diabetes and the dementias. Data from animal and cell culture models have shown that there is a synergistic interaction between most of these stresses in both AD and diabetes, and with the elevated beta-amyloid peptide levels that are also linked to AD. Some of these parameters can be modified by diet and others may require novel drugs. However, because of the multiplicity of physiological pathways involved, conventional drug therapies directed against a single target are not going to be effective in treating AD or the complications of diabetes. It is therefore likely that the only successful therapy will involve the use of drugs with multiple targets in concert with changes in diet and lifestyle.

Integrative nuclear signaling in cell development--a role for FGF receptor-1.

Ontogeny requires the coordinated regulation of multigene programs by a plethora of extracellular and intracellular signals, thereby allowing cells to transition between different states, including proliferation and differentiation. Disruption of this regulation can result in oncogenic transformation in which cells are "arrested" in the proliferative state. This article summarizes our current understanding of a novel "Integrative Nuclear Fibroblast Growth Factor Receptor-1 (FGFR1) Signaling" (INFS) pathway, which influences differentiation of neural progenitor cells and the associated gene activities. Activation of cell surface neurotransmitter, hormonal, or growth factor receptors stimulates the release of FGFR1 from cytoplasmic membranes into the cytosol. This process is enabled by the atypical transmembrane domain of FGFR1 and is facilitated by the interaction with pp90 ribosomal S6 kinase-1. Cytosolic FGFR1 is transported into the nucleus by importin beta and activates transcription in cooperation with CBP (cyclic AMP Responsive Element-Binding Protein) by augmenting RNA polymerase II activity and histone acetylation. To explain the developmental function of FGFR1, a "feed-forward-and-gate" signaling mechanism is presented in which the INFS pathway "feeds forward" the developmental signals to the common and essential transcriptional coactivator, CBP. The coupled activation of CBP (by INFS) and transcription factors (by specific signaling pathways) enables the coordinated regulation of multigene programs by developmental cues. In some cancer cells, in which INFS is inactive, the reconstitution of nuclear FGFR1 signaling may be used to reestablish this coordinated regulation thereby inhibiting tumor cell proliferation and inducing differentiation.

Fibroblast growth factor receptor signaling affects development and function of dopamine neurons - inhibition results in a schizophrenia-like syndrome in transgenic mice.

Developing and mature midbrain dopamine (DA) neurons express fibroblast growth factor (FGF) receptor-1 (FGFR1). To determine the role of FGFR1 signaling in the development of DA neurons, we generated transgenic mice expressing a dominant negative mutant [FGFR1(TK-)] from the catecholaminergic, neuron-specific tyrosine hydroxylase (TH) gene promoter. In homozygous th(tk-)/th(tk-) mice, significant reductions in the size of TH-immunoreactive neurons were found in the substantia nigra compacta (SNc) and the ventral tegmental area (VTA) at postnatal days 0 and 360. Newborn th(tk-)/th(tk-) mice had a reduced density of DA neurons in both SNc and VTA, and the changes in SNc were maintained into adulthood. The reduced density of DA transporter in the striatum further demonstrated an impaired development of the nigro-striatal DA system. Paradoxically, the th(tk-)/th(tk-) mice had increased levels of DA, homovanilic acid and 3-methoxytyramine in the striatum, indicative of excessive DA transmission. These structural and biochemical changes in DA neurons are similar to those reported in human patients with schizophrenia and, furthermore, these th(tk-)/th(tk-) mice displayed an impaired prepulse inhibition that was reversed by a DA receptor antagonist. Thus, this study establishes a new developmental model for a schizophrenia-like disorder in which the inhibition of FGF signaling leads to alterations in DA neurons and DA-mediated behavior.

Factors controlling fibroblast growth factor receptor-1's cytoplasmic trafficking and its regulation as revealed by FRAP analysis.

Biochemical and microscopic studies have indicated that FGFR1 is a transmembrane and soluble protein present in the cytosol and nucleus. How FGFR1 enters the cytosol and subsequently the nucleus to control cell development and associated gene activities has become a compelling question. Analyses of protein synthesis, cytoplasmic subcompartmental distribution and movement of FGFR1-EGFP and FGFR1 mutants showed that FGFR1 exists as three separate populations (a) a newly synthesized, highly mobile, nonglycosylated, cytosolic receptor that is depleted by brefeldin A and resides outside the ER-Golgi lumen, (b) a slowly diffusing membrane receptor population, and (c) an immobile membrane pool increased by brefeldin A. RSK1 increases the highly mobile cytosolic FGFR1 population and its overall diffusion rate leading to increased FGFR1 nuclear accumulation, which coaccumulates with RSK1. A model is proposed in which newly synthesized FGFR1 can enter the (a) "nuclear pathway," where the nonglycosylated receptor is extruded from the pre-Golgi producing highly mobile cytosolic receptor molecules that rapidly accumulate in the nucleus or (b) "membrane pathway," in which FGFR1 is processed through the Golgi, where its movement is spatially restricted to trans-Golgi membranes with limited lateral mobility. Entrance into the nuclear pathway is favored by FGFR1's interaction with kinase active RSK1.

Ligand dependent and independent internalization and nuclear translocation of fibroblast growth factor (FGF) receptor 1.

Basic fibroblast growth factor (FGF-2) is one of the prototype members of a rapidly expanding family of polypeptides. FGF-2 acts on cells via a dual-receptor system consisting of high-affinity tyrosine kinase receptors (FGFR) and low-affinity receptors comprised of heparan sulfate proteoglycans. Following ligand binding and subsequent internalization, both FGF-2 and FGFR1 are translocated to the nucleus where they have activities distinct from those expressed at the cell surface. Despite the growing number of growth factors and receptors shown to translocate to the nucleus, little is known about the mechanisms of internalization and translocation and how these processes are regulated. In the studies reported in this paper, we examined the roles of clathrin-dependent and -independent endocytosis in the uptake of FGFR1 and one of its ligands, FGF-2. While the uptake of FGF-2 occurred at least partly by a caveolar-dependent mechanism, that of FGFR1 was independent of both caveolae and coated pits. Surprisingly, neither the uptake of FGF-2 nor FGFR1 required the activity of the receptor tyrosine kinase. In addition, we identified a cell cycle-dependent pathway of FGFR1 nuclear translocation that appears to be independent of ligand binding.

Integrative nuclear FGFR1 signaling (INFS) as a part of a universal "feed-forward-and-gate" signaling module that controls cell growth and differentiation.

A novel signaling mechanism is described through which extracellular signals and intracellular signaling pathways regulate proliferation, growth, differentiation, and other functions of cells in the nervous system. Upon cell stimulation, fibroblast growth factor receptor-1 (FGFR1), a typically plasma membrane-associated protein, is released from ER membranes into the cytosol and translocates to the cell nucleus by an importin-beta-mediated transport pathway along with its ligand, FGF-2. The nuclear accumulation of FGFR1 is activated by changes in cell contacts and by stimulation of cells with growth factors, neurotransmitters and hormones as well as by a variety of different second messengers and thus was named integrative nuclear FGFR1 signaling (INFS). In the nucleus, FGFR1 localizes specifically within nuclear matrix-attached speckle-domains, which are known to be sites for RNA Pol II-mediated transcription and co-transcriptional pre-mRNA processing. In these domains, nuclear FGFR1 colocalizes with RNA transcription sites, splicing factors, modified histones, phosphorylated RNA Pol II, and signaling kinases. Within the nucleus, FGFR1 serves as a general transcriptional regulator, as indicated by its association with the majority of active nuclear centers of RNA synthesis and processing, by the ability of nuclear FGFR1 to activate structurally distinct genes located on different chromosomes and by its stimulation of multi-gene programs for cell growth and differentiation. We propose that FGFR1 is part of a universal "feed-forward-and-gate" signaling module in which classical signaling cascades initiated by specific membrane receptors transmit signals to sequence specific transcription factors (ssTFs), while INFS elicited by the same stimuli feeds the signal forward to the common coactivator, CREB-binding protein (CBP). Activation of CBP by INFS, along with the activation of ssTFs by classical signaling cascades brings about coordinated responses from structurally different genes located at different genomic loci.

Ectodermal FGFs induce perinodular inhibition of limb chondrogenesis in vitro and in vivo via FGF receptor 2.

The formation of cartilage elements in the developing vertebrate limb, where they serve as primordia for the appendicular skeleton, is preceded by the appearance of discrete cellular condensations. Control of the size and spacing of these condensations is a key aspect of skeletal pattern formation. Limb bud cell cultures grown in the absence of ectoderm formed continuous sheet-like masses of cartilage. With the inclusion of ectoderm, these cultures produced one or more cartilage nodules surrounded by zones of noncartilaginous mesenchyme. Ectodermal fibroblast growth factors (FGF2 and FGF8), but not a mesodermal FGF (FGF7), substituted for ectoderm in inhibiting chondrogenic gene expression, with some combinations of the two ectodermal factors leading to well-spaced cartilage nodules of relatively uniform size. Treatment of cultures with SU5402, an inhibitor FGF receptor tyrosine kinase activity, rendered FGFs ineffective in inducing perinodular inhibition. Inhibition of production of FGF receptor 2 (FGFR2) by transfection of wing and leg cell cultures with antisense oligodeoxynucleotides blocked appearance of ectoderm- or FGF-induced zones of perinodular inhibition of chondrogenesis and, when introduced into the limb buds of developing embryos, led to shorter, thicker, and fused cartilage elements. Because FGFR2 is expressed mainly at sites of precartilage condensation during limb development in vivo and in vitro, these results suggest that activation of FGFR2 by FGFs during development elicits a lateral inhibitor of chondrogenesis that limits the expansion of developing skeletal elements.

Integrative nuclear FGFR1 signaling (INFS) pathway mediates activation of the tyrosine hydroxylase gene by angiotensin II, depolarization and protein kinase C.

The integrative nuclear FGFR1 signaling (INFS) pathway functions in association with cellular growth, differentiation, and regulation of gene expression, and is activated by diverse extracellular signals. Here we show that stimulation of angiotensin II (AII) receptors, depolarization, or activation protein kinase C (PKC) or adenylate cyclase all lead to nuclear accumulation of fibroblast growth factor 2 (FGF-2) and FGFR1, association of FGFR1 with splicing factor-rich domains, and activation of the tyrosine hydroxylase (TH) gene promoter in bovine adrenal medullary cells (BAMC). The up-regulation of endogenous TH protein or a transfected TH promoter-luciferase construct by AII, veratridine, or PMA (but not by forskolin) is abolished by transfection with a dominant negative FGFR1TK-mutant which localizes to the nucleus and plasma membrane, but not by extracellularly acting FGFR1 antagonists suramin and inositolhexakisphosphate (IP6). Mechanism of TH gene activation by FGF-2 and FGFR1 was further investigated in BAMC and human TE671 cultures. TH promoter was activated by co-transfected HMW FGF-2 (which is exclusively nuclear) but not by cytoplasmic FGF-1 or extracellular FGFs. Promoter transactivation by HMWFGF-2 was accompanied by an up-regulation of FGFR1 specifically in the cell nucleus and was prevented FGFR1(TK-) but not by IP6 or suramin. The TH promoter was also transactivated by co-transfected wild-type FGFR1, which localizes to both to the nucleus and the plasma membrane, and by an exclusively nuclear, soluble FGFR1(SP-/NLS) mutant with an inserted nuclear localization signal. Activation of the TH promoter by nuclear FGFR1 and FGF-2 was mediated through the cAMP-responsive element (CRE) and was associated with induction of CREB- and CBP/P-300-containing CRE complexes. We propose a new model for gene regulation in which nuclear FGFR1 acts as a mediator of CRE transactivation by AII, cell depolarization, and PKC.

A novel role for farnesyl pyrophosphate synthase in fibroblast growth factor-mediated signal transduction.

Farnesyl pyrophosphate synthase (FPPS) catalyses the formation of a key cellular intermediate in isoprenoid metabolic pathways. Here we describe a novel role for this enzyme in fibroblast growth factor (FGF)-mediated signalling. We demonstrate the binding of FPPS to FGF receptors (FGFRs) using the yeast two-hybrid assay, pull-down assays and co-immunoprecipitation. The interaction between FPPS and FGFR is regulated by the cellular metabolic state and by treatment with FGF-2. Overexpression of FPPS inhibits FGF-2-induced cell proliferation, accompanied by a failure of the FGF-2-mediated induction of cyclins D1 and E. Overexpression of FPPS in fibroblasts also promotes increased farnesylation of Ras, and temporally extends FGF-2-stimulated activation of the Ras/ERK (extracellular-signal-regulated kinase) cascade. These data suggest that, in addition to its role in isoprenoid biosynthesis, FPPS may function as a modulator of the cellular response to FGF treatment.