Translation Blockers Research Articles

LTP suppression by protein synthesis inhibitors is NO-dependent

For several decades, the ability of protein synthesis inhibitors (PSI) to suppress the long-term potentiation (LTP) of hippocampal responses is known. It is considered that mechanisms of such impairment are related to a cessation of translation and a delayed depletion of the protein pool required for maintenance of synaptic plasticity. The present study demonstrates that cycloheximide or anisomycin applications reduce amplitudes of the field excitatory postsynaptic potentials as well as the presynaptically mediated form of plasticity, the paired-pulse facilitation after LTP induction in neurons of the CA1 area of hippocampus. We showed that nitric oxide signaling could be one of the pathways that cause the LTP decrease induced by cycloheximide or anisomycin. Inhibitor of the NO synthase, L-NNA or the NO scavenger, PTIO, rescued the late-phase LTP and restored the paired-pulse facilitation up to the control levels. For the first time we have directly measured the nitric oxide production induced by application of the translation blockers in hippocampal neurons using the NO-sensitive dye DAF-FM. Inhibitory analysis demonstrated that changes during protein synthesis blockade downstream the NO signaling cascade are cGMP-independent and apparently are implemented through degradation of target proteins. Prolonged application of the NO donor SNAP impaired the LTP maintenance in the same manner as PSI.

Reduction of Huntington's Disease RNA Foci by CAG Repeat-Targeting Reagents.

In several human polyglutamine diseases caused by expansions of CAG repeats in the coding sequence of single genes, mutant transcripts are detained in nuclear RNA foci. In polyglutamine disorders, unlike other repeat-associated diseases, both RNA and proteins exert pathogenic effects; therefore, decreases of both RNA and protein toxicity need to be addressed in proposed treatments. A variety of oligonucleotide-based therapeutic approaches have been developed for polyglutamine diseases, but concomitant assays for RNA foci reduction are lacking. Here, we show that various types of oligonucleotide-based reagents affect RNA foci number in Huntington’s disease cells. We analyzed the effects of reagents targeting either CAG repeat tracts or specific HTT sequences in fibroblasts derived from patients. We tested reagents that either acted as translation blockers or triggered mRNA degradation via the RNA interference pathway or RNase H activation. We also analyzed the effect of chemical modifications of CAG repeat-targeting siRNAs on their efficiency in the foci decline. Our results suggest that the decrease of RNA foci number may be considered as a readout of treatment outcomes for oligonucleotide reagents.

Novel 5′ Untranslated Region Directed Blockers of Iron-Regulatory Protein-1 Dependent Amyloid Precursor Protein Translation: Implications for Down Syndrome and Alzheimer's Disease

We reported that iron influx drives the translational expression of the neuronal amyloid precursor protein (APP), which has a role in iron efflux. This is via a classic release of repressor interaction of APP mRNA with iron-regulatory protein-1 (IRP1) whereas IRP2 controls the mRNAs encoding the L- and H-subunits of the iron storage protein, ferritin. Here, we identified thirteen potent APP translation blockers that acted selectively towards the uniquely configured iron-responsive element (IRE) RNA stem loop in the 5′ untranslated region (UTR) of APP mRNA. These agents were 10-fold less inhibitory of 5′UTR sequences of the related prion protein (PrP) mRNA. Western blotting confirmed that the ‘ninth’ small molecule in the series selectively reduced neural APP production in SH-SY5Y cells at picomolar concentrations without affecting viability or the expression of α-synuclein and ferritin. APP blocker-9 (JTR-009), a benzimidazole, reduced the production of toxic Aβ in SH-SY5Y neuronal cells to a greater extent than other well tolerated APP 5′UTR-directed translation blockers, including posiphen, that were shown to limit amyloid burden in mouse models of Alzheimer's disease (AD). RNA binding assays demonstrated that JTR-009 operated by preventing IRP1 from binding to the IRE in APP mRNA, while maintaining IRP1 interaction with the H-ferritin IRE RNA stem loop. Thus, JTR-009 constitutively repressed translation driven by APP 5′UTR sequences. Calcein staining showed that JTR-009 did not indirectly change iron uptake in neuronal cells suggesting a direct interaction with the APP 5′UTR. These studies provide key data to develop small molecules that selectively reduce neural APP and Aβ production at 10-fold lower concentrations than related previously characterized translation blockers. Our data evidenced a novel therapeutic strategy of potential impact for people with trisomy of the APP gene on chromosome 21, which is a phenotype long associated with Down syndrome (DS) that can also cause familial Alzheimer's disease.

P4‐225: Therapeutic action of translation blockers on expression of the amyloid precursor protein as a central ferroxidase

It is possible to repress amyloid precursor protein (APP) mRNA translation as both an anti-amyloid pharmacotherapy for Alzheimer's disease (AD) and also to tone oxidative ferroxidase activity from APP over-expression.This therapeutic model is consistent with cases where AD pathology is the genetic consequence of duplications of the APP gene. Our pharmacological goal is to reduce APP expression. The antioxidant N-acetyl cysteine selectively reduced APP translation in neural cells while maintaining compensatory APLP-1 /-2 levels, then this drug also limited Aβ production in vivo.Secondly, posiphen is the + enantiomer of the well-tested anti-cholinesterase phenserine, which entered phase II clinical trials for its capacity to limit amyloid in AD subjects via its action as an APP 5'UTR translation blocker. We screened two molecular compound libraries at Harvard and Columbia Universities. From our screens, thirteen top leads shared chemical features pertinent to potently blocking APP 5'UTR directed translation (1nM). Most of our lead APP translation blockers were found to be highly potent intercalators of the RNA stem loop folded from APP 5'untranslated region. All were planar and had either a benzimidazole, benzothiazole or benzoxazole ring in its backbone. Top APP 5'UTR inhibitors were 50-fold more potent blockers of Aβ precursor translation than NAC or posiphen. The most active lead was the ninth compound in the series, APP blocker-9 (“JTR-009”). Critically, JTR-009 effectively reduced APP levels in primary mouse E-18 cortical neurons at single nanomolar concentrations similar to serum concentrations of effective clinical drugs. JTR-009 was anti-amyloid and our calcein assays demonstrated it adjusted cellular iron content to promote cellular viability. Our top APP translation blocker, JTR-009, is a planar tricyclic benzimidazole that intercalates selectively into the single 146 base APP 5'UTR specific RNA stem loop. Confirming specificity and potency, we are checking how JTR-009 blocks ribosome scanning of the APP 5'UTR to impede translation (Aβ output) while not interacting with the 5'UTRs of APP-like protein (APLP-1/-2) mRNAs. This APP translation blocker adjusted the intrinsic levels of APP with its concomitant ferroxidase activity and iron export properties, thus to optimally promote neural cell viability.

The anticholinesterase phenserine and its enantiomer posiphen as 5'untranslated-region-directed translation blockers of the Parkinson's alpha synuclein expression.

There is compelling support for limiting expression of alpha-synuclein (α-syn) in the brains of Parkinson's disease (PD) patients. An increase of SNCA gene copy number can genetically cause familial PD where increased dose of this pathogenic protein correlates with severity of symptoms (triplication of the SNCA gene causes dementia in PD patients). Gene promoter polymorphisms were shown to increase α-synuclein expression as a risk for PD. Cholinesterase inhibitors can clinically slow cognitive decline in the later stages of PD etiology similar to their widespread use in Alzheimer's disease (AD). Pertinent to this, we identified that the well-tolerated anticholinesterase, phenserine, blocked neural SNCA mRNA translation and tested for targeting via its 5′untranslated region (5′UTR) in a manner similar to its action to limit the expression of the AD-specific amyloid precursor protein (APP). Posiphen, its better-tolerated (+) enantiomer (devoid of anticholinesterase action), repressed neural α-synuclein translation. Primary metabolic analogs of posiphen were, likewise, characterized using primary fetal neurons grown ex vivo from the brains of Parkinson's transgenic mice expressing the human SNCA gene.

Mechanism of action of app translation blockers: From model to human

Mechanism of action of app translation blockers: From model to human

The alpha-synuclein 5′untranslated region targeted translation blockers: anti-alpha synuclein efficacy of cardiac glycosides and Posiphen

Increased brain α-synuclein (SNCA) protein expression resulting from gene duplication and triplication can cause a familial form of Parkinson's disease (PD). Dopaminergic neurons exhibit elevated iron levels that can accelerate toxic SNCA fibril formation. Examinations of human post mortem brain have shown that while mRNA levels for SNCA in PD have been shown to be either unchanged or decreased with respect to healthy controls, higher levels of insoluble protein occurs during PD progression. We show evidence that SNCA can be regulated via the 5'untranslated region (5'UTR) of its transcript, which we modeled to fold into a unique RNA stem loop with a CAGUGN apical loop similar to that encoded in the canonical iron-responsive element (IRE) of L- and H-ferritin mRNAs. The SNCA IRE-like stem loop spans the two exons that encode its 5'UTR, whereas, by contrast, the H-ferritin 5'UTR is encoded by a single first exon. We screened a library of 720 natural products (NPs) for their capacity to inhibit SNCA 5'UTR driven luciferase expression. This screen identified several classes of NPs, including the plant cardiac glycosides, mycophenolic acid (an immunosuppressant and Fe chelator), and, additionally, posiphen was identified to repress SNCA 5'UTR conferred translation. Western blotting confirmed that Posiphen and the cardiac glycoside, strophanthidine, selectively blocked SNCA expression (~1 μM IC(50)) in neural cells. For Posiphen this inhibition was accelerated in the presence of iron, thus providing a known APP-directed lead with potential for use as a SNCA blocker for PD therapy. These are candidate drugs with the potential to limit toxic SNCA expression in the brains of PD patients and animal models in vivo.

Specific APP translation blockers as a therapeutic intervention for Alzheimer's disease

Specific APP translation blockers as a therapeutic intervention for Alzheimer's disease

Single-channel analysis of functional epithelial sodium channel (ENaC) stability at the apical membrane of A6 distal kidney cells

Epithelial sodium channels (ENaC) play an essential role in maintaining total body fluid and electrolyte homeostasis. As such, abnormal expression of ENaC at the cell surface is linked to several important human diseases. Although the stability of ENaC subunits has been extensively studied by protein biochemical analysis, the half-life of the functional channel in the apical membrane remains controversial. Because the functional stability of the multisubunit channel may be more physiologically relevant than the stability of individual subunit proteins, we performed studies of functional ENaC channels using A6 epithelial cells, a Xenopus laevis distal nephron cell line. We recorded single-channel activity in over 400 cells with the translation blockers cycloheximide (CHX) or puromycin, as well as the intracellular protein trafficking inhibitors brefeldin A (BFA) or nocodazole. Our cell-attached, single-channel recordings allow us to quantify the channel density in the apical membrane, as well as to determine channel open probability (Po) from control (untreated) cells and from cells at different times of drug treatment. The data suggest that the half-life of ENaC channels is approximately 3.5 h following puromycin, BFA, and nocodazole treatment. Furthermore, these three drugs had no significant effect on the Po of ENaC for at least 6 h after exposure. A decrease in apical channel number and Po was observed following 2 h of CHX inhibition of protein synthesis, and the apparent channel half-life was closer to 1.5 h following CHX treatment. Treatment of cells with the translation inhibitors does not alter the expression of the protease furin, and therefore changes in protease activity cannot explain changes in ENaC Po. Confocal images show that BFA and nocodazole both disrupt most of the Golgi apparatus after 1-h exposure. In cells with the Golgi totally disrupted by overnight exposure to BFA, 20% of apical ENaC channels remained functional. This result suggests that ENaC is delivered to the apical membrane via a pathway that might bypass the Golgi vesicular trafficking pathway, or that there might be two pools of channels with markedly different half-lives in the apical membrane.

EXPRESSION OF HEAT SHOCK PROTEINS UPON THE ADDITION OF GELDANAMYCIN INCREASES PHAGOCYTOSIS OF IGG-OPSONIZED PARTICLES

Cells are constantly subjected to an array of harmful conditions that trigger a widespread response to stress known as the shock response. Heat shock proteins (hsps), which are localized within the cell, are involved in damage repair after stress and further protection from subsequent insults. Recently, hsps have also been found in circulation after severe injury. The extracellular hsps have been proposed to constitute an "alert signal," playing a fundamental role in the modulation of immune response. Moreover, intracellular hsps, constitutive or inducible forms, could also regulate cellular functions important for the immune response. We have observed an increase in phagocytosis of IgGopsonized and non-opsonic particles upon incubation of murine macrophages (J774 cells) with geldanamycin (GA), which is a specific inhibitor of Hsp90 function. The effect of GA treatment of phagocytosis was reversed by the addition of transcription or translation blockers, indicating that gene expression was required. Thus, the increase in phagocytosis is likely due to the expression of hsps as a consequence of GA treatment. This assumption was confirmed by detecting the same increase in phagocytosis after heat shock. The increase in phagocytosis upon stress was not due to elevation of Fcγ receptors on the cell surface. However, it did correlate with an increase in intracellular calcium and a rapid polymerization of actin in proximity to the plasma membrane. The presence of F-actin could be detected even in the absence of foreign ligands, suggesting that hsps could prime macrophages toward phagocytic activity. These results demonstrate that the presence of hsps renders macrophages capable of internalizing pathogens in a highly efficient manner, which may have a tremendous impact on the response to injury and infection.

Growth cones make proteins, too

Axons branch out from neurons as they respond to chemical cues in their extracellular environment. Until recently, many scientists did not believe that elongating axons could synthesize proteins locally. But in the past three years, this view has been largely overturned. One of the first clues that protein synthesis might occur in the growing tips of axons—the growth cones—came from morphological studies conducted over 30 years ago by Virginia Tennyson. In the mid-1960s, several electron microscopy studies of neurons had been published, but few of them focused on the growth cone. Tennyson, then a researcher at Columbia University, decided to examine the axons of fetal rabbit dorsal root neuroblasts at 11–12 days, a time in development when many growth cones are present. “I remember, I wanted to study growth cones,” recalls Tennyson. “I certainly was not expecting to see any ribosomes.” Tennyson observed clusters of particles along the length of an entire axon and in several growth cones. The particles were 150–250 A in diameter and morphologically identical to ribosomes (Tennyson, 1970). “The presence of ribosomes in the early embryonic axons suggests that protein synthesis may continue in these segments at a considerable distance from the perikaryon [neuron cell body],” Tennyson wrote in her 1970 paper. “Of course I had no evidence of protein synthesis at that time, so I did not want to make too much of that observation,” she says. Shortly after Tennyson's study, further ultrastructural analyses confirmed the presence of polyribosomes in growth cones of cultured neurons (Yamada et al., 1971; Bunge, 1973). Figure Ribosomes (arrows) turned up in axons. Since then, several studies have documented ribosomes, mRNA, translational initiation proteins, and protein synthesis in axons and growth cones. Douglas Campbell and Christine Holt (University of Cambridge) demonstrated that molecules that guide the growth of axons rapidly trigger protein synthesis in isolated retinal growth cones (Campbell and Holt, 2001). Inhibition of protein synthesis by translation blockers abolishes the response of these growth cones to guidance molecules. This and other studies (Brittis et al., 2002; Zheng et al., 2001) showed that, at least in vitro, fast, local synthesis of proteins not only occurs but is necessary for guiding axon growth in response to external cues. “In retrospect,” muses Holt, “it is surprising how remarkable everyone thought this was.” Brittis, P.A., et al. 2002. Cell. 110:223–235. [PubMed] Bunge, M.B. 1973. J. Cell Biol. 56:713–735. [PubMed] Campbell, D.S., and C.E. Holt. 2001. Neuron. 32:1013–1026. [PubMed] Tennyson, V.M. 1970. J. Cell Biol. 44:62–79. [PubMed] Yamada, K.M., et al. 1971. J. Cell Biol. 49:614–635. [PubMed] Zheng, J.Q., et al. 2001. J. Neurosci. 21:9291–9303. [PubMed]

Intermediate and long-term memories of associative learning are differentially affected by transcription versus translation blockers in Lymnaea.

Aerial respiratory behaviour in the pond snail, Lymnaea stagnalis, can be operantly conditioned. This associative learning then undergoes consolidation into a long-lasting memory which, depending on the training procedure used, causes intermediate-term memory (ITM; lasting 3 h) or long-term memory (LTM; lasting >6 h) to be formed. We determined the differential susceptibility of these two forms of memory to translation and transcription blockers. The injection of a translation blocker, Anisomycin, 2.5 h before training prevents the establishment of both ITM and LTM. On the other hand, injection of the transcription blocker Actinomycin D, 2.5 h before training, did not prevent the establishment of ITM, but did, however, prevent LTM formation. Thus in Lymnaea, following associative learning, both ITM and LTM are dependent on new protein synthesis. ITM appears to be dependent on protein synthesis from preexisting transcription factors, whilst LTM is dependent on protein synthesis from new transcription messages.

Coupling of the Dolichol-P-P-Oligosaccharide Pathway to Translation by Perturbation-sensitive Regulation of the Initiating Enzyme, GlcNAc-1-P Transferase

In mammalian cells, inhibition of translation interferes with synthesis of the lipid-linked oligosaccharide (LLO) Glc3Man9GlcNAc2-P-P-dolichol as measured with radioactive sugar precursors. Conflicting hypotheses have been proposed, and the fundamental basis for this regulation has remained elusive. Here, fluorophore-assisted carbohydrate electrophoresis (FACE) was used to measure LLO concentrations directly in cells treated with translation blockers. Further, LLO biosynthetic enzymes were assayed in vitro with endogenous acceptor substrates using either cells gently permeabilized with streptolysin-O (SLO) or microsomes from homogenized cells. In Chinese hamster ovary (CHO)-K1 cells treated with translation blockers, FACE did not detect changes in concentrations of Glc3Man9GlcNAc2-P-P-dolichol or early LLO intermediates. These results do not support earlier proposals for feedback repression of LLO initiation by accumulated Glc3Man9GlcNAc2-P-P-dolichol, or inhibition of a GDP-mannose dependent transferase. With microsomes from cells treated with translation blockers, there was no interference with LLO initiation by GlcNAc-1-P transferase (GPT), mannose-P-dolichol synthase, glucose-P-dolichol synthase, or LLO synthesis in vitro, as reported previously. Surprisingly, inhibition of all of these was detected with the SLO in vitro system. Additional experiments with the SLO system showed that the three transferases shared a limited pool of dolichol-P that was trapped as Glc3Man9GlcNAc2-P-P-dolichol by translation arrest. Overexpression of GPT was unable to reverse the effects of translation arrest on LLO initiation, and experiments with FACE and the SLO system showed that overexpressed GPT was not functional in vivo, although it was highly active in microsomal assays. Thus, the combined use of the SLO in vitro system and FACE showed that LLO biosynthesis depends upon a limited primary pool of dolichol-P. Physical perturbation associated with microsome preparation appears to make available a secondary pool of dolichol-P, masking inhibition by translation arrest, as well as activating a nonfunctional fraction of GPT. The implications of these results for the organization of the LLO pathway are discussed.

Neurospecific translation control and the problem of potent danger of drugs and chemicals: a caution.

At present, chemicals are an integral part of the surrounding biosphere. A great number of new xenobiotics for industrial, pharmaceutical, agricultural, or mode-of-life daily use (perfumes, cosmetics, food additives, etc.) may play a role of a danger health hazards. These chemicals may be environmental pollutants and due to this circumstance they promote different ecological disorders including the several epidemic-like processes such as, for instance, Minamata disease (chronic alimentary poisoning with methyl mercury), etc. Also, these chemicals may be industrial hazards promoting the origin and development of various occupational diseases. Finally, the numerous side effects of multiple new drugs may serve as causes of a number of different health disorders. Briefly, we must agree that modern man exists in the world with such inalienable elements as the steadfast increase of chemical soiling of Earths biosphere. Naturally, human health protection in our "chemically soiled" world is very difficult by urgent task. As for the solving of this task, it may be reached only in the case of successful fundamental studies of mechanisms of toxic action of different classes of chemicals performed at different biological levels (systemic, organ/tissue, cellular and molecular levels of research). A separate and special population of modern health-hazardous chemicals is a group of agents which selectively suppress protein synthesis in mammalian cells and tissues due to the direct effect on the translation machinery elements or on the messenger RNA processing steps. The agents of this group ("translation blockers" or, the same but more broadly, "protein synthesis inhibitors" (PSI) usually are quite dangerous for human health since these substances inhibit the central molecular process of life, i.e., the synthesis of protein molecules. The small but important subclass of the PSI is represented by the agents with marked neurotropic properties, i.e., by the agents which are able to promote the effective inhibition of protein synthesis in the brain, while the same agents do not essentially influence the translation processes in the liver and all other organs of the animal (human) organism. The main aim of the present monograph is to review and analyze the data obtained recently in the course of biochemical research of several neurotropic PSI (methyl mercury, lithium salts). However, this is not the single aim of the book. Thus, besides this the present monograph deals with analysis of the general peculiarities of action of some non-neurotrophic inhibitors which are able, nevertheless, to direct a number of specific neurotrophic effects (Cordycepin, tRNA).(ABSTRACT TRUNCATED AT 400 WORDS)

Chemical-induced modulation of ATP and protein synthesis processed inside rat brain mitochondria.

Rates of ATP production and protein synthesis rates were simultaneously studied using the isolated rat brain mitochondria and different types of brain polysomes (total, free, microsomal and mitochondrial membrane-bound). Protein synthesis was measured using a special reticulocyte lysate cell-free incubation medium. Prior to incubation, the intact ("unbroken") mitochondria were pretreated by several xenobiotics and natural metabolic effectors. The mitochondria incubated without these additions were used as controls. Chemically-promoted inhibition of ATP systhesis correlated with a significant activation of protein synthesis directed by the mitochondrial membrane-bound polysomes. The induction of ATP synthesis leads to a marked suppression of mitochondrial translation. Application of the selective translation blockers do not influence the ATP formation inside the isolated mitochondria. In general, mitochondrial membrane-found polysomes isolated from the intact normal organella are least active as compared with other tested polysomal fractions while the releasing of polysomes from mitochondrial membranes by Triton X-100 leads to the essential increase of the activity of these separated translation mechanisms. The separation of polysomes from the microsomal membranes do not change the activity of the former. A possible molecular mechanism, interdependence and biological significance of the established phenomena are discussed.