Pattern detection in the TGFß cascade controls the induction of long-term synaptic plasticity.

Transforming growth factor ß (TGFß) is required for long-term memory (LTM) for sensitization in Aplysia. When LTM is induced using a two-trial training protocol, TGFß inhibition only blocks LTM when administrated at the second, not the first trial. Here, we show that TGFß acts as a "repetition detector" during the induction of two-trial LTM. Secretion of the biologically inert TGFß proligand must coincide with its proteolytic activation by the Bone morphogenetic protein-1 (BMP-1/Tolloid) metalloprotease, which occurs specifically during trial two of our two-trial training paradigm. This paradigm establishes long-term synaptic facilitation (LTF), the cellular correlate of LTM. BMP-1 application paired with a single serotonin (5HT) pulse induced LTF, whereas neither a single 5HT pulse nor BMP-1 alone effectively did so. On the other hand, inhibition of endogenous BMP-1 activity blocked the induction of two-trial LTF. These results suggest a unique role for TGFß in the interaction of repeated trials: during learning, repeated stimuli engage separate steps of the TGFß cascade that together are necessary for the induction of long-lasting memories.

Solid Xenon Carrier Based on α-Cyclodextrin: Properties, Preparation, and Application.

The inert gas xenon (Xe) is increasingly used in medicine as a universal anesthetic, a regulator of cellular metabolism, and a broad-spectrum organoprotector. Commonly utilized Xe inhalation requires expensive equipment that is not universally available. Here we describe the production process and physical characteristics of a solid, highly stable xenon carrier based on α-cyclodextrin (α-CD), developed for oral administration. It was found, that the interaction of α-CD with Xe in an aqueous solution and elevated pressure leads to precipitation of the α-CD-Xe complex. We have discovered three new properties of the resulting complex that promote long-term storage and oral delivery of Xe. (i) At temperatures below 0 °C, the precipitated α-CD-Xe complex containing water is so stable that it allows the removal of water by vacuum freeze-drying (lyophilization). (ii). Lyophilized α-CD-Xe remains stable for months at room temperature. (iii) Upon contact with water, α-CD-Xe rapidly releases gaseous Xe. As revealed in the forced swim test, after oral administration of lyophilized α-CD-Xe to rats, the duration of swimming was significantly increased. The obtained data open up prospects for the development of drugs based on the lyophilized α-CD-Xe complex suitable for storage, transportation, and medical use, including outside the hospital.

Precise timing of ERK phosphorylation/dephosphorylation determines the outcome of trial repetition during long-term memory formation.

Two-trial learning in Aplysia reveals nonlinear interactions between training trials: A single trial has no effect, but two precisely spaced trials induce long-term memory. Extracellularly regulated kinase (ERK) activity is essential for intertrial interactions, but the mechanism remains unresolved. A combination of immunochemical and optogenetic tools reveals unexpected complexity of ERK signaling during the induction of long-term synaptic facilitation by two spaced pulses of serotonin (5-hydroxytryptamine, 5HT). Specifically, dual ERK phosphorylation at its activating TxY motif is accompanied by dephosphorylation at the pT position, leading to a buildup of inactive, singly phosphorylated pY-ERK. Phosphorylation and dephosphorylation occur concurrently but scale differently with varying 5HT concentrations, predicting that mixed two-trial protocols involving both "strong" and "weak" 5HT pulses should be sensitive to the precise order and timing of trials. Indeed, long-term synaptic facilitation is induced only when weak pulses precede strong, not vice versa. This may represent a physiological mechanism to prioritize memory of escalating threats.

Neurotropic and modulatory effects of insulin-like growth factor II in Aplysia.

Insulin-like growth factor II (IGF2) enhances memory in rodents via the mannose-6-phosphate receptor (M6PR), but the underlying mechanisms remain poorly understood. We found that human IGF2 produces an enhancement of both synaptic transmission and neurite outgrowth in the marine mollusk Aplysia californica. These findings were unexpected since Aplysia lack the mammal-specific affinity between insulin-like ligands and M6PR. Surprisingly, this effect was observed in parallel with a suppression of neuronal excitability in a well-understood circuit that supports several temporally and mechanistically distinct forms of memory in the defensive withdrawal reflex, suggesting functional coordination between excitability and memory formation. We hypothesize that these effects represent behavioral adaptations to feeding that are mediated by the endogenous Aplysia insulin-like system. Indeed, the exogenous application of a single recombinant insulin-like peptide cloned from the Aplysia CNS cDNA replicated both the enhancement of synaptic transmission, the reduction of excitability, and promoted clearance of glucose from the hemolymph, a hallmark of bona fide insulin action.

Taking memory beyond the brain: Does tobacco dream of the mosaic virus?

Memory is typically defined through animal behavior, but this point of view may limit our understanding of many related processes in diverse biological systems. The concept of memory can be broadened meaningfully by considering it from the perspective of time and homeostasis. On the one hand, this theoretical angle can help explain and predict the behavior of various non-neural systems such as insulin-secreting cells, plants, or signaling cascades. On the other hand, it emphasizes biological continuity between neural phenomena, such as synaptic plasticity, and their evolutionary precursors in cellular signaling.

Increased Autolysis of µ-Calpain in Skeletal Muscles of Chronic Alcohol-Fed Rats.

BACKGROUND: Proteolysis can proceed via several distinct pathways such as the lysosomal, calcium-dependent, and ubiquitin-proteasome-dependent pathways. Calpains are the main proteases that cleave a large variety of proteins, including the giant sarcomeric proteins, titin and nebulin. Chronic ethanol feeding for 6 weeks did not affect the activities of µ-calpain and m-calpain in the m. gastrocnemius. In our research, changes in µ-calpain activity were studied in the m. gastrocnemius and m. soleus of chronically alcohol-fed rats after 6 months of alcohol intake. METHODS: SDS-PAGE analysis was applied to detect changes in titin and nebulin contents. Titin phosphorylation analysis was performed using the fluorescent dye Pro-Q Diamond. Western blotting was used to determine µ-calpain autolysis as well as µ-calpain and calpastatin contents. The titin and nebulin mRNA levels were assessed by real-time PCR. RESULTS: The amounts of the autolysed isoform (78 kDa) of full-length µ-calpain (80 kDa) increased in the m. gastrocnemius and m. soleus of alcohol-fed rats. The calpastatin content increased in m. gastrocnemius. Decreased intact titin-1 (T1) and increased T2-proteolytic fragment contents were found in the m. gastrocnemius and m. soleus of the alcohol-fed rats. The nebulin content decreased in the rat gastrocnemius muscle of the alcohol-fed group. The phosphorylation levels of T1 and T2 were increased in the m. gastrocnemius and m. soleus, and decreased titin and nebulin mRNA levels were observed in the m. gastrocnemius. The nebulin mRNA level was increased in the soleus muscle of the alcohol-fed rats. CONCLUSIONS: In summary, our data suggest that prolonged chronic alcohol consumption for 6 months resulted in increased autolysis of µ-calpain in rat skeletal muscles. These changes were accompanied by reduced titin and nebulin contents, titin hyperphosphorylation, and development of hindlimb muscle atrophy in the alcohol-fed rats.

Memory Takes Time.

Memory is an adaptation to particular temporal properties of past events, such as the frequency of occurrence of a stimulus or the coincidence of multiple stimuli. In neurons, this adaptation can be understood in terms of a hierarchical system of molecular and cellular time windows, which collectively retain information from the past. We propose that this system makes various timescales of past experience simultaneously available for future adjustment of behavior. More generally, we propose that the ability to detect and respond to temporally structured information underlies the nervous system's capacity to encode and store a memory at molecular, cellular, synaptic, and circuit levels.

Tau-driven 26S proteasome impairment and cognitive dysfunction can be prevented early in disease by activating cAMP-PKA signaling.

The ubiquitin proteasome system (UPS) degrades misfolded proteins including those implicated in neurodegenerative diseases. We investigated the effects of tau accumulation on proteasome function in a mouse model of tauopathy and in a cross to a UPS reporter mouse (line Ub-G76V-GFP). Accumulation of insoluble tau was associated with a decrease in the peptidase activity of brain 26S proteasomes, higher levels of ubiquitinated proteins and undegraded Ub-G76V-GFP. 26S proteasomes from mice with tauopathy were physically associated with tau and were less active in hydrolyzing ubiquitinated proteins, small peptides and ATP. 26S proteasomes from normal mice incubated with recombinant oligomers or fibrils also showed lower hydrolyzing capacity in the same assays, implicating tau as a proteotoxin. Administration of an agent that activates cAMP-protein kinase A (PKA) signaling led to attenuation of proteasome dysfunction, probably through proteasome subunit phosphorylation. In vivo, this led to lower levels of aggregated tau and improvements in cognitive performance.

cAMP-induced phosphorylation of 26S proteasomes on Rpn6/PSMD11 enhances their activity and the degradation of misfolded proteins.

Although rates of protein degradation by the ubiquitin-proteasome pathway (UPS) are determined by their rates of ubiquitination, we show here that the proteasome's capacity to degrade ubiquitinated proteins is also tightly regulated. We studied the effects of cAMP-dependent protein kinase (PKA) on proteolysis by the UPS in several mammalian cell lines. Various agents that raise intracellular cAMP and activate PKA (activators of adenylate cyclase or inhibitors of phosphodiesterase 4) promoted degradation of short-lived (but not long-lived) cell proteins generally, model UPS substrates having different degrons, and aggregation-prone proteins associated with major neurodegenerative diseases, including mutant FUS (Fused in sarcoma), SOD1 (superoxide dismutase 1), TDP43 (TAR DNA-binding protein 43), and tau. 26S proteasomes purified from these treated cells or from control cells and treated with PKA degraded ubiquitinated proteins, small peptides, and ATP more rapidly than controls, but not when treated with protein phosphatase. Raising cAMP levels also increased amounts of doubly capped 26S proteasomes. Activated PKA phosphorylates the 19S subunit, Rpn6/PSMD11 (regulatory particle non-ATPase 6/proteasome subunit D11) at Ser14. Overexpression of a phosphomimetic Rpn6 mutant activated proteasomes similarly, whereas a nonphosphorylatable mutant decreased activity. Thus, proteasome function and protein degradation are regulated by cAMP through PKA and Rpn6, and activation of proteasomes by this mechanism may be useful in treating proteotoxic diseases.

Thiostrepton interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates.

Here, we report a novel mechanism of proteasome inhibition mediated by Thiostrepton (Thsp), which interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates. We identified Thsp in a cell-based high-throughput screen using a fluorescent reporter sensitive to degradation by the ubiquitin-proteasome pathway. Thiostrepton behaves as a proteasome inhibitor in several paradigms, including cell-based reporters, detection of global ubiquitination status, and proteasome-mediated labile protein degradation. In vitro, Thsp does not block the chymotrypsin activity of the 26S proteasome. In a cell-based IκBα degradation assay, Thsp is a slow inhibitor and 4 hrs of treatment achieves the same effects as MG-132 at 30 min. We show that Thsp forms covalent adducts with proteins in human cells and demonstrate their nature by mass spectrometry. Furthermore, the ability of Thsp to interact covalently with the cysteine residues is essential for its proteasome inhibitory function. We further show that a Thsp modified peptide cannot be degraded by proteasomes in vitro. Importantly, we demonstrate that Thsp binds covalently to Rpt subunits of the 19S regulatory particle and forms bridges with a proteasome substrate. Taken together, our results uncover an important role of Thsp in 19S proteasome inhibition.

Autoubiquitination of the 26S proteasome on Rpn13 regulates breakdown of ubiquitin conjugates.

Degradation rates of most proteins in eukaryotic cells are determined by their rates of ubiquitination. However, possible regulation of the proteasome's capacity to degrade ubiquitinated proteins has received little attention, although proteasome inhibitors are widely used in research and cancer treatment. We show here that mammalian 26S proteasomes have five associated ubiquitin ligases and that multiple proteasome subunits are ubiquitinated in cells, especially the ubiquitin receptor subunit, Rpn13. When proteolysis is even partially inhibited in cells or purified 26S proteasomes with various inhibitors, Rpn13 becomes extensively and selectively poly-ubiquitinated by the proteasome-associated ubiquitin ligase, Ube3c/Hul5. This modification also occurs in cells during heat-shock or arsenite treatment, when poly-ubiquitinated proteins accumulate. Rpn13 ubiquitination strongly decreases the proteasome's ability to bind and degrade ubiquitin-conjugated proteins, but not its activity against peptide substrates. This autoinhibitory mechanism presumably evolved to prevent binding of ubiquitin conjugates to defective or stalled proteasomes, but this modification may also be useful as a biomarker indicating the presence of proteotoxic stress and reduced proteasomal capacity in cells or patients.

Non-specific accumulation of glycosphingolipids in GNE myopathy.

BACKGROUND: UDP-GlcNAc 2-epimerase/ManNAc 6-kinase (GNE) is a bifunctional enzyme responsible for the first committed steps in the synthesis of sialic acid, a common terminal monosaccharide in both protein and lipid glycosylation. GNE mutations are responsible for a rare autosomal recessive neuromuscular disorder, GNE myopathy (also called hereditary inclusion body myopathy). The connection between the impairment of sialic acid synthesis and muscle pathology in GNE myopathy remains poorly understood. METHODS: Glycosphingolipid (GSL) analysis was performed by HPLC in multiple models of GNE myopathy, including patients' fibroblasts and plasma, control fibroblasts with inhibited GNE epimerase activity through a novel imino sugar, and tissues of Gne(M712T/M712T) knock-in mice. RESULTS: Not only neutral GSLs, but also sialylated GSLs, were significantly increased compared to controls in all tested models of GNE myopathy. Treatment of GNE myopathy fibroblasts with N-acetylmannosamine (ManNAc), a sialic acid precursor downstream of GNE epimerase activity, ameliorated the increased total GSL concentrations. CONCLUSION: GNE myopathy models have increased total GSL concentrations. ManNAc supplementation results in decrease of GSL levels, linking abnormal increase of total GSLs in GNE myopathy to defects in the sialic acid biosynthetic pathway. These data advocate for further exploring GSL concentrations as an informative biomarker, not only for GNE myopathy, but also for other disorders of sialic acid metabolism.

Glycoprotein misfolding in the endoplasmic reticulum: identification of released oligosaccharides reveals a second ER-associated degradation pathway for Golgi-retrieved proteins.

Endoplasmic reticulum-associated degradation (ERAD) is a key cellular process whereby misfolded proteins are removed from the endoplasmic reticulum (ER) for subsequent degradation by the ubiquitin/proteasome system. In the present work, analysis of the released, free oligosaccharides (FOS) derived from all glycoproteins undergoing ERAD, has allowed a global estimation of the mechanisms of this pathway rather than following model proteins through degradative routes. Examining the FOS produced in endomannosidase-compromised cells following α-glucosidase inhibition has revealed a mechanism for clearing Golgi-retrieved glycoproteins that have failed to enter the ER quality control cycle. The Glc3Man7GlcNAc2 FOS species has been shown to be produced in the ER lumen by a mechanism involving a peptide: N-glycanase-like activity, and its production was sensitive to disruption of Golgi-ER trafficking. The detection of this oligosaccharide was unaffected by the overexpression of EDEM1 or cytosolic mannosidase, both of which increased the production of previously characterised cytosolically localised FOS. The lumenal FOS identified are therefore distinct in their production and regulation compared to FOS produced by the conventional route of misfolded glycoproteins directly removed from the ER. The production of such lumenal FOS is indicative of a novel degradative route for cellular glycoproteins that may exist under certain conditions.

Ubiquitinated proteins activate the proteasomal ATPases by binding to Usp14 or Uch37 homologs.

Degradation of ubiquitinated proteins by 26 S proteasomes requires ATP hydrolysis, but it is unclear how the proteasomal ATPases are regulated and how proteolysis, substrate deubiquitination, degradation, and ATP hydrolysis are coordinated. Polyubiquitinated proteins were shown to stimulate ATP hydrolysis by purified proteasomes, but only if the proteins contain a loosely folded domain. If they were not ubiquitinated, such proteins did not increase ATPase activity. However, they did so upon addition of ubiquitin aldehyde, which mimics the ubiquitin chain and binds to 26 S-associated deubiquitinating enzymes (DUBs): in yeast to Ubp6, which is essential for the ATPase activation, and in mammalian 26 S to the Ubp6 homolog, Usp14, and Uch37. Occupancy of either DUB by a ubiquitin conjugate leads to ATPase stimulation, thereby coupling deubiquitination and ATP hydrolysis. Thus, ubiquitinated loosely folded proteins, after becoming bound to the 26 S, interact with Ubp6/Usp14 or Uch37 to activate ATP hydrolysis and enhance their own destruction.

Restricted processing of glycans by endomannosidase in mammalian cells.

Removal of α-glucose residues from nascent glycoproteins in the early secretory pathway is a requirement for further N-glycan maturation. Although deglucosylation is a stepwise process mediated by endoplasmic reticulum-associated glucosidases I and II for most glycoproteins, Golgi endo-α-mannosidase provides a backup mechanism for glycoprotein deglucosylation. Although conserved in mammals, in certain cell lines, endomannosidase activity in vitro appears to differ from its activity in cells following glucosidase inhibition. Here, we show that in bovine cells this is explained by restricted substrate specificity allowing processing of Glc(1)Man(7)GlcNAc(1/2) and Glc(1)Man(5)GlcNAc(1/2) but not fully glucosylated glycans that build up when glucosidases are inhibited. Our data further demonstrate that such specificity is determined genetically rather than post-translationally. We also demonstrate that the bovine endomannosidase is transcriptionally upregulated by comparison with glucosidase II in Madin-Darby bovine kidney cells and speculate that this is to compensate for the reduced catalytic activity as measured in the recombinant form of the enzyme.

Demonstration that endoplasmic reticulum-associated degradation of glycoproteins can occur downstream of processing by endomannosidase.

During quality control in the ER (endoplasmic reticulum), nascent glycoproteins are deglucosylated by ER glucosidases I and II. In the post-ER compartments, glycoprotein endo-α-mannosidase provides an alternative route for deglucosylation. Previous evidence suggests that endomannosidase non-selectively deglucosylates glycoproteins that escape quality control in the ER, facilitating secretion of aberrantly folded as well as normal glycoproteins. In the present study, we employed FOS (free oligosaccharides) released from degrading glycoproteins as biomarkers of ERAD (ER-associated degradation), allowing us to gain a global rather than single protein-centred view of ERAD. Glucosidase inhibition was used to discriminate between glucosidase- and endomannosidase-mediated ERAD pathways. Endomannosidase expression was manipulated in CHO (Chinese-hamster ovary)-K1 cells, naturally lacking a functional version of the enzyme, and HEK (human embryonic kidney)-293T cells. Endomannosidase was shown to decrease the levels of total FOS, suggesting decreased rates of ERAD. However, following pharmacological inhibition of ER glucosidases I and II, endomannosidase expression resulted in a partial switch between glucosylated FOS, released from ER-confined glycoproteins, to deglucosylated FOS, released from endomannosidase-processed glycoproteins transported from the Golgi/ERGIC (ER/Golgi intermediate compartment) to the ER. Using this approach, we have identified a previously unknown pathway of glycoprotein flow, undetectable by the commonly employed methods, in which secretory cargo is targeted back to the ER after being processed by endomannosidase.

Novel mannosidase inhibitors probe glycoprotein degradation pathways in cells.

Multiple isoforms of mammalian alpha-mannosidases are active in the pathways of N-linked glycoprotein synthesis and catabolism. They differ in specificity, function and location within the cell and can be selectively inhibited by imino sugar monosaccharide mimics. Previously, a series of structurally related novel 7-membered iminocyclitols were synthesised and found to be inhibitors of alpha-mannosidase using in vitro assays. The present study aimed to delineate alpha-mannosidases hydrolytic pathways in azepane inhibitor treated cells by the analysis of free oligosaccharides (FOS) as markers of endoplasmic reticulum (ER), Golgi, lysosomal and cytosolic alpha-mannosidase activities. Two compounds were identified as potent and selective cytosolic alpha-mannosidase inhibitors. Two related compounds were shown to be potent inhibitors of lysosomal alpha-mannosidase with different potencies towards alpha1,6 mannosidase. The specificities of these novel 7-membered imino sugars are related to differences in their structure and D: -mannose-like stereochemistry. Specific ER-mannosidase inhibition by kifunensine also reveals significant non-proteasomal degradation following FOS analysis and appears to be cell line dependent. The availability of more selective inhibitors allows the pathways of N-linked oligosaccharide metabolism to be dissected.

Reversion of structure-activity relationships of antitumor platinum complexes by acetoxime but not hydroxylamine ligands.

The presence of cis-configured exchangeable ligands has long been considered a prerequisite for antitumor activity of platinum complexes, but over the past few years, several examples violating this structure-activity relationship have been recognized. We report here on studies with the geometric isomers of [PtCl2(acetoxime)2], cis-[dichlorobis(acetoxime)platinum(II)] [1 (cis)] and trans-[dichlorobis(acetoxime)platinum(II)] [2 (trans)], as well as those of [PtCl2(hydroxylamine)2], cis-[dichlorobis(hydroxylamine)platinum(II)] [3 (cis)] and trans-[dichlorobis(hydroxylamine)platinum(II)] [4 (trans)]. We found that 2 (trans)is 16 times more cytotoxic than 1 (cis) and as cytotoxic as cisplatin in cisplatin-sensitive ovarian carcinoma cells (CH1). Moreover, 2 (trans) is 15 times more cytotoxic than either cisplatin or 1 (cis) in intrinsically cisplatin-resistant colon carcinoma cells (SW480). Thus, compound 2 (trans) represents a novel type of active platinum(II) complexes of the trans geometry, whereas the hydroxylamine-containing complexes conform to the classic structure-activity relationships. The reactivity of the compounds toward dGMP and DNA and their capacity to alter the structure of double-stranded DNA and form interstrand cross-links were studied by capillary electrophoresis and gel electrophoresis. The slow binding of 2 (trans) to dGMP (tau(1/2) = 50 h versus 8.9 h in the case of cisplatin), the low reactivity toward DNA, the comparatively small impact on DNA secondary structure, and the lack of detectable interstrand cross-linking suggest a mode of action fundamentally different from that of cisplatin. Implications of our findings for the minimal structural requirements (e.g., planarity around the nitrogen donor atom and/or ramified aliphatic moiety attached to the latter) of active trans-configured platinum complexes are discussed.