Mice deficient in synaptic protease neurotrypsin show impaired spaced long-term potentiation and blunted learning-induced modulation of dendritic spines.

Neurotrypsin (NT) is a neuronal trypsin-like serine protease whose mutations cause severe mental retardation in humans. NT is activated in vitro by Hebbian-like conjunction of pre- and postsynaptic activities, which promotes the formation of dendritic filopodia via proteolytic cleavage of the proteoglycan agrin. Here, we investigated the functional importance of this mechanism for synaptic plasticity, learning, and extinction of memory. We report that juvenile neurotrypsin-deficient (NT-/-) mice exhibit impaired long-term potentiation induced by a spaced stimulation protocol designed to probe the generation of new filopodia and their conversion into functional synapses. Behaviorally, juvenile NT-/- mice show impaired contextual fear memory and have a sociability deficit. The latter persists in aged NT-/- mice, which, unlike juvenile mice, show normal recall but impaired extinction of contextual fear memories. Structurally, juvenile mutants exhibit reduced spine density in the CA1 region, fewer thin spines, and no modulation in the density of dendritic spines following fear conditioning and extinction in contrast to wild-type littermates. The head width of thin spines is reduced in both juvenile and aged NT-/- mice. In vivo delivery of adeno-associated virus expressing an NT-generated fragment of agrin, agrin-22, but not a shorter agrin-15, elevates the spine density in NT-/- mice. Moreover, agrin-22 co-aggregates with pre- and postsynaptic markers and increases the density and size of presynaptic boutons and presynaptic puncta, corroborating the view that agrin-22 supports the synaptic growth.

Visual object recognition is facilitated by temporal community structure.

Humans and others primates are highly attuned to temporal consistencies and regularities in their sensory environment and learn to predict such statistical structure. Moreover, in several instances, the presence of temporal structure has been found to facilitate procedural learning and to improve task performance. Here we extend these findings to visual object recognition and to presentation sequences in which mutually predictive objects form distinct clusters or "communities." Our results show that temporal community structure accelerates recognition learning and affects the order in which objects are learned ("onset of familiarity").

Perceptual reversals in binocular rivalry: Improved detection from OKN.

When binocular rivalry is induced by opponent motion displays, perceptual reversals are often associated with changed oculomotor behavior (Frässle, Sommer, Jansen, Naber, & Einhäuser, 2014; Fujiwara et al., 2017). Specifically, the direction of smooth pursuit phases in optokinetic nystagmus typically corresponds to the direction of motion that dominates perceptual appearance at any given time. Here we report an improved analysis that continuously estimates perceived motion in terms of "cumulative smooth pursuit." In essence, smooth pursuit segments are identified, interpolated where necessary, and joined probabilistically into a continuous record of cumulative smooth pursuit (i.e., probability of eye position disregarding blinks, saccades, signal losses, and artefacts). The analysis is fully automated and robust in healthy, developmental, and patient populations. To validate reliability, we compare volitional reports of perceptual reversals in rivalry displays, and of physical reversals in nonrivalrous control displays. Cumulative smooth pursuit detects physical reversals and estimates eye velocity more accurately than existing methods do (Frässle et al., 2014). It also appears to distinguish dominant and transitional perceptual states, detecting changes with a precision of ±100 ms. We conclude that cumulative smooth pursuit significantly improves the monitoring of binocular rivalry by means of recording optokinetic nystagmus.

Perceptual coupling induces co-rotation and speeds up alternations in adjacent bi-stable structure-from-motion objects.

When two bi-stable structure-from-motion (SFM) spheres are presented simultaneously, they tend to rotate in the same direction. This effect reflects a common state bias that is present for various multistable displays. However, it was also reported that when two spheres are positioned so that they touch each other, they tend to counterrotate instead. The latter effect is interpreted as a frictional interaction, indicating the influence of the embedded physics on our visual perception. Here, we examined the interplay between these two biases in two experiments using a wide range of conditions. Those included two SFM shapes, two types of disambiguation cues, the presence or absence of the disambiguation cues, different layout options, and two samples of observers from two different universities (in sum 26 participants). Contrary to the prior report, we observed a robust common state bias for all conditions, including those that were optimized for frictional and "gear meshing" interactions. We found that stronger coupling of perceptual states is accompanied by more frequent synchronous perceptual reversals of the two objects. However, we found that the simultaneity of the individual switches does not predict the duration of the following dominance phase. Finally, we report that stronger perceptual coupling speeds up perceptual alternations.

Regulation of peroxisome proliferator-activated receptors (PPAR) α and -γ of rat brain astrocytes in the course of activation by toll-like receptor agonists.

Peroxisome proliferator-activated receptors (PPAR)-α and -γ in astrocytes play important roles in inflammatory brain pathologies. Understanding the regulation of both activity and expression levels of PPARs is an important neuroscience issue. Toll-like receptor (TLR) agonists are inflammatory stimuli that could modulate PPAR, but the mechanisms of their control in astrocytes are poorly understood. In the present study, we report that lipopolysaccharide, peptidoglycan, and flagellin, which are agonists of TLR4, TLR1/2, and TLR5, respectively, exert time- and nuclear factor kappa-light-chain-enhancer of activated B cells-dependent suppression of mRNA, protein and activity of PPARα and PPARγ. In naïve astrocytes, PPARα and PPARγ mRNA have short turnover time (half-life about 30 min for PPARα, 75 min for PPARγ) with a nearly two-fold stabilization after TLR-activation. p38 inhibition abolished TLR-induced stabilization. The levels of PPARα and PPARγ mRNA, and protein and DNA-binding activity could be modified using c-Jun N-terminal Kinase and p38 inhibitors. In addition, the expression levels of both PPARα and PPARγ isotypes were induced after inhibition of protein synthesis. This induction signifies participation of additional regulatory proteins with short life-time. They are p38-sensitive for PPARα and c-Jun N-terminal Kinase-sensitive for PPARγ. Thus, PPARα and PPARγ are regulated in astrocytes on mRNA and protein levels, mRNA stability, and DNA-binding activity during TLR-mediated responses. Astrocytes have the triad of PPARα, PPARß/δ, and PPARγ in regulation of proinflammatory responses. Activation of Toll-like receptors (TLR) leads to PPARß/δ overexpression, PPARα and PPARγ suppression via TLR/NF-κB pathway on mRNA, protein and activity levels. Mitogen-activated protein kinases (MAPK) p38 and JNK are involved in regulation of PPAR expression. p38 MAPK plays a special role in stabilization of PPAR mRNA.

Peroxisome proliferator-activated receptor ß/δ (PPARß/δ) protects against ceramide-induced cellular toxicity in rat brain astrocytes and neurons by activation of ceramide kinase.

Peroxisome proliferator-activated receptors (PPARs) are important members of the nuclear receptor superfamily. Ligands of these nuclear receptors (PPARα, ß/δ and γ) belong to a wide range of lipophilic substances. In spite of the proven neuroprotective efficacy of PPARß/δ in models of neurological diseases, the biology of PPARß/δ in the brain has been much less investigated than that of PPARα and PPARγ. In the present study, we test the hypothesis that neuroprotection induced by PPARß/δ could rely on the regulation of ceramide metabolism. We found that preincubation of neural cells with the PPARß/δ agonist L-165041 exerts significant protection against ceramide-induced cell death. Most importantly, L-165041 protects against ceramide-induced cell death not only before the insult, but also after the onset of the insult. To identify the mechanism of protection, we show that L-165041 upregulates ceramide kinase (CerK) expression levels in neural cells. Consistent with that, we detected that pharmacological inhibition of CerK reduces the protective effects of L-165041. To further decipher the mechanism of protection, gene knockdown in astrocytes was studied. Knockdown of PPARß/δ and CerK in astrocytes was used to verify that the protective effects of L-165041 are CerK- and PPARß/δ-dependent. We demonstrate that in CerK- or PPARß/δ-knockdown astrocytes, addition of L-165041 has no protective effect. Thus, we conclude that PPARß/δ protects neural cells against ceramide-induced cell death via induction and activation of CerK.

Regulation of peroxisome proliferator-activated receptor ß/δ expression and activity levels by toll-like receptor agonists and MAP kinase inhibitors in rat astrocytes.

Peroxisome proliferator-activated receptor ß/δ (PPARß/δ) is a potential regulator of neuroinflammation. Toll-like receptors (TLR) are innate immunity-related receptors of inflammatory stimuli. In the present report, we evaluate the molecular mechanisms of regulation of mRNA, protein, and transcriptional activity levels of PPARß/δ by agonists of TLR4, TLR1/2, and TLR5, using lipopolysaccharide (LPS), peptidoglycan, and flagellin, respectively. We found that these stimuli increase the PPARß/δ levels in astrocytes. Expression and activity of PPARß/δ are separately regulated by inhibitors of p38, MEK1/2, extracellular signal-regulated kinases 1/2, and c-Jun N-terminal Kinase mitogen-activated protein kinases. The LPS-induced kinetics of PPARß/δ expression is similar to that of the proinflammatory gene cyclooxygenase 2. Moreover, for both genes the expression depends on nuclear factor kappa-light-chain-enhancer of activated B cells and p38, and is induced after inhibition of protein synthesis. The up-regulation of the expression after inhibition of protein synthesis signifies the participation of a labile protein in regulation of PPARß/δ expression. In contrast to cyclooxygenase 2, the cycloheximide-sensitive PPARß/δ expression was not responsive to nuclear factor kappa-light-chain-enhancer of activated B cells inhibition. Measurements of PPARß/δ mRNA stability showed that the PPARß/δ mRNA levels are regulated post-transcriptionally. We found that in LPS-stimulated astrocytes, the half-life of PPARß/δ mRNA was 50 min. Thus, we demonstrate that PPARß/δ expression and activity are regulated in TLR agonist-stimulated astrocytes by mechanisms that are widely used for regulation of proinflammatory genes. Protein expression level of nuclear receptor PPARß/δ is important for functions of this transcription factor. We investigate the regulatory mechanisms of PPARß/δ in rat primary astrocytes stimulated by agonists of toll-like receptors (TLR): TLR4, TLR1/2, and TLR5. Expression, activity, mRNA stability, and superinduction of PPARß/δ were up-regulated after TLR stimulation. These processes are sensitive to MAPKs and NF-kB inhibitors. Superinduction is up-regulation of mRNA expression after inhibition of protein synthesis.

Role of the peroxisome proliferator-activated receptors (PPAR)-α, ß/δ and γ triad in regulation of reactive oxygen species signaling in brain.

Overwhelming evidence shows that oxidative stress is a major cause in development of brain disorders. Low activity of the reactive oxygen species (ROS)-degrading system as well as high levels of oxidative damage markers have been observed in brain tissue of patients with neurodegenerative and other brain diseases to a larger extent than in healthy individuals. Many studies aimed to develop effective and safe antioxidant strategies for the therapy or prevention of brain diseases. Nevertheless, it became clear that rigorous suppression of ROS is deleterious for normal cell functioning. Thus, approaches that can regulate the ROS levels over a wide range, from inhibition to induction, will be a powerful tool for neuroprotection. A most prominent target for such ROS management is the family of peroxisome proliferator-activated receptors (PPARs). All three members (PPAR-α, -ß/δ and -γ) of this nuclear receptor subfamily form a tightly connected triad. For individual PPAR isoforms, neuroprotective properties have been well proven. Their involvement in regulation of ROS production and degradation underlies the therapeutic effects. Nevertheless, the current paradigms of the involvement of PPAR in neuroprotective therapy ignore such interconnections of PPARs and aim at antioxidant effects of individual PPAR isoforms, but do not take into account the necessity of careful regulation of ROS levels. The present review (i) summarizes the data, which support the concept of the PPAR triad in brain, (ii) demonstrates that usage of the PPAR triad allows the regulation of PPAR-dependent genes over a wide range, from inhibition to upregulation, and (iii) summarizes the known data concerning the PPAR triad involvement in regulation of ROS. Our report opens new directions in the field of PPAR/ROS-related neuroscience research.

A shift in the mechanisms controlling hippocampal engram formation during brain maturation.

The ability to form precise, episodic memories develops with age, with young children only able to form gist-like memories that lack precision. The cellular and molecular events in the developing hippocampus that underlie the emergence of precise, episodic-like memory are unclear. In mice, the absence of a competitive neuronal engram allocation process in the immature hippocampus precluded the formation of sparse engrams and precise memories until the fourth postnatal week, when inhibitory circuits in the hippocampus mature. This age-dependent shift in precision of episodic-like memories involved the functional maturation of parvalbumin-expressing interneurons in subfield CA1 through assembly of extracellular perineuronal nets, which is necessary and sufficient for the onset of competitive neuronal allocation, sparse engram formation, and memory precision.

Alternative female and male developmental trajectories in the dynamic balance of human visual perception.

The numerous multistable phenomena in vision, hearing and touch attest that the inner workings of perception are prone to instability. We investigated a visual example-binocular rivalry-with an accurate no-report paradigm, and uncovered developmental and maturational lifespan trajectories that were specific for age and sex. To interpret these trajectories, we hypothesized that conflicting objectives of visual perception-such as stability of appearance, sensitivity to visual detail, and exploration of fundamental alternatives-change in relative importance over the lifespan. Computational modelling of our empirical results allowed us to estimate this putative development of stability, sensitivity, and exploration over the lifespan. Our results confirmed prior findings of developmental psychology and appear to quantify important aspects of neurocognitive phenotype. Additionally, we report atypical function of binocular rivalry in autism spectrum disorder and borderline personality disorder. Our computational approach offers new ways of quantifying neurocognitive phenotypes both in development and in dysfunction.

Aging-Associated Changes in Cognition, Expression and Epigenetic Regulation of Chondroitin 6-Sulfotransferase Chst3.

Understanding changes in the expression of genes involved in regulating various components of the neural extracellular matrix (ECM) during aging can provide an insight into aging-associated decline in synaptic and cognitive functions. Hence, in this study, we compared the expression levels of ECM-related genes in the hippocampus of young, aged and very aged mice. ECM gene expression was downregulated, despite the accumulation of ECM proteoglycans during aging. The most robustly downregulated gene was carbohydrate sulfotransferase 3 (Chst3), the enzyme responsible for the chondroitin 6-sulfation (C6S) of proteoglycans. Further analysis of epigenetic mechanisms revealed a decrease in H3K4me3, three methyl groups at the lysine 4 on the histone H3 proteins, associated with the promoter region of the Chst3 gene, resulting in the downregulation of Chst3 expression in non-neuronal cells. Cluster analysis revealed that the expression of lecticans-substrates of CHST3-is tightly co-regulated with this enzyme. These changes in ECM-related genes were accompanied by an age-confounded decline in cognitive performance. Despite the co-directional impairment in cognitive function and average Chst3 expression in the studied age groups, at the individual level we found a negative correlation between mRNA levels of Chst3 and cognitive performance within the very aged group. An analysis of correlations between the expression of ECM-related genes and cognitive performance in novel object versus novel location recognition tasks revealed an apparent trade-off in the positive gene effects in one task at the expense of another. Further analysis revealed that, despite the reduction in the Chst3 mRNA, the expression of CHST3 protein is increased in glial cells but not in neurons, which, however, does not lead to changes in the absolute level of C6S and even results in the decrease in C6S in perineuronal, perisynaptic and periaxonal ECM relative to the elevated expression of its protein carrier versican.

Binocular rivalry reveals an out-of-equilibrium neural dynamics suited for decision-making.

In ambiguous or conflicting sensory situations, perception is often 'multistable' in that it perpetually changes at irregular intervals, shifting abruptly between distinct alternatives. The interval statistics of these alternations exhibits quasi-universal characteristics, suggesting a general mechanism. Using binocular rivalry, we show that many aspects of this perceptual dynamics are reproduced by a hierarchical model operating out of equilibrium. The constitutive elements of this model idealize the metastability of cortical networks. Independent elements accumulate visual evidence at one level, while groups of coupled elements compete for dominance at another level. As soon as one group dominates perception, feedback inhibition suppresses supporting evidence. Previously unreported features in the serial dependencies of perceptual alternations compellingly corroborate this mechanism. Moreover, the proposed out-of-equilibrium dynamics satisfies normative constraints of continuous decision-making. Thus, multistable perception may reflect decision-making in a volatile world: integrating evidence over space and time, choosing categorically between hypotheses, while concurrently evaluating alternatives.

PPAR activation has dichotomous control on the expression levels of cytosolic and secretory phospholipase A2 in astrocytes; inhibition in naïve, untreated cells and enhancement in LPS-stimulated cells.

Despite the importance of cytosolic phospholipase A(2) type IVA (cPLA(2)) and secretory PLA(2) (sPLA(2)) in physiological and pathological responses of astrocytes in inflammatory conditions, the regulation of the expression of these genes is still unclear. Both genes have peroxisome proliferator-activated receptors (PPAR) binding sites in their promoters. The role of synthetic PPAR agonists in the regulation of gene expression in naïve and lipopolysaccharide (LPS)-stimulated rat astrocytes in culture was investigated. Exposure to LPS resulted in a time-dependent, fourfold transient increase of sPLA(2) expression, with maximum at 4 h; cPLA(2) expression was notably increased after 16-h LPS stimulation. Using selective PPARα, PPARß/δ, and PPARγ agonists, we found that expression of both cPLA(2) and sPLA(2) is under PPAR control, but with different isotypes sensitivity. In naïve astrocytes, all three PPAR agonists significantly suppressed the expression of sPLA(2), while only PPARα and PPARγ activation suppressed cPLA(2) expression. Astonishingly, simultaneous addition of LPS with PPAR agonists evoked the opposite effect. All three PPAR agonists induced potentiation of cPLA(2) expression level. Potentiation of sPLA(2) expression was induced only by simultaneous addition of LPS with PPARγ agonist. By knockdown of PPARα, PPARß/δ, and PPARγ, we confirmed the involvement of PPAR-dependent pathways. The important novelty of our findings is that both sPLA(2) and cPLA(2) are under dichotomous control of PPARs: suppression in naïve control cells, but induction in LPS-stimulated astrocytes.

Peroxisome proliferator-activated receptor (PPAR)-gamma positively controls and PPARalpha negatively controls cyclooxygenase-2 expression in rat brain astrocytes through a convergence on PPARbeta/delta via mutual control of PPAR expression levels.

Peroxisome proliferator-activated receptor (PPAR) transcription factors are pharmaceutical drug targets for treating diabetes, atherosclerosis, and inflammatory degenerative diseases. The possible mechanism of interaction between the three PPAR isotypes (alpha, beta/delta, and gamma) is not yet clear. However, this is important both for understanding transcription factor regulation and for the development of new drugs. The present study was designed to compare the effects of combinations of synthetic agonists of PPARalpha [2-[4-[2-[4-cyclohexylbutyl (cyclohexylcarbamoyl)amino]ethyl]phenyl] sulfanyl-2-methylpropanoic acid (GW7647)], PPARbeta/delta [4-(3-(2-propyl-3-hydroxy-4-acetyl)phenoxy)propyloxyphenoxy acetic acid, (L-165041)], and PPARgamma (rosiglitazone, ciglitazone) on inflammatory gene regulation in rat primary astrocytes. We measured cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) synthesis in lipopolysaccharide (LPS)-stimulated cells. PPARalpha, PPARbeta/delta, and PPARgamma knockdown models served to delineate the contribution of each PPAR isotype. Thiazolidinediones enhanced the LPS-induced COX-2 expression via PPARgamma-dependent pathway, whereas L-165041 and GW7647 had no influence. However, the addition of L-165041 potentiated the effect of PPARgamma activation through PPARbeta/delta-dependent mechanism. On the contrary, PPARalpha activation (GW7647) suppressed the effect of the combined L-165041/rosiglitazone application. The mechanism of the interplay arising from combined applications of PPAR agonists involves changes in PPAR expression levels. A PPARbeta/delta overexpression model confirmed that PPARbeta/delta expression level is the point at which PPARgamma and PPARalpha pathways converge in control of COX-2 gene expression. Thus, we discovered that in primary astrocytes, PPARgamma has a positive influence and PPARalpha has a negative influence on PPARbeta/delta expression and activity. A positive/negative-feedback loop is formed by PPARbeta/delta-dependent increase in PPARalpha expression level. These findings elucidate a novel principle of regulation in the signaling by synthetic PPAR agonists that involves modulating the interaction between PPARalpha,-beta/delta, and -gamma isoforms on the level of their expression.

Peroxisome proliferator-activated receptor (PPAR)ß/δ, a possible nexus of PPARα- and PPARγ-dependent molecular pathways in neurodegenerative diseases: Review and novel hypotheses.

Peroxisome proliferator-activated receptors (PPARα, -ß/δ and -γ) are lipid-activated transcription factors. Synthetic PPARα and PPARγ ligands have neuroprotective properties. Recently, PPARß/δ activation emerged as the focus of a novel approach for the treatment of a wide range of neurodegenerative diseases. To fill the gap of knowledge about the role of PPARß/δ in brain, new hypotheses about PPARß/δ involvement in neuropathological processes are requested. In this paper, we describe a novel hypothesis, claiming the existence of tight interactions between the three PPAR isotypes, which we designate the "PPAR triad". We propose that PPARß/δ has a central control of the PPAR triad. The majority of studies analyze the regulation only by one of the PPAR isotypes. A few reports describe the mutual regulation of expression levels of all three PPAR isotypes by PPAR agonists. Analysis of these studies where pairwise interactions of PPARs were described allows us to support the existence of the PPAR triad with central role for PPARß/δ. In the present review, we propose the hypothesis that in a wide range of brain disorders, PPARß/δ plays a central role between PPARα and PPARγ. Finally, we prove the advantages of the PPAR triad concept by describing hypotheses of PPARß/δ involvement in the regulation of myelination, glutamate-induced neurotoxicity, and signaling pathways of reactive oxygen species/NO/Ca(2+).

Expression of protease-activated receptor (PAR)-2, but not other PARs, is regulated by inflammatory cytokines in rat astrocytes.

Protease-activated receptors (PARs) are widely expressed in the central nervous system (CNS) and are believed to play an important role in normal brain functioning as well as in development of various inflammatory and neurodegenerative disorders. Pathological conditions cause altered expression of PARs in brain cells and therefore altered responsiveness to PAR activation. The exact mechanisms of regulation of PAR expression are not well studied. Here, we evaluated in rat astrocytes the influence of LPS, pro-inflammatory cytokines TNFα and IL-1ß and continuous PAR activation by PAR agonists on the expression levels of PARs. These stimuli are important in inflammatory and neurological disorders, where their levels are increased. We report that LPS as well as cytokines TNFα and IL-1ß affected only the PAR-2 level, but their effects were opposite. LPS and TNFα increased the functional expression of PAR-2, whereas IL-1ß down-regulated the functional response of PAR-2. Agonists of PAR-1 specifically increased mRNA level of PAR-2, but not protein level. Transcript levels of other PARs were not changed after PAR-1 activation. Stimulation of the cells with PAR-2 or PAR-4 agonists did not alter PAR levels. We found that up-regulation of PAR-2 is dependent on PKC activity, mostly via its Ca²âº-sensitive isoforms. Two transcription factors, NFκB and AP-1, are involved in up-regulation of PAR-2. These findings provide new information about the regulation of expression of PAR subtypes in brain cells. This is of importance for targeting PARs, especially PAR-2, for the treatment of CNS disorders.