The Role of Glia in Wilson's Disease: Clinical, Neuroimaging, Neuropathological and Molecular Perspectives.

Wilson's disease (WD) is inherited in an autosomal recessive manner and is caused by pathogenic variants of the ATP7B gene, which are responsible for impaired copper transport in the cell, inhibition of copper binding to apoceruloplasmin, and biliary excretion. This leads to the accumulation of copper in the tissues. Copper accumulation in the CNS leads to the neurological and psychiatric symptoms of WD. Abnormalities of copper metabolism in WD are associated with impaired iron metabolism. Both of these elements are redox active and may contribute to neuropathology. It has long been assumed that among parenchymal cells, astrocytes have the greatest impact on copper and iron homeostasis in the brain. Capillary endothelial cells are separated from the neuropil by astrocyte terminal legs, putting astrocytes in an ideal position to regulate the transport of iron and copper to other brain cells and protect them if metals breach the blood-brain barrier. Astrocytes are responsible for, among other things, maintaining extracellular ion homeostasis, modulating synaptic transmission and plasticity, obtaining metabolites, and protecting the brain against oxidative stress and toxins. However, excess copper and/or iron causes an increase in the number of astrocytes and their morphological changes observed in neuropathological studies, as well as a loss of the copper/iron storage function leading to macromolecule peroxidation and neuronal loss through apoptosis, autophagy, or cuproptosis/ferroptosis. The molecular mechanisms explaining the possible role of glia in copper- and iron-induced neurodegeneration in WD are largely understood from studies of neuropathology in Parkinson's disease and Alzheimer's disease. Understanding the mechanisms of glial involvement in neuroprotection/neurotoxicity is important for explaining the pathomechanisms of neuronal death in WD and, in the future, perhaps for developing more effective diagnostic/treatment methods.

Correction: Babiec et al. Alterations of Purinergic Receptors Levels and Their Involvement in the Glial Cell Morphology in a Pre-Clinical Model of Autism Spectrum Disorders. Brain Sci. 2023, 13, 1088.

In the original publication [...].

Alterations of Purinergic Receptors Levels and Their Involvement in the Glial Cell Morphology in a Pre-Clinical Model of Autism Spectrum Disorders.

Recent data suggest that defects in purinergic signalling are a common denominator of autism spectrum disorders (ASDs), though nothing is known about whether the disorder-related imbalance occurs at the receptor level. In this study, we investigated whether prenatal exposure to valproic acid (VPA) induces changes in purinergic receptor expression in adolescence and whether it corresponds to glial cell activation. Pregnant dams were subjected to an intraperitoneal injection of VPA at embryonic day 12.5. In the hippocampi of adolescent male VPA offspring, we observed an increase in the level of P2X1, with concomitant decreases in P2X7 and P2Y1 receptors. In contrast, in the cortex, the level of P2X1 was significantly reduced. Also, significant increases in cortical P2Y1 and P2Y12 receptors were detected. Additionally, we observed profound alterations in microglial cell numbers and morphology in the cortex of VPA animals, leading to the elevation of pro-inflammatory cytokine expression. The changes in glial cells were partially reduced via a single administration of a non-selective P2 receptor antagonist. These studies show the involvement of purinergic signalling imbalance in the modulation of brain inflammatory response induced via prenatal VPA exposure and may indicate that purinergic receptors are a novel target for pharmacological intervention in ASDs.

Glia-Neurotrophic Factor Relationships: Possible Role in Pathobiology of Neuroinflammation-Related Brain Disorders.

Neurotrophic factors (NTFs) play an important role in maintaining homeostasis of the central nervous system (CNS) by regulating the survival, differentiation, maturation, and development of neurons and by participating in the regeneration of damaged tissues. Disturbances in the level and functioning of NTFs can lead to many diseases of the nervous system, including degenerative diseases, mental diseases, and neurodevelopmental disorders. Each CNS disease is characterized by a unique pathomechanism, however, the involvement of certain processes in its etiology is common, such as neuroinflammation, dysregulation of NTFs levels, or mitochondrial dysfunction. It has been shown that NTFs can control the activation of glial cells by directing them toward a neuroprotective and anti-inflammatory phenotype and activating signaling pathways responsible for neuronal survival. In this review, our goal is to outline the current state of knowledge about the processes affected by NTFs, the crosstalk between NTFs, mitochondria, and the nervous and immune systems, leading to the inhibition of neuroinflammation and oxidative stress, and thus the inhibition of the development and progression of CNS disorders.

Developmental Stage-Dependent Changes in Mitochondrial Function in the Brain of Offspring Following Prenatal Maternal Immune Activation.

Maternal immune activation (MIA) is an important risk factor for neurodevelopmental disorders such as autism. The aim of the current study was to investigate the development-dependent changes in the mitochondrial function of MIA-exposed offspring, which may contribute to autism-like deficits. MIA was evoked by the single intraperitoneal administration of lipopolysaccharide to pregnant rats at gestation day 9.5, and several aspects of mitochondrial function in fetuses and in the brains of seven-day-old pups and adolescent offspring were analyzed along with oxidative stress parameters measurement. It was found that MIA significantly increased the activity of NADPH oxidase (NOX), an enzyme generating reactive oxygen species (ROS) in the fetuses and in the brain of seven-day-old pups, but not in the adolescent offspring. Although a lower mitochondrial membrane potential accompanied by a decreased ATP level was already observed in the fetuses and in the brain of seven-day-old pups, persistent alterations of ROS, mitochondrial membrane depolarization, and lower ATP generation with concomitant electron transport chain complexes downregulation were observed only in the adolescent offspring. We suggest that ROS observed in infancy are most likely of a NOX activity origin, whereas in adolescence, ROS are produced by damaged mitochondria. The accumulation of dysfunctional mitochondria leads to the intense release of free radicals that trigger oxidative stress and neuroinflammation, resulting in an interlinked vicious cascade.

The Role of Bromodomain and Extraterminal (BET) Proteins in Controlling the Phagocytic Activity of Microglia In Vitro: Relevance to Alzheimer's Disease.

The correct phagocytic activity of microglia is a prerequisite for maintaining homeostasis in the brain. In the analysis of mechanisms regulating microglial phagocytosis, we focused on the bromodomain and extraterminal domain (BET) proteins: Brd2, Brd3, and Brd4, the acetylation code readers that control gene expression in cooperation with transcription factors. We used pharmacological (JQ1) and genetic (siRNA) inhibition of BET proteins in murine microglial cell line BV2. Inhibition of BET proteins reduced the phagocytic activity of BV2, as determined by using a fluorescent microspheres-based assay and fluorescently labelled amyloid-beta peptides. Gene silencing experiments demonstrated that all brain-existing BET isoforms control phagocytosis in microglia. From a set of 84 phagocytosis-related genes, we have found the attenuation of the expression of 14: Siglec1, Sirpb1a, Cd36, Clec7a, Itgam, Tlr3, Fcgr1, Cd14, Marco, Pld1, Fcgr2b, Anxa1, Tnf, Nod1, upon BET inhibition. Further analysis of the mRNA level of other phagocytosis-related genes which were involved in the pathomechanism of Alzheimer's disease demonstrated that JQ1 significantly reduced the expression of Cd33, Trem2, and Zyx. Our results indicate the important role of BET proteins in controlling microglial phagocytosis; therefore, targeting BET may be the efficient method of modulating microglial activity.

Prenatal exposure to valproic acid induces alterations in the expression and activity of purinergic receptors in the embryonic rat brain.

Purinergic signalling is involved in the control of several processes related to brain development, such as neurogenesis and gliogenesis, migration and differentiation of neuronal precursors, synaptogenesis and synaptic elimination to achieve a fully wired and efficient mature brain. Therefore, any deregulation of purine-dependent signalling mediated by stimulation of specific adenosine and purinergic receptor subtypes: P1, P2X, or P2Y, can lead to functional deficits and the development of neuropsychiatric disorders, including autism spectrum disorders (ASD). In this study, we investigated the changes in expression and activity of selected purinergic receptors during rat brain development in an animal model of ASD. Pregnant dams received an intraperitoneal injection of valproic acid (VPA; 450 mg/kg body weight) at embryonic day (ED) 12.5, around the time of neural tube closure. Subsequently, changes in the expression and activity of specific purinergic receptor subtypes were analysed at ED19, an important prenatal stage of brain development. Our results suggest that prenatal VPA exposure leads to a significant increase in the level and activity of adenosinergic receptors A1, A2b and A3, which are involved in the regulation of progenitor cell proliferation and nerve growth, and upregulation of purinergic P2X2/P2X3 receptors, which in turn may contribute to the postnatal neuroanatomical abnormalities and synaptic dysfunction. Conversely, the significant downregulation of P2Y1 and P2X7 receptors, together with their reduced activity in the embryonic VPA brain, may indicate disturbances in the processes of neuronal precursor migration and differentiation, dendritic and axonal formation, and glutamate/GABA imbalance, thereby altering neuronal excitability. In conclusion, defects in purinergic signalling induced by prenatal VPA exposure could have a profound impact on brain development during embryogenesis and on intellectual and behavioural functions after birth. These observations could provide clues for future implementation of potential therapeutic strategies for ASD.

Exogenous Alpha-Synuclein Evoked Parkin Downregulation Promotes Mitochondrial Dysfunction in Neuronal Cells. Implications for Parkinson's Disease Pathology.

Aberrant secretion and accumulation of α-synuclein (α-Syn) as well as the loss of parkin function are associated with the pathogenesis of Parkinson's disease (PD). Our previous study suggested a functional interaction between those two proteins, showing that the extracellular α-Syn evoked post-translational modifications of parkin, leading to its autoubiquitination and degradation. While parkin plays an important role in mitochondrial biogenesis and turnover, including mitochondrial fission/fusion as well as mitophagy, the involvement of parkin deregulation in α-Syn-induced mitochondrial damage is largely unknown. In the present study, we demonstrated that treatment with exogenous α-Syn triggers mitochondrial dysfunction, reflected by the depolarization of the mitochondrial membrane, elevated synthesis of the mitochondrial superoxide anion, and a decrease in cellular ATP level. At the same time, we observed a protective effect of parkin overexpression on α-Syn-induced mitochondrial dysfunction. α-Syn-dependent disturbances of mitophagy were also shown to be directly related to reduced parkin levels in mitochondria and decreased ubiquitination of mitochondrial proteins. Also, α-Syn impaired mitochondrial biosynthesis due to the parkin-dependent reduction of PGC-1α protein levels. Finally, loss of parkin function as a result of α-Syn treatment induced an overall breakdown of mitochondrial homeostasis that led to the accumulation of abnormal mitochondria. These findings may thus provide the first compelling evidence for the direct association of α-Syn-mediated parkin depletion to impaired mitochondrial function in PD. We suggest that improvement of parkin function may serve as a novel therapeutic strategy to prevent mitochondrial impairment and neurodegeneration in PD (thereby slowing the progression of the disease).

Maternal Immune Activation Induces Neuroinflammation and Cortical Synaptic Deficits in the Adolescent Rat Offspring.

Maternal immune activation (MIA), induced by infection during pregnancy, is an important risk factor for neuro-developmental disorders, such as autism. Abnormal maternal cytokine signaling may affect fetal brain development and contribute to neurobiological and behavioral changes in the offspring. Here, we examined the effect of lipopolysaccharide-induced MIA on neuro-inflammatory changes, as well as synaptic morphology and key synaptic protein level in cerebral cortex of adolescent male rat offspring. Adolescent MIA offspring showed elevated blood cytokine levels, microglial activation, increased pro-inflammatory cytokines expression and increased oxidative stress in the cerebral cortex. Moreover, pathological changes in synaptic ultrastructure of MIA offspring was detected, along with presynaptic protein deficits and down-regulation of postsynaptic scaffolding proteins. Consequently, ability to unveil MIA-induced long-term alterations in synapses structure and protein level may have consequences on postnatal behavioral changes, associated with, and predisposed to, the development of neuropsychiatric disorders.

P2X7 Receptor is Involved in Mitochondrial Dysfunction Induced by Extracellular Alpha Synuclein in Neuroblastoma SH-SY5Y Cells.

The purinergic P2X7 receptor (P2X7R) belongs to a family of trimeric ion channels that are gated by extracellular adenosine 5'-triphosphate (ATP). Several studies have pointed to a role of P2X7R-dependent signalling in Parkinson's disease (PD)-related neurodegeneration. The pathology of (PD) is characterized by the formation of insoluble alpha-synuclein (α-Syn) aggregates-Lewy bodies, but the mechanisms underlying α-Syn-induced dopaminergic cell death are still partially unclear. Our previous studies indicate that extracellular α-Syn directly interact with neuronal P2X7R and induces intracellular free calcium mobilization in neuronal cells. The main objective of this study was to examine the involvement of P2X7R receptor in α-Syn-induced mitochondrial dysfunction and cell death. We found that P2X7R stimulation is responsible for α-Syn-induced oxidative stress and activation of the molecular pathways of programmed cell death. Exogenous α-Syn treatment led to P2X7R-dependent decrease in mitochondrial membrane potential as well as elevation of mitochondrial ROS production resulting in breakdown of cellular energy production. Moreover, P2X7R-dependent deregulation of AMP-activated protein kinase as well as decrease in parkin protein level could be responsible for α-Syn-induced mitophagy impairment and accumulation of dysfunctional mitochondria. P2X7R might be putative pharmacological targets in molecular mechanism of extracellular α-Syn toxicity.

Detection of ALDH3B2 in Human Placenta.

Aldehyde dehydrogenase 3B2 (ALDH3B2) gene contains a premature termination codon, which can be skipped or suppressed resulting in full-length protein expression. Alternatively, the longest putative open reading frame starting with the second in-frame start codon would encode short isoform. No unequivocal evidence of ALDH3B2 expression in healthy human tissues is available. The aim of this study was to confirm its expression in human placenta characterized by the highest ALDH3B2 mRNA abundance. ALDH3B2 DNA and mRNA were sequenced. The expression was investigated using western blot. The identity of the protein was confirmed using mass spectrometry (MS). The predicted tertiary and quaternary structures, subcellular localization, and phosphorylation sites were assessed using bioinformatic analyses. All DNA and mRNA isolates contained the premature stop codon. In western blot analyses, bands corresponding to the mass of full-length protein were detected. MS analysis led to the identification of two unique peptides, one of which is encoded by the nucleotide sequence located upstream the second start codon. Bioinformatic analyses suggest cytoplasmic localization and several phosphorylation sites. Despite premature stop codon in DNA and mRNA sequences, full-length ALDH3B2 was found. It can be formed as a result of premature stop codon readthrough, complex phenomenon enabling stop codon circumvention.

The interplay between parkin and alpha-synuclein; possible implications for the pathogenesis of Parkinson's disease.

Parkin and alpha-synuclein (α-syn) are two key proteins involved in the pathophysiology of Parkinson's disease (PD). Oligomerization/aggregation and excessive secretion of α-syn contributes to PD through free radical stress, mitochondrial impairment, and synaptic dysfunction. Parkin, an E3 ubiquitin ligase, is considered to be a pleiotropic, neuroprotective protein that modulates metabolic turnover and the accumulation of α-syn. This is in addition to parkin's role in counteracting the more distant effects of α-syn on cellular survival by altering proteasomal, autophagic, and calpain-mediated protein degradation pathways that can reduce α-syn levels. Moreover, parkin regulates mitochondrial turnover, cell survival, and immune phenomena - processes that are all known to be disturbed in PD. In addition, parkin might have an impact on the spreading and propagation of α-syn by controlling its post-translational modifications. On the other hand, recent research has shown that α-syn oligomers affect the expression, post-translational modification, and activity of parkin. This review focuses on the molecular mechanisms of cross-talk between parkin and α-syn in PD. The physical and functional interactions between α-syn and parkin, which have been incompletely characterized to-date, may present a new therapeutic avenue in PD and related synucleinopathies. The development of effective, clinically feasible modulators may offer great hopes for the the rapy of PD.

Extracellular Alpha-Synuclein Oligomers Induce Parkin S-Nitrosylation: Relevance to Sporadic Parkinson's Disease Etiopathology.

α-Synuclein (ASN) and parkin, a multifunctional E3 ubiquitin ligase, are two proteins that are associated with the pathophysiology of Parkinson's disease (PD). Excessive release of ASN, its oligomerization, aggregation, and deposition in the cytoplasm contribute to neuronal injury and cell death through oxidative-nitrosative stress induction, mitochondrial impairment, and synaptic dysfunction. In contrast, overexpression of parkin provides protection against cellular stresses and prevents dopaminergic neural cell loss in several animal models of PD. However, the influence of ASN on the function of parkin is largely unknown. Therefore, the aim of this study was to investigate the effect of extracellular ASN oligomers on parkin expression, S-nitrosylation, as well as its activity. For these investigations, we used rat pheochromocytoma (PC12) cell line treated with exogenous oligomeric ASN as well as PC12 cells with parkin overexpression and parkin knock-down. The experiments were performed using spectrophotometric, spectrofluorometric, and immunochemical methods. We found that exogenous ASN oligomers induce oxidative/nitrosative stress leading to parkin S-nitrosylation. Moreover, this posttranslational modification induced the elevation of parkin autoubiquitination and degradation of the protein. The decreased parkin levels resulted in significant cell death, whereas parkin overexpression protected against toxicity induced by extracellular ASN oligomers. We conclude that lowering parkin levels by extracellular ASN may significantly contribute to the propagation of neurodegeneration in PD pathology through accumulation of defective proteins as a consequence of parkin degradation.

Altered Expression of Urea Cycle Enzymes in Amyloid-ß Protein Precursor Overexpressing PC12 Cells and in Sporadic Alzheimer's Disease Brain.

Urea cycle enzymes may play important yet poorly characterized roles in Alzheimer's disease (AD). Our previous results showed that amyloid-ß (Aß) affects urea cycle enzymes in rat pheochromocytoma (PC12) cells. The aim of the present study was to investigate the changes in arginases, other urea cycle enzymes, and nitric oxide synthases (NOSs) in PC12 cells transfected with AßPP bearing the double 'Swedish' mutation (APPsw, K670M/N671L) and in postmortem sporadic AD brain hippocampus; the mutation intensifies Aß production and strongly associates with AD neuropathology. mRNA expression was analyzed using real-time PCR in cell cultures and DNA microarrays in hippocampal CA1 area of human AD brains. Arginase activity was measured spectrophotometrically, and arginine, ornithine, and citrulline levels by high-performance liquid chromatography. Our data demonstrated that the expression and activity of arginases (Arg1 and Arg2), as well as the expression of argininosuccinate synthase (Ass) were significantly reduced in APPsw cells compared to control. However, argininosuccinate lyase (Asl) was upregulated in APPsw cells. Real-time PCR analysis revealed significant elevation of neuronal nitric oxide synthase (Nnos) mRNA in APPsw cells, without changes in the endothelial Enos, whereas inducible Inos was undetectable. The changes were found to follow closely those observed in the human hippocampal CA1 region of sporadic AD brains. The changes in enzyme expression were accompanied in APPsw cells by significantly elevated citrulline, ornithine, and arginine. Our findings demonstrate that AßPP/Aß alters arginine metabolism and induces a shift of cellular homeostasis that may support the oxidative/nitrosative stress observed in AD.

Inhibition of cyclin-dependent kinase 5 affects early neuroinflammatory signalling in murine model of amyloid beta toxicity.

BACKGROUND: Cyclin-dependent kinase 5 (Cdk5) belongs to the family of proline-directed serine/threonine kinases and plays a critical role in neuronal differentiation, migration, synaptogenesis, plasticity, neurotransmission and apoptosis. The deregulation of Cdk5 activity was observed in post mortem analysis of brain tissue of Alzheimer's disease (AD) patients, suggesting the involvement of Cdk5 in the pathomechanism of this neurodegenerative disease. However, our recent study demonstrated the important function of Cdk5 in regulating inflammatory reaction. METHODS: Since the role of Cdk5 in regulation of inflammatory signalling in AD is unknown, we investigated the involvement of Cdk5 in neuroinflammation induced by single intracerebroventricular (icv) injection of amyloid beta protein (Aß) oligomers in mouse. The brain tissue was analysed up to 35 days post injection. Roscovitine (intraperitoneal administration) was used as a potent Cdk5 inhibitor. The experiments were also performed on human neuroblastoma SH-SY5Y as well as mouse BV2 cell lines treated with exogenous oligomeric Aß. RESULTS: Our results demonstrated that single injection of Aß oligomers induces long-lasting activation of microglia and astrocytes in the hippocampus. We observed also profound, early inflammatory response in the mice hippocampus, leading to the significant elevation of pro-inflammatory cytokines expression (e.g. TNF-α, IL-1ß, IL-6). Moreover, Aß oligomers elevated the formation of truncated protein p25 in mouse hippocampus and induced overactivation of Cdk5 in neuronal cells. Importantly, administration of roscovitine reduced the inflammatory processes evoked by Aß in the hippocampus, leading to the significant decrease of cytokines level. CONCLUSIONS: These studies clearly show the involvement of Cdk5 in modulation of brain inflammatory response induced by Aß and may indicate this kinase as a novel target for pharmacological intervention in AD.

P2X7 receptor-pannexin 1 interaction mediates extracellular alpha-synuclein-induced ATP release in neuroblastoma SH-SY5Y cells.

Abnormalities of alpha-synuclein (ASN), the main component of protein deposits (Lewy bodies), were observed in Parkinson's disease (PD), dementia with Lewy bodies, Alzheimer's disease, and other neurodegenerative disorders. These alterations include increase in the levels of soluble ASN oligomers in the extracellular space. Numerous works have identified several mechanisms of their toxicity, including stimulation of the microglial P2X7 receptor leading to oxidative stress. While the significant role of purinergic signaling-particularly, P2 family receptors-in neurodegenerative disorders is well known, the interaction of extracellular soluble ASN with neuronal purinergic receptors is yet to be studied. Therefore, in this study, we have investigated the effect of ASN on P2 purinergic receptors and ATP-dependent signaling. We used neuroblastoma SH-SY5Y cell line and rat synaptoneurosomes treated with exogenous soluble ASN. The experiments were performed using spectrofluorometric, radiochemical, and immunochemical methods. We found the following: (i) ASN-induced intracellular free calcium mobilization in neuronal cells and nerve endings depends on the activation of purinergic P2X7 receptors; (ii) activation of P2X7 receptors leads to pannexin 1 recruitment to form an active complex responsible for ATP release; and (iii) ASN greatly decreases the activity of extracellular ecto-ATPase responsible for ATP degradation. Thus, it is concluded that purinergic receptors might be putative pharmacological targets in the molecular mechanism of extracellular ASN toxicity. Interference with P2X7 signaling seems to be a promising strategy for the prevention or therapy of PD and other neurodegenerative disorders.

Selol, an organic selenium donor, prevents lipopolysaccharide-induced oxidative stress and inflammatory reaction in the rat brain.

Neuroinflammation and oxidative stress are key intertwined pathological factors in many neurological, particularly neurodegenerative diseases, such as Alzheimer's and Parkinson's disorders as well as autism. The present study was conducted to evaluate the protective effects of Selol, an organic selenium donor, against lipopolysaccharide (LPS)-mediated inflammation in rat brain. The results demonstrated that the peripheral administration of LPS in a dose of 100 µg/kg b.w. evoked typical pathological reaction known as systemic inflammatory response. Moreover, we observed elevated blood levels of thiobarbituric acid-reactive substances (TBARS), a marker of oxidative stress, as well as increased concentration of tumor necrosis factor-α (TNF-α) in LPS-treated animals. Selol significantly prevented these LPS-evoked changes. Subsequently, Selol protected against LPS-induced up-regulation of proinflammatory cytokines (Tnfa, Ifng, Il6) in rat brain cortex. The molecular mechanisms through which Selol prevented the neuroinflammation were associated with the inhibition of oxidized glutathione (GSSG) accumulation and with an increase of glutathione-associated enzymes: glutathione peroxidase (Se-GPx), glutathione reductase (GR) as well as thioredoxin reductase (TrxR) activity and expression. Finally, we observed that Selol administration effectively protected against LPS-induced changes in the expression of brain-derived neurotrophic factor (Bdnf). In conclusion, our studies indicated that Selol effectively protects against LPS-induced neuroinflammation by inhibiting pro-inflammatory cytokine release, by boosting antioxidant systems, and by augmenting BDNF level. Therefore, Selol could be a multi-potent and effective drug useful in the treatment and prevention of brain disorders associated with neuroinflammation.

Protective Effects of Selol Against Sodium Nitroprusside-Induced Cell Death and Oxidative Stress in PC12 Cells.

Selol is an organic selenitetriglyceride formulation containing selenium at +4 oxidation level that can be effectively incorporated into catalytic sites of of Se-dependent antioxidants. In the present study, the potential antioxidative and cytoprotective effects of Selol against sodium nitroprusside (SNP)-evoked oxidative/nitrosative stress were investigated in PC12 cells and the underlying mechanisms analyzed. Spectrophoto- and spectrofluorimetic methods as well as fluorescence microscopy were used in this study; mRNA expression was quantified by real-time PCR. Selol dose-dependently improved the survival and decreased the percentage of apoptosis in PC12 cells exposed to SNP. To determine the mechanism of this protective action, the effect of Selol on free radical generation and on antioxidative potential was evaluated. Selol offered significant protection against the elevation of reactive oxidative species (ROS) evoked by SNP. Moreover, this compound restored glutathione homeostasis by ameliorating the SNP-evoked disturbance of GSH/GSSG ratio. The protective effect exerted by Selol was associated with the prevention of SNP-mediated down-regulation of antioxidative enzymes: glutathione peroxidase (Se-GPx), glutathione reductase (GR), and thioredoxin reductase (TrxR). Finally, GPx inhibition significantly abolished the cytoprotective effect of Selol. In conclusion, these results suggest that Selol effectively protected PC12 cells against SNP-induced oxidative damage and death by adjusting free radical levels and antioxidant system, and suppressing apoptosis. Selol could be successfully used in the treatments of diseases that involve oxidative stress and resulting apoptosis.

Altered Arginine Metabolism in Cells Transfected with Human Wild-Type Beta Amyloid Precursor Protein (ßAPP).

Alterations of enzymes linked to arginine metabolism have been recently implicated in Alzheimer's disease (AD). Despite strong association of arginine changes with nitric oxide (NO) pathway, the impact of amyloid ß (Aß) peptides on arginine degradation and re-synthesis is unknown. In the present study we compared expression levels of arginases (ARG1, ARG2), neuronal, endothelial and inducible NO synthase isoforms (NNOS, ENOS, INOS), enzymes that metabolize arginine or resynthesize it from citrulline and the levels of corresponding amino acids in rat pheochromocytoma (PC12) cells overexpressing human Aß precursor protein (APPwt cells). Moreover, we investigated the changes in miRNAs responsible for modulation of arginine metabolism in AD brains. Real-time PCR analysis revealed in APPwt cells significant decreases of ARG1 and ARG2 which are responsible for lysing arginine into ornithine and urea; this reduction was followed by significantly lower enzyme activity. NNOS and ENOS mRNAs were elevated in APPwt cells while iNOS was undetectable in both cell lines. The expression of argininosuccinate synthase (ASS) that metabolizes citrulline was down-regulated without changes in argininosuccinate lyase (ASL). Ornithine decarboxylase (ODC), which decarboxylates ornithine to form putrescine was also reduced. Arginine, the substrate for both arginases and NOS, was unchanged in APPwt cells. However, citrulline concentration was significantly higher. Elevated miRNA-9 and miRNA-128a found in AD brain tissues might modulate the expression of ASS and NOS, respectively. Our results indicate that Aß affects arginine metabolism and this influence might have important role in the pathomechanism of AD.

The mechanisms regulating cyclin-dependent kinase 5 in hippocampus during systemic inflammatory response: The effect on inflammatory gene expression.

Cyclin-dependent kinase 5 (Cdk5) is critical for nervous system's development and function, and its aberrant activation contributes to pathomechanism of Alzheimer's disease and other neurodegenerative disorders. It was recently suggested that Cdk5 may participate in regulation of inflammatory signalling. The aim of this study was to analyse the mechanisms involved in regulating Cdk5 activity in the brain during systemic inflammatory response (SIR) as well as the involvement of Cdk5 in controlling the expression of inflammatory genes. Genetic and biochemical alterations in hippocampus were analysed 3 and 12 h after intraperitoneal injection of lipopolysaccharide. We observed an increase in both Cdk5 gene expression and protein level. Moreover, phosphorylation of Cdk5 on Ser159 was significantly enhanced. Also transcription of Cdk5-regulatory protein (p35/Cdk5r1) was augmented, and the level of p25, calpain-dependent cleavage product of p35, was increased. All these results demonstrated rapid activation of Cdk5 in the brain during SIR. Hyperactivity of Cdk5 contributed to enhanced phosphorylation of tau and glycogen synthase kinase 3ß. Inhibition of Cdk5 with Roscovitine reduced activation of NF-κB and expression of inflammation-related genes, demonstrating the critical role of Cdk5 in regulation of gene transcription during SIR.