Anna Wilkaniec

Toxicity of extracellular secreted alpha-synuclein: Its role in nitrosative stress and neurodegeneration.

It has been demonstrated that both oligomerisation and accumulation of α-synuclein (ASN) are the key molecular processes involved in the pathophysiology of neurodegenerative diseases such as Parkinsons disease, Alzheimers disease and other synucleinopathies. Alterations of ASN expression and impairment of its degradation can lead to the formation of intracellular deposits of this protein, called Lewy bodies. Overexpressed or misfolded ASN could be secreted to the extracellular space. Today the prion-like transmission of ASN oligomers to neighbouring cells is believed to be responsible for protein modification and propagation of neurodegeneration in the brain. It was presented that oxidative/nitrosative stress may play a key role in ASN secretion and spread of ASN pathology. Moreover, ASN-evoked protein oxidation, nitration and nitrosylation lead to disturbances in synaptic transmission and cell death. The interaction of secreted ASN with other amyloidogenic proteins and its involvement in irreversible mitochondrial disturbances and oxidative stress were also described. A better understanding of the mechanisms of ASN secretion and dysfunction may help to explain the molecular mechanisms of neurodegeneration and may be the basis for the development of novel therapeutic strategies.

Extracellular alpha-synuclein induces calpain-dependent overactivation of cyclin-dependent kinase 5 in vitro.

We found that exposure of PC12 cells to ASN increases Cdk5 activity via calpain‐dependent p25 formation and by enhancement of Cdk5 phosphorylation at Tyr15. Cdk5 and calpain inhibitors prevented ASN‐evoked cell death. Our findings, indicating the participation of Cdk5 in ASN toxicity, provide new insight into how extracellular ASN may trigger dopaminergic cell dysfunction in PD.

Cdk5 at crossroads of protein oligomerization in neurodegenerative diseases: facts and hypotheses

Cyclin‐dependent kinase 5 (Cdk5) is involved in proper neurodevelopment and brain function and serves as a switch between neuronal survival and death. Overactivation of Cdk5 is associated with many neurodegenerative disorders such as Alzheimers or Parkinsons diseases. It is believed that in those diseases Cdk5 may be an important link between disease‐initiating factors and cell death effectors. A common hallmark of neurodegenerative disorders is incorrect folding of specific proteins, thus leading to their intra‐ and extracellular accumulation in the nervous system. Abnormal Cdk5 signaling contributes to dysfunction of individual proteins and has a substantial role in either direct or indirect interactions of proteins common to, and critical in, different neurodegenerative diseases. While the roles of Cdk5 in α‐synuclein (ASN) – tau or β‐amyloid peptide (Aβ) – tau interactions are well documented, its contribution to many other pertinent interactions, such as that of ASN with Aβ, or interactions of the Aβ – ASN – tau triad with prion proteins, did not get beyond plausible hypotheses and remains to be proven. Understanding of the exact position of Cdk5 in the deleterious feed‐forward loop critical for development and progression of neurodegenerative diseases may help designing successful therapeutic strategies of several fatal neurodegenerative diseases.

Selenium in the Therapy of Neurological Diseases. Where is it Going

Selenium (34Se), an antioxidant trace element, is an important regulator of brain function. These beneficial properties that Se possesses are attributed to its ability to be incorporated into selenoproteins as an amino acid. Several selenoproteins are expressed in the brain, in which some of them, e.g. glutathione peroxidases (GPxs), thioredoxin reductases (TrxRs) or selenoprotein P (SelP), are strongly involved in antioxidant defence and in maintaining intercellular reducing conditions. Since increased oxidative stress has been implicated in neurological disorders, including Parkinson’s disease, Alzheimer’s disease, stroke, epilepsy and others, a growing body of evidence suggests that Se depletion followed by decreased activity of Se-dependent enzymes may be important factors connected with those pathologies. Undoubtedly, the remarkable progress that has been made in understanding the biological function of Se in the brain has opened up new potential possibilities for the treatment of neurological diseases by using Se as a potential drug. However, further research in the search for optimal Se donors is necessary in order to achieve an effective and safe therapeutic income.

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 systems development and function, and its aberrant activation contributes to pathomechanism of Alzheimers 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 3β. 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.

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

&NA; Neuroinflammation and oxidative stress are key intertwined pathological factors in many neurological, particularly neurodegenerative diseases, such as Alzheimers and Parkinsons 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 &mgr;g/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‐&agr; (TNF‐&agr;) 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. HighlightsSelol protected against LPS‐induced up‐regulation of brain proinflammatory cytokines.Selol prevented LPS‐evoked oxidized glutathione (GSSG) accumulation.Selol counteracted against LPS‐evoked down‐regulation of antioxidative enzymes.Selol may be therapeutic agent for inflammatory diseases of CNS.

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

Alterations of enzymes linked to arginine metabolism have been recently implicated in Alzheimers disease (AD). Despite strong association of arginine changes with nitric oxide (NO) pathway, the impact of amyloid β (Aβ) 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 Aβ 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 Aβ affects arginine metabolism and this influence might have important role in the pathomechanism of AD.

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.

Alzheimer’s amyloid-β peptide disturbs P2X7 receptor-mediated circadian oscillations of intracellular calcium

Recent data indicate that Alzheimers disease (AD) is associated with disturbances of the circadian rhythm in patients. We examined the effect of amyloid-β (Aβ) peptide, the main component of the senile plaques playing a critical role in the deregulation of calcium (Ca2+) homeostasis in AD, on the circadian oscillation of cytosolic calcium (Ca2+) levels in vitro. The experiments we carried out in human primary skin fibroblasts. This cell line was previously shown to exhibit circadian rhythms of clock genes. Moreover, the basic clock properties of these peripheral cells closely mimic those measured physiologically and behaviorally in human and do not change during aging. In this study we showed that i) cytosolic Ca2+ oscillations depend on the activation of purinergic P2X7 receptors; and ii) these oscillations are abolished in the presence of Aβ. In total, our new findings may help to deepen our understanding of the molecular mechanisms involved in AD-related circadian alterations.