Agata Adamczyk

Poly(ADP-ribose) Polymerase-1 in Amyloid Beta Toxicity and Alzheimer's Disease

Poly(ADP-ribose) polymerase-1 (PARP-1) is a key enzyme responsible for the maintenance of genome stability, transcriptional regulation, and long-term potentiation in neurons. However, the excessive activation of PARP-1 under pathological conditions may lead to an accumulation of poly(ADP-ribose) (PAR), a novel signaling molecule that induces programmed cell death, or to NAD depletion that induces energy crisis and necrotic cell death. PARP-1 is thought to be primarily a nuclear enzyme, but some data indicate that it can also be localized to the mitochondria where it is responsible for posttranslational modification of electron transport chain complexes and alteration of mitochondria function. The enhancement of PARP-1 activity and the accumulation of PAR were demonstrated in the brain of patients with Alzheimers disease (AD), particularly in neurons of the frontal and temporal lobes and in skin fibroblasts and lymphoblasts. Moreover, it has been reported that PARP-1 gene polymorphisms are highly associated with the development of AD. The activation of PARP-1 by oxidative stress seems to be an early and important event in the pathogenesis of AD. It is now widely accepted that the overproduction and oligomerization of amyloid β (Aβ) are responsible for the activation of a free radical cascade and oxidative stress in AD. Interestingly, the activity of PARP-1 is enhanced in AD and is also increased by Aβ peptides. The activation of PARP-1 by Aβ can lead to the PAR-mediated release of apoptosis-inducing factor from the mitochondria and its translocation to the nucleus, which leads to death of some populations of cells. A role of PARP-1 in the regulation of Aβ precursor protein metabolism processing and Aβ liberation has not been described previously. The study presented in this review indicated the relationship between PARP-1 activation, alteration of mitochondria function, and Aβ toxicity. The presented data should stimulate further studies on the role of PARP-1 in AD pathogenesis and thereby engage a new perspective regarding AD therapy.

Alpha-synuclein potentiates Ca2+ influx through voltage-dependent Ca2+ channels

Alpha-synuclein localized in synaptic terminals plays an important role in the pathogenesis of neurodegenerative diseases. The central domain of the protein, the nonamyloid component, is probably responsible for &agr;-synuclein toxicity. Here, we report that &agr;-synuclein and its nonamyloid component induced Ca2+ influx in rat synaptoneurosomes. The effect of &agr;-synuclein was eliminated by the N-type specific Ca2+ channel blocker, ω-conotoxin GVIA. The antioxidant, resveratrol, and the nitric oxide synthase inhibitor, Nω-nitro-L-arginine, did not prevent &agr;-synuclein-induced Ca2+ influx. Our findings indicate that &agr;-synuclein stimulated Ca2+ influx through N-type voltage-dependent Ca2+ channels by a mechanism other than free radicals. A direct interaction between &agr;-synuclein and N-type Ca2+ channels could be responsible for their effects on Ca2+ influx through voltage-dependent Ca2+ channels.

alpha-Synuclein enhances secretion and toxicity of amyloid beta peptides in PC12 cells.

alpha-Synuclein is the fundamental component of Lewy bodies which occur in the brain of 60% of sporadic and familial Alzheimers disease patients. Moreover, a proteolytic fragment of alpha-synuclein, the so-called non-amyloid component of Alzheimers disease amyloid, was found to be an integral part of Alzheimers dementia related plaques. However, the role of alpha-synuclein in pathomechanism of Alzheimers disease remains elusive. In particular, the relationship between alpha-synuclein and amyloid beta is unknown. In the present study we showed the involvement of alpha-synuclein in amyloid beta secretion and in the mechanism of amyloid beta evoked mitochondria dysfunction and cell death. Rat pheochromocytoma PC12 cells transfected with amyloid beta precursor protein bearing Swedish double mutation (APPsw) and control PC12 cells transfected with empty vector were used in this study. alpha-Synuclein (10microM) was found to increase by twofold amyloid beta secretion from control and APPsw PC12 cells. Moreover, alpha-synuclein decreased the viability of PC12 cells by about 50% and potentiated amyloid beta toxicity leading to mitochondrial dysfunction and caspase-dependent programmed cell death. Inhibitor of caspase-3 (Z-DEVD-FMK, 100microM), and a mitochondrial permeability transition pore blocker, cyclosporine A (2microM) protected PC12 cells against alpha-synuclein or amyloid beta evoked cell death. In contrast Z-DEVD-FMK and cyclosporine A were ineffective in APPsw cells containing elevated amount of amyloid beta treated with alpha-synuclein. It was found that the inhibition of neuronal and inducible nitric oxide synthase reversed the toxic effect of alpha-synuclein in control but not in APPsw cells. Our results indicate that alpha-synuclein enhances the release and toxicity of amyloid beta leading to nitric oxide mediated irreversible mitochondria dysfunction and caspase-dependent programmed cell death.

Extracellular α-Synuclein Leads to Microtubule Destabilization via GSK-3β-Dependent Tau Phosphorylation in PC12 Cells

α-Synuclein (ASN) plays an important role in pathogenesis of Parkinsons disease (PD) and other neurodegenerative disorders. Novel and most interesting data showed elevated tauopathy in PD and suggested relationship between ASN and Tau protein. However, the mechanism of ASN-evoked Tau protein modification is not fully elucidated. In this study we investigated the role of extracellular ASN in Tau hyperphosphorylation in rat pheochromocytoma (PC12) cells and the involvement of glycogen synthase kinase-3β (GSK-3β) and cyclin-dependent kinase 5 (CDK5) in ASN-dependent Tau modification. Our results indicated that exogenously added ASN increases Tau phosphorylation at Ser396. Accordingly, the GSK-3β inhibitor (SB-216763) prevented ASN-evoked Tau hyperphosphorylation, but the CDK5 inhibitor had no effect. Moreover, western blot analysis showed that ASN affected GSK-3β via increasing of protein level and activation of this enzyme. GSK-3β activity evaluated by its phosphorylation status assay showed that ASN significantly increased the phosphorylation of this enzyme at Tyr216 with parallel decrease in phosphorylation at Ser9, indicative of stimulation of GSK-3β activity. Moreover, the effect of ASN on microtubule (MT) destabilization and cell death with simultaneous the involvement of GSK-3β in these processes were analyzed. ASN treatment increased the amount of free tubulin and concomitantly reduced the amount of polymerized tubulin and SB-216763 suppressed these ASN-induced changes in tubulin, indicating that GSK-3β is involved in ASN-evoked MT destabilization. ASN-induced apoptotic processes lead to decrease in PC12 cells viability and SB-216763 protected those cells against ASN-evoked cytotoxicity. Concluding, extracellular ASN is involved in GSK-3β-dependent Tau hyperphosphorylation, which leads to microtubule destabilization. GSK-3β inhibition may be an effective strategy for protecting against ASN-induced cytotoxicity.

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.

α-Synuclein induced cell death in mouse hippocampal (HT22) cells is mediated by nitric oxide-dependent activation of caspase-3

Our previous studies indicated that exogenous α‐synuclein (ASN) activates neuronal nitric oxide (NO) synthase (nNOS) in rat brain slices. The present study, carried out on immortalized hippocampal neuronal cells (HT22), was designed to extend the previous results by showing the molecular pathway of NO‐mediated cell death induced by exogenous ASN. Extracellular ASN (10 μM) was found to stimulate nitric oxide synthase (NOS) and increase caspase‐3 activity in HT22 cells, leading to poly(ADP‐ribose) polymerase (PARP‐1) cleavage. The inhibitor of Ca2+‐dependent NOS (N‐nitro‐l‐arginine, 100 μM) prevented ASN‐evoked caspase‐3 activation and PARP‐1 degradation. ASN exposure resulted in apoptotic death of HT22 cells and this effect was reversed by inhibition of NO synthesis and caspase‐3 activity. Our results demonstrated that extracellular ASN induces neuronal cell death by NO‐mediated caspase‐3 activation.

Effect of N-methyl-D-aspartate (NMDA) receptor antagonists on α-synuclein-evoked neuronal nitric oxide synthase activation in the rat brain

alpha-Synuclein (ASN), a small presynaptic protein that is abundant in the brain, is implicated in the pathogenesis of neurodegenerative disorders including Parkinsons and Alzheimers disease. The central domain of alpha-synuclein, the non-amyloid beta component of the Alzheimers disease amyloid (NAC) is probably responsible for its toxicity. However, the molecular mechanism of alpha-synuclein action remains largely elusive. The present study examined the effect of alpha-synuclein and the NAC peptide on nitric oxide synthase (NOS) activity in rat brain cortical and hippocampal slices using a radiochemical technique. Moreover, nitrite levels in brain slices incubated in the presence of alpha-synuclein were measured using the Griess reaction. ASN and the NAC stimulated NOS activity by about 70% and 40%, respectively. beta-Synuclein, a homologous protein of ASN that lacks the NAC domain, had no effect on NOS activity. Under the same experimental conditions, alpha-synuclein increased nitrite levels by 27%. alpha-Synuclein and the NAC affected the activity of constitutive neuronal isoform of NOS, but had no impact on the endothelial or inducible NOS isoforms. The effect of alpha-synuclein and the NAC peptide on NOS activity was inhibited by MK-801 and APV, antagonists of the NMDA receptor. These results indicate that the NMDA receptor plays an important role in alpha-synuclein-evoked nitric oxide synthesis. We suggest that nitric oxide liberated by the over-activated neuronal isoform of NOS could react with superoxide to form peroxynitrite, which modulates the function of a variety of biomolecules including proteins, lipids, and DNA.

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.

Perinatal exposure to lead (Pb) promotes Tau phosphorylation in the rat brain in a GSK-3β and CDK5 dependent manner: Relevance to neurological disorders

Hyperphosphorylation of Tau is involved in the pathomechanism of neurological disorders such as Alzheimers, Parkinsons diseases as well as Autism. Epidemiological data suggest the significance of early life exposure to lead (Pb) in etiology of disorders affecting brain function. However, the precise mechanisms by which Pb exerts neurotoxic effects are not fully elucidated. The purpose of this study was to evaluate the effect of perinatal exposure to low dose of Pb on the Tau pathology in the developing rat brain. Furthermore, the involvement of two major Tau-kinases: glycogen synthase kinase-3 beta (GSK-3β) and cyclin-dependent kinase 5 (CDK5) in Pb-induced Tau modification was evaluated. Pregnant female rats were divided into control and Pb-treated group. The control animals were maintained on drinking water while females from the Pb-treated group received 0.1% lead acetate (PbAc) in drinking water, starting from the first day of gestation until weaning of the offspring. During the feeding of pups, mothers from the Pb-treated group were still receiving PbAc. Pups of both groups were weaned at postnatal day 21 and then until postnatal day 28 received only drinking water. 28-day old pups were sacrificed and Tau mRNA and protein level as well as Tau phosphorylation were analyzed in forebrain cortex (FC), cerebellum (C) and hippocampus (H). Concomitantly, we examined the effect of Pb exposure on GSK-3β and CDK5 activation. Our data revealed that pre- and neonatal exposure to Pb (concentration of Pb in whole blood below 10μg/dL, considered safe for humans) caused significant increase in the phosphorylation of Tau at Ser396 and Ser199/202 with parallel rise in the level of total Tau protein in FC and C. Tau hyperphosphorylation in Pb-treated animals was accompanied by elevated activity of GSK-3β and CDK5. Western blot analysis revealed activation of GSK-3β in FC and C as well as CDK5 in C, via increased phosphorylation of Tyr-216 and calpain-dependent p25 formation, respectively. In conclusion, perinatal exposure to Pb up-regulates Tau protein level and induces Tau hyperphosphorylation in the rat brain cortex and cerebellum. We suggest that neurotoxic effect of Pb might be mediated, at least in part, by GSK-3β and CDK5-dependent Tau hyperphosphorylation, which may lead to the impairment of cytoskeleton stability and neuronal dysfunction.

A novel mechanism of non-Aβ component of Alzheimer's disease amyloid (NAC) neurotoxicity. Interplay between p53 protein and cyclin-dependent kinase 5 (Cdk5).

The non-Aβ component of Alzheimers disease (AD) amyloid (NAC) is produced from the precursor protein NACP/α-synuclein (ASN) by till now unknown mechanism. Previous study showed that like ASN, NAC peptide induced oxidative/nitrosative stress and apoptosis. Our present study focused on the mechanisms of PC12 cells death evoked by NAC peptide, with particular consideration on the role of p53 protein. On the basis of molecular and transmission electron microscopic (TEM) analysis it was found that exogenous NAC peptide (10 μM) caused mitochondria dysfunction, enhanced free radical generation, and induced both apoptotic and autophagic cell death. Morphological and immunocytochemical evidence from TEM showed marked changes in expression and in translocation of proapoptotic protein Bax. We also observed time-dependent enhancement of Tp53 gene expression after NAC treatment. Free radicals scavenger N-tert-butyl-alpha-phenylnitrone (PBN, 1 mM) and p53 inhibitor (α-Pifithrin, 20 μM) significantly protected PC12 cells against NAC peptide-evoked cell death. In addition, exposure to NAC peptide resulted in higher expression of cyclin-dependent kinase 5 (Cdk5), one of the enzymes responsible for p53 phosphorylation and activation. Concomitantly, we observed the increase of expression of Cdk5r1 and Cdk5r2 genes, coding p35 and p39 peptides that are essential regulators of Cdk5 activity. Moreover, the specific Cdk5 inhibitor (BML-259, 10 μM) protected large population of cells against NAC-evoked cell death. Our findings indicate that NAC peptide exerts its toxic effect by activation of p53/Cdk5 and Bax-dependent apoptotic signaling pathway.