Anna Kaźmierczak

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.

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.

[The role of extracellular α-synuclein in molecular mechanisms of cell death].

Recently published data demonstrated that increased release, oligomerization and toxicity of α-synuclein (ASN) is a key molecular process in pathophysiology of neurodegenerative diseases classified as synucleinopathies (e.g. Parkinson disease or Alzheimers disease). It was proved that the excessive release of ASN into the extracellular space, driven by environmental factors as well as neurodegeneration, may have a significant role in the spread of the neurodegeneration process within the brain. Extracellular ASN was shown to be toxic both to neurons and glial cells and the mechanism of its action depends on the concentration of this protein in the extracellular space. Exogenous ASN leads to the activation of plasma membrane receptors, causes increased calcium influx, and stimulates the synthesis of proinflammatory cytokines and nitric oxide, which in turn leads to activation of programmed cell death. These data provide new insights into the involvement of ASN in the neurodegenerative diseases, and thus can serve effectively for the development of their new therapy.

The molecular mechanisms underlying alpha-synuclein-evoked cell death; possible implications for the pathogenesis of Parkinson’s disease

alpha-Synuclein (ASN) play a key role in pathogenesis of Parkinson’s disease (PD) and is implicated in the other neurodegenerative disorders. However, the underlying mechanism by which ASN affects neuronal function and death remains unknown. Our previous data indicated that ASN is secreted from synaptic endings into extracellular space and this pool of the protein is involved in dopaminergic cell death and may have a role in the propagation of synuclein pathology and progression of PD. A number of mechanisms have been proposed to explain the toxic effects of ASN. Our own data indicate that ASN leads to nitric oxide (NO) mediated mitochondria dysfunction and caspase-dependent programmed cell death. Previously, we have shown that ASN enhanced the release and toxicity of amyloid beta peptide and indicated an importance of ASN/amyloid beta interaction in neurodegeneration processes. Novel and most interesting data suggested the significance of ASN in Tau phosphorylation and microtubule instability, which could be involved in the mechanisms of dopaminergic cell death in PD brain. However, the exact relationship between ASN and Tau is unknown. Our preliminary data indicated that extracellular ASN affects glycogen synthase kinase-3beta (Gsk-3beta) and cyclin-dependent kinase 5 (Cdk5), two major protein kinases involved in abnormal phosphorylation of Tau. Herein we show that ASN activates phosphorylation of Cdk5 on tyrosine 15 (Tyr15) that stimulates Cdk5 activity. This phosphorylation has been described as a key point in controlling the activation of Cdk5. In addition, ASN affects Cdk5-dependent phosphorylation of Gsk-3beta. Our data suggest an important functional link between Cdk5 and Gsk-3beta. The specific and non-specific Cdk5 inhibitors (BML-259 and roscovitine) protect dopaminergic PC12 cells against ASN-evoked cells death. Summarizing, our results indicate that extracellular ASN-evoked NO dependent mitochondria dysfunction play important role in dopaminergic cells death. Moreover, our last data demonstrate that proapoptotic effects of ASN might be in part mediated via activation of Cdk5. Thus, extracellulary acting ASN may be an important factor in degeneration processes and it may provide therapeutic target for retarding the progression of the neurodegeneration.