Ceramide in the Molecular Mechanisms of Neuronal Cell Death. The Role of Sphingosine-1-Phosphate

Ceramide and sphingosine-1-phosphate (S1P), two important bioactive sphingolipids, have been suggested as being key players in the pathology of Alzheimer’s disease in inflammation and cancer. However, their role in the molecular mechanisms of neuronal death has not been fully elucidated. Our study indicated that ceramide significantly enhanced the level of free radicals and decreased the viability of the human neuroblastoma cell line (SH-SY5Y) through inhibition of the prosurvival PI3-K/Akt pathway. Ceramide also decreased anti-apoptotic (Bcl-2) and increased pro-apoptotic (Bax, Hrk) mRNA/protein levels. Concomitantly, our study indicated that ceramide induced poly(ADP-ribose) polymerase-1 (PARP-1) activation and accumulation of poly(ADP-ribose) PAR, a signalling molecule involved in mitochondria-nucleus cross-talk and mitochondria integrity. Ceramide treatment significantly decreased the level of apoptosis-inducing factor (AIF) in the mitochondria. The PARP-1 inhibitor (PJ-34) prevented AIF release from the mitochondria. In addition, our data showed that exogenously added S1P increased the viability of SH-SY5Y through the S1P (1,3) receptor-dependent mechanism. It was also revealed that the S1P and PARP-1 inhibitor (PJ-34) decreased oxidative stress, gene expression of the pro-apoptotic Hrk protein and up-regulated the anti-apoptotic Bcl-2 protein. Our data demonstrate that neuronal cell death evoked by ceramide is regulated by PARP/PAR/AIF and by S1P receptor signalling. In summary, our results suggest that PARP-1 inhibitor(s) and modulators of sphingosine-1-phosphate receptor(s) should be considered in potential therapeutic strategies directed at neurodegenerative diseases

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

AbstractOur 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 (10μM) 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, 100μM) 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

Lipoxygenases and Poly(ADP-Ribose) Polymerase in Amyloid Beta Cytotoxicity

The 12/15-lipoxygenase(s) (LOX), poly(ADP-ribose) polymerase (PARP-1) activity and mitochondrial apoptosis inducing factor (AIF) protein in the amyloid β (Aβ) toxicity were investigated in PC12 cells that express either wild-type (APPwt) or double Swedish mutation (APPsw) forms of human Aβ precursor protein. Different levels of Aβ secretion and free radicals formation characterize these cells. The results demonstrated a relationship between the Aβ levels and LOX protein expression and activity. High Aβ concentration in APPsw cells correlated with a significant increase in free radicals and LOX activation, which leads to translocation of p65/NF-κB into the nucleus. An increase in AIF expression in mitochondria was observed concurrently with inhibition of PARP-1 activity in the nuclear fraction of APPsw cells. We suggested that AIF accumulation in mitochondria may be involved in adaptive/protective processes. However, inhibition of PARP-1 may be responsible for the disturbances in transcription and DNA repair as well as the degeneration of APP cells. Under conditions of increased nitrosative stress, evoked by the nitric oxide donor, sodium nitroprusside (SNP, 0.5 mM), 70–80% of all cells types died after 24 h, significantly more in APPsw cells. There was no further significant change in mitochondrial AIF level and PARP-1 activity compared to corresponding non-treated cells. Only one exception was observed in PC12 control, where SNP significantly inhibits PARP-1 activity. Moreover, SNP significantly activated gene expression for 12/15-LOX in all types of investigated cells. Inhibitors of all LOX isoforms and specific inhibitor of 12-LOX enhanced the survival of cells that were subjected to SNP. We conclude that the LOX pathways may play a role in Aβ toxicity and in nitrosative-stress-induced cell death and that inhibition of these pathways offers novel protective strategies

Znaczenie cyklooksygenaz w neurotoksyczności peptydów amyloidu β w chorobie Alzheimera

Przewlekłe procesy zapalne odgrywają istotną rolę w przebiegu choroby Alzheimera (ChA). Dotychczas uważano, że rozwijają się one w odpowiedzi na zewnątrzkomórkowe agregaty peptydów amyloidu β (Aβ) odkładające się w mózgu chorych na ChA. Badania epidemiologiczne wykazały, że długotrwałe stosowanie niesteroidowych leków przeciwzapalnych (NLPZ) zmniejsza ryzyko zachorowania na ChA. Leki te są inhibitorami cyklooksygenaz (COX), enzymów syntetyzujących eikozanoidy, główny składnik odpowiedzi zapalnej w organizmie. Najnowsze badania przyniosły dwa zaskakujące odkrycia: 1) toksyczną formą Aβ nie są agregaty, lecz raczej oligomery Aβ, występujące zarówno wewnątrzkomórkowe, jak i zewnątrzkomórkowo; 2) aktywacja COX-2 zachodzi na wczesnych etapach rozwoju ChA i poprzedza powstawanie blaszek starczych oraz aktywację mikrogleju. Odkrycia te wskazują na możliwy udział COX w toksyczności peptydów Aβ w komórkach nerwowych w I fazie ChA, a nie tylko w rozwoju odpowiedzi zapalnej w komórkach glejowych w zaawansowanym stadium choroby. Uzyskane wyniki wskazują na konieczność dalszych badań nad rolą COX w patogenezie/patomechanizmie ChA

Inhibition of poly(ADP-ribose) polymerase-1 attenuates the toxicity of carbon tetrachloride

Carbon tetrachloride (CCl4) is routinely used as a model compound for eliciting centrilobular hepatotoxicity. It can be bioactivated to the trichloromethyl radical, which causes extensive lipid peroxidation and ultimately cell death by necrosis. Overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) can rapidly reduce the levels of (β-nicotinamide adenine dinucleotide and adenosine triphosphate and ultimately promote necrosis. The aim of this study was to determine whether inhibition of PARP-1 could decrease CCl4-induced hepatotoxicity, as measured by degree of poly(ADP-ribosyl)ation, serum levels of lactate dehydrogenase (LDH), lipid peroxidation,and oxidative DNA damage. For this purpose, male ICR mice were administered intraperitoneally a hepatotoxic dose of CCl4 with or without 6(5H)-phenanthridinone, a potent inhibitor of PARP-1. Animals treated with CCl4 exhibited extensive poly(ADP-ribosyl)ation in centrilobular hepatocytes, elevated serum levels of LDH, and increased lipid peroxidation. In contrast, animals treated concomitantly with CCl4 and 6(5H)-phenanthridinone showed significantly lower levels of poly(ADP-ribosyl) ation, serum LDH, and lipid peroxidation. No changes were observed in the levels of oxidative DNA damage regardless of treatment. These results demonstrated that the hepatotoxicity of CCl4is dependent on the overactivation of PARP-1 and that inhibition of this enzyme attenuates the hepatotoxicity of CCl4

Insights into mitochondrial dysfunction: aging, amyloid-β, and tau-A deleterious trio

Significance: Alzheimer's disease (AD) is an age-related progressive neurodegenerative disorder mainly affecting elderly individuals. The pathology of AD is characterized by amyloid plaques (aggregates of amyloid-β [Aβ]) and neurofibrillary tangles (aggregates of tau), but the mechanisms underlying this dysfunction are still partially unclear. Recent Advances: A growing body of evidence supports mitochondrial dysfunction as a prominent and early, chronic oxidative stress-associated event that contributes to synaptic abnormalities and, ultimately, selective neuronal degeneration in AD. Critical Issues: In this review, we discuss on the one hand whether mitochondrial decline observed in brain aging is a determinant event in the onset of AD and on the other hand the close interrelationship of this organelle with Aβ and tau in the pathogenic process underlying AD. Moreover, we summarize evidence from aging and Alzheimer models showing that the harmful trio "aging, Aβ, and tau protein" triggers mitochondrial dysfunction through a number of pathways, such as impairment of oxidative phosphorylation (OXPHOS), elevation of reactive oxygen species production, and interaction with mitochondrial proteins, contributing to the development and progression of the disease. Future Directions: The aging process may weaken the mitochondrial OXPHOS system in a more general way over many years providing a basis for the specific and destructive effects of Aβ and tau. Establishing strategies involving efforts to protect cells at the mitochondrial level by stabilizing or restoring mitochondrial function and energy homeostasis appears to be challenging, but very promising route on the horizon

PARP1 gene variation and microglial activity on [11C]PBR28 PET in older adults at risk for Alzheimer's disease

Increasing evidence suggests that inflammation is one pathophysio-logical mechanism in Alzheimer's disease (AD). Recent studies have identified an association between the poly (ADP-ribose) polymerase 1 (PARP1) gene and AD. This gene encodes a protein that is involved in many biological functions, including DNA repair and chromatin remodeling, and is a mediator of inflammation. Therefore, we performed a targeted genetic association analysis to investigate the relationship between the PARP1 polymorphisms and brain micro-glial activity as indexed by [11C]PBR28 positron emission tomography (PET). Participants were 26 non-Hispanic Caucasians in the Indiana Memory and Aging Study (IMAS). PET data were intensity-normalized by injected dose/total body weight. Average PBR standardized uptake values (SUV) from 6 bilateral regions of interest (thalamus, frontal, parietal, temporal, and cingulate cortices, and whole brain gray matter) were used as endophenotypes. Single nucleotide polymorphisms (SNPs) with 20% minor allele frequency that were within +/− 20 kb of the PARP1 gene were included in the analyses. Gene-level association analyses were performed using a dominant genetic model with translocator protein (18-kDa) (TSPO) genotype, age at PET scan, and gender as covariates. Analyses were performed with and without APOE ε4 status as a covariate. Associations with PBR SUVs from thalamus and cingulate were significant at corrected p<0.014 and <0.065, respectively. Subsequent multi-marker analysis with cingulate PBR SUV showed that individuals with the “C” allele at rs6677172 and “A” allele at rs61835377 had higher PBR SUV than individuals without these alleles (corrected P<0.03), and individuals with the “G” allele at rs6677172 and “G” allele at rs61835377 displayed the opposite trend (corrected P<0.065). A previous study with the same cohort showed an inverse relationship between PBR SUV and brain atrophy at a follow-up visit, suggesting possible protective effect of microglial activity against cortical atrophy. Interestingly, all 6 AD and 2 of 3 LMCI participants in the current analysis had one or more copies of the “GG” allele combination, associated with lower cingulate PBR SUV, suggesting that this gene variant warrants further investigation

Figure S2: Controls proving the poly-ADP-ribose (PAR) identity in mice sciatic nerves

Background Poly-ADP-ribose (PAR) is a polymer synthesized by poly-ADP-ribose polymerases (PARPs) as a postranslational protein modification and catabolized mainly by poly-ADP-ribose glycohydrolase (PARG). In spite of the existence of cytoplasmic PARPs and PARG, research has been focused on nuclear PARPs and PAR, demonstrating roles in the maintenance of chromatin architecture and the participation in DNA damage responses and transcriptional regulation. We have recently detected non-nuclear PAR structurally and functionally associated to the E-cadherin rich zonula adherens and the actin cytoskeleton of VERO epithelial cells. Myelinating Schwann cells (SC) are stabilized by E-cadherin rich autotypic adherens junctions (AJ). We wondered whether PAR would map to these regions. Besides, we have demonstrated an altered microfilament pattern in peripheral nerves of Trembler-J (Tr-J) model of CMT1-E. We hypothesized that cytoplasmic PAR would accompany such modified F-actin pattern. Methods Wild-type (WT) and Tr-J mice sciatic nerves cryosections were subjected to immunohistofluorescence with anti-PAR antibodies (including antibody validation), F-actin detection with a phalloidin probe and DAPI/DNA counterstaining. Confocal image stacks were subjected to a colocalization highlighter and to semi-quantitative image analysis. Results We have shown for the first time the presence of PAR in sciatic nerves. Cytoplasmic PAR colocalized with F-actin at non-compact myelin regions in WT nerves. Moreover, in Tr-J, cytoplasmic PAR was augmented in close correlation with actin. In addition, nuclear PAR was detected in WT SC and was moderately increased in Tr-J SC. Discussion The presence of PAR associated to non-compact myelin regions (which constitute E-cadherin rich autotypic AJ/actin anchorage regions) and the co-alterations experienced by PAR and the actin cytoskeleton in epithelium and nerves, suggest that PAR may be a constitutive component of AJ/actin anchorage regions. Is PAR stabilizing the AJ-actin complexes? This question has strong implications in structural cell biology and cell signaling networks. Moreover, if PAR played a stabilizing role, such stabilization could participate in the physiological control of axonal branching. PARP and PAR alterations exist in several neurodegenerative pathologies including Alzheimer’s, Parkinson’s and Hungtington’s diseases. Conversely, PARP inhibition decreases PAR and promotes neurite outgrowth in cortical neurons in vitro. Coherently, the PARP inhibitor XAV939 improves myelination in vitro, ex vivo and in vivo. Until now such results have been interpreted in terms of nuclear PARP activity. Our results indicate for the first time the presence of PARylation in peripheral nerve fibers, in a healthy environment. Besides, we have evidenced a PARylation increase in Tr-J, suggesting that the involvement of cytoplasmic PARPs and PARylation in normal and neurodegenerative conditions should be re-evaluated

Oxidative stress induced by the Fe2+/ascorbic acid system or model ischemia in vitro: effect of carvedilol and pyridoindole antioxidant SMe1EC2 in young and adult rat brain tissue

New effective strategies and new highly effective neuroprotective agents are being searched for the therapy of human stroke and cerebral ischemia. The compound SMe1EC2 is a new derivative of stobadine, with enhanced antioxidant properties compared to the maternal drug. Carvedilol, a non-selective beta-blocker, possesses besides its cardioprotective and vasculoprotective properties also an antioxidant effect. We compared the effect of carvedilol and SMe1EC2, antioxidants with a similar chemical structure, in two experimental models of oxidative stress in young and adult rat brain tissue. SMe1EC2 was found to improve the resistance of hippocampal neurons to ischemia in vitro in young and even in 18-month-old rats and inhibited formation of protein carbonyl groups induced by the Fe2+/ascorbic acid pro-oxidative system in brain cortex homogenates of young rats. Carvedilol exerted a protective effect only in the hippocampus of 2-month-old rats and that at the concentration 10-times higher than did SMe1EC2. The inhibitory effect of carvedilol on protein carbonyl formation induced by the pro-oxidative system was not proved in the cortex of either young or adult rats. An increased baseline level of the content of protein carbonyl groups in the adult versus young rat brain cortex confirmed age-related changes in neuronal tissue and may be due to increased production of reactive oxygen species and low antioxidant defense mechanisms in the adult rat brain. The results revealed the new pyridoindole SMe1EC2 to be more effective than carvedilol in neuroprotection of rat brain tissue in both experimental models involving oxidative stress