Mutations in the PTEN-induced kinase 1 (PINK1) gene have recently been implicated in autosomal recessive early onset Parkinson Disease (1, 2). To investigate the role of PINK1 in neurodegeneration, we designed human and murine neuronal cell lines expressing either wild-type PINK1 or PINK1 bearing a mutation associated with Parkinson Disease. We show that under basal and staurosporine-induced conditions, the number of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive cells was lower in wild-type PINK1 expressing SH-SY5Y cells than in mock-transfected cells. This phenotype was due to a PINK1-mediated reduction in cytochrome c release from mitochondria, which prevents subsequent caspase-3 activation. We show that overexpression of wild-type PINK1 strongly reduced both basal and staurosporine-induced caspase 3 activity. Overexpression of wild-type PINK1 also reduced the levels of cleaved caspase-9, caspase-3, caspase-7, and activated poly(ADP-ribose) polymerase under both basal and staurosporine-induced conditions. In contrast, Parkinson disease-related mutations and a kinase-inactive mutation in PINK1 abrogated the protective effect of PINK1. Together, these results suggest that PINK1 reduces the basal neuronal pro-apoptotic activity and protects neurons from staurosporine-induced apoptosis. Loss of this protective function may therefore underlie the degeneration of nigral dopaminergic neurons in patients with PINK1 mutations.
Abstract Inflammation is considered as a major contributor to brain injury following cerebral ischemia. The therapeutic potential of both MLC601/MLC901, which are herbal extract preparations derived from Chinese Medicine, has been reported both in advanced stroke clinical trials and also in animal and cellular models. The aim of this study was to investigate the effects of MLC901 on the different steps of post-ischemic inflammation in focal ischemia in mice. In vivo injury was induced by 60 minutes of middle cerebral artery occlusion (MCAO) followed by reperfusion. MLC901 was administered in post-treatment 90 min after the onset of ischemia and once a day during reperfusion. MLC901 treatment resulted in a reduction in infarct volume, a decrease of Blood Brain Barrier leakage and brain swelling, an improvement in neurological scores and a reduction of mortality rate at 24 hours after MCAO. These beneficial effects of MLC901 were accompanied by an inhibition of astrocytes and microglia/macrophage activation, a drastically decreased neutrophil invasion into the ischemic brain as well as by a negative regulation of pro-inflammatory mediator expression (cytokines, chemokines, matrix metalloproteinases). MLC901 significantly inhibited the expression of Prx6 as well as the transcriptional activity of NFκB and the activation of Toll-like receptor 4 (TLR4) signaling, an important pathway in the immune response in the ischemic brain. MLC901 effects on the neuroinflammation cascade induced by cerebral ischemia probably contribute, in a very significant way, in its potential therapeutic value.
It is widely accepted that Aβ plays a pivotal role in the pathogenesis of Alzheimer's disease (AD) [27]. Attention has been focused mainly on how extracellular Aβ exerts its effects on neuronal cells [7,11,16,32]. However, neuronal degeneration from
Alzheimers disease is characterized by the extracellular deposition of the amyloid β-peptide that derives from its precursor bAPP by sequential actions of β- and γ- secretases, respectively. Recent studies aimed at identifying these enzymes have been reported as it is thougth that their inhibition should hopefully lead to reduce Aβ load in the AD brains. β-secretase seems to be due to BACE1, a novel membrane-bound aspartyl protease. γ-secretase identification is still a matter of controversy. Invalidation of presenilin genes was reported to impair both γ-secretase-mediated Aβ production and Notch cleavage leading to NICD production. This observation together with another biochemical and pharmacological evidences led to suggest that presenilins could be the genuine long-searched γ-secretase that would be responsible for both APP and Notch cleavages. We have designed novel non peptidic potential inhibitors of g-secretase (referred to as JLK inhibitors) and examined their ability to prevent Aβ40 and Aβ42 secretions as well as NICD production. Three out of a series of these agents drastically lower the recoveries of both Aβ40 and Aβ42 produced by bAPP-expressing cell lines and concomitantly protect intracellular C99 and C83 recoveries. These inhibitors also prevent Aβ40/42 productions by C99-expressing cells. Interestingly, these inhibitors were totally unable to affect the DENotch cleavage leading to NICD generation. Here, we also further characterize the pharmacological properties and specificity of these JLK inhibitors. Keywords: amyloidogenic cascade, neurodegeneration, Notch cleavage, c-cadherins, fluorimetric assays, gsk inhibitor
The oxidation of the ribose ring of cytidine-5'-diphospho-choline (CDPC) by periodate to produce reactive dialdehydes was used to couple this activated reagent onto various proteins, onto Ficoll 400 and onto Sepharose 4B. Careful control of parameters of the different steps of the reaction enabled us to synthesize conjugates with a graded number of nucleotide residues. Human serum albumin conjugates with a relatively high degree of substitution were used to demonstrate and to measure anti-phosphorylcholine antibodies and the acute phase reactant, C-reactive protein by precipitation or passive haemagglutination techniques. These methods for measuring C-reactive protein in serum of patient suffering from acute inflammation may be useful for clinicians. CDPC-AH Sepharose was used to purify the phosphorylcholine-binding myeloma protein HOPC8 and to separate C-reactive protein from the bulk of serum proteins. Improvements of this technique will certainly lead to the complete purification of C-reactive protein.
Others and we have shown that T cells have an important role in hippocampal synaptic plasticity, including neurogenesis in the dentate gyrus, spinogenesis, and glutamatergic synaptic function in the CA of the hippocampus. Hippocampus plasticity is particularly involved in the brain effects of the enriched environment (EE), and interestingly CD4+ and CD8+ T cells play essential and differential roles in these effects. However, the precise mechanisms by which they act on the brain remain elusive.We searched for a putative mechanism of action by which CD4+ T cells could influence brain plasticity and hypothesized that they could regulate protein transport at the level of the blood-CSF barrier in the choroid plexus.We compared mice housed in EE and deprived of CD4+ T cells using a depleting antibody with a control group injected with the control isotype. We analyzed in the hippocampus the gene expression profiles using the Agilent system, and the expression of target proteins in plasma, CSF, and the choroid plexus using ELISA.We show that CD4+ T cells may influence EE-induced hippocampus plasticity via thyroid hormone signaling by regulating in the choroid plexus the expression of transthyretin, the major transporter of thyroxine (T4) to the brain parenchyma.Our study highlights the contribution of close interactions between the immune and neuroendocrine systems in brain plasticity and function.
Secreted phospholipase A2-IIA (sPLA2-IIA) hydrolyzes phospholipids to liberate lysophospholipids and fatty acids. Given its poor activity toward eukaryotic cell membranes, its role in the generation of proinflammatory lipid mediators is unclear. Conversely, sPLA2-IIA efficiently hydrolyzes bacterial membranes. Here, we show that sPLA2-IIA affects the immune system by acting on the intestinal microbial flora. Using mice overexpressing transgene-driven human sPLA2-IIA, we found that the intestinal microbiota was critical for both induction of an immune phenotype and promotion of inflammatory arthritis. The expression of sPLA2-IIA led to alterations of the intestinal microbiota composition, but housing in a more stringent pathogen-free facility revealed that its expression could affect the immune system in the absence of changes to the composition of this flora. In contrast, untargeted lipidomic analysis focusing on bacteria-derived lipid mediators revealed that sPLA2-IIA could profoundly alter the fecal lipidome. The data suggest that a singular protein, sPLA2-IIA, produces systemic effects on the immune system through its activity on the microbiota and its lipidome.
