Guoying Bing

Role of the prolyl isomerase Pin1 in protecting against age-dependent neurodegeneration

The neuropathological hallmarks of Alzheimers disease and other tauopathies include senile plaques and/or neurofibrillary tangles. Although mouse models have been created by overexpressing specific proteins including β-amyloid precursor protein, presenilin and tau, no model has been generated by gene knockout. Phosphorylation of tau and other proteins on serine or threonine residues preceding proline seems to precede tangle formation and neurodegeneration in Alzheimers disease. Notably, these phospho(Ser/Thr)-Pro motifs exist in two distinct conformations, whose conversion in some proteins is catalysed by the Pin1 prolyl isomerase. Pin1 activity can directly restore the conformation and function of phosphorylated tau or it can do so indirectly by promoting its dephosphorylation, which suggests that Pin1 is involved in neurodegeneration; however, genetic evidence is lacking. Here we show that Pin1 expression is inversely correlated with predicted neuronal vulnerability and actual neurofibrillary degeneration in Alzheimers disease. Pin1 knockout in mice causes progressive age-dependent neuropathy characterized by motor and behavioural deficits, tau hyperphosphorylation, tau filament formation and neuronal degeneration. Thus, Pin1 is pivotal in protecting against age-dependent neurodegeneration, providing insight into the pathogenesis and treatment of Alzheimers disease and other tauopathies.

p38 kinase is activated in the Alzheimer's disease brain.

Abstract: The p38 mitogen‐activated protein kinase is a stress‐activated enzyme responsible for transducing inflammatory signals and initiating apoptosis. In the Alzheimers disease (AD) brain, increased levels of phosphorylated (active) p38 were detected relative to age‐matched normal brain. Intense phospho‐p38 immunoreactivity was associated with neuritic plaques, neuropil threads, and neurofibrillary tangle‐bearing neurons. The antibody against phosphorylated p38 recognized many of the same structures as an antibody against aberrantly phosphorylated, paired helical filament (PHF) tau, although PHF‐positive tau did not cross‐react with the phospho‐p38 antibody. These findings suggest a neuroinflammatory mechanism in the AD brain, in which aberrant protein phosphorylation affects signal transduction elements, including the p38 kinase cascade, as well as cytoskeletal components.

Inflammation induces mitochondrial dysfunction and dopaminergic neurodegeneration in the nigrostriatal system

Evidence suggests that chronic inflammation, mitochondrial dysfunction, and oxidative stress play significant and perhaps synergistic roles in Parkinson’s disease (PD), where the primary pathology is significant loss of the dopaminergic neurons in the substantia nigra. The use of anti‐inflammatory drugs for PD treatment has been proposed, and inhibition of cyclo‐oxygenase‐2 (COX‐2) or activation of peroxisome proliferator‐activated receptor gamma (PPAR‐γ) yields neuroprotection in MPTP‐induced PD. Lipopolysaccharide (LPS) induces inflammation‐driven dopaminergic neurodegeneration. We tested the hypothesis that celecoxib (Celebrex, COX‐2 inhibitor) or pioglitazone (Actos, PPAR‐γ agonist) will reduce the LPS‐induced inflammatory response, spare mitochondrial bioenergetics, and improve nigral dopaminergic neuronal survival. Rats were treated with vehicle, celecoxib, or pioglitazone and were intrastriatally injected with LPS. Inflammation, mitochondrial dysfunction, oxidative stress, decreased dopamine, and nigral dopaminergic neuronal loss were observed post‐LPS. Celecoxib and pioglitazone provided neuroprotective properties by decreasing inflammation and restoring mitochondrial function. Pioglitazone also attenuated oxidative stress and partially restored striatal dopamine as well as demonstrated dopaminergic neuroprotection and reduced nigral microglial activation. In summary, intrastriatal LPS served as a model for inflammation‐induced dopaminergic neurodegeneration, anti‐inflammatory drugs provided protective properties, and pioglitazone or celecoxib may have therapeutic potential for the treatment of neuro‐inflammation and PD.

Up-regulation of inducible nitric oxide synthase in the substantia nigra by lipopolysaccharide causes microglial activation and neurodegeneration

The present study was designed to examine whether expression of iNOS was involved in LPS-induced neurodegeneration in rat substantia nigra (SN) and to study the role of NO in the loss of the SN dopaminergic neurons. In Western blot analysis, iNOS was induced in the SN after injection of LPS in a time- and dose-dependent manner. Immunofluorescence and immunohistochemical analyses revealed that the iNOS is located in a fully activated microglia with the characteristic amoeboid morphology. Furthermore, LPS-induced loss of dopaminergic neurons was significantly inhibited by the administration of L-N(G)-nitroarginine, a selective inhibitor of NOS, and the glucocorticoid dexamethasone. These inhibiting agents for iNOS reduced LPS-induced microglial activation, suggesting that NO has a role in inflammatory-mediated microglial activation. These results demonstrate that LPS induces the expression of iNOS in activated microglia in the SN, and that NO and/or its metabolites may play a crucial role in inflammation-mediated degeneration of dopaminergic neurons.

Cyclooxygenase-2 mediates microglial activation and secondary dopaminergic cell death in the mouse MPTP model of Parkinson's disease

BackgroundAccumulating evidence suggests that inflammation plays an important role in the progression of Parkinsons disease (PD). Among many inflammatory factors found in the PD brain, cyclooxygenase (COX), specifically the inducible isoform, COX-2, is believed to be a critical enzyme in the inflammatory response. Induction of COX-2 is also found in an experimental model of PD produced by administration of 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).MethodCOX-2-deficient mice or C57BL/6 mice were treated with MPTP to investigate the effects of COX-2 deficiency or by using various doses of valdecoxib, a specific COX-2 inhibitor, which induces inhibition of COX-2 on dopaminergic neuronal toxicity and locomotor activity impairment. Immunohistochemistry, stereological cell counts, immunoblotting, an automated spontaneous locomotor activity recorder and rotarod behavioral testing apparatus were used to assess microglial activation, cell loss, and behavioral impariments.ResultsMPTP reduced tyrosine hydroxylase (TH)-positive cell counts in the substantia nigra pars compacta (SNpc); total distance traveled, vertical activity, and coordination on a rotarod; and increased microglia activation. Valdecoxib alleviated the microglial activation, the loss of TH-positive cells and the decrease in open field and vertical activity. COX-2 deficiency attenuated MPTP-induced microglial activation, degeneration of TH-positive cells, and loss of coordination.ConclusionThese results indicate that reducing COX-2 activity can mitigate the secondary and progressive loss of dopaminergic neurons as well as the motor deficits induced by MPTP, possibly by suppression of microglial activation in the SNpc.

Trichloroethylene: Parkinsonism and complex 1 mitochondrial neurotoxicity

To analyze a cluster of 30 industrial coworkers with Parkinsons disease and parkinsonism subjected to long‐term (8–33 years) chronic exposure to trichloroethylene.

Comparative Analysis of an Improved Thioflavin-S Stain, Gallyas Silver Stain, and Immunohistochemistry for Neurofibrillary Tangle Demonstration on the Same Sections

An improved thioflavin-S stain, Gallyas silver stain, and two immunostainings were quantitatively compared for demonstration of neurofibrillary tangles (NFTs) on the same sections. Sections of hippocampal formation from seven cases of Alzheimers disease (AD) were immunofluorescently stained with a commercially available polyclonal NFT antibody or a PHF-1 monoclonal antibody, followed by an improved thioflavin-S stain, and finally by Gallyas silver staining. The thioflavin-S method was improved by using a combination quenching method that removes background autofluorescence without remarkable tissue damage and by post-treatment with concentrated phosphate buffer, which minimizes photobleaching. PHF-1 or NFT immunostaining is much less sensitive than the improved thioflavin-S staining and Gallyas silver staining, particularly in the transentorhinal region. Moreover PHF-1 immunoreactivity varied greatly among AD individuals. Thioflavin-S staining and Gallyas silver staining show almost the same sensitivity in NFT demonstration, but only the former depends on the secondary protein structure of NFTs. This study suggests that the improved thioflavin-S staining is a simple, sensitive, and consistent method for demonstration of neurofibrillary pathology.

Striatal GDNF administration increases tyrosine hydroxylase phosphorylation in the rat striatum and substantia nigra.

Glial cell line‐derived neurotrophic factor (GDNF) improves motor dysfunction associated with aging in rats and non‐human primates, in animal models of Parkinsons disease, and may improve motoric function in patients with advanced Parkinsons disease. These improvements are associated with increased dopamine function in the nigrostriatal system, but the molecular events associated with this increase are unknown. In these studies, 100 µg of GDNF was injected into the striatum of normal aged (24‐month‐old) male Fischer 344 rats. The protein levels and phosphorylation of TH, ERK1/2, and related proteins were determined by blot‐immunolabeling of striatum and substantia nigra harvested 30 days after injection. In GDNF‐treated rats, TH phosphorylation at Ser31 increased ∼40% in striatum and ∼250% in the substantia nigra. In the substantia nigra, there was a significant increase in ERK1 phosphorylation. In striatum, there was a significant increase in ERK2 phosphorylation. Microdialysis studies in striatum showed that both amphetamine‐ and potassium‐evoked dopamine release in GDNF recipients were significantly increased. These data show that GDNF‐induced increases in dopamine function are associated with a sustained increase in TH phosphorylation at Ser31, which is greatest in the substantia nigra and maintained for at least one month following a single striatal administration of GDNF. These findings, taken from the nigrostriatal system of normal aged rats, may help explain the long lasting effects of GDNF on dopamine function and prior studies supporting that a major effect of GDNF involves its effects on dopamine storage and somatodendritic release of dopamine in the substantia nigra.

Interleukin-10 protects against inflammation-mediated degeneration of dopaminergic neurons in substantia nigra

Inflammation has been increasingly recognized to play an important role in the pathogenesis of Parkinsons disease (PD). Using immunocytochemistry and electron microscopy, we found that intranigral injection of lipopolysaccharide (LPS) caused marked microglial activation and a dose-dependent selective loss of dopaminergic neurons, which was mediated by apoptosis as evidenced by prominent TUNEL labeling. RNase protection assays revealed that mRNA for Bax, Fas and the pro-inflammatory cytokines interleukin (IL)-1α, IL-1β, IL-6 and tumor necrosis factor (TNF)-α were significant increased ipsilaterally in LPS-injected side of SN, while expression of the anti-apoptotic gene Bcl-2 was decreased. Osmotic pump infusion of IL-10, a global inhibitor of cytokine synthesis, protected against LPS-induced cell death of dopaminergic neurons, with a corresponding decrease in the number of activated microglia, suggesting that the reduction in microglia-mediated release of inflammatory mediators may contribute to the anti-inflammatory effect of IL-10. Our results provide evidence that LPS induces apoptotic cell death in SNpc, which is likely through the expression of Fas, Bax, caspase-3, and the pro-inflammatory cytokines.

Noradrenergic activation of immediate early genes in rat cerebral cortex

Previous studies have shown that stimulation of adrenergic receptors in the brain increases the expression of the immediate early gene (IEG), c-fos, in vivo (Mol. Brain Res., 6(1989) 39-45). The present study was undertaken to determine whether this also holds for other IEGs which have been shown to be activated in brain cell culture by adrenergic agonists. Both yohimbine injection and stressful stimulation, two treatments causing brain norepinephrine (NE) release, were found to cause a parallel, transient activation of at least 5 IEGs (c-fos, nur77, tis-7, zif-268 and tis-21) in the rat cortex. Genes that are not immediate early (beta-actin, NGF and HSP70) were found not to be affected in the interval used (6 h). The responses were mediated predominantly by beta-adrenoceptors with some contribution from alpha 1 receptors. The parallel activation of multiple genes by noradrenergic receptors may enable the coding of different biochemical responses to the activation of different receptors.