Jian-Ping Guo

Thrombin and prothrombin are expressed by neurons and glial cells and accumulate in neurofibrillary tangles in Alzheimer disease brain

Abstract Thrombin is a serine protease that is generated by proteolytic cleavage of its precursor, prothrombin. We previously showed that thrombin proteolyses the microtubule-associated protein tau and that phosphorylation of tau inhibits this process. To characterize further the role of thrombin in the brain, we investigated prothrombin and thrombin expression in cultured brain cells and in brains of control, Alzheimer disease (AD) and parkinsonism-dementia complex of Guam (PDCG). We show by reverse transcriptase-polymerase chain reaction that prothrombin mRNA is expressed in brain tissues, neuroblastoma cells, and cultured human astrocytes, oligodendrocytes, and microglial cells. We also show by immunohistochemistry that the proteins prothrombin and thrombin are present in brain using specific monoclonal and polyclonal antibodies for both proteins. All antibodies stained residual serum in blood vessels, as well as normal pyramidal neurons and their processes, and some astrocytes. Additionally, in AD and PDCG cases, all antibodies stained extra- and intracellular neurofibrillary tangles (NFTs), senile plaques, and reactive microglial cells. The ubiquitous expression of prothrombin and thrombin in brain cells suggests that thrombin plays an important physiological role in normal brain. The accumulation of thrombin and prothrombin in NFTs supports the hypothesis that thrombin may be involved in tau proteolysis and that failure to metabolize tau may lead to its aggregation in neurodegenerative diseases.

LRRK2 expression in normal and pathologic human brain and in human cell lines

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) have been recently identified in families with autosomal-dominant late-onset Parkinson disease. We report that by reverse transcriptase-polymerase chain reaction, the mRNA of LRRK2 is expressed in soluble extracts of human brain, liver, and heart and in cultured human astrocytes, microglia, and oligodendroglia as well as in human neuroblastoma cell lines. We find by Western blotting using a polyclonal antibody of the leucine-rich repeat kinase 2 protein (Lrrk2) specific for C-terminal residues 2511-2527 that an apparent full-length protein and several of its fractions are expressed in soluble extracts of normal human brain. By immunocytochemistry, the antibody recognizes neurons, and more weakly astrocytes and microglia, in normal brain tissue. It intensely labels Lewy bodies in Parkinson disease and related neurodegenerative disorders. It also labels a subset of neurofibrillary tangles in Alzheimer disease and the Parkinsonism dementia complex of Guam (PDCG). It labels thorn-shaped astrocytes and oligodendroglial coiled bodies in PDCG; oligodendroglial inclusions in multiple system atrophy; Pick bodies in Pick disease; nuclear and cytoplasmic inclusions in Huntington disease; and intraneuronal and glial inclusions in amyotrophic lateral sclerosis. In summary, LRRK2 is constitutively expressed in neurons and also in glial cells of human brain. It strongly associates with pathological inclusions in several neurodegenerative disorders.

Microglial PHOX and Mac-1 are essential to the enhanced dopaminergic neurodegeneration elicited by A30P and A53T mutant alpha-synuclein.

α‐Synuclein, a gene whose mutations, duplication, and triplication has been linked to autosomal dominant familial Parkinsons disease (fPD), appears to play a central role in the pathogenesis of sporadic PD (sPD) as well. Enhancement of neurodegeneration induced by mutant α‐synuclein has been attributed to date largely to faster formation of α‐synuclein aggregates in neurons. Recently, we reported that microglial activation enhances wild type (WT) α‐synuclein‐elicited dopaminergic neurodegeneration. In the present study, using a primary mesencephalic culture system, we tested whether mutated α‐synuclein could activate microglia more powerfully than WT α‐synuclein, thereby contributing to the accelerated neurodegeneration observed in fPD. The results showed that α‐synuclein with the A30P or A53T mutations caused greater microglial activation than WT α‐synuclein. Furthermore, the extent of microglial activation paralleled the degree of dopaminergic neurotoxicity induced by WT and mutant α‐synuclein. Mutant α‐synuclein also induced greater production of reactive oxygen species than WT α‐synuclein by NADPH oxidase (PHOX), and PHOX activation was linked to direct activation of macrophage antigen‐1 (Mac‐1) receptor, rather than α‐synuclein internalization via scavenger receptors. These results have, for the first time, demonstrated that microglia are also critical in enhanced neurotoxicity induced by mutant α‐synuclein.

SARS corona virus peptides recognized by antibodies in the sera of convalescent cases

Abstract We synthesized on cellulose membranes 4942 ten-amino-acid peptides which included all of the sequences predicted for the severe acute respiratory syndrome (SARS) corona virus. We probed these membranes with four pairs of acute and convalescent sera from recovered SARS cases. We correlated positively reacting peptides with the in vitro SARS-CoV neutralizing activity of the samples. We found that convalescent sera with high neutralizing activity recognized exclusively only a limited number of peptides on the membranes. This suggests that antibodies against the epitopes represented by these peptides could be responsible for much of the SARS-CoV neutralizing activity. The findings have implications for monitoring humoral responses to SARS-CoV as well as for developing a successful SARS vaccine.

Simple In Vitro Assays to Identify Amyloid-β Aggregation Blockers for Alzheimer's Disease Therapy

Compounds that will inhibit buildup of amyloid-beta(Abeta) deposits in Alzheimers disease (AD) brain are potential therapeutic agents. Here we report the development of two simple in vitro screening assays to identify such agents. We use these assays to evaluate the relative potency of some possible candidates. One assay is based on binding of fluorescence-tagged Abeta{1-42} to synthetic Abeta{1-42} plated in wells of fluorescent black-wall microplates. Fluorescence-tagged Abeta{1-42} solutions with and without blockers are then added to the plates, and the amount of bound fluorescence is measured. Another is a tissue type assay, where sections of unfixed AD or AD model transgenic mouse brains are mounted on glass slides. The same solutions assayed in the microplate test are then added to tissue sections. Binding of fluorescence-tagged Abeta{1-42} to the Abeta deposits in AD or transgenic brain tissue is detected with a fluorescence microscope. Good agreement is obtained between the two methods. Most of the tested agents have too low an affinity for Abeta {1-42} to be effective clinically. Agents that may have marginal affinity according to these tests include 1,2,3,4,6-penta-O-galloyl-b-D-glucopyranose (PGG), S-diclofenac, epigallocatechin gallate (EGCG), resveratrol, and extracts of spirulina, ginger, rhubarb, cinnamon, blueberries, and turmeric. Compounds which failed to show binding include scyllo-inositol, myo-inositol, rhamnose, ginkgolide A, emodin, rhein, caryophellene, curcumin, valproic acid, tramiprosate, and garlic extract.

Selective inhibition of the membrane attack complex of complement by low molecular weight components of the aurin tricarboxylic acid synthetic complex

Complement plays a vital role in both the innate and adaptive immune systems. It recognizes a target, opsonizes it, generates anaphylatoxins, and directly kills cells through the membrane attack complex (MAC). This final function, which assembles C5b-9(n) on viable cell surfaces, can kill host cells through bystander lysis. Here we identify for the first time compounds that can inhibit bystander lysis while not interfering with the other essential functions of complement. We show that aurin tricarboxylic acid (ATA), aurin quadracarboxylic acid (AQA), and aurin hexacarboxylic acid (AHA), block the addition of C9 to C5b-8 so that the MAC cannot form. These molecules inhibit hemolysis of human, rat, and mouse red cells with a half maximal inhibitory concentration (IC(50)) in the nanomolar range. When given orally to Alzheimer disease type B6SJL-Tg mice, they inhibit MAC formation in serum and improve memory retention. On autopsy, they show no evidence of harm to any organ. Aurin tricarboxylic acid, aurin quadracarboxylic acid, and aurin hexacarboxylic acid may be effective therapeutic agents in Alzheimer disease and other degenerative disorders where self damage from the MAC occurs.

Aurin tricarboxylic acid self-protects by inhibiting aberrant complement activation at the C3 convertase and C9 binding stages

Aberrant complement activation is known to exacerbate the pathology in a spectrum of degenerative diseases of aging. We previously reported that aurin tricarboxylic acid (ATA) is an orally effective agent which prevents formation of the membrane attack complex of complement. It inhibits C9 attachment to tissue bound C5b678 and thus prevents bystander lysis of host cells. In this study, we investigated the effects of ATA on the alternative complement pathway. We found that ATA prevented cleavage of the tissue bound properdin-C3b-Factor B complex into the active C3 convertase enzyme properdin-C3b-Factor Bb. This inhibition was reversed by adding Factor D to the serum. Using enzyme-linked immunosorbent type assays, we established that ATA binds directly to Factor D and C9 but not to properdin or other complement proteins. We conclude that ATA, by inhibiting at two stages of the alternative pathway, might be a particularly effective therapeutic agent in conditions such as macular degeneration, paroxysmal nocturnal hemoglobinemia, and rheumatoid arthritis, in which activation of the alternative complement pathway initiates self damage.

P2-167: Alzheimer disease tangles and threads display multiple tau phosphorylation sites

ease (AD) there are the neurofibrillary tangles (NFTs) composed of intracellular filamentous aggregates of hyperphosphorylated protein tau. Generally, the level of tau phosphorylation is regulated by the equilibrium between the activities of its protein kinases and phosphatases. An imbalanced regulation in protein kinase and protein phosphatase is proposed to be a reasonable causative factor to the disease process. One protein kinase that play an important role in regulating the phosphorilation state of tau is glycogen synthase kinase (Gsk) 3 . Recent studies show that overexpression of active Gsk3 results in AD. The major phosphatase that dephosphorylates tau is protein phosphatase 2A (PP2A). PP2A is a trimeric serine/threonine protein phosphatase composed of three subunits. Different groups showed that the highly conserved carboxyl-terminal sequence of PP2A C subunit is a focal point for phosphatase regulation. This is the site of a reversible methyl esterification reaction that controls the formation of AB C heterotrimers. Objective(s): The aim of this study is to demonstrate that hypomethylation regulates the equilibrium between protein kinase and phosphatase activities, leading to abnormal hyperphosphorylated tau. Methods: Both SK-N-BE neuroblastoma cell line and TgCRND8 mice were used. In vitro, we used culture media without folate, B12 and B6, whereas in vivo, mice were fed with a diet deficient of the same vitamins. We analyzed expression levels of kinase and phosphatase with quantitative Real-Time PCR, western blot and activity assay. Results: We demonstrated that Gsk3 and PP2A genes were upregulated by inhibiting methylation reactions. Western blot analysis illustrate that methylated PP2A was decreased leading to reduced PP2A activity. By contrast, the levels of Gsk3 protein increased in the same condition. Conclusions: The analysis of Tau and the toxic tau fragments demonstrate that lowered SAM levels are the cause of deregulation of the equilibrium between Gsk3 and PP2A activities.