Andis Klegeris

Reduction of human monocytic cell neurotoxicity and cytokine secretion by ligands of the cannabinoid-type CB2 receptor

Two cannabinoid receptors, CB1 and CB2, have been identified. The CB1 receptor is preferentially expressed in brain, and the CB2 receptor in cells of leukocyte lineage. We identified the mRNA for the CB1 receptor in human neuroblastoma SH‐SY5Y cells, and the mRNA and protein for the CB2 receptor in human microglia and THP‐1 cells. Δ9‐and Δ8‐tetrahydrocannabinol (THC) were toxic when added directly to SH‐SY5Y neuroblastoma cells. The toxicity of Δ9‐ THC was inhibited by the CB1 receptor antagonist SR141716A but not by the CB2 receptor antagonist SR144528. The endogenous ligand anandamide was also toxic, and this toxicity was enhanced by inhibitors of its enzymatic hydrolysis. The selective CB2 receptor ligands JWH‐015 and indomethacin morpholinylamide (BML‐190), when added to THP‐1 cells before stimulation with lipopolysaccharide (LPS) and IFN‐γ, reduced the toxicity of their culture supernatants to SH‐SY5Y cells. JWH‐015 was more effective against neurotoxicity of human microglia than THP‐1 cells. The antineurotoxic activity of JWH‐015 was blocked by the selective CB2 receptor antagonist SR144528, but not by the CB1 receptor antagonist SR141716A. This activity of JWH‐015 was synergistic with that of the 5‐lipoxygenase (5‐LOX) inhibitor REV 5901. Cannabinoids inhibited secretion of IL‐1β and tumor necrosis factor‐α (TNF‐α) by stimulated THP‐1 cells, but these effects could not be directly correlated with their antineurotoxic activity. Specific CB2 receptor ligands could be useful anti‐inflammatory agents, while avoiding the neurotoxic and psychoactive effects of CB1 receptor ligands such as Δ9‐THC.

Therapeutic approaches to inflammation in neurodegenerative disease.

Purpose of reviewAccording to the neuroinflammatory hypothesis of neurodegenerative diseases, drugs with an anti-inflammatory mode of action should slow the disease progression. Here we review recent advances in our understanding of one such disorder, Parkinsons disease, in which anti-inflammatory drugs are now becoming a new therapeutic focus. Recent findingsThe involvement of inflammatory mechanisms in Parkinsons disease has been revealed through in-vitro and in-vivo experimental studies supported by pathological and epidemiological findings. Several of the demonstrated inflammatory mechanisms are shared by other neurodegenerative disorders but some Parkinsons disease-specific mechanisms have also emerged. These include inflammatory stimulation by interaction of α-synuclein with microglia and astrocytes and a suppressive action by nonsteroidal anti-inflammatory drugs on dopamine quinone formation. SummaryIt can be anticipated that a more detailed understanding of neuroinflammatory mechanisms in Parkinsons disease will lead to new cellular and molecular targets, which may, in turn, permit design of Parkinsons disease modifying drugs. Future treatment may involve combination therapies with drugs directed at both inflammatory and non-inflammatory mechanisms.

BETA -AMYLOID PROTEIN ENHANCES MACROPHAGE PRODUCTION OF OXYGEN FREE RADICALS AND GLUTAMATE

Cells of the monocyte phagocytic system can generate superoxide and glutamate anions, both of which are neurotoxic at high levels. We used rat peritoneal macrophages as a model system to test the effects of various stimulants on the production of these molecules. Glutamate production by such cells was enhanced, in a concentration‐dependent manner, by treatment with serum‐opsonized zymosan (OZ), lipopolysaccharide (LPS), phorbol myristate acetate (PMA), and β‐amyloid peptide Aβ (1–40); but not by treatment with the reverse Aβ (40–1) or the Aβ (25–35) subfragment. Superoxide anion production by the cells was stimulated by OZ, PMA, Aβ (1–40), and Aβ (25–35). Moreover, Aβ and its subfragment, when used as priming agents, also enhanced the stimulatory effect of PMA. However, they did not act as priming agents for OZ, suggesting a competition for receptors or intracellular signaling pathways linked to those receptors. Inflammatory mediators, including Aβ, could place glutamate‐sensitive neurons at risk by enhancing glutamate and oxygen free radical production by monocyte‐derived cells. Such mechanisms could contribute to the pathogenesis of neurodegenerative disorders, including Alzheimers disease. J. Neurosci. Res. 49:229–235, 1997. © 1997 Wiley‐Liss, Inc.

Inflammation, the complement system and the diseases of aging

Inflammation is characteristic of neurodegenerative diseases of aging. Neuropathological evidence of activated microglia and activated astrocytes in lesioned areas, combined with epidemiological evidence of sparing of Alzheimers disease (AD), Parkinsons disease (PD) and age-related macular degeneration (AMD) in long-term users of anti-inflammatory agents, indicates that inflammation is autodestructive of neurons. Locally produced autodestructive molecules include the membrane attack complex (MAC) of complement and oxygen-free radicals. Stimulation is provided by a variety of inflammatory cytokines. Agents which reduce the intensity of inflammation should have broad spectrum application in degenerative diseases of aging.

α-Synuclein activates stress signaling protein kinases in THP-1 cells and microglia

Here we show that alpha-synuclein, a major constituent of Lewy bodies, induces inflammation in human microglial and human THP-1 cells. Secretions from such stimulated THP-1 cells contain increased levels of IL-1beta and TNF-alpha. When stimulated by alpha-synuclein in combination with IFN-gamma, secretions from the cells also become toxic towards SH-SY5Y neuroblastoma cells. The A30P, E46K and A53T alpha-synuclein mutations, which induce Parkinsons disease, are more potent than normal alpha-synuclein in the induction of such cytotoxicity. To investigate the signaling mechanisms evoked, protein phosphorylation profiling was applied. At least 81 target phospho-sites were identified. Large increases were induced in the three major mitogen-activated protein (MAP) kinase pathways: p38 MAP kinase, extracellular regulated protein-serine kinase (ERK)1/2 and c-Jun-N-terminal kinase (JNK). Upregulation occurred within minutes following exposure to alpha-synuclein, which is consistent with a receptor-mediated effect. These findings demonstrate that alpha-synuclein acts as a potent inflammatory stimulator of microglial cells, and that inhibitors of such stimulation might be beneficial in the treatment of Parkinsons disease and other synucleinopathies.

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.

Non-steroidal anti-inflammatory drugs (NSAIDs) and other anti-inflammatory agents in the treatment of neurodegenerative disease.

Inflammation is characteristic of a broad spectrum of neurodegenerative diseases. These include Alzheimers (AD), Parkinsons (PD), and Huntingtons diseases, amyotrophic lateral sclerosis, all of the tauopathies, multiple sclerosis and many other less common conditions. Morphologically, the level of inflammation is determined by the concentration and degree of activation of microglial cells. Biochemically, it is judged by the presence of a spectrum of inflammatory mediators. Epidemiological evidence indicates that anti-inflammatory agents such as non-steroidal anti-inflammatory drugs (NSAIDs) have a sparing effect on AD and PD indicating that inflammation exacerbates the pathology in these diseases. NSAIDs are protective in transgenic animal models of AD, providing further evidence of the negative consequences of inflammation. Here we describe an in vitro model, which was used to study the protective effects of NSAIDs in AD. This model is based on neuronal cell killing by stimulated microglia or microglia-like cells. In this model NSAIDs show protective effects at a therapeutically relevant level, which is in the low micromolar range. There are reports suggesting that NSAIDs act independently of cyclooxygenase (COX) inhibition, but only at higher doses. Classical NSAIDs are still the most logical choice for agents that will slow the progression or delay the onset of AD and other neurodegenerative diseases despite failures of naproxen, celecoxib and rofecoxib in AD clinical trials. Several other classes of anti-inflammatory drugs have been identified as potentially beneficial in this and similar assay systems. Therefore combination therapy with other anti-inflammatory agents that work through different mechanisms of action might prove to be a superior therapeutic strategy.

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.

Interaction of Alzheimer β-amyloid peptide with the human monocytic cell line THP-1 results in a protein kinase C-dependent secretion of tumor necrosis factor-α

Immunological mechanisms, including stimulation of brain microglia and elevation of various inflammatory cytokines, have been implicated in the pathogenesis of Alzheimers disease, where accumulation of beta-amyloid peptide (A beta) is one of its main pathological features. In this study we investigated the interaction of human monocyte-like cells with synthetic beta-amyloid peptide A beta (1-40) and its subfragment A beta (25-35). THP-1 cells (a transformed human monocyte cell line) were used with or without prior differentiation by phorbol myristate acetate (PMA), and cell activation was assessed by the secretion of tumor necrosis factor-alpha (TNF-alpha). First, it was shown that THP-1 cells could be induced to secrete significant amounts of TNF-alpha by interleukin-1, lipopolysaccharide, interferon-gamma (IFN-gamma) and PMA alone or in combination with each other. Next it was shown that A beta (1-40) could also induce secretion of TNF-alpha by THP-1 cells, but the effect was diminished when this peptide was applied in combination with IFN-gamma. The A beta subfragment A beta (25-35) was ineffective in inducing TNF-alpha production. The cellular action of A beta (1-40) appears to involve protein kinase C since pretreatment of THP-1 cells by PMA or the protein kinase C inhibitor H-7 diminished the cellular response to A beta (1-40). Identification of the pathway by which extracellular A beta activates the intracellular PKC-dependent secretion of TNF-alpha may help in developing new therapeutic strategies for Alzheimers disease.

Alpha-synuclein and its disease-causing mutants induce ICAM-1 and IL-6 in human astrocytes and astrocytoma cells

Autosomal dominant Parkinson disease (PD) is caused by duplication or triplication of the α‐synuclein gene as well as by the A30P, E46K, and A53T mutations. The mechanisms are unknown. Reactive astrocytes in the substantia nigra of PD and MPTP‐treated monkeys display high levels of the inflammatory mediator intercellular adhesion molecule‐1 (ICAM‐1), indicating that chronic inflammation contributes to the degeneration. Here we report that α‐synuclein strongly stimulates human astrocytes as well as human U‐373 MG astrocytoma cells to up‐regulate both interleukin (IL)‐6 and ICAM‐1 (ED505 µg ml–1). The mutated forms are more potent stimulators than wild‐type (WT) α‐synuclein in these assays. We demonstrate by immunoblotting analysis that this up‐regulation is associated with activation of the major mitogen‐activated protein kinase (MAPK) pathways. It is also attenuated by PD 98059, an inhibitor of the MAPK/extracellular‐regulated kinase kinase MEK1/2, SP 600125, an inhibitor of c‐Jun N‐terminal kinase (JNK), and SB 202190, an inhibitor of p38 MAPK. The inhibitory effects on human astrocytes have IC50 values of 2, 5, and 1.5 M respectively. We hypothesize that the neuroinflammation stimulated by release of an excess of normal α‐synuclein or by release of its mutated forms can be involved in the pathobiology of PD.—Klegeris, A., Giasson, B. I., Zhang, H., Maguire, J., Pelech, S., McGeer, P. L. Alphaα‐synuclein and its disease‐causing mutants induce ICAM‐1 and IL‐6 in human astrocytes and astrocytoma cells. FASEB J. 20, 2000–2008 (2006)