Biomedical subjects

Colin K Combs

Publications and source records attributed to Colin K Combs.

5 recordsLinked to original sources

PubMed2006

Alpha-synuclein expression modulates microglial activation phenotype.

Recent Parkinson's disease research has focused on understanding the function of the cytosolic protein, alpha-synuclein, and its contribution to disease mechanisms. Within neurons, alpha-synuclein is hypothesized to have a role in regulating synaptic plasticity, vesicle release, and trafficking. In contrast, glial-expressed alpha-synuclein remains poorly described. Here, we examine the consequence of a loss of alpha-synuclein expression on microglial activation. Using a postnatal brain-derived culture system, we defined the phenotype of microglia from wild-type and knock-out alpha-synuclein mice (Scna-/-). Scna-/- microglia displayed a basally increased reactive phenotype compared with the wild-type cells and an exacerbated reactive phenotype after stimulation. They also exhibited dramatic morphologic differences compared with wild-type, presenting as large, ramified cells filled with vacuole-like structures. This corresponded with increased protein levels of activation markers, CD68 and beta1 integrin, in the Scna-/- cells. More importantly, Scna-/- microglia, after stimulation, secreted elevated levels of proinflammatory cytokines, TNFalpha (tumor necrosis factor alpha) and IL-6 (interleukin-6), compared with wild type. However, despite the reactive phenotype, Scna-/- cells had impaired phagocytic ability. We demonstrate for the first time that alpha-synuclein plays a critical role in modulating microglial activation state. We suggest that altered microglial alpha-synuclein expression will affect their phenotype as has already been demonstrated in neurons. This has direct ramifications for the contribution of microglia to the pathophysiology of disease, particularly in familial cases linked to altered alpha-synuclein expression.

Animals↗

PubMed2006

Beta-amyloid stimulates murine postnatal and adult microglia cultures in a unique manner.

Reactive microglia are commonly observed in association with the beta-amyloid (Abeta) plaques of Alzheimer's disease brains. This localization supports the hypothesis that Abeta is a specific activating stimulus for microglia. A variety of in vitro studies have used postnatal derived rodent microglia cultures to characterize the ability of Abeta to stimulate these cells. However, it is unclear whether this paradigm accurately models conditions in aged animals. To determine whether Abeta stimulatory phenotypes differ between young and adult microglia, we established cultures of acutely isolated adult murine cortical microglia to compare with postnatal derived microglial cultures. Although cells from both ages expressed robust immunoreactivity for CD68 and CD11b, their responses to activating stimuli differed. Fibrillar Abeta was rapidly phagocytosed by postnatal microglia and both oligomeric and fibrillar peptide stimulated increased tumor necrosis factor alpha (TNFalpha) secretion. However, Abeta oligomers but not fibrils stimulated TNFalpha secretion from adult microglia. More importantly, adult microglia had diminished ability to phagocytose Abeta fibrils. These findings demonstrate that adult microglia respond to Abeta fibril stimulation uniquely from postnatal cells and suggest that adult rather than postnatal microglia cultures are more appropriate for modeling proinflammatory changes in the aged CNS.

Aging↗

PubMed2006

Reactive macrophages increase oxidative stress and alpha-synuclein nitration during death of dopaminergic neuronal cells in co-culture: relevance to Parkinson's disease.

Parkinson's disease (PD) is characterized by progressive degeneration of dopaminergic neurons and a substantial decrease in the neurotransmitter dopamine in the nigro-striatal region of the brain. Increased markers of oxidative stress, activated microglias and elevated levels of pro-inflammatory cytokines have been identified in the brains of patients with PD. Although the precise mechanism of loss of neurons in PD remains unclear, these findings suggest that microglial activation may contribute directly to loss of dopaminergic neurons in PD patients. In the present study, we tested the hypothesis that activated microglia induces nitric oxide-dependent oxidative stress which subsequently causes death of dopaminergic neuronal cells in culture. We employed lipopolysaccharide (LPS) stimulated mouse macrophage cells (RAW 264.7) as a reactive microglial model and SH-SY5Y cells as a model for human dopaminergic neurons. LPS stimulation of macrophages led to increased production of nitric oxide in a time and dose dependent manner as well as subsequent generation of other reactive nitrogen species such as peroxynitrite anions. In co-culture conditions, reactive macrophages stimulated SH-SY5Y cell death characterized by increased peroxynitrite concentrations and nitration of alpha-synuclein within SH-SY5Y cells. Importantly 1,400 W, an inhibitor of the inducible nitric oxide synthase provided protection from cell death via decreasing the levels of nitrated alpha-synuclein. These results suggest that reactive microglias could induce oxidative stress in dopaminergic neurons and such oxidative stress may finally lead to nitration of alpha-synuclein and death of dopaminergic neurons in PD.

Animals↗

PubMed2005

Beta-amyloid-stimulated microglia induce neuron death via synergistic stimulation of tumor necrosis factor alpha and NMDA receptors.

Although abundant reactive microglia are found associated with beta-amyloid (Abeta) plaques in Alzheimer's disease (AD) brains, their contribution to cell loss remains speculative. A variety of studies have documented the ability of Abeta fibrils to directly stimulate microglia in vitro to assume a neurotoxic phenotype characterized by secretion of a plethora of proinflammatory molecules. Collectively, these data suggest that activated microglia play a direct role in contributing to neuron death in AD rather than simply a role in clearance after plaque deposition. Although it is clear the Abeta-stimulated microglia acutely secrete toxic oxidizing species, the identity of longer-lived neurotoxic agents remains less defined. We used Abeta-stimulated conditioned media from primary mouse microglia to identify more stable neurotoxic secretions. The NMDA receptor antagonists memantine and 2-amino-5-phosphopetanoic acid as well as soluble tumor necrosis factor alpha (TNFalpha) receptor protect neurons from microglial-conditioned media-dependent death, implicating the excitatory neurotransmitter glutamate and the proinflammatory cytokine TNFalpha as effectors of microglial-stimulated death. Neuron death occurs in an oxidative damage-dependent manner, requiring activity of inducible nitric oxide synthase. Toxicity results from coincident stimulation of the TNFalpha and NMDA receptors, because stimulations of either alone are insufficient to initiate cell death. These findings suggest the hypothesis that AD brains provide the appropriate microglial-mediated inflammatory environment for TNFalpha and glutamate to synergistically stimulate toxic activation of their respective signaling pathways in neurons as a contributing mechanism of cell death.

Amyloid beta-Peptides↗

PubMed2004

Amyloid precursor protein mediates proinflammatory activation of monocytic lineage cells.

Alzheimer's disease is a progressive neurodegenerative disorder characterized by extracellular deposition of beta-amyloid (Abeta) peptide containing neuritic plaques. Abeta peptides are proteolytically derived from the membrane-bound amyloid precursor protein (APP). Although the function of APP is not entirely clear, previous studies demonstrate that neuronal APP colocalizes with beta(1) integrin receptors at sites of focal adhesion, suggesting that APP is involved in mediating neuronal process adhesion. Integrin-dependent adhesion is also a well-characterized component of immune cell proinflammatory activation. Using primary mouse microglia and the human monocytic cell line, THP-1, we have begun investigating the role of APP in integrin-dependent activation. Co-immunoprecipitation studies demonstrate that APP is recruited into a multi-receptor signaling complex during beta(1) integrin-mediated adhesion of monocytes. Stimulation induces a subsequent, specific recruitment of tyrosine phosphorylated proteins to APP, including Lyn and Syk. Antibody cross-linking of cell surface APP leads to a similar response characterized by activation and recruitment of tyrosine kinases to APP as well as subsequent activation of mitogen-activated protein kinases and increased proinflammatory protein levels. These data demonstrate that APP can act as a proinflammatory receptor in monocytic lineage cells and provide insight into the contribution of this protein to the inflammatory conditions described in Alzheimer's disease.

Amyloid beta-Protein Precursor↗