Porphyromonas gingivalis lipopolysaccharide weakly activates M1 and M2 polarized mouse macrophages but induces inflammatory cytokines.

Porphyromonas gingivalis is associated with chronic periodontitis, an inflammatory disease of the tooth's supporting tissues. Macrophages are important in chronic inflammatory conditions, infiltrating tissue and becoming polarized to an M1 or M2 phenotype. As responses to stimuli differ between these phenotypes, we investigated the effect of P. gingivalis lipopolysaccharide (LPS) on M1 and M2 macrophages. M1 and M2 polarized macrophages were produced from murine bone marrow macrophages (BMMϕ) primed with gamma interferon (IFN-γ) or interleukin-4 (IL-4), respectively, and incubated with a low or high dose of P. gingivalis LPS or control TLR2 and TLR4 ligands. In M1-Mϕ, the high dose of P. gingivalis LPS (10 µg/ml) significantly increased the expression of CD40, CD86, inducible nitric oxide synthase, and nitric oxide secretion. The low dose of P. gingivalis LPS (10 ng/ml) did not induce costimulatory or antibacterial molecules but did increase the secretion of IL-1α, IL-6, IL-12p40, IL-12p70, and tumor necrosis factor alpha (TNF-α). P. gingivalis LPS marginally increased the expression of CD206 and YM-1, but it did enhance arginase expression by M2-Mϕ. Furthermore, the secretion of the chemokines KC, RANTES, eotaxin, and MCP-1 from M1, M2, and nonpolarized Mϕ was enhanced by P. gingivalis LPS. TLR2/4 knockout macrophages combined with the TLR activation assays indicated that TLR2 is the main activating receptor for P. gingivalis LPS and whole cells. In conclusion, although P. gingivalis LPS weakly activated M1-Mϕ or M2-Mϕ compared to control TLR ligands, it induced the secretion of inflammatory cytokines, particularly TNF-α from M1-Mϕ and IL-10 from M2-Mϕ, as well as chemotactic chemokines from polarized macrophages.

Chronic exposure to high levels of zinc or copper has little effect on brain metal homeostasis or Abeta accumulation in transgenic APP-C100 mice.

Aberrant metal homeostasis may enhance the formation of reactive oxygen species and Abeta oligomerization and may therefore be a contributing factor in Alzheimer's disease. This study investigated the effect of chronic high intake of dietary Zn or Cu on brain metal levels and the accumulation and solubility of Abeta in vivo, using a transgenic mouse model that over expresses the C-terminal containing Abeta fragment of human amyloid precursor protein but does not develop amyloid deposits. Exposure to chronic high Zn or Cu in the drinking water resulted in only slight elevations of the respective metals in the brain. Total Abeta levels were unchanged although soluble Abeta levels were slightly decreased, without visible plaque formation, enhanced gliosis, antioxidant upregulation or neuronal loss. This study indicates that brain metal levels are only marginally altered by long term oral exposure to extremely high Cu or Zn levels, and that this does not induce Abeta-amyloid formation in human Abeta expressing, amyloid-free mice, although this is sufficient to modulate Abeta solubility in vivo.

In vitro characterization of Pittsburgh compound-B binding to Lewy bodies.

Dementia with Lewy bodies (DLB) is pathologically characterized by the presence of alpha-synuclein-containing Lewy bodies within the neocortical, limbic, and paralimbic regions. Like Alzheimer's disease (AD), Abeta plaques are also present in most DLB cases. The contribution of Abeta to the development of DLB is unclear. [11C]-Pittsburgh compound B ([11C]-PIB) is a thioflavin-T derivative that has allowed in vivo Abeta burden to be quantified using positron emission tomography (PET). [11C]-PIB PET studies have shown similar high cortical [11C]-PIB binding in AD and DLB subjects. To establish the potential binding of PIB to alpha-synuclein in DLB patients, we characterized the in vitro binding of PIB to recombinant human alpha-synuclein and DLB brain homogenates. Analysis of the in vitro binding studies indicated that [3H]-PIB binds to alpha-synuclein fibrils but with lower affinity than that demonstrated/reported for Abeta(1-42) fibrils. Furthermore, [3H]-PIB was observed to bind to Abeta plaque-containing DLB brain homogenates but failed to bind to DLB homogenates that were Abeta plaque-free ("pure DLB"). Positive PIB fluorescence staining of DLB brain sections colocalized with immunoreactive Abeta plaques but failed to stain Lewy bodies. Moreover, image quantification analysis suggested that given the small size and low density of Lewy bodies within the brains of DLB subjects, any contribution of Lewy bodies to the [11C]-PIB PET signal would be negligible. These studies indicate that PIB retention observed within the cortical gray matter regions of DLB subjects in [11C]-PIB PET studies is largely attributable to PIB binding to Abeta plaques and not Lewy bodies.

Activation of epidermal growth factor receptor by metal-ligand complexes decreases levels of extracellular amyloid beta peptide.

The epidermal growth factor receptor is a receptor tyrosine kinase expressed in a range of tissues and cell-types. Activation of the epidermal growth factor receptor by a number of ligands induces downstream signalling that modulates critical cell functions including growth, survival and differentiation. Abnormal epidermal growth factor receptor expression and activation is also involved in a number of cancers. In addition to its cognate ligands, the epidermal growth factor receptor can be activated by metals such as zinc (Zn) and copper (Cu). Due to the important role of these metals in a number of diseases including neurodegenerative disorders, therapeutic approaches are being developed based on the use of lipid permeable metal-complexing molecules. While these agents are showing promising results in animal models and clinical trials, little is known about the effects of metal-ligand complexes on cell signalling pathways. In this study, we investigated the effects of clioquinol (CQ)-metal complexes on activation of epidermal growth factor receptor. We show here that CQ-Cu complexes induced potent epidermal growth factor receptor phosphorylation resulting in downstream activation of extracellular signal-regulated kinase. Similar levels of epidermal growth factor receptor activation were observed with alternative lipid permeable metal-ligands including neocuproine and pyrrolidine dithiocarbamate. We found that CQ-Cu complexes induced a significant reduction in the level of extracellular Abeta1-40 in cell culture. Inhibition of epidermal growth factor receptor activation by PD153035 blocked extracellular signal-regulated kinase phosphorylation and restored Abeta1-40 levels. Activation of the epidermal growth factor receptor by CQ-Cu was mediated through up-regulation of src kinase activity by a cognate ligand-independent process involving membrane integrins. These findings provide the first evidence that metal-ligand complexes can activate the epidermal growth factor receptor with potentially neuroprotective effects.

Differential modulation of Alzheimer's disease amyloid beta-peptide accumulation by diverse classes of metal ligands.

Biometals have an important role in AD (Alzheimer's disease) and metal ligands have been investigated as potential therapeutic agents for treatment of AD. In recent studies the 8HQ (8-hydroxyquinoline) derivative CQ (clioquinol) has shown promising results in animal models and small clinical trials; however, the actual mode of action in vivo is still being investigated. We previously reported that CQ-metal complexes up-regulated MMP (matrix metalloprotease) activity in vitro by activating PI3K (phosphoinositide 3-kinase) and JNK (c-jun N-terminal kinase), and that the increased MMP activity resulted in enhanced degradation of secreted Abeta (amyloid beta) peptide. In the present study, we have further investigated the biochemical mechanisms by which metal ligands affect Abeta metabolism. To achieve this, we measured the effects of diverse metal ligands on cellular metal uptake and secreted Abeta levels in cell culture. We report that different classes of metal ligands including 8HQ and phenanthroline derivatives and the sulfur compound PDTC (pyrrolidine dithiocarbamate) elevated cellular metal levels (copper and zinc), and resulted in substantial loss of secreted Abeta. Generally, the ability to inhibit Abeta levels correlated with a higher lipid solubility of the ligands and their capacity to increase metal uptake. However, we also identified several ligands that potently inhibited Abeta levels while only inducing minimal change to cellular metal levels. Metal ligands that inhibited Abeta levels [e.g. CQ, 8HQ, NC (neocuproine), 1,10-phenanthroline and PDTC] induced metal-dependent activation of PI3K and JNK, resulting in JNK-mediated up-regulation of metalloprotease activity and subsequent loss of secreted Abeta. The findings in the present study show that diverse metal ligands with high lipid solubility can elevate cellular metal levels resulting in metalloprotease-dependent inhibition of Abeta. Given that a structurally diverse array of ligands was assessed, the results are consistent with the effects being due to metal transport rather than the chelating ligand interacting directly with a receptor.

Overexpression of Abeta is associated with acceleration of onset of motor impairment and superoxide dismutase 1 aggregation in an amyotrophic lateral sclerosis mouse model.

Transgenic mice carrying mutant Cu/Zn superoxide dismutase (SOD1) recapitulate the motor impairment of human amyotrophic lateral sclerosis (ALS). The amyloid-beta (Abeta) peptide associated with Alzheimer's disease is neurotoxic. To investigate the potential role of Abeta in ALS development, we generated a double transgenic mouse line that overexpresses SOD1(G93A) and amyloid precursor protein (APP)-C100. The transgenic mouse C100.SOD1(G93A) overexpresses Abeta and shows earlier onset of motor impairment but has the same lifespan as the single transgenic SOD1(G93A) mouse. To determine the mechanism associated with this early-onset phenotype, we measured copper and zinc levels in brain and spinal cord and found both significantly elevated in the single and double transgenic mice compared with their littermate control mice. Increased glial fibrillary acidic protein and decreased APP levels in the spinal cord of C100.SOD1(G93A) mice compared with the SOD1(G93A) mice agree with the neuronal damage observed by immunohistochemical analysis. In the spinal cords of C100.SOD1(G93A) double transgenic mice, soluble Abeta was elevated in mice at end-stage disease compared with the pre-symptomatic stage. Buffer-insoluble SOD1 aggregates were significantly elevated in the pre-symptomatic mice of C100.SOD1(G93A) compared with the age-matched SOD1(G93A) mice, correlating with the earlier onset of motor impairment in the C100.SOD1(G93A) mice. This study supports abnormal SOD1 protein aggregation as the pathogenic mechanism in ALS, and implicates a potential role for Abeta in the development of ALS by exacerbating SOD1(G93A) aggregation.

Alzheimer's Disease Normative Cerebrospinal Fluid Biomarkers Validated in PET Amyloid-ß Characterized Subjects from the Australian Imaging, Biomarkers and Lifestyle (AIBL) study.

BACKGROUND: The cerebrospinal fluid (CSF) amyloid-ß (Aß)(1-42), total-tau (T-tau), and phosphorylated-tau (P-tau181P) profile has been established as a valuable biomarker for Alzheimer's disease (AD). OBJECTIVE: The current study aimed to determine CSF biomarker cut-points using positron emission tomography (PET) Aß imaging screened subjects from the Australian Imaging, Biomarkers and Lifestyle (AIBL) study of aging, as well as correlate CSF analyte cut-points across a range of PET Aß amyloid ligands. METHODS: Aß pathology was determined by PET imaging, utilizing ¹¹C-Pittsburgh Compound B, ¹8F-flutemetamol, or ¹8F-florbetapir, in 157 AIBL participants who also underwent CSF collection. Using an INNOTEST assay, cut-points were established (Aß(1-42) >544 ng/L, T-tau <407 ng/L, and P-tau181P <78 ng/L) employing a rank based method to define a "positive" CSF in the sub-cohort of amyloid-PET negative healthy participants (n = 97), and compared with the presence of PET demonstrated AD pathology. RESULTS: CSF Aß(1-42) was the strongest individual biomarker, detecting cognitively impaired PET positive mild cognitive impairment (MCI)/AD with 85% sensitivity and 91% specificity. The ratio of P-tau181P or T-tau to Aß(1-42) provided greater accuracy, predicting MCI/AD with Aß pathology with ≥92% sensitivity and specificity. Cross-validated accuracy, using all three biomarkers or the ratio of P-tau or T-tau to Aß(1-42) to predict MCI/AD, reached ≥92% sensitivity and specificity. CONCLUSIONS: CSF Aß(1-42) levels and analyte combination ratios demonstrated very high correlation with PET Aß imaging. Our study offers additional support for CSF biomarkers in the early and accurate detection of AD pathology, including enrichment of patient cohorts for treatment trials even at the pre-symptomatic stage.

Investigation of matrix metalloproteinases, MMP-2 and MMP-9, in plasma reveals a decrease of MMP-2 in Alzheimer's disease.

Pathological changes in the Alzheimer's disease (AD) brain include amyoid-ß (Aß) plaques, and neurofibrillary tangles, as well as neuronal death and synaptic loss. Matrix metalloproteinases MMP-2 and MMP-9 are known to degrade Aß, and their expressions are increased in the AD brain, in particular in the astrocytes surrounding amyloid plaque. To investigate a possible association between plasma metalloproteinases and AD, we quantified MMP-2 and MMP-9 activities in the plasma of healthy controls (HC, n = 56), cases with mild cognitive impairment (MCI, n = 45), and AD (n = 50). All cases had previously been imaged with Pittsburgh compound B (PiB) and had a Mini-Mental Status Examination (MMSE) assessment. MMP-2 and MMP-9 activity was determined using gelatine-zymography. There was a significant 1.5-fold decrease in MMP-2 activity in the AD group compared to HC (p < 0.001) and a 1.4-fold decrease compared to MCI (p < 0.01). There was no difference in MMP-9 levels between the three groups. A positive correlation was identified between MMP-2 plasma activity and MMSE score (r = 0.16, p < 0.05), but there was no association with PiB. This is the first report of a change in MMP-2 activity in AD plasma and these findings may provide some insight into AD pathogenesis.

Serum matrix metalloproteinase-9 activity is dysregulated with disease progression in the mutant SOD1 transgenic mice.

Amyotrophic lateral sclerosis (ALS) is an adult-onset fatal neurodegenerative disorder characterized by progressive deterioration of motor neurons in the spinal cord, brainstem, and cerebral cortex. Matrix metalloproteinase-9 (MMP-9) is proposed to be a biomarker for ALS due to a potential pathological role in the disease. However, despite numerous studies, it is still unclear whether there is a direct correlation between MMP-9 expression in serum and progression of disease. Therefore, we used a TgSOD1(G93A) mouse with a low transgene copy number. This model shows slow disease progression analogous to human ALS and provides a useful model to study biomarker expression at different stages of disease. Using zymography, we found that serum MMP-9 activity was significantly elevated in animals showing early signs of disease when compared to the younger, pre-symptomatic animals. This was followed by a decrease in MMP-9 activity in TgSOD1(G93A) mice with end-stage disease. These results were confirmed in serum of a high copy number strain of TgSOD1(G93A) mice with rapid progression. MMP-9 expression was changed accordingly in spinal motor neurons, glia and neuropil, suggesting a spinal cord contribution to blood MMP-9 activity. Serum MMP-2 activity followed a similar profile as the MMP-9 in these two models. These data indicate that circulating MMP-9 is altered throughout the course of disease progression in mice. Further studies in human ALS may validate the suitability of serum MMP-9 activity as a biomarker for early stage disease.

Plasma amyloid beta42 and amyloid beta40 levels are associated with early cognitive dysfunction after cardiac surgery.

BACKGROUND: Decreased cognitive function associated with coronary artery bypass graft surgery is common. These deficits may be similar to the cognitive dysfunction seen in the spectrum of mild cognitive impairment to Alzheimer's disease, which are believed to result from the accumulation of amyloid beta (Abeta) peptide in the brain. We measured cognition both before and after coronary artery bypass graft surgery and assayed Abeta levels to investigate whether the cognitive dysfunction of cardiac surgery was associated with Abeta levels. METHODS: The plasma of 332 patients, who had undergone neuropsychological testing before and 3 and 12 months after coronary artery bypass graft surgery, was analyzed for Abeta(42) and Abeta(40). Patients were classified as having preexisting cognitive impairment if cognitive function was decreased in two or more tests compared with a healthy control group, and postoperative cognitive dysfunction was defined as a decline in two or more tests compared with the group mean baseline score. RESULTS: Preexisting cognitive impairment was present in 117 patients (35.2%), and postoperative cognitive dysfunction was present in 40 (12%) at 3 months and 41 (13%) at 12 months after surgery. Both plasma Abeta(42) and Abeta(40) levels assessed before the surgery were significantly lower in patients who later had postoperative cognitive dysfunction at 3 months. CONCLUSIONS: Decreased preoperative plasma levels of Abeta(42) and Abeta(40) in patients who exhibit postoperative cognitive dysfunction at 3 months suggest that postoperative cognitive dysfunction at this time may share a common mechanism with mild cognitive impairment and Alzheimer's disease. This process may be exacerbated by anesthesia.

Selective intracellular release of copper and zinc ions from bis(thiosemicarbazonato) complexes reduces levels of Alzheimer disease amyloid-beta peptide.

Copper and zinc play important roles in Alzheimer disease pathology with recent reports describing potential therapeutics based on modulation of metal bioavailability. We examined the ability of a range of metal bis(thiosemicarbazonato) complexes (MII(btsc), where M=CuII or ZnII) to increase intracellular metal levels in Chinese hamster ovary cells overexpressing amyloid precursor protein (APP-CHO) and the subsequent effect on extracellular levels of amyloid-beta peptide (Abeta). The CuII(btsc) complexes were engineered to be either stable to both a change in oxidation state and dissociation of metal or susceptible to intracellular reduction and dissociation of metal. Treatment of APP-CHO cells with stable complexes resulted in elevated levels of intracellular copper with no effect on the detected levels of Abeta. Treatment with complexes susceptible to intracellular reduction increased intracellular copper levels but also resulted in a dose-dependent reduction in the levels of monomeric Abeta. Treatment with less stable ZnII(btsc) complexes increased intracellular zinc levels with a subsequent dose-dependent depletion of monomeric Abeta levels. The increased levels of intracellular bioavailable copper and zinc initiated a signaling cascade involving activation of phosphoinositol 3-kinase and c-Jun N-terminal kinase. Inhibition of these enzymes prevented Abeta depletion induced by the MII(btsc) complexes. Inhibition of metalloproteases also partially restored Abeta levels, implicating metal-driven metalloprotease activation in the extracellular monomeric Abeta depletion. However, a role for alternative metal-induced Abeta metabolism has not been ruled out. These studies demonstrate that MII(btsc) complexes have potential for Alzheimer disease therapy.

Plasma alpha-synuclein is decreased in subjects with Parkinson's disease.

Alpha-synuclein (alphaSN) is implicated in Parkinson's disease (PD) and is the major component of Lewy bodies (LBs). Although alphaSN is mainly expressed in neuronal cells and exists as a cytoplasmic protein, it has been found in body fluids including cerebrospinal fluid and blood. This study explored plasma alphaSN as a diagnostic marker for PD. Western blot analysis was used to characterize plasma alphaSN compared to brain alphaSN. Plasma alphaSN of 16 kDa migrates with the same mobility as its brain counterpart and recombinant alphaSN on denatured polyacrylamide gels and reacted with three different antibodies against the C-terminal and NAC regions of the alphaSN protein. The alphaSN levels in plasma from PD subjects are significantly lower than that in age-matched controls (p=0.001), and the alphaSN levels in patients with early-onset PD are lower than that in both late-onset PD and controls. This initial study indicates that measurement of alphaSN in plasma can provide support for a clinical diagnosis of Parkinson's disease and warrants further study in a larger population.

Chronic exposure to U18666A is associated with oxidative stress in cultured murine cortical neurons.

Findings that antioxidant treatment may be beneficial in Alzheimer's disease indicate that oxidative stress is an important factor in its pathogenesis. Studies have also suggested that cholesterol imbalance in the brain might be related to the development of neurological disorders. Previously, we have reported that U18666A, a cholesterol transport-inhibiting agent, leads to apoptosis and intracellular cholesterol accumulation in primary cortical neurons. In this study, we found that neuronal apoptosis mediated by U18666A is associated with oxidative stress in the treated cortical neurons. Cortical neurons treated with U18666A also showed decreased secretion and increased intraneuronal accumulation of beta-amyloid. The association of neuronal apoptosis with oxidative stress and Abeta accumulation may provide clues to the pathogenesis of Alzheimer's disease, as well as the role oxidative stress plays in other neurodegenerative diseases.

Degradation of the Alzheimer disease amyloid beta-peptide by metal-dependent up-regulation of metalloprotease activity.

Biometals play an important role in Alzheimer disease, and recent reports have described the development of potential therapeutic agents based on modulation of metal bioavailability. The metal ligand clioquinol (CQ) has shown promising results in animal models and small phase clinical trials; however, the actual mode of action in vivo has not been determined. We now report a novel effect of CQ on amyloid beta-peptide (Abeta) metabolism in cell culture. Treatment of Chinese hamster ovary cells overexpressing amyloid precursor protein with CQ and Cu(2+) or Zn(2+) resulted in an approximately 85-90% reduction of secreted Abeta-(1-40) and Abeta-(1-42) compared with untreated controls. Analogous effects were seen in amyloid precursor protein-overexpressing neuroblastoma cells. The secreted Abeta was rapidly degraded through up-regulation of matrix metalloprotease (MMP)-2 and MMP-3 after addition of CQ and Cu(2+). MMP activity was increased through activation of phosphoinositol 3-kinase and JNK. CQ and Cu(2+) also promoted phosphorylation of glycogen synthase kinase-3, and this potentiated activation of JNK and loss of Abeta-(1-40). Our findings identify an alternative mechanism of action for CQ in the reduction of Abeta deposition in the brains of CQ-treated animals and potentially in Alzheimer disease patients.

APP intracellular domain is increased and soluble Abeta is reduced with diet-induced hypercholesterolemia in a transgenic mouse model of Alzheimer disease.

Cholesterol is one of multiple factors, other than familial genetic mutations, that can influence amyloid-beta peptide (Abeta) metabolism and accumulation in Alzheimer disease (AD). The effect of a high-cholesterol diet on amyloid precursor protein (APP) processing in brain has not been thoroughly studied. This study was designed to further investigate the role of cholesterol in the production of Abeta and APP intracellular domain (AICD) in 12-month-old Tg2576 transgenic mice. The mice were maintained on a high-cholesterol diet for 6 weeks. We found that diet-induced hypercholesterolemia increased the APP cytosolic fragment AICD and reduced sAPPalpha in the Tg2576 mice compared to the mice on a control basal diet. In addition, the levels of detergent-extracted Abeta40 were reduced, although no change in guanidine-extracted Abeta levels was observed. Full-length APP, alpha/betaC-terminal fragment (alpha/betaCTF), and beta-secretase (BACE) were not different in the cholesterol-fed mice compared to the control diet-fed mice. This study suggests that a high dietary cholesterol in aged mice may not only influence Abeta metabolism, but also regulate the AICD levels. AICD has a proposed role in signal transduction and apoptosis, hence modulation of AICD production could be an alternative mechanism by which cholesterol contributes to AD pathogenesis.

Brain beta-amyloid accumulation in transgenic mice expressing mutant superoxide dismutase 1.

Oxidative stress is implicated in both the deposition and pathogenesis of beta-amyloid (Abeta) protein in Alzheimer's disease (AD). Accordingly, overexpression of the antioxidant enzyme superoxide dismutase 1 (SOD1) in neuronal cells and transgenic AD mice reduces Abeta toxicity and accumulation. In contrast, mutations in SOD1 associated with amyotrophic lateral sclerosis (ALS) confer enhanced pro-oxidative enzyme activities. We therefore examined whether ALS-linked mutant SOD1 overexpression in motor neuronal cells or transgenic ALS mice modulates Abeta toxicity or its accumulation in the brain. Aggregated, but not freshly solubilised, substrate-bound Abeta peptides induced degenerative morphology and cytotoxicity in motor neuron-like NSC-34 cells. Transfection of NSC-34 cells with human wild-type SOD1 attenuated Abeta-induced toxicity, however this neuroprotective effect was also observed for ALS-linked mutant SOD1. Analysis of the cerebral cortex, brainstem, cerebellum and olfactory bulb from transgenic SOD1G93A mice using enzyme-linked immunosorbent assay of acid-guanidine extracts revealed age-dependent elevations in Abeta levels, although not significantly different from wild-type mouse brain. In addition, brain amyloid protein precursor (APP) levels remained unaltered as a consequence of mutant SOD1 expression. We therefore conclude that mutant SOD1 overexpression promotes neither Abeta toxicity nor brain accumulation in these ALS models.