Are Temporal Differences in GDNF and NOS Isoform Induction Contributors to Neurodegeneration? A Fluorescence Microscopy-Based Study.

BACKGROUND: Specific factors in Parkinson's disease have become targets as to their protective and degenerative effects. We have demonstrated that cytokines and PD-CSF detrimentally affect microglia and astrocyte growth. While glial cell-derived neurotrophic factor (GDNF) has been recognized as a possible neuron-rescue agent, nitric oxide synthase (NOS) has been implicated in neurodegenerative processes. OBJECTIVE: To demonstrate that glial cell activation, cytokine production, and NOS induction, play an intimate role in the loss of dopaminergic signaling, via mechanisms that are a result of inflammation and inflammatory stimuli. METHODS: Study animals were sacrificed following endotoxin treatment and tissue sections were harvested and probed for GDNF and NOS isomers by fluorescence deconvolution microscopy. Fluorescence was mapped and quantified for each probe. RESULTS: An immune cell influx into 'vulnerable' areas of the brain was seen, and three NOS isomers, inducible (iNOS), neuronal (nNOS) and endothelial (eNOS), were synthesized in the brains, a finding which suggests that each isomer has a role in neurodegeneration. eNOS was found associated with blood vessels, while iNOS was associated with glial and matrix cells and nNOS was located with both glia and neurons. Following endotoxin treatment, serum levels of nitric oxide were higher at 6-8 hours, while tissue levels of NOS were elevated for much longer. Thus, induction of NOS occurred earlier than the induction of GDNF. CONCLUSION: Our findings suggest that the protective abilities of GDNF to combat neural destruction are not available rapidly enough, and do not remain at sufficiently high levels long enough to assert its protective effects. (250).

Inflammatory cells and cytokines in the olfactory bulb of a rat model of neuroinflammation; insights into neurodegeneration?

This study examined inflammatory cell and cytokine production in brain tissue from a lipopolysaccharide (LPS)-treated rat model that mimics many of the neuropathologic changes associated with neurodegenerative diseases We also monitored the appearance of a glial cell line-derived neurotrophic factor (GDNF) and circulating nitric oxide (NO) levels, as well as an immune system-associated cells in a selected area of the brain, the olfactory lobe. The studies were based on the hypothesis that LPS treatment stimulates temporal changes within the brain and that these responses include immune cell recruitment, increased tissue levels of immune modulating cytokines and NO, as well as greater glial cell activation resulting in increased production of GDNF. As previously reported by other investigators, our animal model of systemic LPS treatment leads to an increase in the concentrations of circulating cytokines, including TNF-α, IL-Iß, and IL-6, with a maximum response 6 h post LPS administration. Concomitant with cytokine elevations, circulating NO levels were elevated for several hours post LPS administration. The brain content of the GDNF was also elevated over a similar time frame. Lymphocytes, neutrophils, macrophages, plasma cells, and cytokines were all seen in various areas of LPS-treated brains, often around blood vessels associated with the meninges, with these localizations possibly indicating involvement of both the blood-brain and blood-cerebral spinal fluid barriers in these inflammatory episodes. Our results suggest an involvement of both the peripheral and the central nervous system immune components in response to inflammation and inflammatory episodes. This leads us to propose that inflammation initiates an immune response by activating both microglia and astrocytes and that the presence of continuing and increasing proinflammatory mechanisms results in a situation, where cellular protective mechanisms are overcome and the more susceptible cells enter into cell death pathways, initiating a train of events that is a major part of neurodegeneration.

Cytokines disrupt intracellular patterns of Parkinson's disease-associated proteins alpha-synuclein, tau and ubiquitin in cultured glial cells.

The purpose of this study was to determine the effects of specific proinflammatory cytokines interleukin-6 (Il-6), interleukin-1beta (Il-1beta), interferon-gamma (IFN), and tumor necrosis factor-alpha (TNFalpha), on content and distribution of alpha-synuclein (alpha-synuclein), tau and ubiquitin in human derived cultured glial cells. Exposure paradigms mimicked acute (2 h), intermediate (18 h) and prolonged time frames (96 h); consisting of single or repeated low doses (10 ng/ml) or high doses (50 ng/ml), consistent with either mild or serious systemic infectious/inflammatory responses. Images of intracellular protein content and distribution were reconstructed from emission patterns generated by fluorescence deconvolution microscopy. Minor alterations were seen in protein content with IFN; Il-1beta decreased alpha-synuclein and tau at 18 and 96 h; TNFalpha inversely reduced alpha-synuclein and increased ubiquitin content. Combinations of Il-1beta and IFN produced a robust increase of alpha-synuclein and tau at 2 h. Consecutive low doses of Il-6 produced only minor increases in alpha-synuclein and ubiquitin after 4 h, whereas a single high dose resulted in major increases for all three proteins over the first 18 h. Protein localization patterns were distinctly different and were altered dependent upon cytokine treatment. A high dose exposure (2 x 50 ng/ml) with Il-6 and IFN demonstrated that protein increases and dispersals could be sustained and that the normal perinuclear tau and peripheral alpha-synuclein patterns were disrupted. These results support the postulate that specific cytokines affect temporal protein changes with concomitant pattern disruptions, possibly reflecting a mechanism of cell dysfunction in Parkinson's degeneration.

Stimulation of mitochondrial biogenesis and autophagy by lipopolysaccharide in the neonatal rat cardiomyocyte protects against programmed cell death.

Adult rat cardiomyocytes in culture respond to sub-lethal doses of lipopolysaccharides (LPS) by activation of pathways including the production of TNF-alpha and increased apoptosis. We and others have demonstrated a protective phenotype for neonatal rat cardiomyocytes to LPS. Concentrations of LPS far exceeding those necessary to induce TNF-alpha release do not induce apoptosis in the neonatal cells, although these cells are fully capable or inducing apoptosis in response to multiple other stimuli. In neonatal cells, we demonstrate that LPS treatment leads to a loss of mitochondrial membrane potential (Deltapsi) which is temporally associated with an increase in the level of uncoupling protein 3 (UCP3). Cells remain viable with no measurable increase in apoptotic or necrotic cell death. Many markers of mitochondrial biogenesis are also activated. LPS treatment stimulates an increase in the (i) transcription of mitochondrial transcription factor A (Tfam), (ii) nuclear accumulation of redox-sensitive nuclear respiratory factor 1 (NRF-1), and (iii) expression of peroxisome proliferator-activated receptor gamma co-activator 1 (PGC-1). We also observed that LPS increased intracellular autophagy. Autophagy was assessed by monitoring the levels of a mammalian protein specifically associated with autophagosomes, microtubule-associated light chain 3 (LC3). Furthermore, inhibition of autophagy in the presence of LPS stimulates markers of apoptosis. Our data suggest that the protective response of neonatal cells to LPS is multi-faceted at the level of the mitochondrion. Viable cells replace dysfunctional mitochondria by mitochondrial biogenesis and the extent of the damage limited by the rapid removal of damaged organelles by the stimulation of autophagy.

The response of neonatal rat ventricular myocytes to lipopolysaccharide-induced stress.

Sepsis induced by exposure to lipopolysaccharide (LPS) can be life-threatening and lead to multiple-organ dysfunction. Sepsis-associated cardiac dysfunction is a primary cause of mortality. The response of isolated cardiac myocytes to LPS exposure is poorly understood. Cultured neonatal rat ventricular cardiomyocytes were used to evaluate the response to LPS exposure. Other authors have reported that LPS exposure at doses sufficient to induce tumor necrosis factor alpha (TNF-alpha) production and apoptosis in adult cardiomyocytes do not induce apoptosis in neonatal cardiomyocytes. We therefore hypothesized that neonatal cardiomyocytes have innate protective mechanisms that protect from septic damage. Cultured neonatal rat ventricular cardiomyocytes were stimulated by exposure to LPS for varying lengths of time. NFkappaB signaling pathways, TNF-alpha production, and Akt activation were monitored. We also assessed the induction of apoptosis in these cells by monitoring caspase-3 activity. LPS rapidly stimulates nuclear translocation of NFkappaB and Akt activation. TNF-alpha production is also stimulated. However, high doses of LPS are unable to induce apoptosis in these cells, and protection is not a function of Akt activation. LPS treatment also stimulated the levels of cyclooxygenase-2 and the production of downstream metabolites, specifically PGE2 and 15deoxyDelta12-14PGJ2 (15dPGJ2). Specific inhibition of cyclooxygenase-2 activity induced apoptosis in the presence of LPS, whereas direct exposure to 15dPGJ2 at pharmacological levels induced apoptosis. Neonatal rat ventricular cardiomyocytes have innate protective mechanisms that prevent apoptotic cell death after LPS exposure. Metabolic products of arachidonic acid metabolized by the cyclooxygenase pathway can be potentially apoptotic or antiapoptotic. The balance of these products within these cells may define the cellular response to LPS exposure.

Attenuation of fatty acid-induced apoptosis by low-dose alcohol in neonatal rat cardiomyocytes.

Moderate alcohol consumption has been shown to reduce the morbidity and mortality from coronary heart disease. Ethanol elicits its protective effects via mechanisms that include activation of protein kinases linked to growth and survival. Our results in isolated neonatal rat cardiomyocytes demonstrate that repeated short-term, low-dose exposure to ethanol is sufficient to activate the growth and/or survival pathways that involve PKC-epsilon, Akt, and AMP-activated kinase. In addition, we are able to induce apoptosis in these cardiomyocytes using the saturated fatty acid palmitate. Pretreatment with multiple low-dose ethanol exposures attenuates the apoptotic response to palmitate. This protection is manifested by a reduction in caspase-3-like activity, decreased mitochondrial loss of cytochrome c, and decreased loss of the mitochondrial lipid cardiolipin. We previously reported that incubation of cardiomyocytes with palmitate results in decreased production of reactive oxygen species compared with cells incubated with the nonapoptotic fatty acid oleate. In the present study, we observed an increase in the production of superoxide and the rates of fatty acid oxidation in cardiomyocytes pretreated with ethanol and then exposed to fatty acids. The level of superoxide production in palmitate-treated cells returns to the levels observed in oleate-treated cells after ethanol exposure. Taken together with our observed increase in AMP-activated kinase activity, we propose that ethanol pretreatments stimulate oxidative metabolism and electron transport within cardiomyocytes. We postulate that stimulation of palmitate metabolism may protect cardiomyocytes by preventing accumulation of unsaturated precursor molecules of cardiolipin synthesis. Maintaining cardiolipin levels may be sufficient to prevent the mitochondrial loss of cytochrome c and the downstream activation of caspases.

Palmitate-induced apoptosis in neonatal cardiomyocytes is not dependent on the generation of ROS.

The saturated fatty acid palmitate induces apoptosis in neonatal rat cardiomyocytes. This apoptosis is associated with early mitochondrial release of cytochrome c and a subsequent loss of mitochondrial membrane potential. Recent reports implicate a role for reactive oxygen species (ROS) in palmitate-induced apoptosis. We studied the role of ROS in palmitate-induced apoptosis in the neonatal rat cardiomyocyte and report no evidence of ROS involvement. ROS production, nitric oxide production, and nuclear factor-kappaB activation were not increased above those observed using the nonapoptotic fatty acid oleate. Indeed, the production of ROS was significantly higher in cells treated with oleate. Furthermore, the presence of antioxidants and ROS scavengers did not attenuate the induction of apoptosis by palmitate. Variations in the fatty acid-to-albumin ratio from 2:1 to 7:1 had no effect on the absence of ROS production or on the extent of apoptosis. No evidence was found for an increase in oxidative protein modification in palmitate-treated cells. Our results lead us to conclude that oxidative stress does not play a role in palmitate-induced apoptosis.