George A. Smythe

Kynurenine pathway metabolism in human astrocytes: a paradox for neuronal protection.

There is good evidence that the kynurenine pathway (KP) and one of its products, quinolinic acid (QUIN), play a role in the pathogenesis of neurological diseases, in particular AIDS dementia complex. Although QUIN has been shown to be produced in neurotoxic concentrations by macrophages and microglia, the role of astrocytes in QUIN production is controversial. Using cytokine‐stimulated cultures of human astrocytes, we assayed key enzymes and products of the KP. We found that human astrocytes lack kynurenine hydroxylase so that large amounts of kynurenine and the QUIN antagonist kynurenic acid were produced. However, the amounts of QUIN that were synthesized were subsequently completely degraded. We then showed that kynurenine in concentrations comparable with those produced by astrocytes led to significant production of QUIN by macrophages. These results suggest that astrocytes alone are neuroprotective by minimizing QUIN production and maximizing synthesis of kynurenic acid. However, it is likely that, in the presence of macrophages and/or microglia, astrocytes become indirectly neurotoxic by the production of large concentrations of kynurenine that can be secondarily metabolized by neighbouring or infiltrating monocytic cells to form the neurotoxin QUIN.

Expression of indoleamine 2,3‐dioxygenase and production of quinolinic acid by human microglia, astrocytes, and neurons

There is good evidence that the kynurenine pathway (KP) and one of its end products, quinolinic acid (QUIN) play a role in the pathogenesis of several major neurological diseases. While QUIN has been shown to be produced in neurotoxic concentrations by macrophages and microglia, the capacity of astrocytes and neurons to produce QUIN is controversial. Using interferon gamma (IFN‐γ)‐stimulated primary cultures of human mixed brain cells, we assayed expression of the KP regulatory enzyme indoleamine 2,3‐dioxygenase (IDO) and QUIN production by immunocytochemistry. Using IFN‐γ‐stimulated purified cultures of neurons, astrocytes, microglia and macrophages, we studied IDO expression by RT‐PCR and production of QUIN using mass spectrometry. We found that astrocytes, neurons, and microglia expressed IDO but only microglia were able to produce detectable amounts of QUIN. However, astrocytes and neurons had the ability to catabolize QUIN. This study also provides the first evidence of IDO expression and lack of production of QUIN in culture of primary human neurons.

Increased nitric oxide production in heart failure

The role of nitric oxide in heart failure is unknown. The high-capacity inducible isoform of nitric oxide synthase is present in the myocardium of patients with idiopathic dilated cardiomyopathy. Plasma nitrate, the stable end-product of nitric oxide production, was significantly increased in patients with heart failure compared with normal controls (means 51.3 and 24.6 mumol/L). Vasodilation caused by increased nitric oxide may compensate for the vasoconstrictor effect of neurohumoral adaptions to heart failure. Alternatively, excess production may be detrimental to the heart by a direct negative inotropic effect.

Characterization of the Kynurenine Pathway in Human Neurons

The kynurenine pathway is a major route of l-tryptophan catabolism producing neuroactive metabolites implicated in neurodegeneration and immune tolerance. We characterized the kynurenine pathway in human neurons and the human SK-N-SH neuroblastoma cell line and found that the kynurenine pathway enzymes were variably expressed. Picolinic carboxylase was expressed only in primary and some adult neurons but not in SK-N-SH cells. Because of this difference, SK-N-SH cells were able to produce the excitotoxin quinolinic acid, whereas human neurons produced the neuroprotectant picolinic acid. The net result of kynurenine pathway induction in human neurons is therefore predicted to result in neuroprotection, immune regulation, and tumor inhibition, whereas in SK-N-SH cells, it may result in neurotoxicity, immune tolerance, and tumor promotion. This study represents the first comprehensive characterization of the kynurenine pathway in neurons and the first description of the involvement of the kynurenine pathway as a mechanism for controlling both tumor cell neurotoxicity and persistence.

Plasma biomarkers for mild cognitive impairment and Alzheimer's disease

PURPOSE OF REVIEW With the move toward development of disease modifying treatments, there is a need for more specific diagnosis of early Alzheimers disease (AD) and mild cognitive impairment (MCI), plasma biomarkers are likely to play an important role in this. We review the current state of knowledge on plasma biomarkers for MCI and AD, including unbiased proteomics and very recent longitudinal studies. RECENT FINDINGS With the use of proteomics methodologies, some proteins have been identified as potential biomarkers in plasma and serum of AD patients, including alpha-1-antitrypsin, complement factor H, alpha-2-macroglobulin, apolipoprotein J, apolipoprotein A-I. The findings of cross-sectional studies of plasma amyloid beta (A beta) levels are conflicting, but some recent longitudinal studies have shown that low plasma A beta 1-42 or A beta 1-40 levels, or A beta 1-42/A beta 1-40 ratio may be markers of cognitive decline. Other potential biomarkers for MCI and AD reflecting a variety of pathophysiological processes have been assessed, including isoprostanes and homocysteine (oxidative stress), total cholesterol and ApoE4 allele (lipoprotein metabolism), and cytokines and acute phase proteins (inflammation). A panel of 18 signal proteins was reported as markers of MCI and AD. SUMMARY A variety of potential plasma biomarkers for AD and MCI have been identified, however the findings need replication in longitudinal studies. This area of research promises to yield interesting results in the near future.

Expression of the kynurenine pathway enzymes in human microglia and macrophages.

There is good evidence that the kynurenine pathway (KP) and one of its products, quinolinic acid (QUIN) play a role in the pathogenesis of neurological diseases. Monocytic cells are known to be the major producers of QUIN. However, macrophages have the ability to produce approximately 20 to 30-fold more QUIN than microglia. The molecular origin of this difference has not been clarified yet. Using unstimulated and IFN-gamma-stimulated cultures of human fcetal microglia and adult macrophages, we assayed mRNA expression of 8 key enzymes of the KP using RT-PCR and QUIN production using GC-MS. We found that after stimulation with IFN-gamma microglia produced de novo 20-fold less QUIN than macrophages. This quantitative difference in the ability to produce QUIN appears to be associated with a lower expression of 3 important enzymes of the KP in microglia: indoleamine 2,3-dioxygenase (IDO), kynureninase (KYNase) and kynurenine hydroxylase (KYN(OH)ase). These results suggest that activated infiltrating macrophages are the most potent QUIN producers during brain inflammatory diseases with playing a lesser role.

Suppression of Human Growth Hormone Secretion by Melatonin and Cyproheptadine

Pituitary growth hormone (GH) release in the rat is stimulated via serotoninergic pathways and can be inhibited by treatment with compounds that act as serotonin antagonists, such as cyproheptadine or the pineal gland hormone, melatonin. To investigate a possible role for serotonin in the control of human GH release, the effects of cyproheptadine and melatonin administration on the GH responses of normal male subjects were examined. The oral administration of cyproheptadine (8-12 mg daily for 5 days) to normal subjects reduced their GH responses to both insulin-induced hypoglycemia and physical exercise to a highly significant extent. Similarly, the mean GH responses of 10 subjects to insulin-induced hypoglycemia were significantly reduced after the prior oral administration of melatonin (1 g). The data presented show that serotonin antagonism has a similar effect on GH secretion in man to that observed in the rat and provides further evidence for serotoninergic, and possibly pineal, involvement in the control of human GH secretion.

Measurements of protein carbonyls, ortho- and meta-tyrosine and oxidative phosphorylation complex activity in mitochondria from young and old rats.

Mitochondrial bioenergetic function is often reported to decline with age and the accumulation of oxidative damage is thought to contribute. However, there are considerable uncertainties about the amount and significance of mitochondrial oxidative damage in aging. We hypothesized that, as radical production in mitochondria is greater than the rest of the cell, protein oxidative damage should accumulate more in mitochondria than the cytoplasm, and that this relative accumulation should increase with age. To test these hypotheses we measured the accumulation of three markers of protein oxidative damage in liver, brain, and heart from young and old rats. Ortho- and meta-tyrosine levels in protein hydrolysates were measured by a gas chromatography/mass spectrometry assay, and protein carbonyl content was determined by ELISA. Using these assays we found no evidence for increased protein oxidative damage in mitochondria relative to the cytosol. Most increases found in protein oxidative damage on aging were modest for all three tissues and there was no consistent pattern of increased oxidative damage in mitochondrial proteins on aging. Mitochondrial oxidative phosphorylation complex activities were also assessed revealing 39-42% decreases in F0F1--ATP synthase activity in liver and heart on aging, but not in other oxidative phosphorylation complexes. These findings have implications for the contribution of mitochondrial oxidative damage and dysfunction to aging.

Growth Hormone Regulation by Melatonin and Serotonin

THERE is evidence to suggest the existence of a pineal gland substance which moderates growth both in man and the rat. In man, non-parenchymal tumours, such as gliomas or teratomas which result in destructive lesions of the pineal gland, are associated with precocious puberty1,2 and it has been hypothesized that such lesions prevent the pineal from secreting gonadal2 and growth3,4 inhibitory factors. It was recently reported5 that the ability of the pineal gland to influence both growth and gonadal development may be due to two distinct physiological mechanisms. Rats which are blinded or kept in constant darkness show reduced body weight4,5, reduced tibeal length4, and reduced accessory organ weights4 as well as retarded puberty2,5,6. Blinded rats were also found4 to have significantly reduced pituitary gland stores of growth hormone. The effects of blinding or constant darkness on growth and growth hormone stores were abolished by pinealectomy4,7—a procedure also found8 to increase the gain of body weight in otherwise normal rats. Pinealectomy has also been reported to result in increased growth of experimental tumours in rats9. The observation10 of a diurnal fluctuation in the secretion of growth hormone in the rat further suggests that the lighting regime can modify this release. When lighting is reduced, concentrations of the pineal hormone melatonin (N-acetyl-o-methylserotonin) are increased because it is synthesized within the pineal gland from serotonin (5-hydroxytryptamine) by the action of the enzymes serotonin-N-acetyltransferase and hydroxyindole-o-methyltransferase, both of which exhibit their highest activities in the absence of light11,12. The fact that the conditions favouring high melatonin production correlate with those which cause reduced growth (and the other way round) suggests that melatonin has an inhibitory role in growth hormone secretory mechanisms. Collu et al.13 demonstrated that intraventricular injections of serotonin stimulate secretion of growth hormone in the rat, and they proposed that in this animal secretion of growth hormone is controlled through serotoninergic pathways. Serotonin has also been implicated as the stimulus for secretion of growth hormone after the onset of slow-wave (non-rapid eye movement, NREM) sleep in normal humans14. It thus seems that serotonin and its pineal derivative, melatonin, may have opposing effects on the secretion of growth hormone. We describe here experiments which support this contention.

Quinolinic acid is produced by macrophages stimulated by platelet activating factor, Nef and Tat

Activated macrophages produce quinolinic acid (QUIN), a neurotoxin, in several inflammatory brain diseases including AIDS dementia complex. We hypothesized that TL1-β, IL6, transforming growth factor (TGF-β2) and platelet activating factor could increase macrophage QUIN production. And that the HIV-1 proteins Nef, Tat and gp41 may also increase synthesis of QUIN by macrophages. At72 h there were significant increasesin QUIN production in the cells stimulated with PAF (914 ± 50 nM) and Nef (2781 ± 162 nM), with somewhat less production by Tat stimulation (645 ± 240 nM). The increases in QUIN production approximated in vitro concentrations of QUIN shown to be neurotoxic and correlated closely with indoleamine 2,3-dioxygenase induction. IL1-β, IL6, TGF-β2 and gp41 stimulation produced no significant increase in QUIN production. These results suggest that some of the neurotoxicity of PAF, nef and tat may be mediated by QUIN.