Elevated spermidine serum levels in mild cognitive impairment, a potential biomarker of progression to Alzheimer dementia, a pilot study.

BACKGROUND/AIMS: There is a close link between iron and polyamine biosynthesis and metabolism. In a recent study, we reported alterations in the serum levels of hepcidin and other iron-related proteins in Alzheimer's disease (AD) patients (Sternberg et al., 2017). Based on these findings, this pilot study compared serum levels of one of the polyamines, Spermidine, between AD, mild cognitive impairment (MCI), and control subjects, correlating the levels with the existing clinical and neuroimaging data. METHODS: This cross-sectional study measured Spermidine levels in frozen serum samples of 43 AD patients, 12 MCI patients, and 21 age-matched controls, provided by the Oregon Alzheimer's Disease Center Bio-repository, using enzyme-linked immunosorbent assay. RESULTS: MCI patients showed significantly higher mean Spermidine serum levels compared to controls (P = 0.01), with a non-significant trend for higher Spermidine serum levels in pure AD (P = 0.08) participants compared to controls. Spermidine serum levels correlated with the values of cognitive assessment tests including MMSE (r = -0.705, P = 0.003), CDR (r = 0.751, P = 0.002), and CDR-SOB (r = 0.704, P = 0.007), in "pure" AD subgroup, suggesting that higher Spermidine serum levels in MCI can be a potential biomarker of conversion to dementia in subjects with AD underlying pathology. Furthermore, Spermidine serum levels correlated with serum levels of the chief iron regulatory protein, hepcidin in AD participants with a more advanced disease stage, indicated by MMSE (strata of 8-19, P = 0.02), and CDR-SOB (strata of 6-12, P = 0.03). CONCLUSION: Studies with larger cohort are warranted for defining the role of Spermidine in AD pathophysiology, and the utility of polyamines as biomarkers of progression of MCI to AD.

Increased free prostate specific antigen serum levels in Alzheimer's disease, correlation with Cognitive Decline.

BACKGROUND/AIMS: Prostate specific antigen (PSA) is regulated by steroid hormones, such as testosterone, the serum levels of which are altered in patients with Alzheimer's disease (AD).This pilot study compared serum levels of the free (f) PSA between AD, mild cognitive impairment (MCI), and control subjects, and evaluated the relationship between fPSA serum levels and cognitive assessment tests and neuroimaging data. In addition, in a subgroup of AD patients, we correlated fPSA serum levels with the existing data on serum levels of amyloid-beta (Aß), and iron-related proteins, including hepcidin and ferritin. METHODS: Frozen serum samples from the Oregon Tissue Bank were used to measure serum levels of fPSA using enzyme-linked immunosorbent assay. RESULTS: fPSA serum levels calculated as median ±â€¯SD were higher in AD males (663.6 ±â€¯821.0 pg/ml) compared to control males (152.0 ±â€¯207.0 pg/ml), p = 0.003. A similar Pattern emerged when comparing MCI males (310.7 ±â€¯367.0 pg/ml) to control males (P = 0.02). Correlation studies showed a significant association between fPSA and CDR (r = 0.56, P = 0.006) and CDR-SOB (r = 0.54, P = 0.009) in AD males. CONCLUSION: Additional studies in a larger cohort are required for determining whether fPSA can be used as biomarker of AD disease progression and whether it has the potential to identify male subjects at risk of AD dementia.

Cadmium blocks receptor-mediated Jak/STAT signaling in neurons by oxidative stress.

Cadmium is an environmental contaminant producing numerous pathological effects including neurological disorders. The mechanisms through which cadmium produces neurotoxicities are not completely known. We found that divalent cadmium (CdCl2) inhibited ciliary neurotrophic factor (CNTF)-mediated Jak1 and Jak2 tyrosine kinase signaling in human BE(2)-C neuroblastoma cells. CdCl2 concentrations as low as 0.1 microM and for times as brief as 2 h significantly reduced CNTF-induced tyrosine phosphorylation of both STAT1 and STAT3, the principle substrates of Jak kinases in neurons. The phosphorylation of STAT1 by interferon-gamma was also inhibited by CdCl2. However, activation of the fibroblast growth factor receptor tyrosine kinase was not inhibited by CdCl2. Jak/STAT signaling was inhibited by CdCl2 selectively in cultures of chick retina neurons and neuroblastoma cells, whereas signaling in the nonneuronal cells HepG2 and chick skeletal myotubes was not affected. Results using dichlorofluorescein indicated CdCl2 increased cellular oxidative stress, and all of these effects of CdCl2 were protected against by pretreatment with antioxidants. Neuronal inhibition of Jak kinase by CdCl2-induced oxidative stress is a new mechanism of cadmium action which may directly produce neurotoxic symptoms as well as implicate cadmium and related metals as environmental factors in the etiology of neurodegenerative diseases.

Mercury abolishes neurotrophic factor-stimulated Jak-STAT signaling in nerve cells by oxidative stress.

Mercury is a potent neurotoxin that can delay neurological development in neonates, and has been proposed to be an environmental risk factor for several neurodegenerative conditions. The mechanisms by which environmental factors may influence the propagation of neurodegenerative diseases are not yet well delineated. However, it is known that neurons require trophic factor support for maintenance and survival following traumatic physical and toxic insults. We found that divalent mercury (HgCl(2)) inhibited ciliary neurotrophic factor and interferon-gamma receptor-mediated Janus tyrosine kinase (Jak)/signal transducers and activators of transcription (STAT) pathway activation in SK-N-BE(2)-C neuroblastoma cell cultures, but did not inhibit the fibroblast growth factor receptor tyrosine kinase. Results of dichlorofluorescein experiments showed increased levels of oxidative stress in HgCl(2)-treated cells that was similar in magnitude to that caused by treatment with H(2)O(2). The antioxidant agents glutathione, N-acetylcysteine, and sodium ascorbate each protected neurons against HgCl(2)-induced inhibition of STAT activation. HgCl(2) also inhibited Jak-STAT signaling in cultures of chick retina neurons, but did not affect signaling in nonneuronal HepG2 cells and chick skeletal myotubes. The specific inhibition of growth factor-mediated Jak-STAT signaling pathways in neurons by HgCl(2)-induced oxidative stress offers a new mechanism by which mercury may produce neurotoxic symptoms in the developing nervous system, promote neurodegeneration in mature neurons, and inhibit recovery following neurotrauma.

Activation and inactivation of signal transducers and activators of transcription by ciliary neurotrophic factor in neuroblastoma cells.

Neurons in vivo are exposed to a variety of different growth factors and cytokines. A principal signalling pathway for ciliary neurotrophic factor (CNTF)-like cytokines is the Janus kinase (Jak)/signal transducer and activator of transcription (STAT) system of kinases and transcription factors. In the human cell line (SH-SY5Y), STAT1 and STAT3 activation by CNTF-like cytokines showed tyrosine phosphorylation peaking at 0.5 h and inactivating within 2 h. Tyrosine phosphorylation of the receptor-associated tyrosine kinases Jak1 and Jak2 showed a similar time course of activation and inactivation in response to CNTF. The STAT1 response to the non-CNTF-like cytokine, interferon-gamma (IFN-gamma) did not inactivate. Inactivation to CNTF was not due to a decrease in CNTF receptor subunit gp130 or in levels of Jak1 or Jak2. STAT inactivation was inhibited by the protein kinase blocker H7 and a tyrosine phosphatase blocker, but not by inhibitors of protein kinase C, mitogen-activated protein kinase (MAPK) kinase, mTOR-P70/S6 kinase or phosphatidyl inositol-3-kinase (PI-3 kinase). Surprisingly, CNTF caused only a minor increase in levels of suppressors of cytokine signalling, SOCS-1 and SOCS-3. CNTF pretreatment desensitized the cells to the CNTF-like cytokines, leukemia inhibitory factor and oncostatin-M but not to IFN-gamma. These results reveal a complex level of regulation of shared signalling pathways for cytokines that is dependent on both the type of cell and cytokine.

Initiation and maintenance of CNTF-Jak/STAT signaling in neurons is blocked by protein tyrosine phosphatase inhibitors.

Cytokines, including interferon-gamma and ciliary neurotrophic factor (CNTF), act in common through tyrosine kinase-based Jak/STAT signaling pathways. We found that activation of the Jak/STAT pathway by both interferon-gamma and CNTF in nerve cells was rapidly terminated by tyrosine phosphatase inhibitors. Exposure of human neuroblastoma cells, BE(2)-C, first to tyrosine phosphatase inhibitors (either phenylarsine oxide or PTP inhibitor-2) prevented Jak1, STAT1 and STAT3 activation elicited subsequently by either CNTF or interferon-gamma. In contrast, exposure of these cells to phosphatase inhibitors after initial stimulation by CNTF or interferon-gamma prevented the normal time-dependent decrease of total cellular phosphotyrosine-STAT levels as expected, while excluding already formed phosphotyrosine-STAT from the nucleus. Thus, treatment of nerve cells with a tyrosine phosphatase inhibitor blocked nuclear signal transduction. A similar inhibition of CNTF-Jak/STAT signaling was observed following tyrosine phosphatase inhibition in SH-SY5Y human neuroblastoma cells, HMN-1 mouse motor neuron-neuroblastoma hybrid cells, HepG2 human hepatoma cells and embryonic chick ciliary ganglion and retinal neurons. Expression of dominant-negative forms of the tyrosine phosphatases, SHP-1 and/or SHP-2, in BE(2)-C cells had no effect on CNTF activation of STAT or on the ability of phosphatase inhibitors to block signaling. Further, results from H-35 cells expressing gp130 receptor subunits lacking functional SHP-2 binding sites revealed normal cytokine activation of Jak and STAT that was inhibited by phosphatase inhibitors. These findings suggest a critical control for regulating the initiation of Jak/STAT signaling requiring tyrosine phosphatase activity.

Detection of trophic factor activated signaling molecules in cells by a compact fiber-optic sensor.

This paper describes a highly sensitive method to detect trophic factor activated signaling molecules in cells using a compact fiber optic biosensor. The method is demonstrated by quantitative detection of phosphorylation of signal transducers and activators of transcription 3 (STAT3) in neuroblastoma cells. A single fiber-optic probe based on total internal reflection fluorescence sensing system is used. A 405 nm diode laser is used for evanescent wave excitation of immobilized labelled analyte on the probe surface. A compact charged coupled device (CCD) based spectrometer is used for recording the fluorescence signal. The method is two orders of magnitude more sensitive than the Western blotting technique.

Environmental toxicants inhibit neuronal Jak tyrosine kinase by mitochondrial disruption.

Cadmium, mercury and rotenone are environmental pollutants whose neurotoxic mechanisms are not fully understood. We have shown previously that exposure of nerve cells to these agents produces oxidative stress which reversibly blocks growth factor and cytokine-mediated Janus kinase (Jak)/signal transducer and activator of transcription (STAT) signaling. Here we determined a critical role for mitochondrial dysfunction in inhibiting Jak/STAT activity in human BE(2)-C neuroblastoma cells. Exposure of BE(2)-C cells to the heavy metals CdCl(2) and HgCl(2) and to the mitochondrial complex I inhibitor rotenone inhibited interleukin-6, interferon-gamma and ciliary neurotrophic factor-mediated Jak/STAT signaling, reduced Jak1 and Jak2 auto-phosphorylation and induced Jak tyrosine nitration. However, identical exposure of HepG2 hepatoma cells produced no inhibition of these cytokine responses. In contrast, mitochondria in both BE(2)-C and HepG2 cells showed reduced mitochondrial membrane potential and increased superoxide production after exposure to CdCl(2), HgCl(2) and rotenone. Further, in an in vitro Jak auto-phosphorylation assay Jak2 isolated from either BE(2)-C or HepG2 cells was equally inhibited by mitochondria made dysfunctional by treatment with CdCl(2), HgCl(2) and rotenone. Each of these pro-oxidant effects was reversed by the mitochondrial antioxidant alpha-lipoic acid. The actions of cadmium were also blocked by the mitochondrial complex III bypass agent, 2,6-dichloroindophenol. Therefore, in BE(2)-C cells CdCl(2), HgCl(2) and rotenone disrupt mitochondria to increase intracellular ROS, which directly inhibits neuronal Jak tyrosine kinase activity. Non-neuronal cells such as HepG2 cells that are resistant to oxidative stress-mediated inhibition of cytokine signaling possess some as yet unknown mechanism that protects Jak kinases from oxidative insults. Pro-oxidant-induced mitochondrial dysfunction resulting in selective neuronal Jak inhibition provides a potential mechanism for environmental agents to promote neurodegeneration.

Induction of an interferon-gamma Stat3 response in nerve cells by pre-treatment with gp130 cytokines.

Many cytokines mediate their effects through Jak/STAT signaling pathways providing many opportunities for cross-talk between different cytokines. We examined the interaction between two cytokine families, gp130-related cytokines and interferon-gamma (IFN-gamma), which are coexpressed in the nervous system during acute trauma and pathological conditions. Typical nerve cells show an IFN-gamma response that is restricted to activating STAT1, with minor activation of STAT3. IFN-gamma elicited a pronounced STAT3 response in cells pre-treated for 5-7 h with ciliary neurotrophic factor (CNTF), leukemia inhibitory factor or interleukin-6. CNTF or interleukin-6 induced an IFN-gamma STAT3 response in a variety of cells including SH-SY5Y human neuroblastoma, HMN-1 murine motor neuron hybrid cells, rat sympathetic neurons and human hepatoma HepG2 cells. The enhancement was measured as an increase in tyrosine phosphorylated STAT3, in STAT3-DNA binding and in STAT-luciferase reporter gene activity. The enhanced STAT3 response was not due to an increase in overall STAT3 levels but was dependent upon ongoing protein synthesis. The induction by CNTF was inhibited by the protein kinase C inhibitor, BIM, and the MAPK-kinase inhibitor, U0126. Further, H-35 hepatoma cells expressing gp130 receptor chimeras lacking either the SHP-2 docking site or the Box 3 STAT binding sites failed to enhance the IFN-gamma STAT3 response. These results provide evidence for an interaction between gp130 and IFN-gamma cytokines that can significantly alter the final cellular response to IFN-gamma.