Mindfulness-based cancer recovery and supportive-expressive therapy maintain telomere length relative to controls in distressed breast cancer survivors.

BACKGROUND: Group psychosocial interventions including mindfulness-based cancer recovery (MBCR) and supportive-expressive group therapy (SET) can help breast cancer survivors decrease distress and influence cortisol levels. Although telomere length (TL) has been associated with breast cancer prognosis, the impact of these two interventions on TL has not been studied to date. METHODS: The objective of the current study was to compare the effects of MBCR and SET with a minimal intervention control condition (a 1-day stress management seminar) on TL in distressed breast cancer survivors in a randomized controlled trial. MBCR focused on training in mindfulness meditation and gentle Hatha yoga whereas SET focused on emotional expression and group support. The primary outcome measure was relative TL, the telomere/single-copy gene ratio, assessed before and after each intervention. Secondary outcomes were self-reported mood and stress symptoms. RESULTS: Eighty-eight distressed breast cancer survivors with a diagnosis of stage I to III cancer (using the American Joint Committee on Cancer (AJCC) TNM staging system) who had completed treatment at least 3 months prior participated. Using analyses of covariance on a per-protocol sample, there were no differences noted between the MBCR and SET groups with regard to the telomere/single-copy gene ratio, but a trend effect was observed between the combined intervention group and controls (F [1,84], 3.82; P = .054; η(2) = .043); TL in the intervention group was maintained whereas it was found to decrease for control participants. There were no associations noted between changes in TL and changes in mood or stress scores over time. CONCLUSIONS: Psychosocial interventions providing stress reduction and emotional support resulted in trends toward TL maintenance in distressed breast cancer survivors, compared with decreases in usual care.

Telomere length correlates with life span of dog breeds.

Telomeric DNA repeats are lost as normal somatic cells replicate. When telomeres reach a critically short length, a DNA damage signal is initiated, inducing cell senescence. Some studies have indicated that telomere length correlates with mortality, suggesting that telomere length contributes to human life span; however, other studies report no correlation, and thus the issue remains controversial. Domestic dogs show parallels in telomere biology to humans, with similar telomere length, telomere attrition, and absence of somatic cell telomerase activity. Using this model, we find that peripheral blood mononuclear cell (PBMC) telomere length is a strong predictor of average life span among 15 different breeds (p < 0.0001), consistent with telomeres playing a role in life span determination. Dogs lose telomeric DNA ~10-fold faster than humans, which is similar to the ratio of average life spans between these species. Breeds with shorter mean telomere lengths show an increased probability of death from cardiovascular disease, which was previously correlated with short telomere length in humans.

Palmitate alters the rhythmic expression of molecular clock genes and orexigenic neuropeptide Y mRNA levels within immortalized, hypothalamic neurons.

The control of energy homeostasis within the hypothalamus is under the regulated control of homeostatic hormones, nutrients and the expression of neuropeptides that alter feeding behavior. Elevated levels of palmitate, a predominant saturated fatty acid in diet and fatty acid biosynthesis, alter cellular function. For instance, a key mechanism involved in the development of insulin resistance is lipotoxicity, through increased circulating saturated fatty acids. Although many studies have begun to determine the underlying mechanisms of lipotoxicity in peripheral tissues, little is known about the effects of excess lipids in the brain. To determine these mechanisms we used an immortalized, clonal, hypothalamic cell line, mHypoE-44, to demonstrate that palmitate directly alters the expression of molecular clock components, by increasing Bmal1 and Clock, or by decreasing Per2, and Rev-erbα, their mRNA levels and altering their rhythmic period within individual neurons. We found that these neurons endogenously express the orexigenic neuropeptides NPY and AgRP, thus we determined that palmitate administration alters the mRNA expression of these neuropeptides as well. Palmitate treatment causes a significant increase in NPY mRNA levels and significantly alters the phase of rhythmic expression. We explored the link between AMPK and the expression of neuropeptide Y using the AMPK inhibitor compound C and the AMP analog AICAR. AMPK inhibition decreased NPY mRNA. AICAR also elevated basal NPY, but prevented the palmitate-mediated increase in NPY mRNA levels. We postulate that this palmitate-mediated increase in NPY and AgRP synthesis may initiate a detrimental positive feedback loop leading to increased energy consumption.

Interfacial behavior of immortalized hypothalamic mouse neurons detected by acoustic wave propagation.

The attachment of immortalized hypothalamic murine neurons onto the surface of an acoustic wave device yields both positive series resonant frequency (f(s)) and motional resistance (R(m)) shifts as opposed to commonly reported negative f(s) and positive R(m) shifts observed for other cell types. These unique shifts have been confirmed by a variety of experiments in order to verify the source and the validity of the signals. These studies involved monitoring responses to solution flow, the absence of serum proteins, the effect of reducing specific cell -surface interactions and the disruption of the neuronal cytoskeleton components. For the adhesion and deposition of neurons, f(s) and R(m) shifts are positively correlated to the amount of adhered neurons on the sensor surface, whereas non-adhered neurons do not produce any significant change in the monitored parameters. In the absence of serum proteins, initial cell adhesion is followed by subsequent cell death and removal from the sensor surface. The presence of the peptide, GRGDS is observed to significantly reduce cell-surface specific interactions compared to the control of SDGRG and this produces f(s) and R(m) responses that are opposite in direction to that observable for cell adhesion. Cytoskeletal studies, using the drugs nocodazole (10 µM), colchicine (1 µM), cytochalasin B (10 µM) and cytochalasin D (2 µM) all elicit neuronal responses that are validated by phalloidin actin-filament staining. These results indicate that the responses are associated with a wide range of cellular changes that can be monitored and studied using the acoustic wave method in real time, under optimal physiological conditions.

Synchronization of the circadian rhythm generator and the effects of glucagon on hypothalamic mouse neurons detected by acoustic wave propagation.

A thickness shear mode acoustic wave sensor has been used to study the reaction of clonal, immortalized hypothalamic murine neurons in response to glucagon and serum shock in a label free, continuous and real time manner under physiological conditions. Two cell lines were examined; these were the mHypoE-38s and the mHypoE-46s. The technique possesses sufficient sensitivity to detect minor neuronal changes and is capable of discerning subtle differences in cellular behaviors under both stimuli. The kinetics and magnitude of the changes observed here are significantly different compared to those instigated upon causing depolarization, cytoskeletal modifications and surface-adhesion specific interaction alterations with the same cells. Interestingly, this technique has the sensitivity and capability of observing all such changes at the neuronal level without the necessity for invasive interrogation. Under the influence of glucagon, the neurons display both short- and long-term changes, in particular the resonant frequency shifts by -23 ± 8 Hz (n = 13, std. dev.) and the motional resistance decays at a rate of approximately 10 Ω h(-1) over a 2 hour interval. The effect of synchronizing the neurons prior to glucagon stimulation did not influence the cellular changes observed. The process of partial and full synchronization of the cells resulted in different responses. For full synchronization, the addition of the serum bolus triggered resonant frequency and motional resistance shifts of +75 Hz and +18.5 Ω respectively, which decayed back to baseline levels after 30 minutes. The duration of this decay closely matched the time required for full synchronization in a separate study. The changes observed for partial synchronization were significantly different from full synchronization as the baseline levels in both resonant frequency and motional resistance were not re-achieved indicative of the cell-sensor system detecting the difference between full and partial synchronization. Preliminary qualitative immunocytochemistry and RT-PCR studies on these cells support the results obtained with the TSM sensor for the glucagon receptor study.

Nutrient sensing and insulin signaling in neuropeptide-expressing immortalized, hypothalamic neurons: A cellular model of insulin resistance.

Obesity and type 2 diabetes mellitus represent a significant global health crisis. These two interrelated diseases are typified by perturbed insulin signaling in the hypothalamus. Using novel hypothalamic cell lines, we have begun to elucidate the molecular and intracellular mechanisms involved in the hypothalamic control of energy homeostasis and insulin resistance. In this review, we present evidence of insulin and glucose signaling pathways that lead to changes in neuropeptide gene expression. We have identified some of the molecular mechanisms involved in the control of de novo hypothalamic insulin mRNA expression. And finally, we have defined key mechanisms involved in the etiology of cellular insulin resistance in hypothalamic neurons that may play a fundamental role in cases of high levels of insulin or saturated fatty acids, often linked to the exacerbation of obesity and diabetes.

Rhythmic clock and neuropeptide gene expression in hypothalamic mHypoE-44 neurons.

The rhythmic expression of specific clock genes: Bmal1, Per2, Clock and Rev-Erbalpha; and specific hypothalamic neuropeptides: NPY, Crh, AgRP, neurotensin and preproghrelin, expressed in clonal hypothalamic neuronal cell lines, was assayed and analyzed using a novel non-linear least squares statistical analysis to determine rhythmicity in an in vitro milieu. In silico analysis of the neuropeptide promoter regions identified putative E-box motifs and a motif in the NPY promoter is bound to in an oscillatory fashion. Within the mHypoE-44 neurons, we demonstrate that mRNA of four core circadian components: Bmal1, Clock, Per2 and Rev-Erbalpha, oscillate with approximate 24h rhythms. NPY and NT demonstrated significant 24h gene expression. However, CRH and preproghrelin mRNA cycled significantly in an ultradian fashion, oscillating approximately every 18h. AgRP mRNA did not show a significant rhythm. We speculate that endogenous rhythmic neuropeptide expression contributes to neuroendocrine homeostasis, which may include energy balance.

Depolarization of surface-attached hypothalamic mouse neurons studied by acoustic wave (thickness shear mode) detector.

Isolation of neurons from animal tissue is an important aspect of understanding basic biochemical processes such as the action of hormones and neurotransmitters. In the present work, the focus is on an effort to evaluate the utility of acoustic wave physics for the study of such cells. Immortalised hypothalamic neuronal cells from mouse embryos were cultured on the surface of the gold electrode of a 9.0 MHz thickness-shear mode acoustic wave sensor. These cells, which are clonal, are imposed on the surface of the device at a confluence in the range of 80-100%. The coated sensor is incorporated into a flow-injection configuration such that electrolytes can be introduced in order to examine their effects through measurement by network analysis. Both series resonance frequency, fs, and motional resistance, R(m), were measured in a number of experiments involving the injection of KCl and NaCl into the sensor-neuron system. The various responses to these electrolytes were interpreted in terms of changes in cellular structure associated with the depolarization process. The sensor-neuron system was found to elicit different responses to the addition of KCl and NaCl. Preliminary findings indicate that the TSM sensor does not purely measure changes in the membrane potential upon KCl addition. Typical changes in fs for 15 mM, 30 mM and 60 mM KCl additions were 54 +/- 15, 80 +/- 26 and 142 +/- 58 Hz (mean +/- standard deviation) respectively. Typical changes in R(m) for these KCl additions were 7 +/- 3, 13 +/- 4 and 23 +/- 6 Omega, respectively. These results were concluded after 17 runs at each concentration. Despite the large relative standard deviations, the dependence of f(s) and R(m) with respect to concentration was apparent. Controls performed by coating the TSM sensor with laminin or a cell attachment matrix showed no significant changes in either f(s) or R(m) for the same solutions tested on the sensor-neuron system.

Ciliary neurotrophic factor recruitment of glucagon-like peptide-1 mediates neurogenesis, allowing immortalization of adult murine hypothalamic neurons.

The distinct lack of cell lines derived from the adult brain is evident. Ciliary neurotrophic factor (CNTF) triggers neurogenesis in primary culture from adult mouse hypothalamus, as detected by bromodeoxyuridine and Ki67 immunostaining. Using SV-40 T-antigen, we immortalized dividing neurons and generated clonal cell lines expressing neuropeptides and receptors involved in neuroendocrine function. We hypothesized that proglucagon-derived peptides may be the mechanistic downstream effectors of CNTF due to documented neuroprotective and proliferative effects. Indeed, proglucagon gene expression was induced by CNTF, and exposure of primary cells to glucagon-like peptide-1 receptor (GLP-1) agonist, exendin-4, induced cell proliferation. Intracerebroventricular injection of CNTF into adult mice caused increased expression of proglucagon peptide in the hypothalamus. Using a specific GLP-1-receptor antagonist, we found that neurogenesis was significantly attenuated and primary culture from GLP-1-receptor-knockout mice lacked CNTF-mediated neuronal proliferation, thus linking the induction of neurogenesis in the hypothalamus to GLP-1-receptor signaling.

Serine racemase is associated with schizophrenia susceptibility in humans and in a mouse model.

Abnormal N-methyl-d-aspartate receptor (NMDAR) function has been implicated in the pathophysiology of schizophrenia. d-serine is an important NMDAR modulator, and to elucidate the role of the d-serine synthesis enzyme serine racemase (Srr) in schizophrenia, we identified and characterized mice with an ENU-induced mutation that results in a complete loss of Srr activity and dramatically reduced d-serine levels. Mutant mice displayed behaviors relevant to schizophrenia, including impairments in prepulse inhibition, sociability and spatial discrimination. Behavioral deficits were exacerbated by an NMDAR antagonist and ameliorated by d-serine or the atypical antipsychotic clozapine. Expression profiling revealed that the Srr mutation influenced several genes that have been linked to schizophrenia and cognitive ability. Transcript levels altered by the Srr mutation were also normalized by d-serine or clozapine treatment. Furthermore, analysis of SRR genetic variants in humans identified a robust association with schizophrenia. This study demonstrates that aberrant Srr function and diminished d-serine may contribute to schizophrenia pathogenesis.

Hypothalamic cell lines to investigate neuroendocrine control mechanisms.

The hypothalamus is the control center for most physiological processes; yet has been difficult to study due to the inherent heterogeneity of this brain region. For this reason, researchers have turned towards cell models. Primary hypothalamic cultures are difficult to maintain, are heterogeneous neuronal and glial cell populations and often contain a minimal number of viable peptide-secreting neurons. In contrast, immortalized, clonal cell lines represent an unlimited, homogeneous population of neurons that can be manipulated using a number of elegant molecular techniques. Cell line studies and in vivo experimentation are complementary and together provide a powerful tool to drive scientific discovery. This review focuses on three key neuroendocrine systems: energy homeostasis, reproduction, and circadian rhythms; and the use of hypothalamic cell lines to dissect the complex pathways utilized by individual neurons in these systems.