Parkinson's Disease Master Regulators on Substantia Nigra and Frontal Cortex and Their Use for Drug Repositioning.

Parkinson's disease (PD) is among the most prevalent neurodegenerative diseases. Available evidences support the view of PD as a complex disease, being the outcome of interactions between genetic and environmental factors. In face of diagnosis and therapy challenges, and the elusive PD etiology, the use of alternative methodological approaches for the elucidation of the disease pathophysiological mechanisms and proposal of novel potential therapeutic interventions has become increasingly necessary. In the present study, we first reconstructed the transcriptional regulatory networks (TN), centered on transcription factors (TF), of two brain regions affected in PD, the substantia nigra pars compacta (SNc) and the frontal cortex (FCtx). Then, we used case-control studies data from these regions to identify TFs working as master regulators (MR) of the disease, based on region-specific TNs. Twenty-nine regulatory units enriched with differentially expressed genes were identified for the SNc, and twenty for the FCtx, all of which were considered MR candidates for PD. Three consensus MR candidates were found for SNc and FCtx, namely ATF2, SLC30A9, and ZFP69B. In order to search for novel potential therapeutic interventions, we used these consensus MR candidate signatures as input to the Connectivity Map (CMap), a computational drug repositioning webtool. This analysis resulted in the identification of four drugs that reverse the expression pattern of all three MR consensus simultaneously, benperidol, harmaline, tubocurarine chloride, and vorinostat, thus suggested as novel potential PD therapeutic interventions.

Alzheimer's disease master regulators analysis: search for potential molecular targets and drug repositioning candidates.

BACKGROUND: Alzheimer's disease (AD) is a multifactorial and complex neuropathology that involves impairment of many intricate molecular mechanisms. Despite recent advances, AD pathophysiological characterization remains incomplete, which hampers the development of effective treatments. In fact, currently, there are no effective pharmacological treatments for AD. Integrative strategies such as transcription regulatory network and master regulator analyses exemplify promising new approaches to study complex diseases and may help in the identification of potential pharmacological targets. METHODS: In this study, we used transcription regulatory network and master regulator analyses on transcriptomic data of human hippocampus to identify transcription factors (TFs) that can potentially act as master regulators in AD. All expression profiles were obtained from the Gene Expression Omnibus database using the GEOquery package. A normal hippocampus transcription factor-centered regulatory network was reconstructed using the ARACNe algorithm. Master regulator analysis and two-tail gene set enrichment analysis were employed to evaluate the inferred regulatory units in AD case-control studies. Finally, we used a connectivity map adaptation to prospect new potential therapeutic interventions by drug repurposing. RESULTS: We identified TFs with already reported involvement in AD, such as ATF2 and PARK2, as well as possible new targets for future investigations, such as CNOT7, CSRNP2, SLC30A9, and TSC22D1. Furthermore, Connectivity Map Analysis adaptation suggested the repositioning of six FDA-approved drugs that can potentially modulate master regulator candidate regulatory units (Cefuroxime, Cyproterone, Dydrogesterone, Metrizamide, Trimethadione, and Vorinostat). CONCLUSIONS: Using a transcription factor-centered regulatory network reconstruction we were able to identify several potential molecular targets and six drug candidates for repositioning in AD. Our study provides further support for the use of bioinformatics tools as exploratory strategies in neurodegenerative diseases research, and also provides new perspectives on molecular targets and drug therapies for future investigation and validation in AD.

Reduced Bone Stiffness in Women Is Associated with Clinical Attachment and Tooth Loss: The Study of Health in Pomerania.

The authors evaluated the association of reduced bone stiffness of the calcaneus with clinical attachment loss (CAL) and tooth loss. The authors analyzed data from 4,678 subjects (2,384 women), aged 20 to 88 y, from the second follow-up of the population-based Study of Health in Pomerania (SHIP-2) and the baseline examination of the SHIP-Trend cohort. Bone stiffness, characterized by the stiffness index (SI) and the osteoporotic fracture risk (OFR), was assessed by quantitative ultrasound of the heel. SI and OFR were significantly associated with the mean CAL in women. While 1) the SI showed a significant association with the mean CAL and 2) the OFR with the median number of teeth in just the postmenopausal women, the OFR showed a significant association with mean CAL for both pre- and postmenopausal women. In postmenopausal women, a 10-unit increase in the SI was associated with a decrease in the mean CAL of 0.05 mm (95% confidence interval [CI]: -0.10 to 0.00; P = 0.046). Moreover, the adjusted median number of teeth was 21.4 (95% CI: 20.9 to 21.9) among the postmenopausal women with a low OFR, while it was 19.1 (95% CI: 17.8 to 20.3; P = 0.001) among the postmenopausal women with a high OFR. For the premenopausal women with a low OFR, the mean CAL was 1.60 mm (95% CI: 1.53 to 1.66), while for the premenopausal women with a high OFR, it was 2.24 mm (95% CI: 1.78 to 2.69; P = 0.006). Reduced bone stiffness was associated with clinical attachment and tooth loss in women but not in men.

Ultrasound findings in ocular trauma.

OBJECTIVE: To evaluate the frequencies of various eye and/or orbital disorders by ultrasound examination in patients with ocular trauma. MATERIALS AND METHODS: This prospective and descriptive study was conducted on 100 patients with ocular trauma treated in the Conde de Valenciana Institute from March to November 2014. Ultrasound examination was performed primarily using ultrasound B mode, with standardised A mode only used as correlation method. Age, gender, type of trauma, and various ultrasound findings were recorded. RESULTS: Ocular trauma was more frequent in men (83%) compared to women (17%). The left eye was affected in 55%, and right eye in 45%, with 55% being open traumas and 45% blunt traumas. Most cases were young patients with a mean age of 33.7 years, with the group between 41 to 50 years being the most affected. Among the most frequent injuries found was the vitreous haemorrhage (45%) and posterior hyaloid detachment (38%), followed by retinal detachment (32%), and choroidal detachment (18%). CONCLUSION: Ultrasound remains as the investigation method of choice in patients with ocular trauma, since it is a simple, cheap and non-invasive study, and can be very useful in providing diagnostic and prognostic information. This study demonstrated that trauma is more common in young men, with vitreous haemorrhage as the most common finding.

The effect of a ceramide analog, N-acetylsphingosine on the induction of proliferation and IL-2 synthesis in T cells from young and old F344 rats.

Ceramide is a physiological mediator of extracellular signals that control various cellular functions, including proliferation and apoptosis. In the present study, we examined the effects of cell-permeable ceramide analog, N-acetyl-sphingosine (C(2)-ceramide) on the induction of proliferation and interleukin-2 (IL-2) synthesis in T cells from young and old rats. Splenic T cells from 6- and 24-month-old Fischer 344 rats were treated with C(2)-ceramide and then incubated with anti-CD3 antibody for 24 or 48 h. The induction of proliferation and IL-2 production by anti-CD3 was significantly (P<0.001) lower in T cells from old rats compared to T cells from young rats. C(2)-ceramide treatment resulted in suppression of proliferation and IL-2 production in a concentration-dependent manner. The suppressive effect of C(2)-ceramide on proliferation and IL-2 production was greater in T cells from old rats than T cells from young rats. We investigated whether this decreased responsiveness was due to induction of program cell death (apoptosis) and found that there was a significant increase in DNA fragmentation in C(2)-ceramide treated and anti-CD3 stimulated T cells from both young and old rats. The increase in DNA fragmentation was paralleled with an increase in caspase-3 activation. C(2)-ceramide-induced caspase-3 activation and DNA fragmentation was significantly (P<0.5) higher in stimulated T cells from old rats compared to stimulated T cells from young rats. These results suggest that the sphingomyelin-ceramide signaling pathway may play an important regulatory role in the well-documented age-related decline in immune function.

Normal immune function in young and old DNA polymerase-beta deficient mice.

The effect of the DNA polymerase-beta (beta-pol) deficiency on mitogenic response and cytokine production was studied in spleen lymphocytes from 4-5- and 20-22-month-old beta-pol(-/+) mice and their age-matched wild-type littermates. The proliferative response of lymphocytes to Concanavalin A (Con A) and lipopolysaccharide (LPS) was measured by [3H]thymidine incorporation, and the induction of cytokine production (interleukin (IL)-2, IL-4, and interferon necrosis factor (IFN)-gamma) was assessed by enzyme-linked immunosorbent assay. There was no significant difference in Con A- or LPS-induced proliferation or cytokine production in young beta-pol(-/+) mice compared with young wild-type littermates or in old beta-pol(-/+) mice compared with old wild-type littermates. However, mitogen-induced proliferation and cytokine production changed significantly with age. The proliferative response to Con A and to LPS, and the IL-2 production was significantly lower, and IL-4 and IFN-gamma levels were significantly higher in lymphocytes from old beta-pol(-/+) mice and old wild-type mice than in lymphocytes from young beta-pol(-/+) mice and young wild-type littermates. In addition, flow cytometric analysis showed no significant differences between young beta-pol(-/+) mice and young wild-type littermates or between old beta-pol(-/+) mice and old wild-type littermates in the proportion of B- and T-cell populations, and T-cell subsets. However, the number of lymphocytes expressing CD4+ phenotype slightly decreased and the proportion of lymphocytes expressing CD44/Pgp-1 (memory) phenotype increased with age. Thus, we found no evidence for alteration in immune function in DNA polymerase-beta deficient mice, although they exhibit a decline in immunologic function with age.

Influence of aging and caloric restriction on activation of Ras/MAPK, calcineurin, and CaMK-IV activities in rat T cells.

The signaling cascade mediated by Ras (p21ras) and MAPK (mitogen-activated protein kinase) and calcium/calmodulin regulating enzymes, calcineurin (CaN) and CaMK-IV, are considered to be essential for T-cell growth and function. In the present study, the effect of aging and caloric restriction (CR) on the induction of Ras and MAPK activation by concanavalin A (ConA) was studied. Splenic T cells were isolated from young (4-6 months) and old (22-24 months) rats that had free access to food (control group), and from caloric restricted old (22-24 months) rats that beginning at 6 weeks of age were fed 60%(40% caloric restriction) of the diet consumed by the control rats. We found that the induction of Ras activity in T cells isolated from control old rats was lower (P<0.001) than that in control young rats. However, the levels of Ras activity in T cells isolated from CR old rats were similar to the levels in the age-matched control rats. The induction of MAPK activity in T cells isolated from control old rats and CR old rats was significantly less than in T cells isolated from control young rats, and caloric restriction significantly (P<0.05) reduced the age-related decline in MAPK activation. We also measured the induction of CaN and CaMK-IV activities by ConA in T cells from control young and old and CR old rats. The induction of both CaN and CaMK-IV activity decreased with age. Caloric restriction significantly (P<0.05) reduced the age-related decline in CaN activity, but had no significant effect on CaMK-IV activity. The changes in Ras/MAPK activation and in CaN and CaMK-IV activity with age or with CR were not associated with alterations in their corresponding protein levels. Thus, caloric restriction has a differential effect on the activation of the upstream signaling molecules that are altered with age.

Age-related decline in activation of calcium/calmodulin-dependent phosphatase calcineurin and kinase CaMK-IV in rat T cells.

We have previously shown that the DNA binding activity of the transcription factor NFAT which plays a predominant role in IL-2 transcription decreases with age. Because the transactivation (dephosphorylation and nuclear translocation) of the NFAT-c (cytoplasmic component of the NFAT complex) is mediated by the calcium/calmodulin-dependent phosphatase, calcineurin (CaN), and because Ca2+/calmodulin-dependent kinases (CaMK-II and IV/Gr) have been shown to play a critical role in calcium signaling in T cells, it was of interest to determine what effect aging has on the activation and the levels of these calcium regulating enzymes. The induction of calcineurin phosphatase activity, and CaMK-II and IV/Gr activities, were studied in splenic T cells isolated from Fischer 344 rats at 6, 15, and 24 months of age. In addition, the changes in the protein levels of these enzymes were measured by Western blot. The calcineurin phosphatase activity and CaMK-II and IV kinase activities were at a maximum after the cells were incubated with anti-CD3 antibody for 5-10 minutes. The induction of calcineurin activity by anti-CD3 and by calcium ionophore (A23187) declined 65 and 55%, respectively, between 6 and 24 months of age. The induction of CaMK-IV activity, but not CaMK-II activity by anti-CD3, was significantly less (by 54%) in T cells from old rats compared to T cells from young rats. The decline in the activation of these enzymes with age was not associated with changes in their corresponding protein levels. These results demonstrate that alterations in calcineurin phosphatase activity and CaMK-IV activity may contribute to the well-documented age-related decline in T cell function.

The effect of a calcium antagonist on the retention of simple associational learning.

Using a newly developed training paradigm, rats were trained to associate a spatial location and a black interior with mild footshock and another adjacent location with white interior with the absence of footshock in three independent experiments. Retention of these associations was measured 24 and 48 hr after training in situation in which the animals could move freely between the black and white locations over a 90 sec test. Other rats were subjected to a control procedure in which shock was received on both the black and the white sides of the apparatus. In each of the experiments, half of the animals in the experimental and the control groups were trained following the administration of the calcium slow channel blocking agent, nimodipine (5 mg/kg), and the other half after saline administration. In experiment 1, the injections were given 15 min before training. In experiment 2, the injections were given chronically, over a 6 day period before training. In experiment 3, the animals were given a single injection 7 days before training. In all cases, retention was examined both 24 and 48 hr after training. The results were that the experimental procedures produce a strong aversion to the black portion of the apparatus. The greatest amount of retention was found in animals that had received the chronic injections, whether they were of saline or of nimodipine. In every aspect of retention in which the saline-treated rats were less than perfect in retention, the nimodipine animals exhibited superior performance.