Cell death neuroprotection and repair mechanisms in a model of rat spinal cord injury in vitro

Nowadays, new spinal cord injury (SCI) cases are frequently due to non traumatic causes, especially vascular disorders. A prerequisite to developing mechanism-based neuroprotective strategies for acute SCI is a full understanding of the early pathophysiological changes to prevent later disability and paralysis. The immediate damage spreads from the initial site through excitotoxicity and metabolic dysfunction (ischemia, free radicals and neuroinflammation) to surrounding tissue (secondary damage). Using an in vitro neonatal rat spinal cord model, an experimental protocol (pathological medium, PM) has been developed to mimic the profound metabolic perturbation (hypoxia, aglycemia, oxidative stress, acidosis, toxic free radicals) occurring in vivo after ischemic SCI, a condition surprisingly worsened by extracellular Mg2+ (1 mM). The current study sought to identify the cells affected by PM (with Mg2+), and the associated molecular death pathways in the spinal lumbar region which contains the locomotor networks. The results indicated that 1 h PM+Mg2+ application induced delayed pyknosis chiefly in the spinal white matter via overactivation of poly (ADP-ribose) polymerase 1 (PARP1), suggesting cell death mediated by the process of parthanatos and also via caspase 3-dependent apoptosis. Grey matter damage was less intense and concentrated in dorsal horn neurons and motoneurons which became nuclearimmunoreactive for the mitochondrial apoptosis-inducing factor. Moreover, TRPM2 channel expression was enhanced 24 h later in dorsal horn and motoneurons, while TRPM7 channel expression concomitantly decreased. Conversely, TRPM7 expression grew earlier (3 h) in white matter cells, while TRPM2 remained undetectable. Our results show that extracellular Mg2+ amplified the white matter cell death via parthanatos and apoptosis, and motoneuronal degeneration via PARP1-dependent pathways with distinct changes in their TRPM expression. In fact, the PARP-1 inhibitor PJ34, when applied 30 min after the moderate excitotoxic insult, could protect spinal networks controlling locomotion in more than 50 % of preparations. Interestingly, the drug per se strongly increased spontaneous network discharges without cell damage. Glutamate ionotropic receptor blockers suppressed this phenomenon reversibly. Our results suggest that pharmacological inhibition of PARP-1 could prevent damage to the locomotor networks if this procedure had been implemented early after the initial lesion and when the lesion was limited. PJ34 had also a positive effect on PM+Mg2+ treated spinal cords, especially in the white matter after 24 h, both alone or administered together with caspase-3 inhibitor. The neonatal rat in vitro SCI model was also useful to study the activation of endogenous spinal stem cells. We identified the ATF3 transcription factor as a novel dynamic marker for ependymal stem/progenitor cells (nestin, vimentin and SOX2 positive) located around the central canal of the neonatal or adult rat spinal cord. While quiescent ependymal cells showed cytoplasmic ATF3 expression, over 6-24 h in vitro these cells mobilized and acquired intense nuclear ATF3 staining. The migration of ATF3-nuclear positive cells preceded the strong proliferation of ependymal cells occurring after 24 h in vitro. Pharmacological inhibition of MAPK-p38 and JNK/c-Jun, upstream effectors of ATF3 activation, prevented the mobilization of ATF3 nuclear-positive cells. Excitotoxicity or ischemia-like conditions did not enhance migration of ependymal cells at 24 h. ATF3 is, therefore, suggested as a new biomarker of activated migrating stem cells in the rat spinal cord in vitro that represents an advantageous tool to study basic properties of endogenous stem cells

Long-term follow-up of patients with Bartter syndrome type I and II

Background. Little information is available on a long-term follow-up in Bartter syndrome type I and II. Methods. Clinical presentation, treatment and long-term follow-up (5.0-21, median 11years) were evaluated in 15 Italian patients with homozygous (n = 7) or compound heterozygous (n = 8) mutations in the SLC12A1 (n = 10) or KCNJ1 (n = 5) genes. Results. Thirteen new mutations were identified. The 15 children were born pre-term with a normal for gestational age body weight. Medical treatment at the last follow-up control included supplementation with potassium in 13, non-steroidal anti-inflammatory agents in 12 and gastroprotective drugs in five patients. At last follow-up, body weight and height were within normal ranges in the patients. Glomerular filtration rate was <90mL/min/1.73m2 in four patients (one of them with a pathologically increased urinary protein excretion). In three patients, abdominal ultrasound detected gallstones. The group of patients with antenatal Bartter syndrome had a lower renin ratio (P < 0.05) and a higher standard deviation score (SDS) for height (P < 0.05) than a previously studied group of patients with classical Bartter syndrome. Conclusions. Patients with Bartter syndrome type I and II tend to present a satisfactory prognosis after a median follow-up of more than 10years. Gallstones might represent a new complication of antenatal Bartter syndrom

Activation of LXR Receptors and Inhibition of TRAP1 Causes Synthetic Lethality in Solid Tumors

Cholesterol is a pivotal factor for cancer cells to entertain their relentless growth. In this case, we provide a novel strategy to inhibit tumor growth by simultaneous activation of liver-X-receptors and interference with Tumor Necrosis Factor Receptor-associated Protein 1 (TRAP1). Informed by a transcriptomic and subsequent gene set enrichment analysis, we demonstrate that inhibition of TRAP1 results in suppression of the cholesterol synthesis pathway in stem-like and established glioblastoma (GBM) cells by destabilizing the transcription factor SREBP2. Notably, TRAP1 inhibition induced cell death, which was rescued by cholesterol and mevalonate. Activation of liver X receptor (LXR) by a clinically validated LXR agonist, LXR623, along with the TRAP1 inhibitor, gamitrinib (GTPP), results in synergistic reduction of tumor growth and cell death induction in a broad range of solid tumors, which is rescued by exogenous cholesterol. The LXR agonist and TRAP1 inhibitor mediated cell death is regulated at the level of Bcl-2 family proteins with an elevation of pro-apoptotic Noxa. Silencing of Noxa and its effector BAK attenuates cell death mediated by the combination treatment of LXR agonists and TRAP1 inhibition. Combined inhibition of TRAP1 and LXR agonists elicits a synergistic activation of the integrated stress response with an increase in activating transcription factor 4 (ATF4) driven by protein kinase RNA-like endoplasmic reticulum kinase (PERK). Silencing of ATF4 attenuates the increase of Noxa by using the combination treatment. Lastly, we demonstrate in patient-derived xenografts that the combination treatment of LXR623 and gamitrinib reduces tumor growth more potent than each compound. Taken together, these results suggest that TRAP1 inhibition and simultaneous activation of LXR might be a potent novel treatment strategy for solid malignancies

Long-term follow-up of patients with Bartter syndrome type I and II

BACKGROUND: Little information is available on a long-term follow-up in Bartter syndrome type I and II. METHODS: Clinical presentation, treatment and long-term follow-up (5.0-21, median 11 years) were evaluated in 15 Italian patients with homozygous (n = 7) or compound heterozygous (n = 8) mutations in the SLC12A1 (n = 10) or KCNJ1 (n = 5) genes. RESULTS: Thirteen new mutations were identified. The 15 children were born pre-term with a normal for gestational age body weight. Medical treatment at the last follow-up control included supplementation with potassium in 13, non-steroidal anti-inflammatory agents in 12 and gastroprotective drugs in five patients. At last follow-up, body weight and height were within normal ranges in the patients. Glomerular filtration rate was <90 mL/min/1.73 m(2) in four patients (one of them with a pathologically increased urinary protein excretion). In three patients, abdominal ultrasound detected gallstones. The group of patients with antenatal Bartter syndrome had a lower renin ratio (P < 0.05) and a higher standard deviation score (SDS) for height (P < 0.05) than a previously studied group of patients with classical Bartter syndrome. CONCLUSIONS: Patients with Bartter syndrome type I and II tend to present a satisfactory prognosis after a median follow-up of more than 10 years. Gallstones might represent a new complication of antenatal Bartter syndrome

ATF3 is a novel nuclear marker for migrating ependymal stem cells in the rat spinal cord

The present study identified ATF3 as a novel dynamic marker for ependymal stem/progenitor cells (nestin, vimentin and SOX2 positive) around the central canal of the neonatal or adult rat spinal cord. While quiescent ependymal cells showed cytoplasmic ATF3 expression, during 6-24. h in vitro these cells mobilized and acquired intense nuclear ATF3 staining. Their migratory pattern followed a centrifugal pathway toward the dorsal and ventral funiculi, reminiscent of the rostral migratory stream of the brain subventricular stem cells. Thus, the chain cell formation was, by analogy, termed funicular migratory stream (FMS). The FMS process preceded the strong proliferation of ependymal cells occurring only after 24. h in vitro. Pharmacological inhibition of MAPK-p38 and JNK/c-Jun (upstream effectors of ATF3 activation) prevented the FMS mobilization of ATF3 nuclear-positive cells. Excitotoxicity or ischemia-like conditions, reported to evoke neuronal and glial injury, did not further enhance migration of ependymal cells at 24. h, suggesting that, at this early stage of damage, the FMS phenomenon had peaked and that more extensive repair processes are delayed beyond this time point. ATF3 is, therefore, useful to identify activation and migration of endogenous stem cells of the rat spinal cord in vitro. \ua9 2014

Dual Inhibition of Bcl-2/Bcl-xL and XPO1 is synthetically lethal in glioblastoma model systems

XPO1 has recently emerged as a viable treatment target for solid malignancies, including glioblastoma (GBM), the most common primary malignant brain tumor in adults. However, given that tumors become commonly resistant to single treatments, the identification of combination therapies is critical. Therefore, we tested the hypothesis that inhibition of anti-apoptotic Bcl-2 family members and XPO1 are synthetically lethal. To this purpose, two clinically validated drug compounds, the BH3-mimetic, ABT263, and the XPO1 inhibitor, Selinexor, were used in preclinical GBM model systems. Our results show that inhibition of XPO1 reduces cellular viability in glioblastoma cell cultures. Moreover, addition of ABT263 significantly enhances the efficacy of XPO1 inhibition on the reduction of cellular viability, which occurs in a synergistic manner. While selinexor inhibits the proliferation of glioblastoma cells, the combination treatment of ABT263 and selinexor results in substantial induction of cell death, which is accompanied by activation of effector- initiator caspases and cleavage of PARP. Mechanistically we find that XPO1 inhibition results in down-regulation of anti-apoptotic Mcl-1 and attenuates ABT263 driven Mcl-1 up-regulation. Consistently, siRNA mediated silencing of Mcl-1 sensitizes for ABT263 mediated cell death and partially for the combination treatment. By using a human patient-derived xenograft model of glioblastoma in mice, we demonstrate that the combination treatment of ABT263 and Selinexor reduces tumor growth significantly more than each compound alone. Collectively, these results suggest that inhibition of XPO1 and Bcl-2/Bcl-xL might be a potential strategy for the treatment of malignant glial tumors

Activation of LXRβ inhibits tumor respiration and is synthetically lethal with Bcl-xL inhibition

Liver-X-receptor (LXR) agonists are known to bear anti-tumor activity. However, their efficacy is limited and additional insights regarding the underlying mechanism are necessary. By performing transcriptome analysis coupled with global polar metabolite screening, we show that LXR agonists, LXR623 and GW3965, enhance synergistically the anti-proliferative effect of BH3 mimetics in solid tumor malignancies, which is predominantly mediated by cell death with features of apoptosis and is rescued by exogenous cholesterol. Extracellular flux analysis and carbon tracing experiments (U-13C-glucose and U-13C-glutamine) reveal that within 5 h, activation of LXRβ results in reprogramming of tumor cell metabolism, leading to suppression of mitochondrial respiration, a phenomenon not observed in normal human astrocytes. LXR activation elicits a suppression of respiratory complexes at the protein level by reducing their stability. In turn, energy starvation drives an integrated stress response (ISR) that up-regulates pro-apoptotic Noxa in an ATF4-dependent manner. Cholesterol and nucleotides rescue from the ISR elicited by LXR agonists and from cell death induced by LXR agonists and BH3 mimetics. In conventional and patient-derived xenograft models of colon carcinoma, melanoma, and glioblastoma, the combination treatment of ABT263 and LXR agonists reduces tumor sizes significantly stronger than single treatments. Therefore, the combination treatment of LXR agonists and BH3 mimetics might be a viable efficacious treatment approach for solid malignancies

The association of indwelling urinary catheter with delirium in hospitalized patients and nursing home residents: an explorative analysis from the "Delirium Day 2015"

Backround: Use of indwelling urinary catheter (IUC) in older adults has negative consequences, including delirium. Aim: This analysis, from the "Delirium Day 2015", a nationwide multicenter prevalence study, aim to evaluate the association of IUC with delirium in hospitalized and Nursing Homes (NHs) patients. Methods: Patients underwent a comprehensive geriatric assessment, including the presence of IUC; inclusion criteria were age &gt; 65 years, being Italian speaker and providing informed consent; exclusion criteria were coma, aphasia, end-of-life status. Delirium was assessed using the 4AT test (score ≥ 4: possible delirium; scores 1-3: possible cognitive impairment). Results: Among 1867 hospitalized patients (mean age 82.0 ± 7.5 years, 58% female), 539 (28.9%) had IUC, 429 (22.9%) delirium and 675 (36.1%) cognitive impairment. IUC was significantly associated with cognitive impairment (OR 1.60, 95% CI 1.19-2.16) and delirium (2.45, 95% CI 1.73-3.47), this latter being significant also in the subset of patients without dementia (OR 2.28, 95% CI 1.52-3.43). Inattention and impaired alertness were also independently associated with IUC. Among 1454 NHs residents (mean age 84.4 ± 7.4 years, 70.% female), 63 (4.3%) had IUC, 535 (36.8%) a 4AT score ≥ 4, and 653 (44.9%) a 4AT score 1-3. The multivariate logistic regression analysis did not show a significant association between 4AT test or its specific items with IUC, neither in the subset of patients without dementia. Discussion: We confirmed a significant association between IUC and delirium in hospitalized patients but not in NHs residents. Conclusion: Environmental and clinical factors of acute setting might contribute to IUC-associated delirium occurrence

&quot;Delirium Day&quot;: A nationwide point prevalence study of delirium in older hospitalized patients using an easy standardized diagnostic tool

Background: To date, delirium prevalence in adult acute hospital populations has been estimated generally from pooled findings of single-center studies and/or among specific patient populations. Furthermore, the number of participants in these studies has not exceeded a few hundred. To overcome these limitations, we have determined, in a multicenter study, the prevalence of delirium over a single day among a large population of patients admitted to acute and rehabilitation hospital wards in Italy. Methods: This is a point prevalence study (called "Delirium Day") including 1867 older patients (aged 65 years or more) across 108 acute and 12 rehabilitation wards in Italian hospitals. Delirium was assessed on the same day in all patients using the 4AT, a validated and briefly administered tool which does not require training. We also collected data regarding motoric subtypes of delirium, functional and nutritional status, dementia, comorbidity, medications, feeding tubes, peripheral venous and urinary catheters, and physical restraints. Results: The mean sample age was 82.0 ± 7.5 years (58 % female). Overall, 429 patients (22.9 %) had delirium. Hypoactive was the commonest subtype (132/344 patients, 38.5 %), followed by mixed, hyperactive, and nonmotoric delirium. The prevalence was highest in Neurology (28.5 %) and Geriatrics (24.7 %), lowest in Rehabilitation (14.0 %), and intermediate in Orthopedic (20.6 %) and Internal Medicine wards (21.4 %). In a multivariable logistic regression, age (odds ratio [OR] 1.03, 95 % confidence interval [CI] 1.01-1.05), Activities of Daily Living dependence (OR 1.19, 95 % CI 1.12-1.27), dementia (OR 3.25, 95 % CI 2.41-4.38), malnutrition (OR 2.01, 95 % CI 1.29-3.14), and use of antipsychotics (OR 2.03, 95 % CI 1.45-2.82), feeding tubes (OR 2.51, 95 % CI 1.11-5.66), peripheral venous catheters (OR 1.41, 95 % CI 1.06-1.87), urinary catheters (OR 1.73, 95 % CI 1.30-2.29), and physical restraints (OR 1.84, 95 % CI 1.40-2.40) were associated with delirium. Admission to Neurology wards was also associated with delirium (OR 2.00, 95 % CI 1.29-3.14), while admission to other settings was not. Conclusions: Delirium occurred in more than one out of five patients in acute and rehabilitation hospital wards. Prevalence was highest in Neurology and lowest in Rehabilitation divisions. The "Delirium Day" project might become a useful method to assess delirium across hospital settings and a benchmarking platform for future surveys